Photosystem II: The Light-Driven Water:Plastoquinone Oxidoreductase (Advances in Photosynthesis and Respiration, 22) 1402042493, 9781402042492

Citation preview

Photosystem II

Advances in Photosynthesis and Respiration VOLUME 22 Series Editor: GOVINDJEE University of Illinois, Urbana, Illinois, U.S.A.

Consulting Editors: Julian EATON-RYE, Dunedin, New Zealand Christine H. FOYER, Harpenden, U.K. David B. KNAFF, Lubbock, Texas, U.S.A. Anthony L. MOORE, Brighton, U.K. Sabeeha MERCHANT, Los Angeles, California, U.S.A. Krishna NIYOGI, Berkeley, California, U.S.A. William PARSON, Seatle, Washington, U.S.A. Agepati RAGHAVENDRA, Hyderabad, India Gernot RENGER, Berlin, Germany

The scope of our series, beginning with volume 11, reflects the concept that photosynthesis and respiration are intertwined with respect to both the protein complexes involved and to the entire bioenergetic machinery of all life. Advances in Photosynthesis and Respiration is a book series that provides a comprehensive and state-of-the-art account of research in photosynthesis and respiration. Photosynthesis is the process by which higher plants, algae, and certain species of bacteria transform and store solar energy in the form of energy-rich organic molecules. These compounds are in turn used as the energy source for all growth and reproduction in these and almost all other organisms. As such, virtually all life on the planet ultimately depends on photosynthetic energy conversion. Respiration, which occurs in mitochondrial and bacterial membranes, utilizes energy present in organic molecules to fuel a wide range of metabolic reactions critical for cell growth and development. In addition, many photosynthetic organisms engage in energetically wasteful photorespiration that begins in the chloroplast with an oxygenation reaction catalyzed by the same enzyme responsible for capturing carbon dioxide in photosynthesis. This series of books spans topics from physics to agronomy and medicine, from femtosecond processes to season long production, from the photophysics of reaction centers, through the electrochemistry of intermediate electron transfer, to the physiology of whole orgamisms, and from X-ray christallography of proteins to the morphology or organelles and intact organisms. The goal of the series is to offer beginning researchers, advanced undergraduate students, graduate students, and even research specialists, a comprehensive, up-to-date picture of the remarkable advances across the full scope of research on photosynthesis, respiration and related processes. The titles published in this series are listed at the end of this volume and those of forthcoming volumes on the back cover.

Photosystem II The Light-Driven Water:Plastoquinone Oxidoreductase

Edited by

Thomas J. Wydrzynski The Australian National University, Canberra, Australia

and

Kimiyuki Satoh Okayama University, Okayama, Japan

Assistant Editor

Joel A. Freeman

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN-10 ISBN-13 ISBN-10 ISBN-13

1-4020-4249-3 (HB) 978-1-4020-4249-2 (HB) 1-4020-4254-X (e-book) 978-1-4020-4254-6 (e-book)

Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands.

www.springeronline.com Front Cover Image. An underwater scene of sp. from Ewans Ponds conservation area, V South Australia. Note the stream of oxygen gas bubbles being produced by Photosystem II. Photograph was taken by Warwick Hillier. The camera ready text was prepared by Lawrence A. Orr, Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizon 85287-1604, U.S.A.

Printed on acid-free paper

All Rights Reserved © 2005 Springer No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed in the Netherlands.

'SPN
UIF
4FSJFT
&EJUPS "EWBODFT
JO
1IPUPTZOUIFTJT
BOE
3FTQJSBUJPO 7PMVNF

1IPUPTZTUFN
**
5IF
-JH J IU%SJWFO 8BUFS1MBTUPRVJOPOF 0YJEPSFEVDUBTF ) AM DELIGHTED D TO ANNOUNCE THE PUBLI U CATION IN D HOTOSYNTH T ESISAND D 2ESPIRATION !)0( !DVANCESIN0 3ERIES OF 0HOTOSYSTEM))4HE,IGHT $RIVEN7ATER 0LASTOQUINONE/XIDORE D DUCTASE D E ABOOKCOVER K INGTHE F LIFEON F CENTRAL ROLEOFF THEOXYGEN EVOLVINGSYSTEMFOR EARTHITDEALSWITHBOTHTHESTRUCTUREANDTHEFUNCTION D AUTHORITIES OFF THISUNIQUEPROCESS4WO DISTINGUISHED HAVEEDITED D THISVOLUME4HOMAS*7YDRZYNSKIOF !USTRALIAAND+IMIYUKI3ATOHOF*APAN3OMEOFTHE EARLIERVOLUMESHAVE INCLUDED DESCRIPTIONSOFF 0HOTO SYSTEM))6OLUME /XYGENIC0HOTOSYNTH T ESIS4H 4 E ,IGHT2EACTIONS EDITEDBY$ONALD2/RTAND#HARLES &9OCUM 6OLUME  0HOTOSYNTHESIS 0 0 0HOTOBIO CHEMISTRYANDD 0HOTOBIOPHYSICS H AUTHORED BY"ACON + +E AND6OLUME # #HLOROPHYLLA&LUORESCENCE OF 0HOTOSY S NTH T ESIS EDITED BY 'EORGE# !3IG IGNATUREO 0APAGEORGIOUAND'OV D INDJEE 4HECURRENTVOLUME FOLLOWSTHE  VOLUMES LISTED BELOW

% 6OLUME,IPIDSIN0 D HOTOSY S NTH T ESIS3TRUCTURE & &UNCTIONAN D 'ENETICS #HAPTERSPAGES  EDITED BY 0AUL !NDRÏ 3IEGENTHALER AND .ORIO -URATA FFROM 3WITZERLAND AND *APAN )3".     % 6OLUME  4HE -OLECULAR "IOLOGY OF #HLORO R PLASTS ANDD -ITOCHONDRIA D IN #HLAMYD M DOMONAS #HAPTERSPAGESEDITED BY*EAN $AVID2OCHAIX -ICHEL'OLDSCHMIDT #LERMONT ANDD 3ABEEHA -ERCHANT FFROM 3WITZERLANDD AND 53!)3".     % 6OLUME 4HE 0HOTOCHEMISTRY OF #AROTENOIDS D #HAPTERSPAGESEDITED BY(ARRY !&RANK !NDREW*9OUNG 'EORGE"RITTONAND 2ICHARD*#OGDELL FROM53!AND5+)3".      % 6OLUME0HOTOSYNTH T ESIS0HYSIO H LOGYANDD -E TABOLISM #HAPTERSPAGESEDITED BY 2ICHARD#,EE D GOOD 4 D HOMAS$3HARK R EYAND 3USANNE VON #AEMMERER FFROM 5+ 53! AND !USTRALIA)3".     % 6OLUME0HOTOSY S NTH T ESIS0HOTOBIOCHEMISTRY R ANDD 0HOTOBIOPHYSICS H #HAPTERSPAGES AUTHOREDBY"ACON+E FROM53!)3".      % 6OLUME  2EGULATION OF OF 0HOTOSYNTH T ESIS  #HAPTERSPAGESEDITEDBY%VA -ARI !RO ANDD "ERTIL !NDERSSON FFROM &INLANDD AND 3WEDEN)3".     % 6OLUME0HOTOSYNTHETIC.ITROGEN!SSIMILATION AND!SSOCIATE D D #ARBONANDD 2ESPIRATORRY-ETABO LISM #HAPTERSPAGESEDITED BY #HRISTINE&OYERAND'RAHAM.OCTOR FROM5+ ANDD &RANCE  % 6OLUME  ,IGHT (ARVESTING !NTENNAS  #HAPTERSPAGESEDITEDBY"EVERLEY 'REEN AND D 7ILLIAM 0ARSON FFROM #ANADA AND 53!)3".     % 6OLUME0HOTOSYNTHESISIN!LGAEE #HAPTERS  PAGES  EDITED BY !NTHONY N ,ARK RKUM

1VCMJTIFE
7PMVNFT m

% 6 6OLUME -OLECULAR"IOLOGYOF OF #YANOBACTERIA #HAPTERSPAGESEDITEDBY$ONALD !"RYANT FROM53!)3".    F  % 6O 6 LUME!NOXYGENIC0HOTOSYNTH T ETIC"ACTERIA #HAPTERSPAGESEDITEDBY2OBERT %"LANKENSHIP -ICHAEL 4-ADIGANAND#AR D L % "AUER FROM53!)3".    F  6

% 6OLUME"IOPHYSICAL4ECHNIQUESIN0HOTOSYN THESIS #HAPTERSPAGESEDITED BY THELATE*AN!MESZAND D THELATE!RNOLD*(O D FF F FROM4HE.ETHERLANDS)3".     6 LUME/XYG X GENIC0HOTOSY S NTH T ESIS4H 4 E,IGH I T % 6O 2EACTIONS #HAPTERSPAGESEDITED BY$ONALD2/RTAND#HARLES&9OCUM FROM 53!)3".     % 6O 6 LUME0HOTOSYNTH T ESISAND D THE%NVIRONMENT #HAPTERSPAGESEDITEDBY.EIL 2"AKER FROM5+)3".    F  V

OF F EVOLUTION ONE COULDNT HAVE MODELED IT n YEARS AGO DESPITE ALL THE ADVANCES IN CHEMISTRY PHYSICSAND BIOLOGY)TCONS Y ISTSOFF A LIGHT HARVESTING UNITANTENNA ANDD AREACTIONCENTERUNITTHATOPER ATESATANUNUSUALLYHIGHREDOXPOTENTIALITISTHIS LATTERUNIQUECHARACTERISTICTHATALLOWS ITTOOXIDIZE WATERTOOXYGENAT ITS@OXYGEN EVOLVINGCOMPLEX! BOOKONTHISUNIQUESYSTEMHASBEENEDITEDBYTWO F HESTRUCTURE OUTSTANDINGAUTHORITIESINTHEAREAOFT F IONOFF THEOXYGEN EVOLVING0HOTOSYSTEM ANDD THEFUNCT ))4HOMAS*7YDRZYNSKIOFTHE2ESEARCH3CHOOL OFF "IOLOGICAL 3CIENCES 4HE !USTRALIAN .ATIONAL 5NIVERSITY #ANBERRA !USTRALIA ANDD +IMIYUKI3ATOH OFTHE$EPARTMENTOF"IOLOGY /KAYAMA5NIVERSITY /KAY A AMA *APAN  2ESPIRING ORGANISMS INCLUDING HUMANS ON THIS PLANETDEPENDONTHEOXYGENTHATGREENPLANTSGENER ATETHROUGH0HOTOSYSTEM))4HUS THISBOOK ISAVERY IMPORTANTADDITIONTOTHEALREADYPUBLI U SHED BOOKSIN THE!)0(3ERIES)TESSENTIALLYADDRESSESWATERlRST AS A SOURCE OF F THE ELECTRONS THAT ARE NECESSARY FO F R THEREDUCT D IVESYNTHESESOFORGAN F ICMATTER ANDD THEN ASASOURCEOFMOLECULAROXYGENTHATISNECESSARYFOR THEENERGY PRODUC D ING CATABOLICOXIDATIONS INCLUDING RESPIRATION $URINGTHELASTDECADE ORSO DRAMATICADVANCES HAVE BEEN MADE IN ELUCIDATING THE STRUCTURE OF 0HOTOSYSTEM))TONEARATOMICSCALEWITH THE8 RAY CRYSTALLOGRAPHY ANDINRELATINGITTOITSBIOPHYSICAL BIOCHEMICAL ANDD MOLECULAR BIOLOGICAL PROPERTIES F HAPTERS AUTHORED BY INTERNATIONALLY 4HIRTY FOURC ACKNOWLEDGEDEXPERTS SUMMARIZETHISEXTRAORDINARY F SCIENTIlC PROGRESS COVERING AREAS THAT RANGE FROM THECAPTUREOF mEETINGPHOTONS THEIRCONVERSION INTO CHEMICAL ENERGY OXIDATION REDUCTION TO THE DY NAMICREGULATORY PROCESSESTHATSUSTAINANDOPT D IMIZE THEPHOTOSYNTHETICOXIDATIONOFF WATER!DISCUSSION ISALSOPROVIDEDONTHEBEGINNINGSOF0HOTOSYSTEM)) ANDD PHOTOSYNTHESISMORETHANBILLIONYEARSAGO IN THE!RCHAEAN%RA ANDD ON ITSEVENTFU F LEVOLUTIONTOTHE PRESENTDAYDIVERSITYOFMICROBIALANDHIGHERPLANTS ,ASTLY T Y HEDESIGNOFF ARTIlCIAL BIOMIMETIC 0HOTOSYS TEMS)) ISALSODISCUSSED7HOKNOWS ONE K DAYTHESE SYSTEMSMAYSERVETHENEEDSOFHUMANITYEITHERON %ARTH ORONSOMEDISTANTOUTPOST INSPACE D 4HE BOOK IS DESIGNEDD TO BE USED BY GRADUATE STUDENTS BEGINNING RESEARCHERS AND ADVANCED UN DERGRADUATESTU D DENTSINTHEAREASOFF PLANTSCIENCES MICROBIOLOGY CELLANDD MOLECULARBIOLOGY BIOCHEM ISTRY BIOPHYSICS BIOENERGETICSANDCHEMISTRY ASWELL ASTHOSEINAGRICULTUREAND BIOTECHNOLOGY

3USAN$OUGLASAND D *OHN2AVEN FROM!USTRA F LIA #ANADAAND5+)3".     % 6O 6 LUME2ESPIRATIONIN!RCHAEAAND D "ACTE RIA $IVERSITYOF OF 0ROKARYOTIC% K LECTRON4RANSPORT # #ARRIERS #HAPTERSPAGESEDITED BY$AVIDE:ANNONI FROM)TA F LY)3".     6 SPIRATIONIN!RCHAEAAND"ACTERIA % 6OLUME2ES OF 0ROKAR K RYOTIC2ESPIRATORY R 3Y 3YSTEMS $IVERSITY T OF CHAPTERSPAGESEDITED BY$AVIDE :ANNONI FROM)TALY)3".     6 LUME0LANT-ITOC L HONDRIA&ROM'ENOMETO D % 6O &UNCTION #HAPTERSPAGESEDITED BY$AVID!$AY !(ARVEY-ILLARAND*AMES 7HELAN FFROM !USTRALIA )3".      6OLUME  0LANT 2ESSPIRATION &ROM #ELL TO % 6 %COSY SYSTEM #HAPTERSPAGESEDITED BY(ANS,AMB M ERS AND D -IQUEL 2IBAS #ARB R OFROM F !USTRALIAAND3PAIN)3".    % 6O 6 LUME  # #HLOROPHYLLL A &LUORESCENCE ! T ESIS #HAPTERS 3IGNATUREOF OF 0HOTOSYNTH PAGESEDITEDBY'EORGE#0APAGEORGIOU AND D 'OVINDJEE FROM'REECEAN F D 53!)3".    8  % 6OLUME$ISCOVERIESIN0HOTOSYNTHESIS 6  #HAPTERS   XXX PAGES  EDITED BY 'OVINDJEE *4HOMAS"EATTY (OWARD D 'ESTAND *OHN&!LLEN FROM53! #ANADAAND3WEDEN 5+ )3".    AND % 6O 6 LUME  0HOTOP O ROTECTION 0HOTOINHIBITION 'ENE2EGULATION,AND%NVIRONMENTT #HAPTERS ^PAGESEDITED BY "ARB R ARA$EMMIG 7 AM 7 !DAMS ))) AND D !UTAR +. !DAMS 7ILLI -ATTOO ALLFROM53!)3".    &ORA DESCRIPTIONOFT F HESCOPEOFF THE!)0(3ERIES K COVEROF THISBOOK&URTHERINFORMAT F ION SEETHEBACK ONTHESEBOOKSANDORDERINGINSTRUCTIONSCANBEFOUND ATHTTPWWWSPRINGERONLINECOMUNDERTHE"OOK D 2ESPIRATION 3ERIES@!DVANCES IN0HOTOSYNTHESISAND 3PECIALDISCOUNTSAREAVAILABLEFORMEMBERSOFTHE )NTERNATIONAL 3OCIETY OFF 0HOTOSYNTHESIS 2ESEARCH )302HTTPWWWP  HOTOSYNTH T ESISRESEARCHORG 4O ENSUREYOURDISCOUNT PLEASEORDERTHROUGH.OELINE 'IBSON SPRINGER SBMCOM 1IPUPTZTUFN
**
5IF
-JHIU%SJWFO
8BUFS1MBTUPRVJOPOF
0YJEPSFEVDUBTF 0HOTOSYSTEM))ISTRULYANUNPRECEDENTED DISCOVERY VI

4HISBOOK ISAPPROPRIATELYDEDICATEDTOAP D IONEER IN THE lELD 'ERALD 4 "ABCOCK THE DEDICATION IS AUTHORED BY#HARLES&9OCUM 2OBERT"LANKENSHIP D 3HELAGH&ERGUSON -ILLER ALL OFF 53! +IMIYUKI AND 3ATOH *APAN 4HOMAS * 7YDRZYNSKI !USTRALIA D 'OVINDJEE 53! PROVIDE AN )NTR TRODUCTION TO AND 0HOTOSYSTEM))AND D THECHAPTERS INTHI T SVOLUME#H # AP A TER )TISFOLLOWEDBYlVECHAPTERSTHATDEALWITH THE 0ROTEIN#ONSTITUENTSOF OF 0HOTOSY SYSTEM)))"EVERLEY 'REEN #ANADA AND D %LISABETH 'ANTT53! DISCUSS THEDISTALANDEXTRINSICANTENNA#HAP A TER *ULIAN %ATON 2Y 2 E.EW:EALAND AND D #INDY0UTNAM %VANS 53! SUMMARIZEOURUNDERSTANDINGOFT F THE#0AND #0COREANTENNACOMPONENTS#HA # APTER  0ETER .IXON5+ -ARY3ARCINA 5+ AND D "RUCE$INER 53! PROVIDEANACCOUNTOFF THE$AND D $CORE PROTEINS #HAP A TER 4HISISFOLLOWEDBY #HA # APTER BY4ERRY"RICKER53! AND D 2OBERT"URNAP53! ON THEOXYGENENHANCINGEXTRINSICPROTEINS AND #HAP A TER  BY,EEANN4HORNTON53! *OHNNA2OOSE53! (IMADR D I0AK AKRASI 53! AND D -ASAAHIKO)KEUCHI *APAN ONTHE LOWMOLECULARWEIGHTCOMPONENTS 4HENEXTNINECHAP A TERSFOCUSONTHE /RGANIZATIONOF THE&UNCTIONAL3ITESIN0HOTOSYSTEM))'ERNOT2ENGER ) ) AND D !LFRE F D (OLZWARTH BOTHOFF 'ERMANY DISCUSSTHE PRIMARYELECTRONTRANSFER#HAP A TER 6ASILI0ETROU 

LEAS 'REECE AND D !NTHONY N #ROFTS F 53! SUMMARIZE INFORMAT F IONONTHE QU Q INONE IRON ACCEPTORCOMPLEX #HAP A TER  "RUCE$INERAND$AVID"RITTBOTHOF 53! DISCUSSTHEREDOXACTIVETYROSINES9: AND 9$ #H # AP A TER   6I 6 TTAL 9ACHANDRA 53! SUMMARIZES THECURRENTUNDERSTANDINGABOUTTHEORGANIZATIONOF THEMANGANESEIONSOFT F HEMANGANESECLUSTEROFF THE / EVOLVINGCOMPLEX #HAP A TER 2ICHARD D $EBUS 53! SUMMARIZES WHAT IS KNOWN ABOUT PROTEIN LIGANDS OFF THE MANGANESE CLUSTER #H # APTER   +ARIN¯HRLING!USTRALIA 2ONALD0ACE!USTRALIA AND -ICHAEL %VANS 5+ PROVIDE INFORMATION ON D THEIRIMPLICATIONSON SPECTROSCOPICOBSERVATIONSAND F IONAL DETAILSOFCATA F LYTICMANGA STRUCTURAL AND FUNCT NESECLUSTER#HAP A TER (ANSVAN'ORKOM4HE .ETHERLANDS AND# D HARLES9OCUM 53! DISCUSSTHE ROLES OF F #ALCIUM AND D #HLORIDE IONS #HAP A TER   *ACKVAN2ENSEN4HE.ETHERLANDS AND6YACHESLAV +LIMOV2USSIA ADDRESST D HEUNIQUEROLEOF BICARB R ON ATEONTHEACCEPTORSIDEAND D THEDONORSIDEOFF 0HO TOSYSTEM))REACTIONCENTER#HAP A TER AND0ETER &ALLER #HRISTIAN&UFEZANAN F D 7ILLIAM2UTHERFOR F D ALL F OFF &RANCE EXAMINETHESECONDARYELECTRONTRANSFER PATHWAYSAROUNDTHE0HOTOSYSTEM))REACTIONCENTER #H # APTER 

3UBSEQUENTLY INTHENEXTSIXCHAPTERS THEFOCUS SHIFTS TO THE 3TRUCTURAL "ASIS FOR 0HOTOSYSTEM )) ) 4AK AKUMI.OGUCHI *APAN ANDD #ATTHERINE"ERTH T OMIEU &RANCE ANALYZET L THEMOLECULARSTRUCTUREOFT F THE INTERME DIATESOFTHESYSTEM USINGINFORMATION OBTAINEDD FROM # APTER 2OBERT"ITTL  VIBRATIONALSPECTROSCOPY #H 'ERMANY AND!SA D KO+AWAMORI *APAN SUMMARIZE THECONlGURATIONOFTHEELECTRONTRANSPORTINTERMEDI ATESOFF 0HOTOSYSTEM)) ASOBTAINED BYELECTRONPARA MAGNETICRESONANCESPECTROSCOPY #H # AP A TER "EN  (ANKAMER!USTRALIA *AMES"ARBER5+ AND*ON .IELD 5+ DESCRIBETHESTRUCTUREOFF THECOREANTENNA HOLOCOMPLEXASVISUALIZED BYELECTRONMICROSCOPY #HAP A TER (ORST7ITT 'ERMANY N DISCUSSESTHE lRSTTHREE DIMENSIONAL STRUCTUREOFF 0HOTOSYSTEM)) OBTAINED BY8 RAYCRYSTALLOGRAPHYANDOTHERBIOPHYSI CALMETHODS#HAP A TER *IAN 2EN3HENAND.OBUO +AMIYABOTHOF*APAN F DISCUSSTHISSTRUCTURE USING ALSO8 RAYCRYSTALLOGRAPHY H #H # AP A TER ANDD *AMES "ARBERAND3O)WATABOTHOF5+ DISCUSSASOMEWHAT D D ITSIMPLICATIONSTOTHEFUNCT F ION RElNEDSTRUCTURE AN # AP A TER  OFF 0HOTOSYSTEM)) #H 4HESECHAPTERSONTHESTRUCTUREAREFOLLOWEDBYFOUR U $ NAMICSOF OF 0HOTOSY SYSTEM)) CHAPTERSON-OLECULAR$Y ,AURA "ARTER 5+ $AVID D +LUG 5+ ANDD 2IENK VAN 'RONDELLE 4HE .ETHERLANDS SUMMARIZE OUR UNDERSTANDING ABOUTEXCITATIONENERGY TRAPPINGAND ITSEQUILIBRATION#HAP A TER "ARRY0OGSON!US TRALIA (EATHER2ISSLER!USTRALIA AND(ARRY&RANK 53! ELABORATE THE ROLE OF F CAROTENOIDS IN ENERGY QUENCHING#HAP A TER 6LADIMIR3HINKAREV 53! DISCUSSESTHEPATTERNANDTHEANALYSESOFTHE/ EVO LUTION INATRAINOF LIGHTmASHES#HAPTER AND 7 ICKK (ILLIER!USTRALIA AND*O 7ARW D HANNES-ESSINGER 'ERMANY PRESENTANOVERVIEWONTHEMECHANISM OFWATEROX F IDATION#HAPTER  4HISIS FO F LLOWED BYA DISCUSSIONOF !SSEMBLYAN L D "IODYNAMICS OF 0HOTOSYSTEM ))) IN FOUR CHAPTERS #HARLES$ISMUKES 'ENNADY!NANY N EVANDD 2ICHARD 7 ALL OF 7ATT F 53! DISCUSS THE PHOTOASSEMBLY OF THECATALYTICMANGANESECLUSTER#HAP A TER 7AH   3OON#HOW !USTRALIA ANDD %VA -ARI !RO&INLAND SUMMARIZEOURUNDERSTANDINGOFF PHOTOINACTIVATION ANDMECHANISMSOFRECOVERY#HA # APTER +ENICHI  9 9AMA GUCHI 53! 3TEPHEN -AYl A ELD 53! AND -AMORU 3UGITA *APAN PRESENT A CURRENT PICTURE OF TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF GENEEXPRESSION #HAPTER ANDD 3TEVEN4HEG AND ,AN 8IN 3HI BOTH OFF 53! DISCUSS TRANSPORT AND POST TRANSLATIONAL PROCESSING IN BIOSYNTHESIS AND HOMEOSTATIS#H # APTER  VII

PROVIDEDD NOTONLYTHEEVIDENCEFORT F HESERIESSCHEME OFPHOTOSYNTHESIS BUTWASTHElRSTPAPERTOCALLTHE D D CYTOCHROME SYSTEMTHATOXIDIZEDD WATERAND REDUCE L HOTOSYSTEM)) WH W EREASTH T E AS@3YSTEMCURRENTLY 0 OTHERSYSTEMTHATOXIDIZEDCYTOCHROMEAS@3YSTEM CURRENTLY Y 0HOTOSYSTEM )  )N THIS SEMINAL PAPER $UYSENSANDD COLLEAGUES HADRECOGN D IZEDD NOTONLYTH T EIR OWNWORK BUTTHATOF2OBERT%MERSONWITH-ARCIA "RODY %UGENE 2ABINOWITCH WITH %MERSON AND A NI 'OVINDJEEANDD *AN"4HOMAS #3TACY WITH 2AJ &RENCH WITH 6+ 9OUNG AND WITH *ACK -YERS .ORMAN"ISHOP AND,EO6ERNON D WITH ,0:AUGG  3INCEINSUNLIGHT BOTH SYSTEMSAREEXCITEDD SIMULTANE OUSLYANDBEGINTOFUNCTIONALMOSTSIMULTANEOUSLY ITISNOTAQUESTIONOFW F WHICH STARTS lRST4HENAMING OFT F HESYSTEM ISTHUSARBI R TRARY Y

4HISISFO F LLOWED BYA DISCUSSIONOFF THE #OM # MPARI SONOF0HOTOSYSTEM))WITH/THER.ATURAL!RTIlCIAL 3YSTEM Y S IN FFOUR CHAPTERS #HARLES $ISMUKES AND 2OBERT "LANKENSHIP BOTH OF F 53! DESCRIBE THE ORIGINSANDTHEEVOLUTIONOFOXYGENICPHOTOSYNTHE SIS #HAPTER 'ARY"RUDVIG 53! AND D -ÌRTEN 7IKSTRÚMPRESENTMECHANISTICCOMPARISONS BETWEEN 0HOTOSYSTEM))AND#YTOCHROME C OXIDASE#HA # APTER   ,ÈZLØ +ÈLMÈN (UNGARY *O!NN 7ILLIAMS 53! AND*AMES! D LLEN53! SUMMARIZERESEARCH ON MIMICKING THE PROPERTIES OF 0HOTOSYSTEM )) IN PURPLEBACTERIALREACTIONCENTERS#H # APTER "RIAN 'IBNEY53! AND#EC D ILIA4OMMOS 3WEDEN DISCUSS DENOVOPROTEINDESIGNINRESPIRATIONANDPHOTOSYN THESIS#H # APTER AND D !NN-AGNUSON 3TENBJÚRN D ,EIF(AMMARSTRÚM F ALLOFF 3WEDEN END 3TYRINGAND THISBOOKWITHANUNDERSTANDINGOF0HOTOSYSTEM)) THROUGH ARTIlCIALPHOTOSYNTHESIS

'VUVSF "*1) #PPLT "
#JU
PG
)JTUPSZ
'JSTU
$MFBS
&WJEFODF
PG
UIF 4FSJFT
4DIFNF
BOE
UIF
/BNJOH
PG 4ZTUFN  /PX
1IPUPTZTUFN
** CZ
-PVJT
/.
%VZ TFOT
+BO
"NFT[
BOE
#
.
,BNQ
JO


4HEREADERSOFT F HECURRENTSERIESAREENCOURAGEDD TO WATCH FORT F HEPUBLI U CATIONOFF THE FORT F HCOMINGBOOKS NOTNECESSARILYARRANGEDINTHEORDEROFFUTUREAP PEARANCE  % 4HE3TRUCTUREANDD &UNCTIONOOF 0LASTIDS D %DITORS 2OBERT7ISEAND*+ENNETH(OOBEREXPECTED TOCONTAIN#HAPTERSANDD ^PAGES)3".      % "IOCHEMISTRY "IOPHYSICSAND"IOLOGICAL&UNC TIONSOF OF #HLOROPHYLLS %DITORS"ERNHARDD 'RIMM 2OBERT * 0ORRA 7OLFHART 2àDIGER AND D (UGO 3CHEER  % 0HOTOSY SYSTEM)4 4HE,IGH I T $RIVEN0LASTOCY C ANIN & &ERRE D DOXIN / DORE /XI D DUCTASE D %DITOR *OHN 'OL BECK  % "IOPHYSICAL 4ECHNIQUES ))) %DITORS  4HIJS * !ARTSMAAND*ÚRG-ATYS D IK  % 0HOTOSYNTHESIS!#OMPREHENSIVE4REATISE"IO CHEMISTRY RY "IOPHYSICS0 Y HYSIO Y LOGY O ANDD -OLECULAR L "IOLOGY 0ART %DITORS*ULIAN%ATON 2YEAND "AISHNAB4RIPATHY AND % 0HOTOSY S NTH T ESIS!#OMPREHENSIVE4REATISE"IO CHEMISTRY "IOPHYSICS0 H H HYSIO LOGYANDD -OLECULAR L "IOLOGY 0ART %DITORS"AISHNAB4RIPATHYAND 2 E  *ULIAN%ATON 2Y )N ADDITION TO THESE CONTRACTED BOOKS WE ARE ALREADY IN TOUCH WITH PROSPECTIVE %DITORS FOR THE F LLOWING BOOKS FO % -OLECULAR"IOLOGYOF#YANO F BACTERIA)) % 0ROTONATIONAND!403YNTHASES % 'ENOMICSANDD 0ROTEOMICS

!RECENTLYPUBLISHEDTIME LINEONOXYGENICPHOTOSYN THESISCOVERSMANY N ASPECTSOFF THE HISTORY OFF @0HOTO SYSTEM)) SEE'OVINDJEEAND D $+ROGMANN $ISCOVERIESINOXYGENICPHOTOSYNTHESISn  !PERSPECTIVE0HOTOSYNTH 2ESn #HAPTER OFF THISBOOK BY+3ATOH 4*7YDRZYNSKIAND D 'OVIN DJEEINCLUDESAHISTORICALACCOUNTOF0HOTOSYSTEM)) FORRE F F FERENCES SEET HISCHAPTER )NAPAPER PUBLI U SHED ON-AY  ,OUIS.-$UYSENS *AN!MESZ AND "- +AMP 4WO PHOTOCHEMICAL SYSTEMS IN PHOTOSYNTHESIS.ATURE  USED FORT F HE lRSTTIMETHENAME@3YSTEMFORT F HEPHOTOSYSTEM RESPONSIBLEFORTHEACTIONSPECTRUMOFCHLOROPHYLL A mUORESCENCE ITWASTHESYSTEMTHATWASSUGGESTED TOOXIDIZEWATERTOOXYGEN AND D REDUCECYTOC D HROME @3YSTEM ONTHEOTHERHAND OXIDIZEDCYTOCHROME AND D REDUCE D D PYRIDINENUCLEOTIDE$UYSENSAND D COL LEAGUESADDED lRSTRED LIGHT NM LIGHTABSORB R ED MAINLYBYCHLOROPHYLL ANDOBSERVEDOXIDATIONOFA D ALGA 0ORP 0 HYRIDIUM CRUENTUM CYTOCHROME IN A RED D THENTHEYADDEDGREEN D LIGHTNMABSORB R ED AND MAINLYINPHYCOERYTHRIN ANDOBSERVEDREDUCTIONOF THISOXIDIZED D CYTOCHROME!DDITIONOFF THE HERBI R CIDE $#-5ELIMINATED D THEREDUCT D IONOFCYTOC F HROME BY GREENLIGHT BUTNOTITSOXIDATIONBYREDLIGHT4HIS F F LIGHTAND D ONCYTOCHROME ANTAGONISTICEFFECTO VIII

'IBSONBOTH OFF 3PRINGER FORT F HEIRFR F IENDLYWORKI R NG RELATIONSHIPWITHUSTHATLEDTOTHEPRODUCTIONOFTHIS BOOK ) THANKK *EFFF (AAS $IRECTOR OFF )NFORMAT F ION F 3CIENCES 5NIVERSITY OFF )LLINOIS 4ECHNOLOGY ,IFE AT 5RBANA #HAMPAIGN 5)5# AND %VAN $E,UCIA (EAD $EPARTMENTO D F 0LANT"IOLOGY 5)5# Y FORT F HEIR SUPPORT-YFAM F ILYMYWIFE2A F AJNI'OVINDJEEOUR DAUGHTER!NITA HER HUSBAND -ORTEN #HRISTIANSEN N AY AY HIS ANDD OUR GRAND DAUGHTER3UNITAOURSON3ANJ WIFE-AR F ILYN ANDD OURGRANDSONS!RJ R UNANDD 2AAJIV HASBEENVERYSUPPORTIVEDURINGTHEPREPARATIONOF THIS BOOK ) AMGRATEFU F LTO'EORGE0APAGEORGIOUFOR F HISSUG GESTIONSTOIMPROVETHIS%DITORIAL 3PECIAL THANKSGOTO,ARRY /RRFOR F HISWONDERFU F L WORK R INTYPESETTINGTHIS BOOK(ISCONSTANTADVISE TOTHEEDITORSANDHISOUTSTANDINGINTERACTIONSWITH ALL THOSE INVOLVED IN IN THIS BOOK K ARE A SOURCE OF INSPIRATIONTOALL OFUS F 

% 0ROTEIN#OMPLEXESOFF 2ESPIRATIONAND D 0HOTOSYN THESIS % 3ULFUR-ETA F BOLISMIN0HOTOSYNTHETIC3YSTEMS % -OLECULAR"IOLOGYOF3TRESS F IN 0LANTS % 'LOBAL!SPECTSOF0HOTOSYNTHESISAND2ESPIRA TION VOLUMES % !RTIlCIAL 0HOTOSYNTHESIS 2EADERSAREENCOURAGEDTOSENDTHEIRSUGGESTIONS F FUTUREVO FOR F LUMESTOPICS NAMESOF FUTUREE F DITORS D OF FUTUREAUT F HORS TOME BY% MAIL GOV UIUC AND EDU ORFAX     )N VIEW OFF THE INTERDISCIPLINARY CHARACTER OFF RE SEARCH IN PHOTOSYNTHESIS AND D RESPIRATION IT IS MY EARNESTHOPETHATTHISSERIESOFBOOKSWILLBEUSEDIN D INGSTUDENTSAND D RESEARCHERSNOTONLY IN0LANT EDUCAT 3CIENCES -OLECULAR AND D #ELL "IOLOGY )NTEGRATIVE "IOLOGY "IOTECHNOLOGY !GRICULTURAL3CIENCES -I CROBIOLOGY " Y IOCHEMISTRY AN Y D "IOPHYSICS BUTALSO IN"IOENGINEERING #HEMISTRY AND D 0HYS H ICS

!UGUST  'OVINDJEE 3ERIES%DITOR !DVANCESIN0HOTOSYNTHESISAND 2ESPIRATION R ANA #HAMPAIGN 5NIVERSITYOFF )LLINOISAT5RB $EPARTMENTOF0LANT"IOLOGY 5RB R ANA ),  53! % MAILGOV UIUCEDU 52,HTTPWWWLIFEUIUCEDUGOVINDJEE

"DLOPXMFEHNFOUT ) TAKETHISOPPORTUNITY TOTHANK K 4HOMAS*7YD 7 RZYN SKI AND D +IMIYUKI 3ATOH FFOR THEIR OUTSTANDING AND PAINSTAKINGEDITORIALWORK7EAREGRATEFULTOTHEM F PERSONALLY SUB FOR U SIDIZINGOFF THECOLORPLATES INTHISVOLUME)THANKA K LLTHEAUTHORSOFVO F LUME WITHOUTTHEIRAUTHORITATIVECHAPTERS THEREWOULD BENOSUCH VOLUME)THANK K *ACCO&LIPSENAND D .OELINE

IX

'OVINDJEEIN3OUTH !FR F ICA 

IN .!$0 REDUCTION IN CHLOROPLASTS    4OGETHERWITH HISGRADUATESTU D DENTS HEDISCOVERED AnNMEMISSIONBANDD WHENPHOTOSYNTHESIS WAS SATURATED OR BLOCKED MADE THE lRST MEASURE MENTSONTHETEMPERATURE DEPENDENCE DOWNTO+ F HE OF #HL A mUORESCENCE PROVIDEDD EXPLANATIONFORT mUORESCENCETRANSIENTCURVEINTERMSOFATRAFlCJAM OFE F LECTRONSONTHEACCEPTORSIDEOFF 03) ASWELL AS IN TERMS OFF NON 1! RELATEDD CHANGES !GAIN WITH SEVERAL GRADUATE STUDENTS HE FOCUSED ON THE ROLE ANDD THESITEOF BICARB R ONATE IONSON03)) PARTICULARLY ON ITS ACCEPTOR SIDE A ROLE OF BICARB R ONATE ON THE PROTONATIONATTHE1" SITEWASSUGGESTED(ISOTHER MAJ A ORCONTRIBUTIONS HAVEBEENTHElRSTPICOSECOND MEASUREMENTSONTH T EPRIMARYCHARGESEPARATIONIN03 )) ANDTHETHEORYOF@THERMOLUMINESCENCEFROM03 )))T ISWORTHMENTIONING THAT'EORGE#0APAGEOR GIOU ANDD 4HOMAS*7YDRZYNSKI EDITOROFF VOLUME ANDTHISVOLUME RESPECTIVELY HADWORKEDWITH 'OVINDJEE FORT F HEIRDOCTORATE DEGREESATTHE5)5# (ISCURRENT INTERESTSARE )MAGINGANDREGU D LATION OFLIFETIMEOF#HL A mUORESCENCEINSINGLEALGALOR CYANOBACTERIAL CELLS (ISTORYOFF PHOTOSYNTHESIS RESEARCHAND  0HOTOSYNTHESISEDUCAT D ION(EISA MEMBEROFTHE!MERICAN3OCIETYOF0LANT"IOLOGY !MERICAN 3OCIETY FFOR 0HOTOBIOLOGY Y "IOPHYSICAL 3OCIETY OFF !MERICA ANDD THE )NTERNATIONAL 3OCIETY OF 0HOTOSYNTHESIS 2ESEARCH )302  &OR FURTHER INFORMAT F ION SEEHISWEB PAGEATHTTPWWWLIFE F D GOVINDJEE (ECANBEREACHED BYE MAILAT UIUCEDU GOV LIFEUIUCEDU

'OVINDJEE BORNIN HAS BEEN 0ROFESSO F R %MERI TUSOFF "IOCHEMISTRY " Y IOPHYSICSAND D 0LANT"IOLOGY AT THE 5NIVERSITY OFF )LLINOIS AT 5RB R ANA #HAMPAIGN 5)5# )LLINOIS 53! SINCE(ERECEIVEDHIS "3C #HEMISTRY AND D "IOLOGY AND-3C D "OTANY N  0LANT0HYS H IOLOGY FROMT F HE5NIVERSITYOFF !LLAHABAD D )NDIA INAND RESPECTIVELY(EEARNEDHIS 0H$IN"IOPHYSICSFROMT F HE5)5#IN WORK R ING lRSTWITH2OBERT%MERSONAND D THENWITH%UGENE 2ABINOWITCH(ESERVEDONTHEFACULTYOFTHE5NI VERSITYOFF !LLAHABAD FROMnAN F D THE5)5# F FROMn( ISHONORSINCLUDE&ELLOWOFF THE !MERICAN!SSOCIATIONOF!DVANCEMENTOF3CIENCE !!!3  &ELLOW OFF THE .ATIONAL !CADEMY M OF F 3CI ENCES )NDIA $ISTINGUISHED D ,ECTUREROFT F HE3CHOOL OF,IFE3CIENCESAT5)5# 0RESIDENTOFTHE !MERICAN 3OCIETY FFOR 0HOTOBIOLOGY n  &ULBRIGHT3ENIOR,ECTURERn AND D (ONOR ARY0RESIDENTOFTHE)NTERNATIONAL0HOTOSYNTHESIS #ONGRESS-ONTREAL #ANADA 'OVINDJEESRESEARCH INTERESTSHAVE BEEN SINCE ONTHE FUNCT F IONOF 0HOTOSYSTEM 03 )) EVEN BEFORE IT WAS CALLED AS SUCH-OSTOF HISWORK R HASBEENCOLLABORATIVE BUT D DUETO LACKO K FS F PACE THENAMESOF HISCOLLABORATORS ARE NOT MENTIONED HERE THEY ARE AVAILABLE ON HIS WEB PAGEHTTPWWWLIFEU F IUCEDU D GOVINDJEE (E DISCOVERED D THAT IN ADDITION TO AUXILIARY PIGMENTS ASHORT WAVELENGTHFORMOF#HLOROPHYLL#HL A IS PRESENT INWH W AT ISNOWCALLED03)) D  DISCOVERY F IN#HL A OFF THETWO LIGHTTWO PIGMENTSYSTEMEFFECT mUORESCENCE %MERSON%NHANCEMENT%FFECT X

$POUFOUT &EJUPSJBM

W

$POUFOUT

YJ

1SFGBDF

YYJ

"VUIPS *OEFY

YYWJJ

$PMPS
1MBUFT

$1m$1

%FEJDBUJPO1FSTQFDUJWF
"
USJCVUF
UP
+FSSZ
#BCDPDL

m

1BSU
*
1FSTQFDUJWF
PG
1IPUPTZTUFN
**
3FTFBSDI 

*OUSPEVDUJPO
UP
1IPUPTZTUFN
** , ,JNJZVLJ
4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

m

4VNNBSZ *

%JTDPWFSZ
PG
0YZHFO
BOE
0 1SPEVDUJPO CZ 1MBOUT ** $PODFQUVBM
%FWFMPQNFOU
PG
1IPUPTZTUFN ** *** 0 1SPEVDUJPO ‡ 1IFOPNFOPMPHZ *7

*TPMBUJPO
PG
UIF
$IFNJDBM
&OUJUZ‡4USVDUVSBM
0SHBOJ[BUJPO
PG
1IPUPTZTUFN
**
7

'VODUJPOBM
4JUFT ‡ $BUBMZUJD
3PMF
PG 1IPUPTZTUFN ** 7 7*

'VUVSF
1FSTQFDUJWFT "DLOPXMFEHNFOUT
3FGFSFODFT

        

1BSU
**
1SPUFJO
$POTUJUVFOUT
PG
1IPUPTZTUFN
** 

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN ** "OUFOOBT #FWFSMFZ
3
(SFFO
BOE
&MJTBCFUI (BOUU U 4VNNBSZ *
*OUSPEVDUJPO ** 1IZDPCJMJQSPUFJOT
BOE 1IZDPCJMJTPNFT ***

1SPDIMPSPQIZUF
"OUFOOBT
BOE
UIF
*TJ"
1SPUFJOT
*7

5IF
-)$
4VQFSGBNJMZ PG
$IMPSPQMBTUT "DLOPXMFEHNFOUT
3FGFSFODFT



5IF $1
BOE $1 $PSF
"OUFOOB $PNQPOFOUT +VMJBO
+
&BUPO3ZF
BOE $JOEZ
1VUOBN&WBOT 4VNNBSZ *
*OUSPEVDUJPO

m       

m  

XI

** 5IF QTC# BOE QTC$ (FOFT ***

0WFSWJFX
PG
&OFSHZ
5SBOTGFS
BOE
$IMPSPQIZMM #JOEJOH *7

5IF
)ZESPQIJMJD
%PNBJOT
PG $1
BOE $1 7 $PODMVTJPOT 7 "DLOPXMFEHNFOUT
3FGFSFODFT



     

5IF % BOE % $PSF 1SPUFJOT 1FUFS
+
/JYPO
.BSZ SZ
4BSDJOB
BOE
#SVDF
"
%JOFS

m

4VNNBSZ *
*OUSPEVDUJPO **

*EFOUJàDBUJPO
PG UIF %
BOE
%
1SPUFJOT ***

5IF
1SJNBSZ 4USVDUVSFT
PG
%
BOE
% *7
*EFOUJàDBUJPO
PG
UIF
%
BOE
%
1SPUFJOT
BT
UIF
1IPUPTZTUFN
**
3FBDUJPO $FOUFS
4VCVOJUT
7

.VUBHFOFTJT
PG UIF
%
BOE
%
1SPUFJOT 7 7*

$PODMVEJOH
3FNBSLT "DLOPXMFEHNFOUT
3FGFSFODFT



5IF &YUSJOTJD
1SPUFJOT
PG
1IPUPTZTUFN
** 5 5FSSZ
. #SJ SJDLFS
BO L E 3PCFSU
- #VSOBQ 4VNNBSZ *
*OUSPEVDUJPO **

5IF

L%B
.BOHBOFTF4UBCJMJ[JOH 1SPUFJO
1TC0

***

5IF

L%B
BOE

L%B
1SPUFJOT
1TC1
BOE
1TC2
JO
)JHIFS
1MBOUT
*7

.VMUJQMF
&YQSFTTFE
(FOFT
PG
UIF
&YUSJOTJD
1SPUFJOT
JO
)JHIFS
1MBOUT
7 $ZUPDISPNF D 1TC7
JO $ZBOPCBDUFSJB 7 7*

5IF
1TC6
1SPUFJO
JO
$ZBOPCBDUFSJB 7**
$PODMVTJPOT "DLOPXMFEHNFOUT
3FGFSFODFT



5IF -PX
.PMFDVMBS
8FJHIU
1SPUFJOT
PG
1IPUPTZTUFN
** -FFBOO
&
5IPSOUPO
+PIOOB
-
3PPTF
)JNBESJ
#
1BLSBTJ
BOE .BTBIJLP
*LFVDIJ 4VNNBSZ * *OUSPEVDUJPO ** .FNCSBOF
4QBOOJOH
4VCVOJUT
***

&YUSJOTJD
4VCVOJUT *7 $PODMVTJPO "DLOPXMFEHNFOUT
3FGFSFODFT

XII

          

m          

m

      

1BSU
***
0SHBOJ[BUJPO
PG
'VODUJPOBM
4JUFT
JO
1IPUPTZTUFN
** 

1SJNBSZ
&MFDUSPO
5SBOTGFS (FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI 4VNNBSZ *
*OUSPEVDUJPO ** $PGBDUPST
PG
4UBCMF $IBSHF 4FQBSBUJPO
JO
1IPUPTZTUFN
** *** 1IPUPQIZTJDBM
1SPQFSUJFT
PG
1JHNFOU
1SPUFJO
$PNQMFYFT *7

/BUVSF
BOE
1SPQFSUJFT
PG 1
BOE
1IFP
7

,JOFUJDT
BOE
&OFSHFUJDT
PG
$IBSHF 4FQBSBUJPO 7 7*

'PSXBSE
#BDL
BOE
4JEF
3FBDUJPOT
PG
3BEJDBM
*PO
1BJS
1 r 2"mrm
7**
$PODMVEJOH
3FNBSLT
BOE
'VUVSF 1FSTQFDUJWFT "DLOPXMFEHNFOUT
3FGFSFODFT



5IF *SPO2VJOPOF
"DDFQUPS $PNQMFY 7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z Z
3
$SPGUT 4VNNBSZ *

*OUSPEVDUJPO

**

1SPCJOH
UIF
*SPO2VJOPOF
$PNQMFY
5ISPVHI
UIF
*SPO
4JUF ***

0SHBOJ[BUJPO
PG
UIF
2VJOPOF
#JOEJOH
4JUFT
5IF
5XP&MFDUSPO
(BUF
*7 $PODMVTJPOT "DLOPXMFEHNFOUT
3FGFSFODFT



5IF
3FEPY"DUJWF
5Z 5 SPTJOFT :; BOE :% #SVDF " %JO J FS BOE 3 %BWJE W #SJ S UU U 4VNNBSZ *

*OUSPEVDUJPO **

$IFNJDBM
/BUVSF
PG
4JHOBM
** ***

1SPUPOBUJPO
4UBUFT
PG
UIF 0YJEJ[FE
BOE
3FEVDFE
'PSNT
PG
5ZSPTJOF
*7 -PDBMJ[BUJPO
PG
:; BOE :% 7 5IF
1SPUPO "DDFQUPS
BOE )ZESPHFO#POEJOH 7
7*

,JOFUJDT
PG :; 0YJEBUJPO
BOE
3FEVDUJPO 7**

,JOFUJDT
PG
:% 0YJEBUJPO
BOE
3FEVDUJPO
BOE
$PNQBSJTPO 7***

.FDIBOJTNT GPS :; 0YJEBUJPO
BOE 3FEVDUJPO *9 $PODMVEJOH
3FNBSLT "DLOPXMFEHNFOUT
3FGFSFODFT

m          

m       

m            

 5IF
$BUBMZUJD
.BOHBOFTF
$MVTUFS 0SHBOJ[BUJPO
PG
UIF
.FUBM
*POT
m 7JUUBM U , , :BDIBOESB S 4VNNBSZ *

*OUSPEVDUJPO **

0YJEBUJPO
4UBUFT
PG
UIF
.BOHBOFTF *** 4USVDUVSF
PG
UIF
.BOHBOFTF
$MVTUFS *7 4USVDUVSBM
3PMF
PG
UIF $BMDJVN
$PGBDUPS

XIII

    

7 4USVDUVSBM
3PMF
PG
UIF $IMPSJEF
$PGBDUPS 7 7*

.FDIBOJTN
PG
8BUFS
0YJEBUJPO
BOE 0 &WPMVUJPO "DLOPXMFEHNFOUT
3FGFSFODFT

 5IF
$BUBMZUJD
.BOHBOFTF
$MVTUFS 1SPUFJO
-JHBUJPO 3JD J IBSE S + %FCVT

   

m

4VNNBSZ *
*OUSPEVDUJPO ** 5IF
% 1PMZQFQUJEF ***

5IF
$1
1PMZQFQUJEF *7
$PODMVEJOH
3FNBSLT "DLOPXMFEHNFOUT
3FGFSFODFT

      

 5IF $BUBMZUJD
.BOHBOFTF $MVTUFS
*NQMJDBUJPOT GSPN
4QFDUSPTDPQZ , ,BSJO
"
ªISMJO H 3POBME
+
1BDF
BOE
.JDIBFM
$
8
&WBOT

m

4VNNBSZ * *OUSPEVDUJPO **

44UBUF
4QFDUSPTDPQZ ***

" 4QFDUSPTDPQJD
.PEFM
GPS
UIF
$BUBMZUJD
4JUF 3FGFSFODFT

 5IF $BMDJVN
BOE $IMPSJEF
$PGBDUPST )BOT +
WBO (PSLPN
BOE
$IBSMFT
'
:PDVN

    

m

4VNNBSZ *
*OUSPEVDUJPO ** $IMPSJEF

*** $BMDJVN *7
$PODMVEJOH
3FNBSLT "DLOPXMFEHNFOUT
3FGFSFODFT

      

 #JDBSCPOBUF
*OUFSBDUJPOT +BDL
+
4
WBO
3FOTFO
BOE
7Z 7ZBDIFTMBW
7
,MJNPW

m

4VNNBSZ * *OUSPEVDUJPO **

#JDBSCPOBUF
3FRVJSFNFOU
PO
UIF
&MFDUSPO
"DDFQUPS
4JEF
PG
1IPUPTZTUFN
**
*** #JDBSCPOBUF
3FRVJSFNFOU
PO
UIF &MFDUSPO %POPS
4JEF
PG
1IPUPTZTUFN
**
*7 $PODMVTJPOT "DLOPXMFEHNFOUT
3FGFSFODFT

XIV

      

 4JEF1BUI
&MFDUSPO
%POPST $ZUPDISPNF C $IMPSPQIZMM ; BOE B$BSPUFOF 1FUFS
'BMMFS $ISJTUJBO
'VGF[BO
BOE
"
8JMMJBN
3VUIFSGPSE 4VNNBSZ *
*OUSPEVDUJPO ** -PDBUJPO
PG
"DDFTTPSZ
&MFDUSPO %POPST *** 4QFDUSPTDPQJD
4UVEJFT *7

&MFDUSPO
5SBOTGFS
1BUIXBZT 7
'VODUJPO
PG
UIF
"MUFSOBUJWF
&MFDUSPO
5SBOTGFS
1BUIXBZ 7 7** $PODMVTJPOT
"DLOPXMFEHNFOUT
3FGFSFODFT

m         

1BSU
*7
4USVDUVSBM
#BTJT
GPS
1IPUPTZTUFN
** 
.PMFDVMBS
"OBMZTJT
CZ
7JCSBUJPOBM 4QFDUSPTDPQZ 5 5BLVNJ
/PHVDIJ
BOE
$BUIFSJOF
#FSUIPNJFV 4VNNBSZ *
*OUSPEVDUJPO ** -JHIU*OEVDFE
'PVSJFS
5SBOTGPSN
*OGSBSFE
'5*3 %JGGFSFODF
5FDIOJRVF
***

$PGBDUPST
PO
UIF &MFDUSPO
%POPS
4JEF *7
$PGBDUPST
PO
UIF &MFDUSPO"DDFQUPS 4JEF
7 $PGBDUPST
JO 4FDPOEBSZ
&MFDUSPO5SBOTGFS 1BUIXBZT 7 "DLOPXMFEHNFOUT
3FGFSFODFT

 $POàHVSBUJPO
PG
&MFDUSPO
5SBOTGFS
$PNQPOFOUT 4UVEJFE
CZ &13
4QFDUSPTDPQZ 3PCFSU
#JUUM U BOE "TBLP ,BXB X NPSJ S 4VNNBSZ *
*OUSPEVDUJPO **

4QFDUSPTDPQJD
#BDLHSPVOE *** 0SJFOUBUJPO
PG
$PGBDUPS
.PMFDVMFT *7

%JTUBODFT
#FUXFFO
$PGBDUPS
.PMFDVMFT 7 $PODMVEJOH
3FNBSLT 7
"DLOPXMFEHNFOUT
3FGFSFODFT


 4USVDUVSBM
"OBMZTJT
PG
UIF
1IPUPTZTUFN
** $PSF F"OUFOOB )PMPDPNQMFY
CZ
&MFDUSPO
.JDSPTDPQZ #FO
)BOLBNFS
+BNFT
#BSCFS
BOE
+PO
/JFME 4VNNBSZ *

*OUSPEVDUJPO

**

&MFDUSPO
$SZP.JDSPTDPQZ
5FDIOJRVFT ***

4USVDUVSF
PG
)JHIFS
1MBOU
1IPUPTZTUFN
**
BOE
*UT
"OUFOOB
4ZTUFN



XV

m        

m        

m    

*7 0SHBOJ[BUJPO
BOE
%ZOBNJDT
PG
)JHIFS
1MBOU
1IPUPTZTUFN
**
BOE *UT
"OUFOOB
*O
7JWP 7

'VUVSF
1SPTQFDUT
BOE
$PODMVEJOH
3FNBSLT 7 "DLOPXMFEHNFOUT
3FGFSFODFT

   

 1IPUPTZTUFN
** 4USVDUVSBM
&MFNFOUT
UIF
'JSTU
% $SZTUBM
4USVDUVSF BOE
'VODUJPOBM
*NQMJDBUJPOT m )PSTU
5
8JUU U 4VNNBSZ * *OUSPEVDUJPO **

5SBOTNFNCSBOF
$IBSHF
4FQBSBUJPO
&WFOUT
BT
1SJNBSZ
"DUT
PG
-JHIU &OFSHZ $POWFSTJPO BOE 4QBUJBM
0SHBOJ[BUJPO
PG
UIF
&MFDUSPO
%POPST BOE "DDFQUPST ‡ "OBMZTJT CZ
B
.PMFDVMBS 7P 7 MUNFUFS ***

5IF
1SJNBSZ
&MFDUSPO
%POPS
$IMPSPQIZMM
1
BOE
*UT
4UBCMF
&MFDUSPO "DDFQUPS
1MBTUPRVJOPOF 2" ‡ 5IF
&OHJOF
%SJWJOH
8BUFS
0YJEBUJPO

*7

5IF
.FNCSBOF4QBOOJOH $IMPSPQIZMM2VJOPOF
$PVQMF BT B 3FBDUJPO $FOUFS
.PEFM
GPS %JGGFSFOU
1IPUPTZTUFNT 7

5XP
$IMPSJOT
CFUXFFO
UIF $IMPSPQIZMM2VJOPOF $PVQMF
BT
B
'BTU
1BUI 7 GPS
&MFDUSPOT $SPTTJOH
UIF
.FNCSBOF 7*

5IF
1MBTUPRVJOPOF
1PPM BT
UIF 1BUIXBZ
GPS
5SBOTGFS
PG
&MFDUSPOT
GSPN 2" UP 1IPUPTZTUFN
*
BOE
PG
1SPUPOT
GSPN
UIF
0VUFS
"RVFPVT
1IBTF
UP
UIF .FNCSBOF -VNFO 7**

1SJNBSZ
&MFDUSPO
%POPST
0SHBOJ[FE
BT
$IMPSPQIZMM
1BJST 7***

*EFOUJàDBUJPO
PG 1IPUPTZTUFN
**
BT
B
%JNFS
BOE
1IPUPTZTUFN
*
BT
B
5SJNFS
*9

)PNPMPHZ
PG
UIF
1IPUPTZTUFN
** $PSF
$PNQMFY
XJUI
1IPUPTZTUFN
*
BOE UIF
#BDUFSJBM
3FBDUJPO $FOUFS 9

'JSTU
% $SZTUBMT
PG
1IPUPTZTUFN
**
$BQBCMF
PG 8BUFS 0YJEBUJPO BOE 93BZ
4USVDUVSF "OBMZTJT BU
m
BOE
 ª 3FTPMVUJPO 9*

.BOHBOFTF
7BMFODFT
1SPUPO
3FMFBTFT
BOE
8BUFS
4UBUFT
PG
UIF 2VBUFSOBSZ 44UBUF
$ZDMF
PG
UIF
-JHIU
%SJWFO
&OHJOF 9** 'VODUJPOBM *NQMJDBUJPOT "DLOPXMFEHNFOUT
3FGFSFODFT

 %
$SZTUBM
4USVDUVSF
PG
UIF 1IPUPTZTUFN
**
$PSF
+JBO3FO
4IFO
BOE
/PCVP
,BNJZ JZB 4VNNBSZ *

*OUSPEVDUJPO **

$SZTUBMMJ[BUJPO *** $SZTUBM
4USVDUVSF
PG
1IPUPTZTUFN
**
GSPN
5IFSNPQIJMJD $ZBOPCBDUFSJB
*7

'VUVSF
1SPTQFDUT
BOE
$PODMVEJOH 3FNBSLT "DLOPXMFEHNFOUT
3FGFSFODFT

XVI

 

   

        

m       

 3FàOFE
93BZ 4USVDUVSF
PG
1IPUPTZTUFN
** BOE *UT *NQMJDBUJPOT
+BNFT
#BSCFS
BOE 4P
*XBUB 4VNNBSZ *
*OUSPEVDUJPO **

93BZ
$SZTUBMMPHSBQIZ

***

.BKPS
%JGGFSFODFT
GSPN
&BSMJFS 4USVDUVSFT *7
0WFSBMM 4USVDUVSF 7

1SPUFJO
4VCVOJUT 7 7*

1JHNFOUT
BOE
$PGBDUPST
7**

5IF 0YZHFO
&WPMWJOH $FOUFS
7*** (FOFSBM
*NQMJDBUJPOT
PG
UIF
4USVDUVSF *9

8BUFS
0YJEBUJPO
.FDIBOJTN 9
1FSTQFDUJWFT
"DLOPXMFEHNFOUT
3FGFSFODFT

m             

1BSU
7
.PMFDVMBS
%ZOBNJDT
PG
1IPUPTZTUFN
**  &OFSHZ
5SBQQJOH
BOE
&RVJMJCSBUJPO
"
#BMBODF
PG 3FHVMBUJPO BOE
&GàDJFODZ -BVSB
.
$
#BSUFS S %BWJE
3
,MVH BOE
3JFOL
WBO
(SPOEFMMF 4VNNBSZ *
*OUSPEVDUJPO ** 5IF
$POUFYU
GPS
4PMBS
&OFSHZ
$POWFSTJPO
JO
1IPUPTZTUFN
** ***
3BQJE
&OFSHZ
5SBOTGFS
BOE
&RVJMJCSBUJPO
XJUIJO
*TPMBUFE $PNQMFYFT
*7
$POWFSTJPO
PG
&YDJUFE
4UBUFT
JOUP
$IBSHF
4FQBSBUFE
4UBUFT
7

$PODMVEJOH
3FNBSLT 7 "DLOPXMFEHNFOUT
3FGFSFODFT

 5IF
3PMF
PG $BSPUFOPJET
JO
&OFSHZ
2VFODIJOH #BSSZ
+ 1PHTPO 1

)FBUI U FS
. 3JTT J MFS
BOE )BSSZ
" 'SBO ' L 4VNNBSZ *
*OUSPEVDUJPO **

#JPTZOUIFTJT
BOE
1IPUPTZTUFN "TTFNCMZ ***

$BSPUFOPJET
BOE
1IPUPQSPUFDUJPO "DLOPXMFEHNFOUT
3FGFSFODFT

 'MBTI*OEVDFE 0YZHFO
&WPMVUJPO
BOE 0UIFS
0TDJMMBUPSZ 1SPDFTTFT
7 7MBEJNJS
4IJOLBSFW
4VNNBSZ *
*OUSPEVDUJPO **

5IF
,PL
.PEFM
PG
0YZHFO
&WPMVUJPO ***

#JOBSZ 0TDJMMBUJPOT
JO
UIF
,PL
.PEFM *7
$PODMVTJPOT

XVII

m        

m      

m     

"DLOPXMFEHNFOUT 3FGFSFODFT

 

 .FDIBOJTN
PG
1IPUPTZOUIFUJD 0YZHFO
1SPEVDUJPO 8 8BSXJDL
)JMMJFS
BOE
+PIBOOFT
.FTTJO HFS

m

4VNNBSZ * *OUSPEVDUJPO **

1IPUPTZOUIFUJD
0 &WPMVUJPO
1BUUFSOT BOE
UIF
,PL
.PEFM ***

4USVDUVSFT
BOE 0YJEBUJPO
4UBUFT
PG
UIF
.O0Y$B
$PNQMFY *7 4VCTUSBUF
*OUFSBDUJPOT
7

&OFSHFUJD
BOE
,JOFUJD
$POTJEFSBUJPOT 7 7*

.FDIBOJTUJD
0WFSWJFX
PG
00
#POE 'PSNBUJPO 3FBDUJPOT 7**

"
/FX
.FDIBOJTUJD
3FOEJUJPO
PG
1IPUPTZOUIFUJD
0YJEBUJPO
1SPEVDUJPO
"DLOPXMFEHNFOUT
3FGFSFODFT


         

1BSU
7*
"TTFNCMZ
BOE
#JPEZOBNJDT
PG
1IPUPTZTUFN
**  1IPUP"TTFNCMZ
PG
UIF
$BUBMZUJD
.BOHBOFTF
$MVTUFS (
$IBSMFT
%JTNVLFT
(FOOBEZ EZ .
"OBOZFW
BOE
3JDIBSE
8BUU


m

4VNNBSZ *

*OUSPEVDUJPO **

'VODUJPO
PG
1IPUPTZTUFN
**
4VCVOJUT
JO
8BUFS 4QMJUUJOH
BOE
1IPUP"TTFNCMZ
*** #JPHFOFTJT
PG
UIF
8BUFS
0YJEJ[JOH $PNQMFY *7

3PMFT
PG
UIF
*OPSHBOJD
$PGBDUPST
GSPN
1IPUP"TTFNCMZ 7 $PODMVEJOH
3FNBSLT
7
"DLOPXMFEHNFOUT
3FGFSFODFT

 1IPUPJOBDUJWBUJPO
BOE
.FDIBOJTNT
PG
3FDPWFSZ 8 4PPO
$IPX
BOE
&WB.BSJ
"SP 8BI


m

4VNNBSZ * *OUSPEVDUJPO **

5IF
*OFWJUBCJMJUZ
PG
1IPUPJOBDUJWBUJPO
PG
1IPUPTZTUFN ** ***

1PUFOUJBM
"HFOUT
PG
1IPUPTZTUFN
** 1IPUPJOBDUJWBUJPO
*7

5IF
7BSJBCJMJUZ
PG
UIF
&YUFOU
PG 1IPUPTZTUFN
**
1IPUPJOBDUJWBUJPO
7 .PMFDVMBS
3FBSSBOHFNFOUT
1SFDFEJOH
UIF
%FHSBEBUJPO
PG
UIF
%
1SPUFJO
7*

%FHSBEBUJPO
PG UIF
%BNBHFE
%
1SPUFJO
7**

#JPHFOFTJT
BOE
"TTFNCMZ
PG
UIF
/FX
% $PQZ
JOUP
1IPUPTZTUFN
**
"DLOPXMFEHNFOUT
3FGFSFODFT


 5 5SBOTDSJQUJPOBM
BOE
5SBOTMBUJPOBM
3FHVMBUJPO
PG 1IPUPTZTUFN
** (FOF
&YQSFTTJPO , ,FOJDIJ
:BNB HVDIJ
4UFQIFO
1
.BZGJFME
BOE
.BNPSV
4V Z HJUB 4VNNBSZ *

*OUSPEVDUJPO

       

         

m  

XVIII

**

3FHVMBUJPO
PG
1IPUPTZTUFN
**
(FOF &YQSFTTJPO JO "MHBF ***

3FHVMBUJPO
PG
1IPUPTZTUFN
**
(FOF
&YQSFTTJPO
JO
)JHIFS
1MBOUT "DLOPXMFEHNFOUT
3FGFSFODFT

 1SPUFJO
5SBOTQPSU
BOE
1PTUUSBOTMBUJPOBM
1SPDFTTJOH JO 1IPUPTZTUFN
**
#JPTZOUIFTJT
BOE
)PNFPTUBTJT 4UFWFO
.
5IFH
BOE
-BO9JO 4IJ 4VNNBSZ *
*OUSPEVDUJPO **

5BSHFUJOH
1BUIXBZT
6UJMJ[FE
CZ
%JGGFSFOU
1IPUPTZTUFN
** 4VCVOJUT ***

"TTFNCMZ
PG
4VCVOJUT
JOUP 1IPUPTZTUFN
** *7 1PTU5S 5 BOTMBUJPOBM .PEJàDBUJPOT 7 $PODMVEJOH
3FNBSLT 7 "DLOPXMFEHNFOUT
3FGFSFODFT


   

m           

1BSU
7**
$PNQBSJTPO
PG
1IPUPTZTUFN
**
XJUI
0UIFS
/BUVSBM"SUJGJDJBM
4ZTUFNT  5IF 0SJHJO
BOE
&WPMVUJPO
PG
1IPUPTZOUIFUJD 0YZHFO 1SPEVDUJPO
(
$IBSMFT
%JTNVLFT
BOE
3PCFSU
&
#MBOLFOTIJQ J

m

4VNNBSZ * 5IF 5JNFUBCMF BOE #JPHFPDIFNJDBM
$POTFRVFODFT
PG
0YZHFOJD 1IPUPTZOUIFTJT **

.JOJNBM
$PGBDUPS
%JWFSTJUZ
JO 8BUFS 0YJEJ[JOH $PNQMFYFT

*** 5SBOTJUJPOBM &MFDUSPO
%POPST BOE A.JTTJOH -JOLT *7

1PTTJCMF
&WPMVUJPO
1BUIXBZT
GPS
UIF
1IPUPTZTUFN
**
8BUFS
0YJEJ[JOH $PNQMFY 7 $PODMVEJOH
3FNBSLT 7 "DLOPXMFEHNFOUT
3FGFSFODFT




 .FDIBOJTUJD
$PNQBSJTPOT
#FUXFFO
1IPUPTZTUFN
** BOE $ZUPDISPNF D 0YJEBTF (BSZ SZ
8
#SVEWJJH
BOE
.ÇSUFO
8JLTUSÕN Ç 4VNNBSZ *

*OUSPEVDUJPO **

1SPUFJO
4USVDUVSF
BOE $PGBDUPST ***

&OFSHFUJDT
PG
8BUFS
0YJEBUJPO
BOE 0YZHFO
3FEVDUJPO
*7

$BUBMZUJD
.FDIBOJTNT 7

"OBMPHJFT
CFUXFFO
UIF
0YZHFO $IFNJTUSZ
PG
1IPUPTZTUFN
**
BOE 7 $ZUPDISPNF D 0YJEBTF
"DLOPXMFEHNFOUT
3FGFSFODFT

XIX

      

m        

 .JNJDLJOH
UIF
1SPQFSUJFT
PG 1IPUPTZTUFN
**
JO #BDUFSJBM 3FBDUJPO
$FOUFST -ÃT[MÓ
,ÃMNÃO
+P"OO $
8JMMJBNT
BOE
+BNFT
1
"MMFO 4VNNBSZ *
&WPMVUJPOBSZ %FWFMPQNFOUT **

"DIJFWJOH
B
)JHIMZ
0YJEJ[JOH
&MFDUSPO
%POPS ***

0YJEBUJPO
PG
5Z 5 SPTJOF
3FTJEVFT
BOE
.FUBMT *7

%FTJHOJOH
B
.BOHBOFTF $MVTUFS "DLOPXMFEHNFOUT
3FGFSFODFT

 %F
/PWP W 1SPUFJO
%FTJHO JO
3FTQJSBUJPO
BOE
1IPUPTZOUIFTJT
#SJBO
3
(JCOFZ Z BOE
$FDJMJB
5PNNPT

m       

m

4VNNBSZ  *

*OUSPEVDUJPO

 **

$POTUSVDUJPO
PG
1SPUFJOT
$POUBJOJOH
$PGBDUPST
*OWPMWFE
JO
&OFSHZ
$POWFSTJPO
 ***

1FSTQFDUJWF  "DLOPXMFEHNFOUT
 3FGFSFODFT 

 6OEFSTUBOEJOH
1IPUPTZTUFN
**
'VODUJPO
CZ "SUJàDJBM 1IPUPTZOUIFTJT "OO
.BHOVTPO
4UFOCK C ÕSO
4UZ UZSJOH
BOE
-FJG
)BNNBSTUSÕN 4VNNBSZ *

*OUSPEVDUJPO

**

.JNJDLJOH
1IPUPTZTUFN ** 3FBDUJPOT ***

3FEPY
1SPQFSUJFT
JO
/BUVSBM
BOE
"SUJàDJBM
1IPUPTZOUIFUJD 4ZTUFNT
*7

'VUVSF 0VUMPPL "DLOPXMFEHNFOUT
3FGFSFODFT



*OEFY

m       



XX

1SFGBDF LYZES A SERIES OF REACTION EVENTS FROM THE CAPTURE ANDD TRANSFERO F F LIGHTENERGY OCCURRINGONTHE FE F M TOSECONDD OR THE n S TIME SCALE TO THE EFlCIENT CONVERSION OF EXCITATION ENERGY INTO THE CHEMICAL ENERGY OFF A STABILIZEDD CHARGE SEPARATION ANDD THE F OFF ELECTRONS FFROM WATER TO PLASTOQU Q INONE TRANSFER OCCURRINGONAMILLISECONDORTHEn STIMESCALE  )NRECENT YEARSTHERE HASBEENADRAMATICSURGEINOUR K KNOW LEDGEOFF THEMOLECULARORGANIZATION DYNAMICS ANDREACTIONPATHSOF03)))N PARTICULAR THEOVERALL ATOMICSTRUCTUREOFF 03)) HAS BEENSUCCESSFU F LLYELU CIDATED BY8 RAYCRYSTALLOGRAPHICANALYSIS DOWNTO An¯RESOLUTION4HISLASTACCOMPLISHMENTIS NOTRIVIAL FEATCONS F IDERING THESUB U UNITCOMPLEXITY AND MOLECULARSIZEOFF THE03))CORECOMPLEXMOLECULAR MASS^ K$A 3IMULTANEOUSLY THEBIOPHYSICS F HASALSO BEENGREATLY ANDD MOLECULARBIOLOGY OF03)) ADVANCE D D4HUS T H THETIMEWASRIPE FORACOM F MPREHENSIVE I BOOKDEDICATEDTO03))TOAPPEARINTHELITERATUREAND TOSUMMARIZEOURCURRENT KNOW K LEDGEASABASISFOR F F FUTURERESEARC H AND FORT F HEAPPLICATIONOFT F HEUNIQUE Q 03 )) CHEMISTRY IN BIOTECHNOLOGY AND RENEWABLE ENERGY SCHEMES 4OBEGINTHEBOOK WEPROVIDEASPECIAL PERSPECTIVE DEDICATEDTOTHELATE'ERALD*ERRY 4"ABCOCKASONE OFT F HETRULYOUTSTANDINGPIONEERSIN03))RESEARCH (ISUNTIMELY DEATH INOCCURRED JUSTASHEWAS ADVOCATING A NEW PROPOSAL FOR THE MECHANISM OF WATEROXIDATION!STHEREADERSOFT F HISBOOKK WILLSOON F IDEASCONTINUETOPROVIDETHE REALIZE MANYOF*ERRYS UNDERLYINGBASISOFCURRENTTHINKING4HEFOLLOWING K ITTEN BYAUTHORSWH W O CHAPTERS INTHE BOOKWEREWR WERE INVITEDD AS LEADING EXPERTS IN THEIR RESPECTIVE lELDSOF03))RESEARCH-ANYCHAPTERSAREJOINTLY F DIFFERENTPARTSO F F THEWORLD AUTHORED BY EXPERTSFROM SOASTOPROVIDETHEREADERWITH THEMOST INDEPTH PERSPECTIVEONEACHTOPIC!TOTALOFAUTHORSFROM COUNTRIESHAVECONTRIBUTEDTOTHEBOOK REPRESENT INGMAJ A ORCENTERSOFF 03))RESEARCH IN%UROPE .ORTH !MERICA !SIAAND!USTRALIAANDMAKINGTHISBOOKA TRULY INTERNATIONAL EFFORT4 F HEBOOK ISDIVIDED INTOSIX DISTINCT PARTSWHI W CH ARESUMMARIZEDD ASFO F LLOWS 0ART) !0ERSPECTIVEOF0HOTOSYSTEM))2ESEARCH PROVIDES AN INTRODUCT D ION TO 03 )) CORRELATING OUR K LEDGEOF03))W F ITH IMPORTANTHISTORI CURRENT KNOW CAL DEVELOPMENTS IN PHOTOSYNTHESIS RESEARCH 4HE F IONAL PROPERTIES OF F 03 )) THAT STRUCTURAL AND FUNCT

0HOTOSYSTEM )) 4HE ,IGHT $RIVEN 7ATER0LAS TOQUINONE /XIDORE D DUCTAS D E IS THE NDD VOLUME IN THE !DVANCES D IN 0HOTOSYNTH T ESIS AND D 2ESPIRATION !)0( SERIES3ERIES%DITOR 'OVINDJEE PUBLISHED BY 3PRINGER FORMER F LY +LUWER!CADEMIC 0UBLI U SH ERS 0HOTOSYSTEM))03)) ISTHEHEARTOFOXYGEN F IC PHOTOSYNTHESIS CATALYZING THE OXIDATION OF WATER TO MOLECULAR OXYGEN -ANY N VOLUMES IN THE!)0( H ICS BIOCHEMISTRY SERIESHAVEDEALTWITH THE BIOPHYS ANDMOLECULARBIOLOGYOF03))BUTALWAYSWITHINTHE F FRAMEWOR RKOF AMUCH BROADERPICTURE)N6OLUME T ESIS4HE,IGH G T2EACTIONS 03 )) /XYGENIC0HOTOSYNTH WASDISCUSSEDALONGWITH0HOTOSYSTEM)ANDTHYLAKOID STRUCTUREWHIL W E IN6OLUME 0HOTOSY S NTH T ESIS0HOTO BIOCHEMISTRYAND D 0HOTOBIOPHYSICS H 03))WAS INCLUDED WITHDISCUSSIONSONBACTERIALPHOTOSYNTHESIS)NOTHER S NTH T ESISAND D THE VOLUMESSUCHAS6OLUME 0HOTOSY % %NVIRONMENT THEROLEOFF 03)) INSTRESSRESPONSEAND PHOTO INHIBITIONWASDISCUSSEDIN6OLUME ,IGHT ( (ARVESTIN G G!NTENNAS TH T EVARIOUSLIGHTHARVESTINGPIG MENT PROTEINSOFF 03))ARE DOCUMENTEDAND IN6OLUME  #HLOROPHYLLA&LUORESCENCE!3IGNATUREOF0HO TOSY S NTH T ESIS THEUSEOF03))C F HLOROPHYLL mUORESCENCE ASAMOLECULARPROBEWASTAKENTOAGLOBAL SCALE4HE PRESENTVOLUMEISUNIQUEINTHE!)0(SERIESINTHAT Q ITCOVERSALLASPECTSOFF THEBIOPHYSICS BIOCHEMISTRY AND D MOLECULARBIOLOGYOF03)) MA F KING ITTHEMOST COMPREHENSIVETEXTTOBEPUBLISHEDONTHISSUBJECT TODATE!COMPANIONVOLUMEON 0HOTOSYSTEM)IS ) CURRENTLYBEINGEDITED BY*OHN'OLBECK !LL AEROBIC ORGANISMS ON EARTH WHETHER PLANT D ONTHE FUNCT F IONOF03)) F ANIMAL ORMICROBIAL DEPEND TOMAINTAINTHEATMOSPHERIC/ THATSUSTAINS LIFE4 F HE STRONGESTOXIDANTKNOWNINBIOLOGYISCREATEDWITHIN 03)) BYVISIBLE LIGHTAND ISUSED D TOOXIDIZEWATERAS THEULTIMATESOURCEOFE F LECTRONSFORT F HECARB R ON lXING REACTIONSIE THE#ALVIN "ENSON "ASSHAMCYCLE  4HECARB R ON lXINGREACTIONSSTORECHEMICAL ENERGY IN THEFORMO F F ORGANIC FOO F D STUFFF ANDASCONSEQUENCE D MOLECULAROXYGENISRELEASEDASABY PRODUCT3EVERAL OTHERBOOKS6OLUMES    INTHE!)0( SERIESCOVERTHEEQUALLYIMPORTANT REVERSEPROCESSOF PHOTOSYNTHESIS IE RESPIRATION INWHICHMOLECULAR OXYGEN ISREDUCE D D TOWATERBY METABOLICALLY BURNING THEORGANICFOO F D STUFFRE F LEASINGTHESTORED D CHEMICAL ENERGYTOPOWERALLOTHERLIFEPROCESSES 03)) ISANEXTREMELY COMPLEXENZYMETHATCATA XXI

AREDISCUSSED IN DETAIL INTHEFO F LLOWINGCHAPTERSARE OUTLINEDANDPUTINTOTHEPERSPECTIVEOF03))ASA F FUNCT IONAL UNIT INTHE BROADERFRAMEWOR F RK OF OXYGENIC PHOTOSYNTHESIS4HE FUTUREO F F 03))RESEARCH AND ITS ROLEINBIOTECHNOLOGICALAPPLICATIONSAREALSOSUM MARIZED 0ART )) 0ROTEIN #ONSTITUENTS OF 0HOTOSYSTEM )) ) IS DEVOTED TO A COMPREHENSIVE DESCRIPTION OF THE PROTEIN SUB U UNIT COMPOSITION OFF 03 )) FOCUS F ING ON THEKNOWN K F FUNCT IONS OFF EACH COMPONENT%MPHASIS IS PLACEDONTHEDIVERSEDISTALANDEXTRINSICANTENNA PIGMENT PROTEINS INVOLVED IN THE LIGHT HARVESTING F FUNCT ION OF F 03 )) FOUN F D ACROSS THE MANY DIFFEREN F T CLASSESOFPHOTOSYNTHETICORGANISMSTHE#0AND #0 PROXIMAL ANTENNA PIGMENT PROTEINS WHI W CH AREUNIVERSALCOMPONENTSOFF 03))THATCHANNEL THE EXCITATIONENERGYINTHELIGHT HARVESTINGARRAYTOTHE PHOTOCHEMICALREACTIONCENTERTHEINTRINSIC$AND $SUB U UNITS WHI W CH FORMT F HEREACTIONCOREAND HARB R OR THEPRIMARYPHOTO REACTANTSANDTHEELECTRONCARRIERS THATCATALYZETHEOXIDATIONOFF WATERAND THEREDUCT D ION Q INONETHE/ ENHANCINGEXTRINSICPROTEINS OF PLASTOQU OFTHETHYLAKOIDMEMBRANE WHICHAREIMPORTANTFOR D IONBY03)) OPTIMIZINGMOLECULAROXYGENPRODUCT D THE IDENTIlCATION AND PROPOSED D STRUCTURE AND AND F FUNCTIONOFMINOR PROTEINCOMPONENTSCONTAINEDIN THE03))COMPLEXESFROMC F YANOBACTERIA ALGAEAND HIGHERPLANTS 0ART))) /RGANIZATIONOFTHE&UNCTIONAL3ITESIN 0HOTOSYSTEM)) DISCUSSESINDEPTHTHEARRANGEMENT ) AND PROPERTIESOFF THEELECTRONTRANSFERCOM F PONENTS IN03))4HESEFUNCTIONALCOMPONENTSINCLUDETHE REACTIONCENTER0 CHLOROPHYLL PHEOPHYTINCOM PLEXTHAT FORMST F HESITEOFF PRIMARYCHARGESEPARATION Q PTOR INTHE03))THE1! AND1" IRON QUINONEACCE COMPLEX THAT LINKS THE ONE ELECTRON REACTIONS AT THEREACTIONCENTERWITHTHETWO ELECTRONREDUCT D ION Q 9: REDOX ACTIVETYROSINETHAT OFPLASTOQUINONETHE MEDIATESAPROTON COUPLED D ELECTRONTRANSFER F FROMT F HE MANGANESE CALCIUM-N#A CLUSTERTOTHEREACTION CENTERANDTHESYMMETRICAL 9$ REDOX ACTIVETYROSINE THAT INTERACTSWITHTHE03)) DONORSIDETHECATALYTIC -N#ACLUSTERTHATSEQUENTIALLYACCUMULATESOXIDIZ INGEQUIVALENTSFROMTHEREACTIONCENTERANDCATALYZES THEPRODUCT D IONOFF MOLECULAR/ FROMWATERAN F D THE H LL : CYTOCHROME SIDE PATHELECTRON DONORSCHLOROPHY B AND ` CAROTENE WHICHPROVIDEALTERNATEELECTRON TRANSFERROUTES F F HE DISSIPATIONOFF EXCESSENERGY FORT IN03))%MPHASISISALSOGIVEN INTHISSECTIONTOTHE F FUNCTIONOFTHECALCIUMANDCHLORIDEINTHE / PRODUC INGREACTIONSAND D THEROLEOF BICARB R ONATEINELECTRON

TRANSFERON F BOTH THEDONORANDD ACCEPTORSIDESOFT F HE 03 ))REACTIONCENTER 0ART )6 6 3TRUCTURAL "ASIS FOR 0HOTOSYSTEM )) ) PROVIDES THE LATEST INFORMAT F ION ON THE ATOMIC AND MOLECULARSTRUCTUREOF03))INBOTHPROKARYOTICAND EUKARYOTIC PHOTOSYNTHETIC ORGANISMS ! RANGE OF BIOPHYS H ICALTECHNIQUESAREEMPLOYED INTHESTRUCTURAL STUDIES WHICHINCLUDETHEMOLECULARANALYSISOFTHE 03))FUNCT F IONALSITES BY Y VIBRATIONALSPECTROSCOPY EG &OURIERTRANSFORM F INFRARE F D DOR&4)2SPECTROSCOPY AND ELECTRON PARAMAGNETIC RESONANCE %02 SPECTROS COPYTHE DETERMINATIONOFF THESUB U UNITORGANIZATION IN03))COREANDD ANTENNA HOLOCOMPLEXESBYELECTRON CRYOMICROSCOPYANDTHERESOLUTIONOFATOMICSTRUC TURE OFF THE 03 )) CORE COMPLEX FFROM THERMOPHILIC H SPECIES OFF CYANOBACTERIA BY 8 RAY CRYSTALLOGRAPHY DOWNTO n ¯ 0ART6 6 -OLECULAR $YNAMICS OF 0HOTOSYSTEM )) ) PROVIDESDETAILEDD ACCOUNTSOF THEMOLECULAREVENTS THAT DElNE THE ROLE OF 03 )) FUNCTION IN OXYGENIC PHOTOSYNTHESIS 4HESE INCLUDE AN ANALYSIS OFF THE ENERGYTRAPPINGANDD EQUILIBRATIONWITHTHE03))REAC TIONCENTERTHESPECIALROLEOFCAROTENOIDPIGMENTS INTHEQUENCHING OFF THEEXCESSENERGYABSORB R ED BY 03))THEUNIQUEmASH INDUCE D D OSCILLATORYPROCESSES IN03))THATARECHARACTERISTICINPROMOTINGELECTRON T TRANS F FROMWATERTOP FER F LASTOQUINONEANDD THEULTIMATE Q QUEST IONOF HOWTHEOXIDATIONOFWATERTA F KESPLACETO PRODUCEMOLECULAROXYGENWITHOUTTHEFORMATIONOF HIGHLYREACTIVEOXYGENRADICAL INTERMEDIATES 0ART 6) !SSEMBLY L AND "IODDYNAMICS OF 0HOTO SYSTEM)) ) IS DEVOTED TO DISCUSSIONS OF THE UNIQUE ) BIODYNAMICPROCESSESTHATSUSTAIN03))FUNCT F ION IN F HE LIGHT VIVO4OPICS INCLUDETHESPECIAL PROCESSFORT INDUCED ASSEMBLY OF THE CATALYTIC -N#A CLUSTER THEMECHANISMSOFF RECOVERY FROMT F HE LIGHT INDUCE D D INACTIVATIONOF03)) F INPARTICULAR THESPECIAL DAMAGE ANDREPAIRCYCLEOFTHE$PROTEINTRANSCRIPTIONALAND D ING03)) GENEEXPRESSION TRANSLATIONAL REGULATION DUR D LATIONALPROCESSING ANDD THETRANSPORTANDPOST TRANS IN03))PROTEINBIOSYNTHESIS 0ART6)) #OMPARISONO # OF 0HOTOSY SYSTEM))WITTH /TH T ER . .ATURA L!RTI  IlCIALL 3YSTEMS ADDRESSEST D HEROLEOFF 03)) WITH RESPECT TO LIFE ON EARTH IN GENERAL AND MORE SPECIlCALLY TO APPLICATIONS IN BIOTECHNOLOGY Y 4HE K F DIVERSITY INTHESTRUCTURALFUNCT F IONAL PECULIARLACKO ORGANIZATIONOFTHECATALYTICSITEFORWATEROXIDATION IN03))OFPRESENT F DAY A PHOTOSYNTHETICORGANISMSIS CENTRAL TOTHEROLE03 )) HASHAD INTHEEVOLUTIONOF LIFE3IMILARLY ACOMPARISONBETWEEN03))ANDTHE CYTOCHROME C OXIDASEENZYME WHI W CH CATALYZESTHE XXII

MISTAKESWECAUSEDMANYPRO D BLEMSFOR,ARRY AN F D WEWISHTOEXPRESSOURSINCEREAPPRECIATIONOFHIS PERSEVERANCE EXPERTASSISTANCEANDD UNLIMITED GUID ANCEINBRINGINGTHIS BOOKTOPR K INT)NTERMSOFT F HE PRACTICAL ASPECTS IN PUBLISHING THE BOOK WE THANK THESTAFFAT3PR F INGER INPARTICULAR*ACCO&LIPSENAND .OELINE'IBSON ,ASTBUTNOTLEASTWEGIVEOURVERYSPECIALTHANKS TO'OVINDJEE(EHASPROVIDED GENEROUSADVICEAND ENCOURAGEMENT TO US ANDD TO THE AUTHORS OVER THE SEVERALYEARSFROMTHEINCEPTIONOFTHISPROJECTTOITS lNAL OUTCOME!STHE!)0(3ERIES%DITOR 'OVINDJEE HAS PROMOTEDD THEMOSTCOMPREHENSIVESETOF BOOKS ONPHOTOSYNTHESISYETTOAPPEAR COVERINGSUBJECTS F FROMT HEHISTORICAL PERSPECTIVESOFF PHOTOSYNTHESISTO ITSRELATIONSHIPWITH RESPIRATIONANDD THEFUNCT F IONING OFTHEBIOSPHERE'OVINDJEEHASPROVIDEDATREATISE F FOROURT IMECOMPARABLETOTHE%UGENE)2ABINOWICH F  TREATISEON0HOTOSYNTHESISOFYEARSAGO &INALLY 47 THANKS ALL THOSE IN THE 0HOTOBIOEN ERGETICS'ROUPFORT F HEIRUNDERSTANDING ANDD SUPPORT D INGTHISPROJ DUR O ECT INPARTICULARTO3AM(AY "RETT 7ALLACE !DELE 7ILLIAMSON )AIN -C#ONNELL AND 7 "RENDON#ONLAN!SPECIALTHANKSFROM47 F GOESTO 7 ICKK (ILLIER FOR 7ARW F BEINGANOUTSTANDINGSCIENTIlC COLLEAGUEANDAGOODMATEOVERTHEYEARS+3THANKS HISWIFE F 4AMIKO3ATOH FOR F HERSUPPORT

REVERSEREACTIONBYREDUC D ING/ TOWATER REVEALSVERY LITTLE MECHANISTIC COMMONALITY IMPLYING THAT THE APPEARANCEOFF 03))WASAUNIQUE ONE TIMEEVENT IN THEHISTORYOFT F HEEARTH&INALLY ATTEMPTSTOMIMIC 03))REACTIONSINNATURALBACTERIALREACTIONCENTERS SYNTHETICPROTEINS AND BIOINORGANICCOMPLEXESSTAND Q INTHECONSTRUCTIONAND D APPLICATIONOFF ARTIlCIAL UNIQUE PHOTOSYNTHESIS 4HROUGHOUTTHECONCEPTUALIZATION RElNEMENTAND lNALREALIZATIONOFT F HISBOOK WE HAVEMANYPEOPLETO THANK&IRSTOFALLWEWISHTOSINCERELYTHANKALLOFTHE F ITUDEAND D WILLINGNESSTO AUTHORS4HEIRPATIENCE FORT PROVIDEINDEPTHACCOUNTSCOVERINGALLASPECTSOF03 F ))STRUCTUREANDFUNCTIONHAVEMADETHISBOOKATRULY OUTSTANDING ADDITIONTOTHESCIENTIlC LITERATURE ,IKEWISE WITHOUTTHEEXPERTADVICEOFF THEMANY OUTSIDEREVIEWERS ABOOKOFTHISSCOPEWOULDNOTHAVE BEENPOSSIBLE)NTHISCAPACITY WES Y INCERELY THANK *AN-!NDERSON 4HE!USTRALIAN.ATIONAL 5NIVERSITY ,ARS %RIC !NDREASSON 5NIVERSITY OF 'OTHENBURG 4ERRY -"RICKER ,OUISIANA3TATE5NIVERSITY'ARY 7 "RUDVIG 9ALE 5NIVERSITY 7AH 3OON &RED 7 #HOW 4HE!USTRALIAN.ATIONAL5NIVERSITY2ICHARD $EBUS 5NIVERSITY OF#A F LIFORN F IAAT2IVERSIDE"RUCE $INER %) DU0ONT D DE.EMOURS#OMPANY*ULIAN %ATON 2YE 5NIVERSITYOF/TAGO'RAHAM&LEMMING F #ALIFORN F IA AT "ERK R ELEY 'OVINDJEE 5NIVERSITY OF R ANA #HAMPAIGN 3AM 5NIVERSITY OFF )LLINOIS AT 5RB (AY 4HE!USTRALIAN.ATIONAL5NIVERSITY7ARWICK (ILLIER 4HE!USTRALIAN.ATIONAL5NIVERSITY6AUGHAN (URRY 5N Y IVERSITY OF5MEÌ7O F LFGANG *UNGE5NI VERSITYOF/SNABRàCK(IROYUKI+OIKE 5NIVERSITYOF (YOGO*OHANNES-ESSINGER -AX0LANCK)NST K ITUTE FFOR "IOINORGANIC #HEMISTRY -AMORU -IMURO +YOTO 5NIVERSITY -ELVIN9 /KAMURA 5NIVERSITY + F IAAT3AN$IEGO4AKAAKI /NO )NSTITUTE OFF #ALIFORN H ICAL AND D #HEMICAL 2ESEARCH2ONALD D *0ACE OFF 0HYS 4HE !USTRALIAN .ATIONAL 5NIVERSITY *EAN $AVID 2OCHAIX 5NIVERSITY OF F 'ENEVA -ICHAEL 3EIBERT .ATIONAL 2ENEWABLE %NERGY ,ABORATORY *IAN 2EN 3HEN /KAYAMA5NIVERSITY-ICHAEL27ASILEWSKI .ORTHWESTERN5NIVERSITY6I 6 TTAL+9ACHANDRA ,AW A RENCE"ERK R ELEY.ATIONAL ,ABORATORY 7 7EALSOWISHTOTHANKTHOSEWHOWERECRITICALFOR lNALIZING THE BOOK FFOR PU UBLICATION )N THIS REGARD WESINCERELYTHANK*OE K L &REEMANFOR F HISEXPERTAS SISTANCEINTHEEDITINGOFTHEBOOK7ITHOUTHISHELP THElNALSTAGESWOULD HAVETAKENALOT LONGERTOCOME TOFRU F ITION!ND D WEOFFERAVERYSPEC F IALTHANKSTOTHE PERSONWHOWASMOSTCRUCIALINBRINGINGTHISBOOKTO THEPUBLI U CATIONSTAGE ,ARRY/RR4HROUGH OURMANY N

!UGUST  4HOMAS*7YDRZYNSKI 4HE!USTRALIAN.ATIONAL5NIVERSITY #ANBERRA !USTRALIA %MAILTOMWYDRZYNSKI ANUEDUAU +IMIYUKI3ATOH /KAYAMA5NIVERSITY /KAYAMA *APAN %MAIL KIMIYUKI CCOKAYAMA UACJP 

XXIII

'ERNOT2ENGERATTHE-AX 6OLMER)NSTITUTEFOR0 F H I HYS CALAND"IOPHYSICAL#HEMISTRY 4ECHNICAL5NIVERSITY N 4ORE6ÊNNGÌRD INTH T E$EPARTMENTOF "ERLIN 'ERMANY H ICS #HALMERS5NIVERSITY "IOCHEMISTRYANDD "IOPHYS 'ÚTEBORG 3WEDENAND9ORINAO)NOUEINTHE3OLAR%N ERGY R Y2ESEARCH'ROUP T H THE2)+%.)NSTITUTEOFF 0HYSICAL ANDD #HEMICAL 2ESEARCH 3AITAMA *APAN$URINGTHIS TIMEHEPURSUEDTHEIDEATHATTHECONTROLLEDACCESSOF SOLVENTWATERTHROUGH THEPROTEINSUPERSTRUCTURETO THECATALYTICSITEWASCRITICAL NOTONLYFORMA F INTAINING THESTABILITYOFTHEFUNCTIONALMANGANESEBUTALSOIN / / BOND FORMAT F ION DUR D ING /PRODUCT D ION)N HE JOINEDD THE2ESEARCH3CHOOLOFF "IOLOGICAL 3CIENCE 23"3 AT4HE!USTRALIAN.ATIONAL5NIVERSITYIN#AN BERRAAND DUR D ING  BECAMETHE lRST(EADD OF THENEWLYCREATEDD 0HOTOBIOENERGETICS'ROUP!TTHE 23"3HEEMBARKEDONSOMEOFHISMOSTSIGNIlCANT WORK R BY DEVELOPINGWITH HIS0H$STUDENTSTHE lRST RAPID MILLISECONDD TIMERANGE MASSSPECTROMETRIC METHODSOFOXYGENISOTOPICEXCHANGEFORMONITOR ING THESUB U STRATEWATERDIRECTLY ATTHECATALYTICSITEIN U 0HOTOSYSTEM))4HE BINDINGPROPERTIESOFF THESUB STRATEWATERASDETERMINEDBYTHESEMEASUREMENTSARE CRITICAL FORT F HECHEMICALMECHANISMOF / FORMAT F ION )N HESETUPARESEARCHPROGRAMONTHEDESIGN ANDSYNTHESISOFPHOTO ACTIVEPROTEINSINWHICHTHE lRSTARTIlCIAL QUINONE BASEDD PHOTOSYNTHETICREACTION CENTER WAS ENGINEERED FFROM A NATURAL CYTOCHROME PROTEIN4HEOVERALLGOALOFTHESESTUDIESISTOESTABLISH THEPRACTICALEXPERIMENTALPRINCIPLES FORT F HEFUTURE F DEVELOPMENT OF F ARTIlCIAL SYSTEMS FOR F LIGHT ENERGY CONVERSION PROCESSES )N  HE SET UP U ANOTHER RESEARCHPROGRAMUNDERA(UMAN&RONTIER3CIENTIlC 0ROGRAMGRANTTOSTUDYTHEEVOLUTIONARYCHEMISTRY OFOXYGENICPHOTOSYNTHESISINPRESENT DAYORGANISMS F FROMEXTREMEAN D UNUSUALENVIRONMENTSANDD THEPOS SIBLEROLEOF BICARB R ONATEASANALTERNATIVESUB U STRATE F / PRODUCTION7ITHRESPECTTOOTHERPROFESSIONAL FOR ACTIVITIES HEWASCHAIROFF THEORGANIZINGCOMMITTEES F HE 2OBERTSON3YMPOSIUMON#HL FORT # OROPHY H LLL &LUO RESCENCE IN  ANDTH D E NDD!NNUAL-EETINGOFTHE !USTRALIAN3OCIETY T FOR"IOP F HYSIC Y S IN CO CHAIR F FORTWOSYMPOS IAATTHE TH )NTERNATIONALL 0HOTOSYN THESIS#ONGRESS IN ANDWASONTHEORGANIZING COMMITTEEANDD CO EDITORFORT F HESPECIAL SYMPOSIUM &ROM"IOPHYSICSTO-O H LECULAR"IOLOGY!0ATH T INTH T E 2ESEARCHOF O 0HOTOSYSTEM))) IN

5IPNBT
+PIO
8ZES[ZOTLJ

4HOMAS *7YDRZYNSKI WAS BORN IN  IN 3AINT ,OUIS -ISSOURI ANDISCURRENTLY A3ENIOR&ELLOWIN THE2ESEARCH3C H HOOL OFF "IOLOGICAL 3CIENCES23"3 AT THE!USTRALIAN.ATIONAL5NIVERSITY!.5 #ANB N ERRA !USTRALIA(ECOMPLETEDHIS0H$0LANT0HYSIOLOGY INATTHE5NIVERSITYOFF )LLINOISAT5RB R ANA #HAM PAIGN 5)5# UNDERTHESUPERVISIONOFF 'OVINDJEE &ORHIS0H$THESISRESEARCHHEWASTHElRSTTOAPPLY SOLVENT WATER PROTON NUCLEAR MAGNETIC RESONANCE .-2 RELAXATIONMEASUREMENTSASA DYNAMICPROBE OFTHEFUNCTIONALMANGANESEIN0HOTOSYSTEM))INSITU !LSO WHIL W EHEWASA0H$STUDENT HEPROVIDED D THE lRSTCLEAREXPERIMENTALEVIDENCE IN FORAS F ITE OFBICARBONATEACTIONONTHEELECTRONACCEPTORSITEIN D A.ATIONAL3CIENCE 0HOTOSYSTEM))(EWASAWARDED &OUNDATION.3& %NERGY 2ELATED D 0OSTDOCTORAL2E SEARCH&ELLOWSHIPINANDWORKEDWITH+ENNETH 3AUER AT THE ,AWRENCE "ERK R ELEY .ATIONAL ,ABORA TORYFROMTO W F WHERE HEOBTAINED D SOMEOF THElRSTEXPERIMENTALEVIDENCETOINDICATETHATTHE F FUNCT IONAL MANGANESECHANGESOXIDATIONSTATEDUR D ING / PRODUCT D ION(ETHEN JOINED INDUSTRYAN D D WORRKED F FROMTOINTHE"IOTECHNOLOGY$IVISION D IL #OMPANY N )NDIANA #ORPORATE2ESEARCH 3TANDARD/ IN#HICAGO NOWAPARTOFF THE"RITISH0ETROLEUM"0 #ORPORATION 3UBSEQUENTLY HERE JOINEDACADEMIA AND D RECEIVED D RESEARCH FE F LLOWSHIPSFROMT F THE!LEXANDER VON (UMBOLDT &OUNDATION 'ERMANY  THE 3CIENCEAND4ECHNOLOGY!GENCY *APAN THE 7ENNERGREN&OUNDATION 3WEDEN THE.ORD ISKA&ORSENINGSRADET .&2 3WEDEN AND D THE $EUTSCHEFORSCHUNGSGEMEINSCHAFT$&' 'ERMANY  /VERTHISPERIOD INHISCAREERHEWORK R ED D WITH XXIV

,JNJZVLJ
4BUPI

THE03))CORECOMPLEXCONTAINSTWOPOLYPEPTIDESOF APPARENTMOLECULARMASSESOFABOUTK$A WHICH EVENTUALLYWEREIDENTIlEDD ASTHE$ANDD $PROTEINS F F 0LANT0HYS H IOLOGYAT )N HE BECAME0ROFESSORO T THE/KAYAMA5NIVERSITY WHEREHECONTINUEDHISWORK ONTHE03))CORECOMPLEX/NEOFF THESUCCESSESIN HIS LABORATORY WASTHEISOLATIONOFF AN/ EVOLVING03 ))CORECOMPLEX WHICHWASTHElRSTDEMONSTRATION THAT / EVOLUTION COULD D TAKE PLACE IN THE ISOLATED PROTEIN PIGMENT COMPLEX ! MAJ A OR ACHIEVEMENT F FROMHISLABORATORYATTHE/KAYAMA5NIVERSITYWAS THEISOLATIONOFT F HE$ $ CYTOCHROME B COMPLEX 4HISSTUDYESTABLISHEDD THATTHESITEOFF PRIMARYCHARGE SEPARATIONINTHE03))ISLOCATEDINTHECOMPLEXTHAT CONTAINS$ANDD $PROTEINS THEPROTEINSTHATARE HOMOLOGOUSTOTHE,ANDD -SUUBUNITSOFF THEPURPLE BACTERIALREACTIONCENTER(ETHENPURIlEDTHECARBOXY TERMINAL PROCESSING PROTEASE FFOR THE $ PRECURSOR PROTEIN FROMSP F INACH ANDD EXTENSIVELYANALYZEDD THE ENZYMATICPROCESS&ROMTO HESERVED ASAN!DJUNCT0ROFESSOR F INTHE.ATIONAL )NSTITUTEFOR F "ASIC"IOLOGYAT/KAZAKI3INCETHEN HEHASBEEN ENGAGEDINTHERANDOMMUTAGENESISOFTHE$PROTEIN F FROMT THECYANOBACTERIUM3Y 3 NECHOCY CYSTIS SP0## WITHTHEGOALOFF OBTAININGPHOTO TOLERANTMUTANTSAND MUTANTSDElCIENTINAUTOTROPHYFORUSEINDETAILED STRUCTUREFUNCT F IONANALYSESOFT F HE03))REACTIONCEN TER+IMIYUKI 3ATOH HASSERVEDD ASTHE0RESIDENTOFT F HE *APANESE3OCIETYOF0LANT0HYSIOLOGISTSn ANDD ASTHE3ECRETARY OFF THE)NTERNATIONAL3OCIETYOF 0HOTOSYNTHESIS2ESEARCH n 

+IMIYUKI 3ATOH IS AN %MERITUS 0ROFESSOR AT THE /KAYAMA5NIVERSITYIN*APAN(ERECEIVEDHISUN DERGRADUATE D DEGREE FFROM /KAY A AMA 5NIVERSITY AND OBTAINED HIS 0H$ $OCTOR OF F 3CIENCE FFROM THE 5NIVERSITY OF4OKYO IN  IN "IOCHEMISTRY AND RKONPHOTOSYNTHESIS "IOPHYSICS(ESTARTED HISWORK UNDERTHEGUIDANCEOF0RO F F FESSOR( IROSI (UZISIGEOF /KAYAMA 5NIVERSITY!T THAT TIME HE CONTRIBUTED TOTHEIDENTIlCATIONOF FERRE F DOXIN DEPENDENTNITRITE REDUCTASE D IN CHLOROPLASTS AND D WAS INVOLVED IN THE ANALYSISOFPHOTOINACTIVATIONOFPHOTOCHEMICALACTIVI TIES INCHLOROPLASTS&ROMTO HEWASAPOST DOCTORAL FE F LLOWINTHELABORATORYOF0RO F F FESSOR7ARREN "UTLERATTHE5NIVERSITYOF#ALIFORNIAAT3AN$IEGO ,A *OLLA #ALIFORN F IA(ERE HE INITIATED D WORRKONTHEBIO CHEMICAL SEPARATIONOFC F HLOROPHY H LL PROTEINCOMPLEXES AIMING TO IDENTIFY THE PIGMENT PROTEIN COMPLEXES RESPONSIBLE FFOR EACH ONE OF F THE THREE mUORESCENCE EMISSION BANDS&&AND D & OBSERVEDD AT +4HROUGHTHISSTUDY HEBECAMEDEEPLYINVOLVED INTHE BIOCHEMICALSTUDY OFF 0HOTOSYSTEM))03)) AND D SUCCEEDED INPURIFY F INGTHE03))CORECOMPLEX F FROMSPINACHBYINTRODUCINGISOELECTRICFOCUSING(E THENWORK R ED INTHE LABORATORIESOF*0 F HILIP4HORNBER ATTHE5NIVERSITYOF#A F LIFORN F IAAT,OS!NGELES AND D OF #HARLES!RNTZENATTHE0LANT2ESEARCH,ABORATORYAT -ICHIGAN3ATE5NIVERSITY %AST,ANS Y ING -ICHIGAN 4HERE HEIDENTIlED D THEPOLYPEPTIDECOMPOSITIONOF THE PURIlED 03 )) CORE COMPLEX )N COLLABORATION WITH!RNTZENSRESEARCH GROUP HE DEMONSTRATED D THAT

XXV

+PFM
'SFFNBO

-BSSZ 0SS

*OEL &REEMAN BORN IN WASTHETECHNICAL OFlCER FFOR THE 0HOTOBIOENERGETICS 'ROUP IN THE 2ESEARCH 3CHOOL OF "IOLOGICAL 3CIENCES AT THE !USTRALIAN .ATIONAL5NIVERSITY n (ECOMPLETED HIS "ACHELOROFF !QUACU Q LTUREWITH HONOURSINFROM F 4HE5NIVERSITYOF4ASMANIA$URINGTHISPERIOD HE WAS GRANTED D MEMBERSHIP INTHE'OLDEN+EY.ATIONAL (ONOUR 3OCIETY  AND D PLACED D ON THE $EANS 2OLLOF%XCELLENCE   (ETHENJOINEDTHE 0HOTOBIOENERGETICS'ROUPWH W EREHEHAS DEVELOPED APROTOCOL FORT F HEEXTRACTIONOFT F HE FUNCT F IONAL MAN GANESE AND CALCIUM FROM 0HOTOSYSTEM )) AND HAS SINCE WORK R ED D ON PRO OJECTS INCLUDING SPECTROSCOPIC STUDIESOFF 0HOTOSYSTEM)) INCHLOROPHY H LL D CONTAINING D !CARYOCHLORISMARINA HYDROGENPRODUCTIONMECHA NISM INMICROALGAEAND ITSINDUSTR D IAL POTENTIAL AND D THE EVOLUTIONARY BIOLOGYOFF OXYGENICPHOTOSYNTHESIS IN PARTICULAR THEROLEOFBICARBONATEIN0HOTOSYSTEM)) STEADY STATEELECTRONTRANSPORT IN 3PIRU 3 LINAMAXIMA *OEL NOWWORK R SASAPOLICYAND D TECHNICAL OFlCERWITH 0LANT 0ROGRAMS AT THE !USTRALIAN 1UARANTINE AND )NSPECTION 3ERVICE (E ASSISTS IN THE DEVELOPMENT AND IMPLEMENTATION OFF OPERATIONAL STANDARDS AND PROCEDURESFORTHEIMPORTATIONOFGRAINS SEEDSAND NURSERYSTOCK INTO!USTRALIA

,ARRY /RRWASBORNINANDD RAISED INATRAVELING CARNIVAL UNTIL HIS FAM F ILY SETTLED DOWN IN NORTHERN 7ISCONSIN9EARS LATER HE GRADUATED FROM!RIZONA 7 3TATE5NIVERSITY !35 WITH A"!IN%NGLISH LITERA TURE NOTGRAMMAR ,IKEMOST,IBERAL !RTSMAJ A ORS HEWORKEDATAVARIETYOFODDJOBSUNTILHELANDEDA POSITION INAS!DMINISTRATIVE#OORDINATORFOR F THE .3& OFlCE IN -C-URDO !NTARCTICA4HERE HE BECAMEVERYINTERESTEDINWORKINGWITHTHESCIENTISTS THATCAMETO@4HE)CE FROMA F LLOVERTHEWORLD D AND D D IZZAPARLOR USINGONEOF HISODDD SKILLSLEARNEDASAP MANAGER PRODUCEDTHEMOSTFAMOUSPIZZAPARTIESON THECONTINENT!FTER F LEAVING THE!NTARCTICPROGRAM HE WAS HIRED FFOR A SIMILAR POSITION WITH THE!35 #ENTERFORTHE3TUDYOF%ARLY%VENTSIN0HOTOSYNTHE SIS IN.OPIZZASWERENEEDEDD THERE BUTOTHER PASTJOBSKILLSPROVEDD USEFU F LWRITING PHOTOGRAPHY H PURCHASING PROOF READING OFlCE ADMINISTRATION D INGANDD TYPESETTING3TILL ATTHE EDITING WORDPROCESS 0HOTOSYNTHESIS #ENTER HE IS INTERESTED IN SCIENCE EDUCATION ESPECIALLYPHOTOSYNTHESIS FORTHEGENERAL PUBLI U C ,ARRY HAS PLAY A EDD A KEY ROLE IN THE SUCCESS OFF THE!)0(!DVANCES IN0HOTOSYNTHESISANDD 2ES PIRATION 3ERIESSINCEITSINCEPTIONIN(EHAS GUIDED W D ITH HUMOR THE%DITORSASWELLASTHEAUTHORS IN@STAYINGONTRACK4OGETHERWITH'OVINDJEE ,ARRY HAS PRODUCEDONEOFTHEMOSTUSEDWEBARTICLESON @0HOTOSYNTHESISANDD THE7EB)T ISAVAILABLEAT HTTP PHOTOSCIENCELAASUEDU DUPHOTOSYN NPHOTOWEBASWELL ASATHTTPWWWLIFEUIUCEDUGOVINDJEEPHOTOWEB )TISCURRENTLY UNDERGOING REVISIONTHUS READERSARE WELCOMETOSENDD SUGGESTIONSTOLARRYORR ASUEDU ANDGOV LIFEUIUCEDU

XXVI

"VUIPS
*OEFY +LUG $AVID2n

¯HRLING +ARIN!n !LLEN *AMES0n !NANYEV 'ENNADY-n !RO %VA -ARIn

-AGNUSON !NNn -AYlELD 3TEPHEN0n -ESSINGER *OHANNESn

"ARB R ER *AMESn n "ARTER ,AURA-#n "ERTHOMIEU #ATHERINEn "ITTL 2OBERTn "LANKENSHIP 2OBERT%n n "RICKER 4ERRY-n "RITT 2$AVID D n "RUDVIG 'ARY7n "URNAP 2OBERT,n

.IELD *ONn .IXON 0ETER*n .OGUCHI 4AKUM K In 0ACE 2ONALD*n D  0AKRAS K I (IMADRI"n 0ETROULEAS 6ASILIn 0OGSON "ARRY*n 0UTNAM %VANS #INDYn

#HOW 7AH3OONn #ROFTS !NTON F NY 2n

2ENGER 'ERNOTn 2ISSLER (EATHER-n 2OOSE *OHNNA,n 2 HERFOR 2UT F D !7 D 7ILLIAMn

$EBUS 2ICHARD*n $INER "RUCE!n n $ISMUKES '#HARLESn n

3ARCINA -ARYn 3ATOH +IMIYUKIn 3HEN *IAN 2ENn 3HI ,AN 8INn 3HINKAREV 6LADIMIRn 3TYRING 3TENBJÚRNn 3UGITA -AMORUn

%ATON 2Y 2 E *ULIAN*n %VANS -ICHAEL #7n &ALLER 0ETERn &ERGUSON -ILLER 3HELAGH n &RANK (ARRY!n &UFEZAN # F HRISTIANn

4HEG 3TEVEN-n 4HORNTON ,EEANN%n 4OMMOS #ECILIAn

'ANTT %LISABETHn 'IBNEY "R Y IAN2n 'OVINDJEEn 'REEN "EVERLEY2n

VAN'ORKOM (ANS*n VAN'RONDELLE 2IENKn K  VAN2ENSEN *ACKK *3n

(AMMARSTRÚM ,EIFF n (ANKAMER "ENn (ILLIER 7ARWICKn K  (OLZWARTH !LFRE F D 2n

7ATT 2ICHARDD n 7 7IKSTRÚM -ÌRTENn 7 7ILLIAMS *O!NN#n 7ITT (ORST4n 7 DRZYNSKI 4HOMAS*n 7Y

)KEUCHI -ASAHIKOn )WATA 3On

9ACHANDRA 6I 9 6 TTAL +n 9 9AMAGUC HI +ENICHIn 9 9OCUM #HARLES&n n 

+ÈLMÈN ,ÈSZLØn +AMIYA .OBUOn +AWAMORI !SAKOn +LIMOV 6YACHESLAV6n XXVII

$PMPS
1MBUFT

&IIG  0ROCHLORON 03))DIMERSURROUNDED BY PROCHLOROPHYTE#HL AB 0CB MONOMERS#OLORCODEGREEN #HL A OR#HL B BLUE TRANS & MEMBRANEHELICESYELLOW $ORANGE $DARK R GREEN #0RED #0PURP D LE LOWMOLECULARWEIGHTSUB U UNITSCYAN CYTOCHROME B 3CALEBAR  ¯#OURTESY OF*"ARBER3EE#HAPTER P

&IIG  3TEREOGRAMOFF ,(#))TRIMERINDIVIDUA & D L MONOMERSINDICATED BY ) ))AND D ))) VIEWED FROMT F HESTROMALSIDE SHOWING POSITIONSOF #HLSAND D CAROTENOIDS#OLORCODE GREEN #HL ABLUE #HL B ORANGE NEOXANTHINMAGENTA XANTHOPHYLL CYCLECAROTENOIDSGREY RIBBONS HELICES#OURTESYOF7# F HANG3EE#HAPTER P 47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PP#0n#0 D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D

$PMPS
1MBUFT

&IIG  4HEMODELEDD COFACTORS & F INTHE03))REACTIONCENTER!DAPTED FROM6AN2ENSEN F  AND D ORIGINALLY FROM8 F IONGETAL A  3EE#HAPTER P

&IG  3TEREOVIEWSOFELECTRONDENSITYMAPSFORPARTOFTHEWATER OXIDIZINGCOMPLEXAPUTATIVENON PROTEINLIGANDMODELEDASBICAR &I BONATE ISSHOWNINBLUE2EPRODUCEDWITHPERMISSIONFROM&ERREIRAETAL 3EE#HAPTER P

#0

$PMPS
1MBUFT

&IG #ARTOONSOF03))ANDITSASSOCIATEDLIGHT HARVESTINGCOMPONENTS! OFHIGHERPLANTSANDGREENALGAEAND" OFCYANOBACTERIA & EMPHASIZINGSUBUNITCOMPOSITIONANDPRIMARYANDSECONDARYELECTRONTRANSFERSTEPSTHATOCCURINTHEREACTIONCENTER$AND$PRO TEINS)NTHECASEOFT F HE HIGHERPLANTSANDD GREENALGAE ! NOTETHATTHESECONDARYANTENNAISMADEUPOFF #HL AB A BINDING,HCB PROTEINS INTRINSICTOTHEMEMBRANE WHIL W EIN " THECYANOBACTERIAL03)) ISSERVICED BY ALARGEEXTRINSIC SOLUBL U EPHYCOBILIPROTEINMASSONITS STROMALSIDE!LSONOTE WH W ENCOMPARINGTHETWOSYSTEMS THECOMPOSITIONAL DIFFERENCESPRESENT F F HE LUMENALLY BOUND FORT D EXTRINSIC SUB U UNITSOFT F HE/%#3EE#HAPTER P 

&IG IG  #OMPARISONOFF THE03))CORE DIMEROF ! SPINACH (ANKAMERETAL B AND " 3ELONGATUS:OUNI ETAL  AND RESPECTIVEASSIGNMENTSOFTHELOWMOLECULARWEIGHT,-7 SINGLE TRANSMEMBRANEHELICALSUBUNITS4HEDARKBLUEREGIONIN! SHOWS THEHELIXORGANIZATIONOFTHESPINACH#0 2#SUBCOMPLEX2HEEETAL  WHICHHADTO BEMODIlED TWOTRANSMEMBRANEHELICES ADJACENTTOHELIX"OFTHE$AND$PROTEINSBEINGREMOVED4HE REMAININGCYANREGIONSCONTAINTHEADDITIONALTRANSMEMBRANEHELICES OF#0ANDOTHER,-7COREDIMERSUBUNITS#OLORCODERED #0GREEN #0YELLOW $ORANGE $PURPLE #YT B WHITE ,-7SUB U UNITS3EE#HAPTER P

#0

&IG ! 3IDEVIEWOFTHE$MAPOFTHESPINACH03))COREDIMER STROMALSIDEUPPERMOST EMPHASIZINGTHETRANSMEMBRANEREGION ANDLUMENALEXTENSIONSASSIGNEDTOTHELOOPS JOININGHELICESANDOF#0REDARROWS AND#0GREEN ARROWS (ANKAMERETAL B #OLORCODEASFORPANEL# BELOW" 0REDICTEDFOLDINGFROMHYDROPATHYANALYSES W OF#0AND#0(ANKAMERETAL B # 4RANSMEMBRANE VIEWOFASECTIONOFTHE$MAPOFTHESPINACH03))COREDIMER (ANKAMERETAL B 4HEMAPISSAMPLEDAT ^¯ANDISDISPLAYEDASCHICKEN WIRECONTOURS WITHCOMPENSATIONFORHIGH RESOLUTION FA F DEOUT WHI W TECONTOURS TEMPERATUREFACTORn  m ANDD WITHNOCOMPENSATIONBLUE F D BRANE HELICESCOLORCODE YELLOW $ORANGE $ RED #0 D GREEN #0CYAN _ AND ` SUB U UNITSOF# F YTBMAGENTA ,-7SUB U UNITS $ !N CONTOURS  m &ITTEDTRANSMEM Q IEWOFT F HE$STRUCTURALMODELOFT F LECTRONCRYSTALLOGRAPHY#O F HESPINACH 03))CORE DIMERFROME Y LORCODEASFORPANE U UNITSOF# F L # ABOVE EXCEPTPURPLE _ AND ` SUB F YTB OBLIQUEV WHITE ,-7SUB U UNITS(ANKAMERETAL A "ARFORPANE F LS ! AND # REPRESENTSNM3EE#HAPTER P

$PMPS
1MBUFT

#0

$PMPS
1MBUFT

&IIG  3TRUCTUREOFTHEMONOMERICPARTOF03))WITHASSIGNMENTOFHELICES COFACTORSANDSUBUNITSAT¯ RESOLUTIONADAPTEDFROM & :OUNIETALA ! 3IDEVIEWOF03))MONOMERA F LONG THE LONGAXISOFF $$SUB U UNITSWITHSLIGHTLYTILTED D MEMBRANEPLANE4HE lVETRANSMEMBRANEHELICESOFSUBUNIT$$AREINDICATEDINRED D ANDYELLOW4HESIXTRANSMEMBRANEHELICESOFTHEANTENNASUBUNIT #0#0ARECOLOREDDARKBLUEANDGREEN4HETWELVETRANSMEMBRANEHELICESOFEIGHTSMALLSUBUNITSARELIGHT BLUEANDHAVENOT BEENASSIGNEDHERE4HE _ AND ` HELICESOF#YT B AREDARKGREEN4HEEXTRINSICSUBUNIT0SB/K$A ISSHOWNASA ` SHEETSTRUCTURE AND#YT C K$A ASHELICALMODEL4HE-NCLUSTERWITHINTHEMEMBRANE ISLOCATEDCLOSETOTHEDOMAINOFTHEEXTRINSICSUBUNITS &ORCLARITYTHE#HLCOFACTORSAN F D OTHERSHAVEBEENOMITTED" 4HESTRUCTURE IN ! WITH VIEWDIRECTION FROMT F HESTROMALSIDEONTO F BETWEENTHETWOMONOMERICPARTSOFT F HE DIMERAREINDICATED D ASWELL ASTHEVIEW THEMEMBRANEPLANE4HELOCAL C AXISATTHE INTERFACE DIRECTIONOF#O F LOR0LATE &IG!3EE#HAPTER P

#0

$PMPS
1MBUFT

&IG  4OP!RRANGEMENTOFCOFACTORSOFTHEELECTRONTRANSPORTCHAIN %4# LOCATEDINSUBUNIT$$AT¯RESOLUTIONVIEWDIREC &I TIONASINDICATEDIN#OLOR0LATE &IG"ANDALONGTHEMEMBRANEPLANE 4HECENTER TO CENTERDISTANCES¯ AREINDICATEDASWELLAS THEDOTTEDPSEUDO C AXISWHICHISPARALLELTOTHELOCALC AXIS4HESYMMETRY OFTHE%4#ISBROKENWITHTHELOCATIONOFTHE#YTB TOWARDSTHESTROMALSIDEANDTHE-NCLUSTERTOWARDSTHELUMENAL SIDE3EEALSOTHECLOSEUPVIEWOF#YT B ALONGTHEMEMBRANEPLANE "OTTOMLEFTA -NCLUSTERWITHVIEWALONGTHEMEMBRANEPLANE ITSLONGAXISPOINTSTOWARDSTHELUMENTILTED^ AGAINSTTHE PLANE B VIEWFROMTHELUMENONTOTHEPLANE"OTTOMRIGHT-NCLUSTERSURROUNDEDBYHELICESANDLOOPSOFSUBUNIT$AT¯ RESOLUTION m ANDOF9: AT m :OUNIET 4HEPOSITIONOF0$ OF 0 ANDD 9: OFTHE%4#AREINDICATED$ENSITY OFTHE-NCLUSTERCONTOUREDATm AL A B 3EE#HAPTER P

#0

$PMPS
1MBUFT

&IIG  #RYSTALSTRUCTUREOFF 03)) FROM & F 4VULCANUS! 3IDEVIEWOFF ONE03))MONOMER " 4OPVIEWOFF THETRANSMEMBRANEREGION F FROMT HE LUMENAL SIDEOFF ONE03))MONOMER TOGETHERWITH PARTOFF ANEIGHBORING03))MONOMERSHOWING THEFORMAT F IONOFA03)) F DIMER INTHECRYSTAL4RANSMEMBRANE HELICES 4-(S OFF $$SUB U UNITSARENUMBERED D ! % AND 4-(SOF#0 F #0ARENUMBERED D ) 6) 4 STRUCTUREBUTNOTIN 4ELONGATUS 4 S STRUCTURE# 3TRUCTURES !NARROWIN" INDICATESONEADDITIONAL#HLFOUNDIN#0OF4VULCANUSS OF$$SUBUNITS&OUR#HLSANDTWO0HEOSARESHOWN TOGETHERWITH9: 9$ ANDTHE-N CLUSTER2ESIDUESPOSSIBLYPROVIDINGLIGANDS TOTHE-N CLUSTERARELABELED$ 3TRUCTURESOF#0 #0SUBUNITS% 3TRUCTUREOFTHEK$APROTEIN& 3TRUCTUREOF#YT C' 3TRUCTUREOFTHEK$A PROTEIN3EE#HAPTER P

#0

#0

D

T

$PMPS
1MBUFT

$PMPS
1MBUFT

&IG /VERALLSTRUCTUREOF03))! 6IEWOFTHE03))DIMERPERPENDICULARTOTHEMEMBRANENORMAL(ELICESAREREPRESENTEDASCYLINDERS WITH$INYELLOW $INORANGE #0INRED #0INGREEN #YT BINWINERED 0SB, 0SB- AND0SB4INMEDIUMBLUE AND0SB( 0SB) 0SB* 0SB+ 0SB8 0SB:ANDTHEPUTATIVE0SB.INGREY4HEEXTRINSICPROTEINSARE0SB/INBLUE 0SB5INMAGENTA AND0SB6IN CYAN#HLOROPHYLLSOFTHE$$REACTIONCENTERARELIGHTGREEN PHEOPHYTINSAREBLUE CHLOROPHYLLSOFTHEANTENNACOMPLEXESAREDARK GREEN ` CAROTENESAREORANGE HEMESANDNON HEME&EARERED 1!AND1"AREMAGENTA4HEOXYGENEVOLVINGCENTER/%# ISSHOWNAS THEREDOXYGENATOMS MAGENTA-NIONS ANDCYAN#A BALLS" 6IEWOFTHE03))MONOMERALONGTHEMEMBRANENORMALFROMTHE LUMENALSIDE!PARTOFTHEOTHERMONOMERINTHEDIMERISSHOWNTOEMPHASIZETHEREGIONOFMONOMERMONOMERINTERACTIONALONGTHE DOTTEDLINE4HEPSEUDO TWO FOLDAXISPERPENDICULARTOTHEMEMBRANEPLANEPASSINGTHROUGHTHENON HEME&ERELATESTHETRANSMEMBRANE HELICESOFTHE$$HETERODIMER THELOWMOLECULARWEIGHTSUBUNITS 0SB)AND0SB8AND#0AND#0ASEMPHASIZEDBYTHEBLACK LINESENCIRCLINGTHESESUBUNITS#OLORCODINGISTHESAMEASIN! 3EE#HAPTER P

#0

$PMPS
1MBUFT

&IG  3TRUCTUREOFT &I F HE LARGEEXTRINSIC DOMAINSOF ! #0AND " #04HEMEMBRANEEXTRINSICDOMAINOFF #0CONTAINSTWO LONG RESIDUESTOTO  ANDTWOSHORT _ HELICESRESIDUESTOTO ASTRAND` SHEETRESIDUESTO TO ANDAHELIXRESIDUESTO 4HEHELIX COMPOSEDOF''%4-2&7$RESIDUESTO ISFULLYCONSERVED D IN ALL#0SEQUENCESANDCONTAINSRESIDUESINVOLVEDINWATEROXIDATIONATTHE/%#'LUAND!RG ISSHOWNINPINK4HELUMENAL DOMAINOF#0CONTAINSTWOLONGRESIDUESTOTO ANDFOURSHORTHELICESRESIDUESTOTOTO F DED RESIDUESTOTO D TOTO ATHREE STRANDED RESIDUESTO D TO ANDD AFOUR STRAN D 4HECONSERVED D &&%4&36,6SEQUENCE RESIDUES D TOTO ANDD ATWO STRANDED ` SHEETRESIDUESTOTO F HYDROPHOBICENVIRONMENTAROUND 9$ $ 4Y 4 R 3EE#HAPTER P TO FORMSA

&IIG  !VIEWOFF THE LUMENALSURFACES & F HOWING THEEXTRINSICPROTEINS0SB/ 0SB5AND D 0SB6 THE LARGEEXTRINSIC LOOPSOFF #0AND #0 AND D THELUMENAL PORTIONSOFF THE$AND$PROTE D INS4OGETHERTHESEPOLYPEPTIDESFORMA@PROTE F IN CAPOVERTHE/%#3EE#HAPTER P

#0

$PMPS
1MBUFT

&IG  #OFACTORS &I F INVOLVED INELECTRONTRANSFER F ! %LECTRONTRANSFERCO F F FACTORSS HOWNPERPENDICULARTOTHE INTERNAL PSEUDO TWO FO F LD D HEOPHYTINS HAVE BEENREMOVED FORC F LARITY4 Y HESIDECHAINSOF 9: $ 4Y 4 R AND D $ (IS 4HEPHYTYL TAILSOFF THECHLOROPHYLLSANDP ARESHOWN IN YELLOWAND 9$ $ 4Y 4 R ANDD $ (IS INORANGE4HE FOURC F HLOROPHYLLSCOMPRISING0ARE INDIRECTVANDER7AALS CONTACT ANDOTHERELECTRONTRANSFERDISTANCESAREGIVENIN ¯ " 4HE0DIMEROFCHLOROPHY H LLS 0$ AND 0$ AND D ACCESSORY CHLOROPHYLLS #HL$ AND#HL$ 4HEPROTEINMAINCHAINISDEPICTEDINLIGHTGREYWHILETHESIDECHAINBONDSANDCARBONATOMSFOLLOWTHECOLORINGOF THEPROTEINSUBUNITSYELLOW $ORANGE $ 4HEHISTIDINELIGANDS $ (ISAND$ (IS ARESHOWNASWELLASTHEREDOXACTIVE 9: $ (ISAND 9$ $ (ISPAIRS4HEVIEWISDOWNTHEPSEUDO TWO FOLDAXISFROMTHESTROMALSIDE3EE#HAPTER P

#0

$PMPS
1MBUFT

&IG4HEOXYGENEVOLVINGCENTER/%# ! 6IEWOFTHE/%#WITHSIDECHAINLIGANDSANDPOSSIBLECATALYTICALLYIMPORTANTSIDECHAIN RESIDUES-NIONS #A ANDOXYGENATOMSARESHOWNINMAGENTA CYANANDRED RESPECTIVELY/NEUNIDENTIlEDNON PROTEINLIGANDTO THE/%#ISCOLOREDINBLUE0ROTEINMAINCHAINISDEPICTEDINLIGHTGREYWHILETHESIDECHAINBONDSANDCARBONATOMSFOLLOWTHECOLOR CODINGOFTHEPROTEINSUBUNITSYELLOW $GREEN #0  m!WEIGHTED\&O\ \&C\DENSITYISSHOWNASALIGHTBLUEWIREMESHCONTOURED ATm!NOMALOUSDIFFERENCE&OURIERMAPSAT¯-NEDGE CONTOUREDATm AND¯HIGHLIGHTS#A CONTOUREDAT m WAVELENGTHSARESHOWNINMAGENTAANDBLUE GREEN RESPECTIVELY" 4HESAMEAS! BUTWITHAROTATIONAROUNDTHEY AXISOF  ANDWITHOUTELECTRONDENSITYANDANOMALOUSDIFFERENCEMAPS# 3CHEMATICVIEWOFTHE/%#2ESIDUESIN$ $AND#0SUBUNITS ARESHOWNINYELLOW ORANGEANDGREEN RESPECTIVELY8 8AND8AREPOSSIBLESUBSTRATEWATERBINDINGPOSITIONSTO-N 8 ANDTO #A 8 AND8 IDENTIlEDFROMTHEPOSITIONOFTHENON PROTEINLIGANDANDCOORDINATIONPATTERNOF-NAND#AIONS 0OSSIBLEWATER MOLECULES WHICHARENOTVISIBLEATTHECURRENTRESOLUTION AREINDICATEDAS7$ (YDROPHILICPATHWAYBETWEENTHEACTIVESITEANDLUMEN BLUEARROW 4HERESIDUECOLORINGISTHESAMEASIN" 3EE#HAPTER P

#0

$PMPS
1MBUFT

&IG 3PECTRAL EVOLUTIONIN,(#))AT+FO & F LLOWINGEXCITATIONWITH AFS F LASERPULSEATNM4HEEXPERIMENTAL SPECTRAASAFUNCT F ION OF DELAY A TIMEWEREANALYZEDD WITHA LINEARSCHEME! " # $ !WITH INCREASING DECAY A TIMESTOVISUALIZETHESPECTRALEVOLUTION INTHE SYSTEMAND D THERESULTING3PECIES!SSOCIATEDD $IFFERENCE3PECTRA F 3!$3 HAVEBEENPLOTTEDBLACK LIFET F IME FS F RED LIFET F IME PS BLUE LIFET F IMEPS YELLOW LIFET F IMENS4HEINSERTSHOWSTHEEXPERIMENTAL KINETICSATSOMESELECTED D WAVELENGTHS INCLUDING D ONTHISMODEL ADAPTED FROM'RA F DINARUETAL 3EE#HAPTER P THE lTS BASED

&IG #AROTENOIDSAND,(#)) & D B ASSEMBLY3PECIlCITYOFF EACH BINDINGSITEISINDICATED , LUTEIN6 VIOLAXANTHIN. NEOXANTHIN: ZEA XANTHIN !N,(#))B MONOMERBINDSAPPROXIMATELY #HL A MOLECULESBLUE AND# D HL B MOLECULESGREEN +àHLBRANDT ETAL  D #AFFARR F I  )NAN,(#))B MONOMER THE,SITE ISOCCUPIED BY LUTEIN ORANGE WHIL W ETHE,SITE ISTYPICALLY OCCUPIED BY "ASSI AND EITHERLUTEINORVIOLAXANTHINPURPLE 4HE.SITEISSPECIlC FOR F BINDINGNEOXANTHINYELLOW 6I 6 OLAXANTHINISPROPOSED D TO BIND LOOSELY D INGTHEXANTHOPHYLL CYCLE#AFFARR F I ETAL  !DAPTED FROM#ROCEETA F L TOTHE6SITEAND ISINTER EXCHANGEABLEWITHZEAXANTHINDUR  AND"ASSIAND#AFFARRI 3EE#HAPTER P

#0

$PMPS
1MBUFT

& &IG 0HOTOINHIBITION REPAIRCYCLEOFTHE03 ))COMPLEXES3EE#HAPTER P TEXTFORDETAILS

#0

D BACTERSPHAEROIDES &IG IG  2HODO D #C/ 3VENSSON %KETAL  INAMODEL MEMBRANE4WOSUBUNITSARESHOWNINDIFFERENTCOLORS) GREEN)) GREY4HE REDOXCENTERSAREMARKEDWITHHEMESINPURPLEANDCOPPERSORANGE4HESTRUCTURE ISDRAWNFROM-PDBANDAMODELMEMBRANE BYTHEPROGRAM-/,3#2)04 +RAULIS  4HEPROTONTRANSFERPATHWAYS$AND+AREROUGHLY OUTLINED ASARETHE KEYPROTONTRANSFERRES F IDUES!SP ' D LUAND D + NUMBERING ACCORDING TOTHE BOVINEHEARTENZYME !HYDROPHOBICCAVITY BETWEEN'LU AND HEME A YELLOW MAY A TRANSIENTLY CONTAINANUMBEROFF WATERMOLECULES 3EE#HAPTER P

&IG /VERALLFUNCTIONOF#C/(EMEGROUPSARESHOWNINPURPLEANDCOPPERIONS INORANGE4HEHISTIDINELIGANDSOFTHEHEMEGROUPSANDOF#U" ARESHOWNINGREEN ASISTHETYROSINERESIDUEINTHEBINUCLEARSITETHATISCOVALENTLYBONDEDTO ONE OFTHE HISTIDINELIGANDSOF#U"4HEBLUEARROWSDEPICTHOWTHECHEMISTRYOF/ REDUCTIONTO WATERBY ELECTRONAND D PROTONTRANSFER F ISORIENTEDW D ITH RESPECTTOTHEPOSITIVELY CHARGED 0 SIDE AND D NEGATIVELY CHARGEDD . SIDE OF F THE MEMBRANE 2ED ARROWS DEPICT PROTON F HEMOLECULARGRAPHICS(UMPHREYET TRANSLOCATION4HEPROGRAM6-$WASUSED FORT AL  3EE#HAPTER P

$PMPS
1MBUFT

#0

$PMPS
1MBUFT

&IG  0HOTOSYSTEM))VIEWED FROMT &I F HESIDETOSHOWTHETOPOLOGYOFF THEACCEPTORSIDE#OORDINATESWERETAKENFROMT F HESTRUCTURE BY &ERREIRAETAL 0$")$SL BYABSTRACTIONOFAMONOMEROFTHEMAINFUNCTIONALSUBUNITSANDCHROMOPHORES4HE$SUBUNIT ISINBLUEWITHTHE@0%34 LIKELOOPINMAGENTA $INGREEN SHOWNBYRIBBONS0ROSTHETICGROUPSARESHOWNASSTICKMODELS WITH # ATOMSOF1! INORANGE 1" INYELLOW ANDBICARBONATEANDHEMEB INGRAY OTHERATOMSIN#0+ COLORS/THERSTRUCTURALFEATURES DISCUSSEDINTHETEXTAREINDICATED3EE#HAPTER P 

&IIG4HE: SCHEMEOFOX & F YGENICPHOTOSYNTHESIS FORE F LECTRONTRANSFER F FROMWATERTON F ICOTINAMIDEADENINEDINUCLEOTIDEPHOSPHATE .!$0 F IGOF'OV F INDJEE &ROMLEFTTOR F IGHT-NSTANDSFOR-NC F LUSTER9Z FORT F YROSINE ONTHE$PROTEIN0 FOR F MODIlED FROM& F HEOPHYTIN1! FORT F HElRSTPLASTOQU Q INONEELECTRONACCEPTOROFF 03))1" FORT F HESECOND THEPRIMARYELECTRONDONOROFF 03))0HEO FORP PLASTOQUINONEELECTRONACCEPTOROFF 03))01 FORT F HEPLASTOQUINONEPOOL#YTB, AND D #YT B( FOR F LOWAND HIGH POTENTIALCYTOCHROMEB #YT B F FORCYTOCHROME F F B F COMPLEX#YT F FORCYTOCHROME F F &E3 FOR2IESKEIRONSULFUR0# FORPLASTOCYANIN0 FORTHEPRIMARY ELECTRONDONORFOR03) ! FORAMONOMER#HL ! FORVITAMIN+ &X & &! AND&" FORIRONSULFURCENTERS@X @!AND@" RESPECTIVELY AND&D FORFERREDOXIN!LSOSHOWNARETHEAPPROXIMATEHALF TIMESFORR MOSTOFTHEREACTIONS THEEXISTENCEOFTHE@1 CYCLE THECYCLIC REACTIONAROUND03) ANDTHE POSSIBLESITESFORCALCIUM CHLORIDEANDBICARBONATEIONSIN03))3EE#HAPTER P

#0

(FSBME
5
#BCDPDL
m

%FEJDBUJPO1FSTQFDUJWF
"
USJCVUF
UP
+FSSZ
#BCDPDL 4HEASSEMBLYANDPUBLICATIONOFABOOKCOVERINGALL ASPECTSOFF 0HOTOSYSTEM))03)) INCLUDING ANALO GIES BETWEEN THIS ENZYME SYSTEM AND D CYTOCHROME OXIDASE WOULDHAVETHRILLEDANDDELIGHTED*ERRY)F F HISVOLUME ITTAKESVERY ONELOOKSATTHECONTENTSOFT LITTLETIMETODISCOVERANUMBEROFF CHAPTERS FORE F X AMPLE n   AND WHERE*ERRYWOULD HAVE BEENALEADINGCHOICEASANAUTHOR!CURSORY INSPECTIONOFF THECITATIONSATTHEEND D OF MANYMOREOF THESECHAPTERSWILLTURNUP U HISNAMEASACONTRIBUTOR OF IMPORTANTRESULTSTOOURUNDERSTANDINGOFF MECHA NISTICAND D STRUCTURAL ASPECTSOFF 03))ANDCYTOC D HROME OXIDASE)TISTHISBREADTHOFIMPACTOF*ERRYSRESEARCH THATMAKESTHIS BOOKSUC K H A lTTINGTRIBUTETOOURCOL LEAGUE WH W OHAD D EVENMORETOCONTRIBUTE WH W EN HIS UNTIMELYDEATHENDEDANOUTSTANDINGSCIENTIlCCAREER 0HOTOSY SYSTEM))4 4HE,IGH I T$RIVEN7ATER0LASTOQUI NONE /XI / DORE D DUCTASE D STANDSASALASTINGTRIBUTETOTHE LIFEANDWORKOF*ERRY"ABCOCK ANDITISOURHONOR AND D PRIVILEGETODEDICATEITTO HISMEMORY !NOBITUARYOFF *ERRYAPPEARED IN 0HOTOSYNTH T ESIS 2ESEARCH ; n  = 7E HAVE EXPANDED ON MATERIAL FFROM THAT PAPER FFOR THIS DEDICATION TO F HATREAD ENLARGEONSOMEASPECTSOFF *ERRYS LIFESOT ERS INCLUDINGHISMANYFRIENDSANDCOLLEAGUES MAY GAINSOMELARGERSENSEOFT F HESCOPEOFF *ERRYSMANY N

ACTIVITIES AND THE IMPRESSION HE MADE ON PEOPLE F THROUGHOUTHISLIFE M ER *ERRYSIMPACTONSCIENCEWASSIGNALED BYANUMB OF EVENTSTHATWERE HELDIN HIS HONOR AFTER HIS DEATH 4HE lRSTOFT F HESE ORGANIZED BY,ES$UTTON WASA SYMPOSIUM ENTITLEDD @%TPCET(!4PT 9EARS OF 4UNNELINGIN"IOLOGYTHATWASHELDATTHE*OHNSON2E SEARCH &OUNDATIONOFT F HE5NIVERSITYOF0ENNS F YLVANIA IN-AY 4HESECONDD SYMPOSIUM ORGANIZED BY ONEOFUS3& - AND*OHN-C#RACKENAT-ICHIGAN 3TATE5NIVERSITY WAS Y HELD IN*UNE AND FOCUSE F D D D INHIS LIFET F IME ONRESEARCH TOPICSTHAT*ERRYADDRESSE 4HE THIRD SYMPOSIUM WAS A SATELLITE CONFERENCE F FEATUR ING TALKS ON PHOTOSYNTHETIC ELECTRON TRANSFE F R HELDAT#OURAN#OVE !USTRA D LIA ORGANIZED BY+ARIN ¯HRLINGAND2ON0ACE PRECEDINGTHE)NTERNATIONAL 0HOTOSYNTHESIS#ONGRESS HELD IN"RISBANE IN!UGUST F F FOURT H EVENT ASYMPOSIUMORGANIZED BY OF! 'ERNOT 2ENGER AND4OM7YDRZYNSKI WAS HELD AS PARTOFF THE)NTERNATIONAL0HOTOSYNTH T ESIS#ONGRESSAND F FEATURE D TALKSON03 ))ANDD THE / EVOLVINGREACTION )NADDITION ASPECIALLECTURE @4HE'4"ABCOCK-E MORIAL,ECTURE WASDESIGNATEDD ATTHE)NTERNATIONAL 

%LECTRONTRANSFERPROTONCOUP F LED D ELECTRONTRANSFER( F YDROGEN F PROTONTRANSFE F R !TOM4RANSFER

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D

 #ONGRESS OFF "IOINORGANIC #HEMISTRY IN &LORENCE )TALY IN AND GIVEN BY ONE OF US 3& - ON ELECTRONTRANSPORT INCYTOCHROME C OXIDASE!SPECIAL ISSUEOF "IOCHIMICAET"IOPHYSICA H !CTA ;n  = EDITEDBY0ETER"RZEZINSKI #ECELIA4OMMOS D !NDERS%HRENBERG WASDEDICATEDTO*ERR D Y Y AND K WAS THE lRSTB T ORN SON OF F %MELDA *ERRY "ABCOCK -EL AND&RANK"ABCOCK(EWASBORNON&EBRUARY D RECEIVED HIS  IN-INNEAPOLIS -INNESOTA AND PRIMARYAND D SECONDARYSCHOOL EDUCAT D IONTHERE(E ALSOBEGANHISATHLETICCAREERASABASKETBALLPLAYER IN THE -INNEAPOLIS SCHOOL SYSTEM *ERRY ENROLLED F R AT #REIGHTON 5NIVERSITY /MAHA .EBRASKA FO HIS UNDERGRADUATE EDUCATION (E RECEIVED HIS "3 DEGREEWITH HONORS IN#HEMISTRY AN Y D HEWASALSOA MEMBEROFF THEVARSITYBASKETB T ALLTEAM PLAYINGON A.ATIONAL#OLLEGIATE!THLETIC!SSOCIATIONSCHOLAR SHIP0AUL -ICHAEL A FORMERC F LASSMATEAT#REIGHTON D DAYS AND INDOING REMEMBERS*ERRYSUNDERGRADUATE SOALSOEXPLAINSFORUS*ERRYSLATERFASCINATIONWITH D ITION IN PICKUP K BASKETB T ALL GAMESATMANY N ANDCOMPET 'ORDON2ESEARCH #ONFERENCES0AU F L4-ICHAEL OF !RLINGTON 6IRGINIA WRITES @7EBOTH T GRA G DUATE D D FROM#REI F IGHTONINEARLY R SUM MEROF OF AND *ERRY ˆ WHOPRACTICALLYEVERYONE L CALLED@"ABS ˆ BEGANARAPIDASCENTINACADEMIA T ATULTIMATELY LED HIMTOTH TH T E-ICHIG IGAN3TATE5NI VERSITYACADEMICCOMMUNITY) D )F )REMEMBERCORRECTLY L "ABS OBTAINED HIS ADVANCED DEGREES FROM 5 #AL "ERK RKELEY E AND DID D @POSTDOCWOR D RK AT2ICE5NIVERSITTY BEF EFORE HE ARRIVED D IN %AST ,ANSING "UT IT MUST BE UNDERSTOOD THAT "ABS WAS A #REIGHTON 5NIVERSITY @ LUEJ @" E AY A LONG BEF EFORE HEBECAMEA-ICHIG IGAN3TATE 5NIVERSITY@3PARTAN4 5 4HAT DISTINCTION IS IMPORTANT "EINGA"LUEJAYMEANT"ABSHADTHECHANCETOBEAN ATHL T ETEANDP D LAY A ATTH T E HIGH I ESTLEVELL OF COMPETITION K BALLL ATTTHATTIME,IKEMANY*ESUI K T INCOLLEGEBASKET SCHOOLS #REIGHTONTAKESITSBASKETBALLSERIOUSLYAND ITHAD D SOMEO OF THEMOSTCOMPETITIVESCHEDU D LESINTH T E NATION DURING"A D BSCOLLEGEDAYS D @@"ABS STARTED HIS ATHLETIC CAREER AT #REIGHTON ASAWALK ON K F FORA&RES HMANBASKET K BALLL TEAMTTHAT D D  ATTHLETIC SCHOLARSHIP PLAYERS (E TH T EN INCLUDE MADETHEVARSITYTEAMHISSOPHOMOREYEAR ALSOAS A WALK ON K AND PLAY A ED D IN A NUMBER OF O G GAMES (E MADET D THEVARSITYAGAININ HISJUNIORYEAR ASAWALK K ON BUTHISPLAYINGTIMEINCREASEDSUBSTANTIALLY!S ASENIOR "ABSMADET D THEVARSITY T ONCEAG AGAIN"UTTTHAT YEARHEWASA"LUEJ E AY A BASKET K BALLP L LAY A ERWHO HAD D AN ATHLETICSCHOLARSHIPANDSTARTEDINEVERYGAME SAVE ONE DUE TO ILLNESS

@ @"ABSHADARRIVED%VERYONEKNEWTHATHISINTEL

LIGENCE WAS EXTRAORDINARY "UT IN HIS SENIOR YEAR HEBECAMEAFU F LL m

mEDGE G D @JOCK K4 4HATIMAG AGEMIIGHTBE DIFl I CULTTOACCEPTORCOMPREHEND FORSOMEONEW F HO ONLYCAMETOKNOW"ABSWHENHEWASANADULT ESPE CIALLY INANACADEMICENVIRONMENT"UT)AMPOSITIVE D T AT"ABSWASAS HAPPYANDD THRILLEDD WITTH HISATHL TH T ETIC ACCOMPLISHMENTS AT #REIGHTON AS HE WAS WITH HIS ACADEMICAC D HIEVEMENTS(EPERSEVEREDD AND EARNED AN ATHL T ETICSCHOLARSHIP BY B HONINGHISSKI K LLSAN L D TALENTS ( (ESTARTEDHISSENIORYEARANDPLAYEDAGAINSTSOME OF THE BESTCOLLEG OF EGEPLAY A ERSINTH T ETH T CENTURY RY4 4HOSE WITH T AWORK RKINGKNOW K LEDGEO D OF BASKET K BALLL WILLAPPREC L I ATETHEFACTTHAT"ABSWENT TOUSEACLICHE TOE TO TOE WITH T TH T ELIKESO K OF %LVIN(AY A ESANDD "OB ,ANIERBOTH T T E."!S(ALLL OF &AMEE )NFACT F ) OF WHOMAREINTH OF HAVEAVIVIDMEMORYOF"ABSINACONFRONTATIONDUR R INGAROA G D GAMETOWAR G RDSTTHEENDD OF OURSENIORYEAR "ABSWASSTANDING ROUGHL G LYSPEAKING NOSE K HIS S TO BELLY BUTTON BELONGING TO g   LB SIZE  % %%%SNEA K KER "O B,ANIER OF OF 3T"ONAVENTUREE AND T EOWNEROF OF THEBELLY L BUTTONINQUESTION SHOUTINGATTH APERSONWHOSESIZEWASBLOTTINGOUTTHEOVERHEAD LIGH I TINGG IN"ABSIMMEDIATEPERSONALL SPACEE TOSHUT D  UPANDD GOTO(ADES @4HERE IS NO DOUBT THAT "ABS INTELLIGENCE WAS OVERWHELMING G (E WOULD HAVE ATTAINEDD A PERRFECT GRADEAVERAGE D F HISENTIRECOLLEGECAREERHAD FOR ITNOTBEENFORTHE"INAMANDATORY2/4#COURSE ONE SEMESTER "UT ) KNOW K HE WAS JUST AS PROUDD OF TWOOTH T ERNUMBERS HEPOSTEDD INTTHATPERIODAND 4HOSEWEREHISAVERAGESINHISSENIORYEAR THIS BRAINY N GUY U FROM3T,OUIS0AR F RK -INNESOTAWHOHAD BIGDREAMS INPOINTS D   ANDD REBOUNDS  D    OVER A FULLVARSITYBASKETBALLSEASONASASTARTERAGAINST A ERSWHOWOULD LATEREARNTH T EIRLIVINGINPRO G OFES PLAY SIONAL BASKET K BALL!NOTH T ERTH T INGISWORTH T MENTION INGABOUTHISYEARASA@FORMALJOCKAT#REIGHTON ) BELIEVEALLL OF HIS FRIEN F D DSIN%AST,ANSIN G WOULD HAVE D BSBLUSH TURNINGBRIGH G TREDD ONTTHE ENJOYEDSEEING"A COURTINTHElNALHOMEGAMEOFHISSENIORYEARWHEN T ESTUDENT TH D BODY BEG EGANTOCHANT@*ERRY RY *ERRRY *ERRRY OF WHAT TOWARD RDSTTHEENDD OF THEGAMEINRECOGNITIONOF "ABSHADACHIEVEDASANATHLETE @4WOOlNALL THOUGH U TSMAY A HELPTO L O mESHOUTTH T EPERSON ) AMATTEMPTINGTO DESCRI D BE!BOUTORWEEKS K BEF EFORE OURGRADUATIONIN "ABSSHOWEDMEALETTERHE HADD RECEIVED FROMT F THE$ALLAS#OWBOY OYSPROOFESSIONAL F BALLL TEAM7HILE$ALLASTYPICALLY FOOT L HADGREATTEAMS D INTHOSEYEARS ITALSOHADTHEREPUTATIONOFHAVING D LEXSY SYSTEMOOF PLAY AYSAND T EMOSTCOMPLICATED COMP TH

 FFORMATIONSUSED INTTHE.ATIONALL &OOTBALL,EAGUE&OR L THATREASON $ALLASSOUGHTINDIVIDUALSWHOWEREGREAT ATHL T ETESAND I D IF POSSIBLE ALSO L HAD D SHOWNALEVELL OF ACADEMICAC D HIEVEMENTNOTUSUALLYASSOCIATE L D WITTH COLLEGEATHLETES)TWASRUMOREDTHAT$ALLASMAIN TAINED D A DATA D BASEONACOMPUTER NO  LESS S BYWHICH ITKEPTTA K BSONINTELLIGENTATHL T ETESWHOMIGH G THAVE POTENTIAL EVENIFTHEYWERENOTFOOTBALLPLAYERS4HAT WASTH T EPURPOSEOF OF THELETTER"ABSRECEIVED4 D 4HE$AL LAS#OWBOYSHAD D INVITED HIMTOASPECIALCAMPEAR L RLY THATSUMMERFORTHEPROVERBIALTRYOUT EVENTHOUGH "ABS HADNOTP D LAY A ED FOOT F BALLL SINCE HIGH I SCHOOL!S) R LL T RECA L THELETTERINDICATED D THE#OWBOYSTH T OUGH G THE MIGHTHAVESOMEPOTENTIALASATIGHTEND SINCEHE WASABOUTgAND D WEIIGHED D A LITTLEOVER LBS 4HOSEWHO KNEW K HIMMAYRECALLL THATHEALSO L HAD D VERY LARGEHANDSWITHLONGlNGERS EVENFORSOMEONEOF HISSIZE)F )F YOUTH T INKK ABOUTIT "ABSMIGH I THAVEENDE D D T 2OGER3TAUBACH7EURGED HIMTO UPPLAYINGWITH ACCEPTTHEINVITATION BUTHEJUSTCHUCKLEDANDSAID HE HAD D OTTHERTH T INGSIN G LIF IFETOPURSUE F D IONFROM#RE DUAT F IGHTON *ERRYJOINED D +EN !FTERGRA NETH3AUERSGROUPATTHE5NIVERSITYOF#ALIFORNIAAT "ERK R ELEY ASA0H$STUDENT IN"ERK R ELEY WAS THEN ASNOW ACENTERFORT F HEAPPLICATIONOF BIOPHYS H I CALTECHNIQUESTOPROBLEMSINPHOTOSYNTHESIS4HESE WERE EXCITING YEARS FFOR PROGRESS IN UNDERSTANDING THEMECHANISMOFP F HOTOSYNTHETIC/ EVOLUTION WITH THE mASHING LIGHT EXPERIMENTS OF 0IERRE *OLIOT AND "ESSEL+OKT K HATESTABLISHED D THEPERIOD FOURPATTERN F OF / RELEASE*ERRYTOOK K THISAREAASHISTHESISTOPIC ANDCONTINUEDRESEARCHINGITFORHISENTIRESCIENTIlC CAREER!LONG WITH#HARLES7EISS APOSTDOC INTHE 3AUERGROUP HEBUILTA*OLIOT STYLEOXYGENELECTRODE THATWASACOMPLEXMARVELOFPUMPS HOSESANDA HODGE PODGEOFF ELECTRONICSSALVAGED FROMOT F HERUSES )TWASNOTPRETTY BUTITWORK R ED D REMARRKABLYWELL4HIS @MAKEITWORKPHILOSOPHYWASCHARACTERISTICOF*ERRY AND HISAPPROACHTOSC H IENCE3OON*ERRYSUPP Y LEMENTED HISWORK RKWITHTHEOXYGENELECTRODEWITH STUDIESON %02SIGNALSASSOCIATEDWITHPHOTOSYNTHESIS"ARRY #OMMONER HAD lRST OBSERVED D THESE SIGNALS SOME YEARSEARLIERAND D THE LABORATORYOFF -ELVIN#ALVINHAD DONESOMEWORKONTHEMINPHOTOSYNTHETICBACTERIA BUT*ERRYWASTHE lRSTAT"ERK R ELEYTOSTUDY3IGNAL )) WHICHWASASSOCIATED D WITH03)))TSSLUGGISH KINET ICSINITIALLYSUGGESTEDTHATITWASNOTAMAINSTREAM ELECTRON CARRIER AND D THEREFORE F DIDNT RECEIVE MUCH ATTENTION5NDER+ENSGUIDANCE *ERRYUNDERTOOK KA SYSTEMATIC INVESTIGATION OF THE @3IGNAL )) SPECIES AND D SHOWED D THATTHEREWAS IN FACT MORET F HANONE

CONTRIBUTINGRADICALCOMPONENT INTHESIGNAL "AB COCKAND3AUER  "YAVARIETYOFTREATMENTSTHAT AFFECTE F D / EVOLUTION *ERRY WASABLETOSHOWTHAT F HESESIGNALSCOINCIDEDD WITH THEKINETICBEHAVIORSOFT THEPRESENCEORABSENCEOFAFUNCTIONAL/ EVOLVING REACTION/UTOFT F HESEINVESTIGATIONSEMERGEDD THElRST SYSTEMATIC NOMENCLATURE FFOR THESE IMPORTANT 03)) REDOXINTERMEDIATES 3IGNAL))S THEDARK STABLEFORM OFT F HESIGNAL 3IGNAL ))F T F HE %02SIGNAL FROMASECON F D F IDATION SPECIESDETECTEDD UPONINHIBITIONOFWATEROX ANDLASTLY 3IGNAL))VF AVERYRAPIDLYDECAYINGSPECIES F LLY FUNCT F IONAL / EVOLVINGREAC ASSOCIATEDD WITH THEFU TION)TWASONTHEBASISOFF WORRK BY*ERRYANDD ONEOF US2" THAT+EN3AUERSGROUPWASABLETOPOSITION THE 3IGNAL ))F))VF F SPECIES AS A REDOX INTERMEDIATE BETWEEN THE / EVOLVING REACTION AND THE PRIMARY ELECTRONDONOR 0 OF03))"LANKENSHIPETAL  "UTTHEMOLECULARIDENTITYOF3 F IGNAL))LATER RENAMEDD @: WAS STILL ELUSIVE 3OME EXPERIMENTS INDICATED THAT IT MIGHT BE A QUINONE BUT THE DATA D RETURNTOTHIS WERENOTVERY CONVINCING*ERRYWOULD Q QUEST ION LATERIN HIS INDEPENDENTSCIENTIlCCAREER )NTHEAUTUMNOF ANOTEWASFOUNDINTHE DRAWEROFF THE DESKT K HAT*ERRY OCCUPIED FROM F n INTHE#ALVIN LABORATORY)T ISCLEARLYWRITTEN IN *ERRYSHANDWRITINGANDREADS @ @)TISVER RYIMPORTANTTO HAVEALONGRAN G G GOA GE G L L RD ITS ONCETH T ISISREALIZEDD THESTEPSNECESSARYTOWARD ATTAINMENT BECOME LOGICAL PROGRESSIONS AND NOT R PLUNGESINTOT G THEVOID D ARBITRARY 7 HAVENOTBEENABL 7E A ETODETERMINE IFF *ERRYWASTHE Q AUTHOROFTHIS QUOTEORIFHEFOUNDITSOMEWHEREELSE BUT ITTYPIlESHISTHOUGHTFU F LAPPROACH TOSCIENCE $URING THE TIME THAT *ERRY WAS A GRADUATE D STU DENT HE WAS THE NATURAL LEADER BOTH SCIENTIlCALLY Y OFF THE 3AUER GROUP WHI W CH CONSISTED ANDD SOCIALLY F A SUUBSTANTIAL NUMBER OFF STUDENTS POSTDOCS AND OF OTHERVISITORSWHOCAMEANDWENT!MONGTHEMOST MEMORABLERECURRINGEVENTSWERETHEABALONEDIV INGEXPEDITIONSTHATTOOKK PLACEWITH SOMEREGULARITY ONTHE.ORTHERN#ALIFORNIACOASTNEAR3ALT0OINT4O GETTOTHEABALONE ITWASNECESSARYTOFREE F DIVETOA SIGNIlCANTDEPTH INCOLD D AND INVARIABLYMURK RKYWATERS INTHEPRESENCEOFTHICKSTANDSOFGIANTKELP!FTER REACHING THEBOTTOM YOUHADD TOSWIMAROUNDD AND lND ANABALONE MEASUREITWITH AGAUGETODETERMINE IF ITWASOFLEGALSIZEANDTHENPRYITOFFTHEROCKWITH AMETAL BARAKINTOATIRE IRON)FF THEABALONESENSED ITSIMPENDING FATE F ITWOULD D CLAMPDOWNlRMLYON THEROCKANDBECOMEALMOSTIMPOSSIBLETOREMOVE *ERRY WASTHEMASTEROFF THISVERYCHALLENGINGACTIV

 ITY(ECOULD D ROUTINELYSTAY DOWNMUCH LONGERTHAN ANYONEELSE DESPITEHISHEAVYSMOKINGHABIT WHICH D TOSIGNIlCANTAMOUNTSOF#/ F HEMOGLOBIN CAN LEAD INTHE BLOOD (EALSO HAD D THE KNAC K K TOPOPTHEABA LONEOFFTHEROCKSQUICKLYANDEASILY3OMETIMESHE WOULD D SURFACEW F ITH TWOORTHREEIN HISARMS FROMA F F HEDIVINGEXPEDITION THEABALONE SINGLE DIVE
!FTERT WOULDBEBROUGHTBACKTO"ERKELEYANDTHATEVENING K BALONE FEE F D WOULD D TAKEPLACE WITH A HUGEPOT LUCKA POUNDED D BREADED D AND FR F IED D ABALONEASCENTERSTAGE BUTWITHAWIDERANGEOFOTHERDISHESPROVIDEDBY OTHERGUESTS /NEOFF US2" WASASTUDENTINTHE3AUERGROUP ATTHATTIME BEGINNINGSTUDIESTWOYEARSAFTER*ERRY ARRIVED)TISOFTENCUSTOMAR F YTOHAVEAMORESENIOR GRADUATE D STUDENT SERVE AS AN INFORMA F L MENTOR FOR F NEWSTUDENTS*ERRYSERVEDTHATROLEFOR2"ANDTHEY SPENT MANY N HOURS TOGETHER DOING EXPERIMENTS AND DISCUSSINGBOTH THEDETAILSOFT F HEWORK RKANDGENERA D L ASPECTSOFSCIENCEANDITSRELATIONTOSOCIETY F YRECEIVED HIS0H$IN HESPENTA !FTER*ERR YEARASAPOST DOCTORAL FE F LLOW INTHE3AUERGROUP)N  HEJOINED'RAHAM0ALMERSLABORATORYAT2ICE W ERE HE BEGANRESEARCHONCYTOCHROME 5NIVERSITYWH A ORPARTOF HIS OXIDASETHATWASTOLATERBECOMEAMAJ INDEPENDENT SCIENTIlC CAREER (IS WEEKDAYS WERE A OUTCYTOCHROMEOXIDASE WITH T FE F LLOW SPENTLEARNINGAB POSTDOC,ARRY6I 6 CKERY RESULTINGINSEVERALGROUND BREAKINGANDSTILLOFTENQUOTED PAPERSONTHEELEC TRONICPROPERTIESOFF THE HEMECENTERSOFC F YTOCHROME K L   4WEEDLEETAL OXIDASE"ABCOCKETA   $URING THIS TIME HE AUGMENTED HIS EXPERI MENTAL EXPERTISEWITHTECHNIQUESSUCHAS RESONANCE 2AMAN SPECTROSCOPY THAT WOULD BE CRITICAL TO THE SUCCESSOFHISLATERRESEARCH)TISLESSWIDELYKNOWN THATMANY N WEEKENDSWH W EN'RAHAMWASOUTOFF TOWN WERESPENTmASHINGTHY H LAKOID D MEM MBRANESUSPENSIONS THATWEREQUICKLYTRANSFERREDINTOAN %02 2 CRYOSTAT INSEARCHOFF PARAMAGNETICSIGNALSTHAT*ERRY BELIEVED W LD BEASSOCIATED WOU D WITH TH THE3 STATEOFT F THE+OK *OLIOT #YCLE)TREMAINEDFOR'##HUCK $ISMUKESAND 9 9ONA3 IDERERTO lND D THISELUSIVESIGNAL $ISMUK U ESAND 3IDERER  THEWELL KNOWN3 K  MULTILINESIGNALOF 0HOTOSYSTEM)) #ANOEING WAS A FAVOR F ITE MODE OF F RELAXATION FO F R F DENTSAND D POST DOCS *ERRY ASMANYOF HISFORMERSTU WILLTESTIFY!T2ICE *ERRYORGANIZEDSEVERALMEMO RABLEOUTINGS(OWEVER INONECASE HENEGLECTED D TO D TOCHECK K ONTHEWATERLEVEL INTHEEASTFOR F RK CALL AHEAD OFTHE3AN*ACINTO2IVER ANDMEMBERSOFTHE0ALMER

D INGTHEIRCANOESSOME DISTANCE GROUPENDEDUPCARRY DOWNADRYRIVERBEDTOTHEPOINTWHERETHEYWERETO BEPICKEDD UP *ERRYWASRECRUITED BYTHE#HEMISTRY$EPARTMENT AT-ICHIGAN3TATE5NIVERSITY-35 WHEREHEJOINED THE FACU F LTY TYASANASSISTANTPROFESSOR F IN ANDD WHERE HESPENTTHERESTOF HISCAREER(EWAS PROMOTEDD TO ASSOCIATEPROFESSORWITHTENUREINANDTOFULL PROFESSOR F IN FROMTO F HEWASCHAIR F HE#HEMISTRY$EPARTMENT)N HERECEIVED OFT THEHIGHESTHONORHISUNIVERSITYCANBESTOW THETITLE OF5N F IVERSITY$ISTINGUISHEDD 0ROFESSOR7 F HENHEAR RIVEDAT-35 *ERRYSETA D BOUTESTABLISHINGAlRST RATE F FACILITYFORTHEAPPLICATIONOFPHYSICALMETHODSTOTHE F YTOCHROMEOX H IDASEANDP D HOTOSYN CHARACTERIZATIONOFC THETICSYSTEMS(E BEGANTHEPROCESSOFRECRU F ITINGA RESEARCHGROUPOFENTHUSIASTICGRADUATESTUDENTSAND INITIATEDD ANUM MBEROF INNOVATIVERESEARCHPRO H OJECTSTHAT CONTINUEDD TOTHEENDD OF HISCAREER)N HIS lRSTWORK R ONCYTOCHROMEOXIDASE HECOLLABORATEDWITH)RVING D NY AND# D HRIS#HANG AT 3ALMEEN&ORD-OTOR#OMPAN -35 TOCARRYOUTASERIESOFF RESONANCE2AMANSTUDIES ONOXIDASEANDMODELHEMECOMPOUNDS3ALMEENET D HANG "ABCOCKK AND AL "ABCOCKK AND# 3ALMEEN  4HESEANDD SU UBSEQUENTSTUDIESWERE CRITICALTODElNINGTHECOMPLEXVIBRATIONALSPECTRUM F HEENZYME KNOW K LEDGETHATWOULD BEKEYTOLATER OFT INCISIVE INVESTIGATIONSOFT F HEMECHANISMOFOXYGEN F REDUCTIONANDTHENATUREOFTHEMETALLIGANDS!PAR TICULARLY IMPORTANTCONTRIBUTION FROMMO F DELSTUDIES 7ARDD ETAL  ANDD SPECTRALANALYSISOFOX F IDASE #ALLAHANAND"ABCOCK  WASONEOFTHElRST EXPLICITMOLECULARMODELSOF HOWAREDOXREACTION COULD BELINKEDD TOPROTONPUMPING INCYTOCHROME OXIDASE "ABCOCK AND #ALLAHAN   $URING A SABBATICAL WITH"ILL7OODRUFF IN!USTIN 4EXAS THE lRSTTIME RESOLVEDD RESONANCE2AMANSTUDIESONCY TOCHROMEOXIDASEWERECARRIEDOUT"ABCOCKETAL   4HISBEGANA YEARINTENSIVEEFFOR F T TOPIN DOWNTHEELUSIVEOXYGENINTERMEDIATESOFT F HE CYTOCHROME OXIDASE REACTION AN EFFORT THAT WOULD REQUIREPUSHING RESONANCE 2AMANSPECTROSCOPY TO ITSLIMITS 7ITH THE OXIDASE EXPERIMENTS UP AND RUNNING 7 *ERRY RETURNEDD TO INVESTIGATIONSOFF THE 3IGNAL))SPE CIESASSOCIATEDD WITH0HOTOSYSTEM))THATINVOLVED HIS lRSTGRADUATESTUDENTSINTHISAREA#HRISTINE9ERKES $EMETRIOS'HANOTAKIS ANDD APOST DOCTORAL FE F LLOW 0ATRICKK /g-ALLEY4HE FOCUSO F F HISRESEARCHWASTO FT EXTENDD THE CHARACTERIZATION OFTHE %02SIGNAL9ERKES

 AND D "ABCOCK 'HANOTAKISETAL  ANDD TO DISCOVERITSMOLECULARORIGINS)NTHElRSTOFTHESEEX PERIMENTS ASEARCHWASMADEFORMO F DELCOMPOUNDS THATWOULDD SIMULATETHE %02SPECTRUMOF 3IGNAL )) BYNOWCALLED@:4HISAPPROACHIDENTIlEDQUINONE D "AAB RADICALSASPROMISINGCANDIDATES/-ALLEY AND COCK A BUTTHESPECTROSCOPICDATAFROMT F HESE COMPOUNDSWASNEVERENTIRELYSATISFACTORY ANDTHE PLASTOQUINONECONTENTOFF 0HOTOSYSTEM))PREPARATIONS WAS INSUFlCIENTTOACCOUNT FORT F HE PRESENCEOF 1! 1" ANDTHETWOADDITIONAL QUINONESNEEDEDFORTHE Q TWO %02SIGNALSCOMPRISING THE DIFFEREN F T 3IGNAL)) SPECIES4HEALTERNATEAPPROACHWASTOAPPLYISOTO PICLABELINGTECHNIQUESANDASSESSTHERESULTSUSING %024HElRSTEXPERIMENTS BY"RIDGETTE"ARRY W Y WHO WASA JOINTPOST DOCWITH*ERRYAND D ,EE-C)NTOSH EXPLORED INCORPORATION OF DEUTERATED TYROSINE INTO CYSTIS 0##THE %02PROPERTIES CELLSOF 3YNECHOCY OFF @:WERE DRAMATICALLYMODIlED ALTERED LINESHAPE NARROWEDLINEWIDTH ARESULTSHOWINGTHAT@:WASIN F FACTAT YROSINERADICAL "ARRYAND D "ABCOCK   F LLOWED BYTHESITE DI 4HIS KEYRESULTWASQUICKLY FO RECTEDMUTAGENESISEXPERIMENTS INCOLLABORATIONWITH 2ICK K $EBUSAND D ,EE-C)NTOSH WHI W CH IDENTIlED D THE REDOXACTIVETYROSINESOFF 03))AS4YROF $AND 4YROF$$EBUSETAL A B 4HESE D ONE LASTREVISIONOFF THE SEMINAL lNDINGSPROMPTED F HESE IMPORTANTREDOXCONSTITUENTS NOMENCLATURE FORT OF03)) TO 9: AND 9$ /THERWORK RK ON03)) DERIVED FROMACO F LLABORATION WITHONEOFT F HEAUTHORS #9 WH W OSPENTASABBATICAL YEARINTHE"ABCOCKLAB4HEYHADHADANINFORMALCOL LAB A ORATIONOVERTH T EPRECEDING YEARS BUTTH T E INABILI A TY TYTO R ORCEMENTED D THEIRSCIENTIlC GET %02TIME IN !NN !RB ANDPERSONALRELATIONSHIPSFORTHEFORESEEABLEFUTURE *ERRY WASTHEIDEAL HOST BUTTHEREWERESOMESTRAINS D INGA"ABCOCK ALONGTHEWAY7HILEHOUSESITTINGDUR F FORAYTO%GYPTAFTERTHE0HOTOSYNTHESIS#ONGRESS IN'REECE TWOFAM F ILY CATSPASSED D AWAAY SEQUENTIALLY Y D OUTTOBEA FE F LINE DISTEMPER 4HEACTUAL CAUSETURNED VIRUSRAMPANTIN%AST,ANSINGATTHETIME BUTUNTIL THISWASPROVENTOBETHECASE THENEWSABBATICAL VISITORINTHE"ABCOCK LAB WAS INTRODUCE D D TOONEAND ALL ASTHE @CATKILLER 4HE HALLMARK OFINTERACTIONS WITH*ERRY WASANEASY GOING GIVE AND TAKE INWHI W CH IDEASWEREmOATED DEMOLISHED RElNED D AND REmOATED UNTILEXPERIMENTSGOTDONE ORTHE@REmOATELEMENT INTHECYCLEEXPIRED!LTHOUGH THE BASICELEMENTS FO F R THE03))PREPARATIONNOW KNOWNAS@""9 K SHORT F FOR"ERTHOLD"ABCOCK9OCUM HADBEENCOMPLETED

WHILE#9WASAPOST DOCAT#ORNELL PUBLI U CATION HAD BEENDEFERREDOWINGTOAGENERALATTITUDEOFDISBELIEF ABOUTTHEPOSSIBILITYOF ISOLATING ACTIVE03))AMONG THESENIORPEOPLE INTHEPHOTOSYNTHESISlELD*ERRYS COMPLAINTS ABOUT THE DIFlCULTIES OF MAKING %02 MEASUREMENTSON 9$ŠAND9:Š INTHEPRESENCEOFAVERY LARGESIGNAL FROM0 F  ACCELERATEDD THEPUBLI U CATION PROCESS"YCOUPLINGAREVISEDBIOCHEMICALMETHOD THSOME %02MEASUREMENTSTTHATREVEALEDD THE OLOGY WITH ORIENTATIONOF9 9$Š INTHENEWPREPARATION "ERTHOLDET AL MANAGEDTOGETTHEPAPERACCEPTEDIN&%"3 ,ETTERS*ERRYSONLYMISGIVING ABOUTTHIS JOINTEFFORT F WASTHATNOONECITED IT FORT F HE%02RESULTS /NTHE OTHERHAND HEHADANONGOINGFASCINATIONWITHTHE CITATION@HITSTHEPAPERGARNERED ITSABOUTAT THISPOINT ANDD WEHADD ASMALL PARTYONTHEOCCASION OF CITATION@HIT 1UANTIlCATION OFF THE -N CONTENT OF F 03 )) AND CHARACTERIZATIONOF ITSINHIBITORSENSITIVITY 9OCUMET AL  WEREOTHEREARLYRESULTSOFOURCOLLABORA TION,ONGERTERM INTERACTIONSBETWEENTHE"ABCOCK ANDD 9OCUMGROUPSWERECENTRAL TOTHEDISCOVERYTHAT #A WAS AN ESSENTIAL COFAC F TOR FO F R WATER OXIDATION 'HANOTAKISETAL A B ANDD TOTHEISOLATIONAND F BLE03))REACTIONCENTERPREPA CHARACTERIZATIONOFSTA RATIONSTHATRESULTEDINAREEVALUATIONOFTHEPIGMENT CONTENT OFF THE REACTION CENTER 'HANOTAKIS ET AL  *ERRYSGENEROSITYWASA KEYELEMENTINTHIS WORK ASWASMUTUALRESPECTFORONEANOTHERSPOINTS OFV F IEW)NSTRUMENTTIMECOULD D ALWA L AYSBE FOUN F D NO D MATTERHOWPRESSINGOTHERRESEARCHPROJ O ECTSMIGHTBE ANDHENEVERDEMANDEDTOBEADDEDTOAPAPERASAN AUTHORONTHE BASISOFF USEOF ONEOF HIS INSTRUMENTS 4HISWASTRUEOF HIS INTERACTIONSWITHALLOF HISCOL LEAGUESANDCOLLABORATORS /THERRESEARCH BY *ERRYgS GROUPINTHISPERIODD EX AMINEDD THESPECTROSCOPICPROPERTIESOFF 0HOTOSYSTEM) 03) 0RIORRESEARCHHADESTABLISHEDTHATTHEREACTION F HLOROPHYLL A DIMER BUT CENTERWASCOMPRISEDD OFAC THATIN0 TH T EUNP N AIREDD SPINWASLOCALIZED PREDOMI NANTLYONASINGLECHLOROPHYLLMOLECULE/-ALLEY ANDD "ABCOCK B !SABBATICAL VISITTO,EIDEN 5NIVERSITY 4HE.ETHERLANDS INBROUGHT*ERRY TOTHE"IOPHYSICS$EPARTMENTOFTHE(UYGENS,ABORA TORY W Y WHERE HEWORK R EDD WITH!RNO H LD D (OFF n TOAPPLY ELECTRONSPINECHOTECHNIQUESTOASTUDYOF 3IGNAL))DE 'ROOTETAL  ANDWITH(ANSVAN 'ORK R OMS GROUP$EKKERETAL  ON56ABSOR BANCECHANGESASSOCIATEDD WITHTHE3 STATES 4HEOXIDASERESEARCHEVOLVEDTOAPOINTWHERE*ERRY

 AND HISGRADUATESTU D DENT #OSTAS6AROTSIS HAD D RElNED THE TECHNOLOGY TO DETECT THE OXYGEN INTERMEDIATES IN THE ENERGY TRANSDUC D ING OXYGEN REDUCT D ION REAC TIONOFCYTOC F HROMEOXIDASE 6AROTSISETAL   4IME RESOLVEDRESONANCE2AMANWASA POWERFULBUT CHALLENGING METH T ODOLOGY DEMANDING LARG R EQUANTITIES OFF ENZYME AND YIELDING VERY LOW SIGNAL TO NOISE .EVERTHELESS *ERRYAND#OSTASFAMOUSFORMAKING HEARTPREPARATIONSOF BOVINECYTOCHROMEOXIDASE WEREABLETORESOLVEAND IDENTIFYT F HETRANSIENTVIBRA TIONALMODESFOROXYGENBOUNDTOREDUCEDHEME A AND D THE FERR F YL OXOINTERMEDIATEAFTERT F HEOXYGENWAS D "ABCOCK 6AROTSISETAL CLEAVED 6AROTSISAND   !PREDICTEDPEROXYINTERMEDIATEWAS F FRUSTRAT INGLYUNDETECTABLE AFACTT F HATBECAMEMORE F HETHEORYTHAT*ERRYAND UNDERSTANDABLEINTERMSOFT HISCOLLABORATORSDEVELOPEDINTHELASTFEWYEARSBE F FORE HISDEATH SUGGESTING ACONCERTED D FOURE F LECTRON D IONOFF THEOXYGEN0ROSHLYAKOVETAL  REDUCT  INVOLVING FORMATION OF A TYROSINE RADICAL AT THEACTIVESITE 4HE POWEROFF RESONANCE2AMANSPECTROSCOPYAND ALLOFTHEGROUNDWORKDONEBY*ERRYANDCOWORKERS INIDENTIFYING MANY N OFT F HEVIBRATIONAL MODES INOXI DASEALLOWED HIMTOPLAYA KEYROLEINTHEANALYSIS OF MUTANTFO F RMS OF A BACTERIAL OXIDASE FROM 2HODO D BACTERSPHAEROIDE D S INCOLLABORATIONWITHONEOFF US 3& - 2OBERT'ENNISAND D THEIRCOLLEAGUES4HESE STUDIES IDENTIlED THE LIGANDS OF THE METAL CENTERS INTHEENZYME 3HAPLEIGHETAL  AND GAVEAN AMAZINGLYACCURATEMODEL OFT F HEIRARRANGEMENTIN THEACTIVESITE#ALHOUNETAL (OSLERETAL  SEVERAL YEARS BEFORET F HECRYSTAL STRUCTUREWAS D ACTIVEINVOLVEMENTIN2A OBTAINED*ERRYgSCONTINUED MAN AND %02STUDIESOFMUTANTFORMSOFCYTOCHROME C OXIDASE UNTIL THE TIME OF HIS DEATH WAS CRUCIAL TOTHECHARACTERIZATIONAND D UNDERSTANDINGOFMANY F ALTERED FORMS OF OXIDASE LEADING TO NEW INSIGHTS INTOTHEMECHANISMOFE F LECTRONANDPROTONTRANS D F FER &ETTERETAL 1IANETAL  4HEDETAILED %02ANALYSISOFTHEMANGANESE SUBSTITUTED-GSITE IN OXIDASE CARRIED D OUT BY *ERRY AND D COLLEAGUES IN COLLABORATION WITH 3& - AND D *ON (OSLER %SPE ET AL (OSLERETAL  CONTINUESTOPROVIDE APOWERFU F LTOOL FORUN F DERSTANDINGTHESTRUCTUREAND F FUNCT IONOFT F HISREGIONOFOX F IDASEABOVETHEHEMES W WHEREPROTONSANDWATEREXIT&LORENSETAL    /VER THIS PERIOD D OFF NEARLY  YEARS OF F COL LABORATION BETWEEN *ERRY AND D 3& - REGULAR JOINT GROUPMEETINGSWEREHELD WHICHUSUALLYLEFTEITHER

THECHEMISTRYSTUDENTSORTHEBIOCHEMISTRYSTUDENTS SOMEWHATBEMUSED(OWEVER *ERRYgSSKILLFULTUTORING INSPECTROSCOPY AND DIPLOMATIC@REPHRASINGOF INFO F R MATION INVARIABLYLEDD TOCONSIDERABLEENLIGHTENMENT ANDWASACONSTANTJOYTOBEHOLD *ERRYS CONTINUING INTEREST IN THE CHEMISTRY AND F FUNCT IONOFF TYROSINERADICALSGRADUA D LLYEVOLVED INTO ARETHINKINGOFTHEPOSSIBLEROLESOFTHESESPECIESIN THEMECHANISMOFP F HOTOSYNTHETICOXYGENEVOLUTION F LINGOF ! DIFlCULTYWITHCONVENTIONAL MODELS FORCYC THE3 STATESISTHATIFONEASSUMESTHATTHEMECHANISM INVOLVES SEQUENTIAL OXIDATIONS OF F THE -N CLUSTER TWOPROBLEMSAREENCOUNTERED&IRST METAL CENTERED OXIDATIONS WOULD IN THEORY PRODUCE A POTENTIALLY DAMAGING ACCUMULATIONOFF POSITIVECHARGESWITHIN THEWATER OXIDIZINGSITE ANDD SECOND T D HElNALSTEPOF THECYCLE3 A 3 A 3 MIGHTREQUIREFORMATION  F SPECIES GENERATIONOFT F HISOXIDATIONSTATE OFA-N ISGENERALLYBELIEVEDD TO BE BEYONDD THECAPABILITIES OF BIOLOGICAL REDOX SYSTEMS!S *ERRY VIEWED THIS PROBLEM THE LATTERISSUECOULD BERESOLVED IFF THE 3 STATEINVOLVEDD THECOMBINATIONOF 3 ANDD THE NEUTRAL TYROSINERADICAL 9 9:s )FTHISWERESO ITWOULDINVOLVE 9:s DIRECTLY IN(/OXIDATIONCHEMISTRY4 Y HEFORME F R DIFlCULTY AMELIORATING POSSIBLE DAMAGE TO 03 )) STABILITY FROM THE ACCUMULATION OF EXCESSIVE POSI TIVECHARGE COULD BEACCOMPLISHED IFT F HECHEMICAL MECHANISM FOR F (/ OXIDATIONWEREREFORMU F LATEDD TO AVOIDCHARGEBUILDUPONTHE-NCLUSTER*ERRY HIS POST DOCS #URT(OGANSON +URT7ARNECKE AND HIS COLLAB A ORATORS"RUCE$INER *OHN-C#RACKEN #ECELIA 4OMMOS DEVISEDATHEORETICALMECHANISM"ABCOCK (OGANSONETAL (OGANSONANDD "AABCOCK  "ABCOCKETA K L "LOMBERGETAL  , ,YDAKIS 3IMANTIRISETAL 4OMMOSETAL  4OMMOSANDD "ABCOCK  FOROX F YGENEVOLUTION INWHI W CH -N BOUND (/WASOXIDIZEDBY9 9:s VIAAN ( ATOM ABSTRACTION REACTION 4HIS REFO F RMULATION OF F HEOXIDIZINGSIDEOF03))WOU F LD ELECTRONTRANSFERONT Q IRETHATTHE-NCLUSTERAND 9: BE INRELATIVELY REQU CLOSEPROXIMITYTOONEANOTHERTOFACILITATETHE( F ION0ULSEDD MAGNETICRESONANCE ATOMTRANSFERREACT MEASUREMENTSONMODIlED FORMSO F F 03))THATAC CUMULATETHE39 9:s STATE,AKSHMIETAL  HAVE PLACEDTHETYROSINEWITHINn ¯ OFTHE-NCLUSTER F F FUNCT IONING( ATOMAB ASWOULD BEREQUIRED FORA STRACTIONMECHANISM4HISDISTANCEISSUPPORTEDBY THESOLUTIONOFT F HE03))CRYSTAL STRUCTURE :OUNI ET AL +AMIYAANDD 3HEN &ERREIRA ETAL  WHERE A -N 9: SEPARATION OF ABOUT  ¯ IS

 PROPOSED4HE@CHARGE NEUTRALMODEL HASSTIMULATED NEW DEBATES ABOUTTHE MECHANISM OF (/OXIDATION AND ISNOWBEING TESTED INANUMBEROF LABORATORIES AROUND D THEWORLD 4HENEUTRALRADICALPROPOSALILLUSTRATESHOW*ERRYS WIDE RANGING INTERESTS MESHED D WITH HIS ABILITY TO DRAWTOGETHERINFORMAT F ION FROMANUM F BEROF lELDS IN ORDER TO SYNTHESIZE NEW IDEAS AND MODELS FOR F DAMENTALPROCESSESINELECTRONTRANSFERS FUN F YSTEMS )NADDITIONTOHISRESEARCH ONREACTIONCENTERSAND CYTOCHROME OXIDASE *ERRY ALSO COLLABORATED ON A F BLEMS INRELATED D SYSTEMS SUCH ASMY M NUMBEROFPRO ELOPEROXIDASE GREEN HEME PROTEIN RIBONUCLEOTIDE REDUCTASE HYDROXYLAMINEOXIDOREDUCTASE GALACTOSE OXIDASE $.!0HOTOLYASEAND GUANYL N ATECYCLASE(IS COLLABORATORS INTHISWORK R INCLUDED D !%HRENBERG ! (OOPER --ARLETTA !3ANCAR " -3JÚBERG * D *7HITTAKER-EMBERSOFT F HE 3TUBB U E 27EVERAND "ABCOCK K GROUP WHO WORK R ED D ON THESE AND D CLOSELY RELATEDPROJECTSINCLUDE'UERT$EINUM 2ENE&LORIS -ATT'ARDNER 9VONNE'INDT 4ONY N /ERTLING (ANS 3CHELVIS 3TEVE 3IEBOLD AND D %STHER 6OLLENBROEK AMONGOTHERS.EARTHEENDOFHISLIFE HEBEGANAN OTH T ERNEWRESEARCH INITIATIVE WHI W CH H EMPLOY O ED D &OURIER TRANSFORM F INFRARE F D SPECTROSCOPYTOCHARACTERIZETHE 03))3 STATES(ILLIERAND"ABCOCK   D ANUMBEROFAWAR F DSAND HONORS IN *ERRY RECEIVED THECOURSEOF HISCAREER!MONGTHEMWASA$ISTIN GUISHED&ACULTY!WARDFROM-ICHIGAN3TATE5NIVER SITY  AVISITING PROFESSORS F HIPATTHE#OLLÒGE DE&RANCE  THE0HILIPS,ECTUREAT(AVERFOR F D #OLLEGE A3IGMA8I3ENIOR2ESEARCH!WARD  A -ICHIGAN !CADEMIC 'OVERNING "OARD D THE#HARLES&+ETTERING!WARD FOR F !WARD  AND EXCELLENCEINPHOTOSYNTHESISRESEARCH PRESENTEDBY THE!MERICAN3OCIETY OFF 0LANT0HYSIOLOGISTS IN (EWASSOUGHTAFTERASAMEM F BEROF FE F DERAL ADVISORY PANELS ANDSERVEDINTHISCAPACITYFORANUMBEROF AGENCIES INCLUDING53$!5NITED D 3TATES$EPARTMENT F LTH OFF !GRICULTURE .)(.ATIONAL )NSTITUTESOF(EA 53! AND$/%$EPARTMENTOF%NERGY 53! (E WASAMEMBEROFF SEVERAL EDITORIAL BOARDS INCLUDING 0HOTOCHEMISTRYAND D 0HOTOBIOLOGY !NNUAL 2EVIEWS OF0HYSICAL#HEMISTRY "IOCHIMICA"IOPHYSICA!CTA 2EVIEWSON"IOENERG R ETICS "IOCHEMISTRY AN Y D*OURNA D L OFF "IOLOGICAL#HEMISTRY(ECHAIRED D THE'ORDON 2ESEARCH#ONFERENCEON0HYSICOCHEMICAL!SPECTSOF 0HOTOSYNTHESISAND D WASCHAIR ELECTOFA F FORT F HCOMING 'ORDON #ONFERENCEON2A F DICALS(EWASANINVITED SPEAKERATNUMEROUSREGIONAL NATIONALANDINTERNA

TIONAL MEETINGS)TWASNOTUNCOMMONFOR*ERRYTO F PREPARETHEOVERHEADSFORTHESETALKSTHENIGHTBEFORE INLONGHAND AN D D MAN NY OFF USREMEMBERHISPOSTERS W CH WEREOFTEN F HANDD WRITTEN ATVARIOUSMEETINGS WHI ONBROWNPAPERTHATWASINMANYINSTANCESDERIVED F FROM GROCERY BAGS !LTH T OUGHH THESEPROFESS F IONALACTIV I ITIESCONSUMEDD AN ENORMOUSAMOUNTOFTIME *ERRYWASABLETOMENTOR GRADUATESTU D DENTS ANDD POST DOCTORAL FE F LLOWS 4HERESEARCH EXPERTISEOFT F HESESTUDENTSWASDIVIDED ABOUT EQUALLY BETWEEN PHOTOSYNTHETIC AND CYTO CHROMEOX H IDASERESEARCHH PRO OJECTS(EWASALSOAGIFTE F D TEACHERINTHECLASSROOM SOMETHINGTHATWASEASILY DEDUCEDFROMLISTENINGTOONEOF*ERRYSSYMPOSIUM PRESENTATIONS)T ISSIGNIlCANTTHATEVENWH W ENHEHAD BEENPROMOTEDD TOTHETOP FACU F LTYRANKSHECONTINUED TOTEACHACOURSEININTRODUCTORYCHEMISTRY Y 7HENWELOOK BACKK ON*ERRYSPROFESS F IONAL CAREER THERE ARE SEVERAL THEMES THAT EMERGE4HE lRST OF THESEIS*ERRYSSUCCESSASATEACHER(EWASSUPERB INTHECLASSROOM BUTHEALSOHADD AREAL LOVE FOR F IM PARTINGCLEAREXPLANATIONSOF HISRESEARCH ANDD OF THE TECHNIQUESTHATHEUSED TOHIS Q PEERS&OREXAMPLE HE COULD D EXPLAINTHE BASICSOFF %02TOBIOCHEMISTS AND EVENTOMOLECULARBIOLOGISTS4HESECONDD THEMEWAS ANUNENDINGQUESTFORACOMPLETEUNDERSTANDINGOFTHE F DAMENTALCHEMISTRY INVOLVED INALLOFT FUN F HESYSTEMS THAT HESTUDIED(EWASMOST DEMANDINGOF HIMSELF INTHISARENA ANDONECAN SEEINHISPAPERSASTEADY PROGRESSION TOWARDS THE BASIC WORKI R NG PRINCIPLES F HROMEOXIDASEANDD 0HOTOSYSTEM))!THIRD OFCYTOC THEMEIS*ERRYSUNUSUALCAPACITYTODRAWTOGETHER PEOPLEWORKI R NG INVERY DIFFERENTAREAS AN F D TOFOCUS F THEM ON A PARTICULAR GOAL (IS COLLABORATIVE WORK R WITH-AARTEN7IKSTROM ,EE-C)NTOSH "RUCE$INER "ILL7OODRUFF 0ER3 F IEGBAHNANDD -ARGARETA"LOM BERG (ARRY 'RAY -IKE -ARLETTA $AVE"RITT 2ON 7 7EVER 7ILLIAM3MITH ,ES$UTTON THEAUTHORSOFTHIS @DEDICATION ANDD MANNYOTHERSTESTIlESNOTONLY TOTHE BREADTHOF HISINTERESTS BUTALSOTO HISPERSONAL SKILLS INDRAWINGADIVERSEGROUPOFPEOPLEINTOCOMMON F ENTERPRISES7EWOULD BEREMISSASHISTORIANS IFWE DID D NOTPOINTOUT HERETHATTHEATMOSPHEREANDD MENU AT $AGWOODS"ARAND'RILLIN,ANSINGWASACRITICAL F FACTOR INLUB U RICATINGMANY N OF*ERR F YSCOLLABORATIONS *ERRY"AB A COCKS LIFENOWSEEMS F LIKEA@BRIEFCAN F DLE TOTHOSEOFUSWHOHADTHEPRIVILEGETOINTERACTCLOSELY WITH HIM$EATH HASINTERRUPTEDD ACAREERTHATWASAT ONEANDD THESAMETIMEINAMATUREPHASE ALLOWINGUS ALLTOBENElTFROMHISWISDOM KEENINSIGHTANDBROAD

 BASED KNOW K LEDGE AND D YET STILL MOVING IN EXCITING NEWDIRECTIONSTHATWEREONLYBEGINNINGTOBEARFRUIT 7 HAVECITED 7E D ONLYASMALLPORTIONOF*ERR F YSOUTPUT INTHIS@DEDICATION(EAUTHORED D ORCOAUTHORED D  PAPERSANDREVIEWSONHISRESEARCH ANDWEWILLNEVER K KNOW HOWMUCH FURT F HER*ERRYSEVER INQUIRING MIND AND INNOVATIONWOULD HAVETAKEN HIM7HATWE DO KNOWISTHATOURlELDSOFRESEARCHPROlTEDIMMENSELY F FROM*ERR YSEXCEPTIONAL GIFTS AN F D THATWEAREALL FA F R F HETIMETHATWEHAD D WITH HIM RICHERFORT "DLOPXMFEHNFOUT 0ETER"RZEZINSKI GENEROUSLYPROVIDED D THEPHOTOGRAPH OFF *ERRYWEHAVEUSED HEREAND INTHE 0HOTOSYNTHESIS 2ESEARCH OBITUARY7ETHANK-S6ADA/$ONNELL Y FOR F HER WORK R IN COLLECTING *ERRYS LAST SECRETARY MATERIALTHATWASASSEMBLED FORAMEMOR F IAL BOOKLET DISTRIBUTED AT THE -ICHIGAN 3TATE 5NIVERSITY SYM POSIUM HELD IN HIS HONOR 7E HAVE USED D MATERIAL F FROMT HATPUBLI U CATIONINWRITINGTHISCHAPTER7EARE GRATEFULTO0ROF*.3IEDOW$UKE5NIVERSITY FOR HISRECOLLECTIONSAND D ASSISTANCE AND D WETHANK K 0AUL -ICHAEL FOR F PERMISSIONTOUSEHISREMINISCENCEOF *ERRY)TISINEVITABLE GIVENBOTHSPACELIMITATIONSAND THEWIDERANGEOFF *ERRYSINTERESTSAND D COLLABORATIONS THATWE HAVENOT BEENABLETOMENTIONEVERYONEWH W O WAS PART OF HIS SCIENTIlC CAREER7E APOLOGIZE FOR THESEOMISSIONS 3FGFSFODFT "ABCOCK K '4  0HOTOSYSTEM))ASAMETALLO RADICALENZYME )N-ATHIS0 ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL )) PP n+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT "ABCOCK K '4AND#A D LLAHAN0-  2EDOX LINKED HYDROGEN BOND D STRENGTH CHANGES IN CYTOCHROME A )MPLICATIONS FOR F THE CYTOCHROME OXIDASE PROTON PUMP "IOCHEMISTRY  n "ABCOCK'4AND#HANG#+ /XYGENBINDINGTOFERROUS HEME A ANDASYNTHETICANALOG&%"3,ETTn K '4AND 3ALMEEN)  2ESONANCE2AMANSPECTRA "ABCOCK AND D OPTICALPROPERTIESOFF OXIDIZEDD CYTOCHROMEOXIDASE"IO CHEMISTRY n K '4AND3AUER+ D  4HERAPIDD COMPONENTOFE F LEC "ABCOCK TRON PARAMAGNETIC RESONANCE SIGNAL ))! CANDIDATE FFOR THE PHYSIOLOGICALDONORTO0HOTOSYSTEM))INSPINACHCHLOROPLASTS "IOCHIM"IOPHYS!CTAn "ABCOCK'4 6ICKERY,%AND0ALMER' 4HEELECTRONIC STATE OF HEME IN CYTOCHROME OXIDASE ) -AGNETIC CIRCULAR DICHROISMOFT F HE ISOLATEDD ENZYMEAND ITSDERIVATIVES*"IOL #HEMn

"AB A COCK K '4 6ICKERY Y ,%AND 0ALMER' 4HEELECTRONICSTATE OFHEMEINCYTOCHROMEOXIDASE))2EDOXPOTENTIALINTERACTION ANDHEMEIRONSPINSTATEBEHAVIOROBSERVEDDURINGREDUCTIVE TITRATIONS*"IOL#HEMn "ABCOCK '4 *EAN *- *OHNSTON ,. 0ALMER ' AND7OODRUFF 7(  4IME RESOLVEDRESONANCE2AMANSPECTROSCOP D Y OF F D DUR D INGTHEOXIDATIONOFC F YTOCHROME TRANSIENTSPECIES FORME OXIDASE BY DIOXYGEN*!M#HEM3OCn "ABCOCK K '4 4 *EAN*- *OHNSTON,. 0ALMER'AND7OO D DRUFF7( F  &LOW mASH H TIME RESOLVEDRESONANCE2AMANSPECTROSCOP D PY OFTHEOXIDATIONOFREDUCEDANDOFMIXEDVALENCECYTOCHROME OXIDASEBYDIOXYGEN*)NORG"IOCHEMn "ABCOCK '4 %SPE - (OGANSON #7 ,YDAKIS 3IMANTIRIS . -C#RACKEN* 3HI7 3TYRING3 4OMMOS#AND7ARNCKE+  4Y 4 ROSYLRADICALS INENZYMECATALYSIS3OMEPROPERTIES AND D A FFOCUS ON PHOTOSYNTHETIC WATER OXIDATION!CTA #HEM 3CAND D n "ARRY "!AND D "ABCOCK K '4 4Y 4 ROSINERADICALSAREINVOLVED INTHEPHOTOSYNTHETICOXYGEN EVOLVINGSYSTEM0ROC.ATL !CAD 3CI53!n "ERTHOLD $! "ABCOCK '4 AND 9OCUM #&  ! HIGHLY RESOLVED OXYGEN EVOLVING 0HOTOSYSTEM )) PREPARATION FROM SPINACH THYLAKOID MEMBRANES %02 AND ELECTRON TRANSPORT PROPERTIES&%"3,ETTn "LANKENSHIP2% "ABCOCK'4 7AR K DEN*4AND3AUER+ D  /BSERVATIONOFF ANEW%02TRANSIENT INCHLOROPLASTSTHATMAY A REmECTTH T EELECTRONDONORTO0HOTOSYSTEM))ATROOMTEMPERATURE &%"3,ETTn "LOMBERG-2! 3IEGBAHN0%- 3TYRING3 "ABCOCK'4 ¯KER MARK " AND +ORALL 0  ! QUANTUM CHEMICAL STUDY OF HYDROGENABSTRACTIONFROMMANGANESECOORDINATEDWATERBYA TYROSYLRADICAL!MODELFORWATEROXIDATIONIN0HOTOSYSTEM )) * !M #HEM3OCn #ALHOUN-7 4HOMAS*7 (ILL** (OSLER*0 3HAPLEIGH *0 4ECK LENBERG--* &ERGUSON -ILLER3 "ABCOCK'4 ! K LBEN*/AND F HEAXIAL LIGAND D OF THE HIGH SPIN 'ENNIS2"  )DENTITYOFT HEMEINCYTOCHROMEOXIDASE3PECTROSCOPICCHARACTERIZATION OFMUTANTSINTHE BO TYPEOXIDASEOF %SCHERICHIACOLI % ANDTHE AA TYPEOXIDASEOF 2HODOBACTERSPHAEROIDES"IOCHEMISTRY n #ALLAHAN0-AND"ABCOCK'4 4HEORIGINOFTHECYTO CHROME A ABSORPTION RED SHIFT! F P( DEPENDENT INTERACTION BETWEEN ITS HEME A FORM F MYLANDPROTE D IN INCYTOCHROMEOXIDASE "IOCHEMISTRY n $EBUS2* "ARRY"! "ABCOCK K '4AND -C)NTOSH , A 3ITE DIRECTED D MUTAGENESISIDENTIlESATYROSINERADICAL INVOLVED IN THEPHOTOSYNTHETICOXYGENEVOLVINGSYSTEM0ROC.ATL!CAD 3CI53!n $EBUS2* "ARRY"! 3ITHOLE) "ABCOCK'4AND-C)NTOSH, B $IRECTEDMUTAGENESISINDICATESTHATTHEDONORTO0 IN 0HOTOSYSTEM))IS4YR OFT F HE$POLYPEPTIDE"IOCHEM ISTRYn $E'ROOT! 0LIJTER** %VELO2 "ABCOCK'4AN K D (OFFF !* F HEOXIDATIONSTATEOFT F HEOXYGEN EVOLVING 4HEINmUENCEOFT COMPLEXOFF 0HOTOSYSTEM))ONTHESPIN LATTICERELAXATIONTIME OFSIGNAL))ASDETERMINEDBYELECTRONSPINECHOSPECTROSCOPY "IOCHIM"IOPHYS!CTAn $EKKER*0 'HANOTAKIS$& 0LIJTER** VAN'ORKOM(*AND"AB COCK'4 +INETICSOFTHEOXYGEN EVOLVINGCOMPLEXIN SALT WASHED D 0HOTOSYSTEM )) PREPARATIONS "IOCHIM "IOPHYS H !CTA  n

 $ISMUK U ES'#AND3 D IDERER9 )NTERMEDIATESOFAPO F LYNUCLEAR MANGANESECENTERINVOLVEDINPHOTOSYNTHETICOXIDATIONOFWATER 0ROC.ATL !CAD3CI53!n %SPE- (OSLER* &ERGUSON -ILLER3 "ABCOCK'4 AND-C#RACKEN * !#7 ANDPULSED %02CHARACTERIZATIONOFTHE-N BINDING SITE IN 2HODO D BACTERSPH S AEROIDE D S CYTOCHROME C OXIDASE "IOCHEMISTRYn &ETTER * 1IAN * 3HAPLEIGH * 4HOMAS * 'ARCIA (ORSMAN ! 3CHMIDT% (OSLER* * "ABCOCKK '4 4 4 'ENNIS2AND&ERGUSON -ILLER3 0OSSIBLEPROTONRELAY A PATHWAY A S INCYTOCHROME C OXIDASE0ROC.ATL!CAD3CI53!n &LORENS, (OGANSON# -C#RACKEN* &ETTER* -ILLS$ "ABCOCK '4 AND &ERGUSON -ILLER 3  4HE ROLE OF MAGNESIUM AND ITS ASSOCIATED WATER CHANNEL IN ACTIVITY AND REGULATION OFF CYTOCHROME C OXIDASE )N0ESCHEK' ,OE K FFE F LHARDT7AND 3CHMETTERER'EDS 4HE0HOTOTROPIC0ROKARYOTES PPn +LUWER!CADEMIC0LENUM0RESS $ORDRECHT &LORENS, 3CHMIDT" -C#RAC D KEN* ANDD &ERRGUSON -ILLER3 &ASTDEUTERIUMACCESSTOTHE BURIEDD MAGNESIUMMANGANESESITE INCYTOCHROME C OXIDASE"IOCHEMISTRYn 'HANOTAKIS $& 9ERKES #4 AND "ABCOCK '4  4HE ROLE OF!$29 REAGENTS IN DESTABILIZING HIGH POTENTIAL OXIDIZING EQUIVALENTSGENERATEDINCHLOROPLAST0HOTOSYSTEM))"IOCHIM H "IOPHYS!CTAn K D 9OCUM#& A #ALCIUM 'HANOTAKIS$& "ABCOCK'4AN F OXYGEN EVOLUTION IN POLYPEPTIDE RECONSTITUTES HIGH RATES OF DEPLETED 0HOTOSYSTEM )) PREPARATIONS &%"3 ,ETT  n 'HANOTAKIS$& 4OPPER*. "ABCOCK'4AND9OCUM#&B 7ATER SOLUBLE  AND  K$A POLYPEPTIDES RESTORE OXYGEN 7 EVOLUTIONACTIVITYBYCREATINGAHIGH AFlNITYSITEFOR#A ONTHE OXIDIZINGSIDEOF0HOTOSYSTEM))&%"3,ETTn 'HANOTAKIS $& DE 0AULA *# $EMETRIOU $- "OWLBY .2 0ETERSEN* "ABCOCKK '4AND 9OCUM#&  )SOLATIONAND CHARACTERIZATIONOFT F HE K$APROTEINANDD THE$ $ CYTO CHROME B COMPLEX"IOCHIM"IOPHYS!CTAn D "ABCOCK'4 K  3 STATE DEPENDENT&4)2DIFFE F R (ILLIER7AND ENCESPECTRAFORTHE0HOTOSYSTEM))OXYGEN EVOLVINGCOMPLEX "IOCHEMISTRYn (OGANSON#7AND"ABCOCK'4 !METALLORADICALMECHA NISMFORTHEGENERATIONOFOXYGENFROMWATERINPHOTOSYNTHESIS 3CIENCEn (OGANSON #7 ,YD , AKIS 3IMANTIRIS . 4ANG 8 3 4OMMOS # 7 7ARNC KE+ "ABCOCK'4 $ K INER"! -C#RACKEN*AND3T D YRING 3  ! HYDROGEN ATOMABSTRACTIONMODEL FORT F HEFUNCT F ION OFF 9: IN PHOTOSYNTHETIC OXYGEN EVOLUTION 0HOTOSYNTH 2ES n (OSLER*0 &ERGUSON -ILLER3 #ALHOUN-7 4HOMAS*7 (ILL* ,EMIEUX, -A* 'EORGIOU* &ETTER* 3HAPLEIGH* 4ECKLEN BURG--* "ABCOCK'4AND'ENNIS2" )NSIGHTINTO ACTIVE SITE STRUCTURE AND FUNCT F ION OF F CYTOCHROME OXIDASE BY ANALYSISOFS F ITE DIRECTEDD MUTANTSOF BACTERIAL CYTOCHROME AA AND D CYTOCHROME BO*"IOENERG"IOMEMBRn K D &ERGUSON -ILLER3 (OSLER* %SPE- :HEN9 "ABCOCK'4AN  !NALYSISOFF SITE DIRECTEDD MUTANTS LOCATESANON REDOX ACTIVEMETALNEARTHEACTIVESITEOFCYTOCHROME C OXIDASEIN 2HODOBACTERSPHAEROIDES"IOCHEMISTRYn ,AKSHMI+6 %ASTON33 %ATON'2 &RANK(!AND"RUDVIG'7  !NALYSISOFDIPOLARANDEXCHANGEINTERACTIONSBETWEEN D INE:INTHE39:ŠSTATEOFACETATE INHIBITED MANGANESEANDTYROS 0HOTOSYSTEM))VIA%02SPECTRALSIMULATIONSAT8 AND1 D BANDS

*0HY H S#HEM"n ,YDAKIS 3IMANTIRIS. 'HANOTAKIS$&AND"ABCOCK'4 , +INETICISOTOPEEFFECTSONTHEREDUCTIONOFTHE9: RADICALIN OXYGEN EVOLVINGANDTRIS WASHED0HOTOSYSTEM))MEMBRANESBY TIME RESOLVED%02"IOCHIM"IOPHYS!CTAn /-ALLEY0*AND D "ABCOCK'4 K A %020ROPERTIESOF IM MOBILIZED D QUINONE CATION RADICALS AND D THE MOLECULAR ORIGIN OFF SIGNAL )) IN SPINACH CHLOROPLASTS "IOCHIM "IOPHYS!CTA  n /-ALLEY0*AND D "ABCOCK K '4 B %.$/2EVIDENCESUP PORTINGAMONOMERICNATUREFOR0 INSPINACHCHLOROPLASTS 0ROC.ATL !CAD3CI53!n 0ROSHLYAKOV$! 0RESSLER-!AND"ABCOCK'4 $IOXYGEN ACTIVATIONANDBONDCLEAVAGEBYMIXED VALENCECYTOCHROME C OXIDASE0ROC.ATL!CAD 3CI 53!n 0ROSHLYAKOV$! 0RESSLER-! $E-ASO# ,EYKAM*& $E7I 7 TT $,AND D "ABCOCK K '4 /XYGENACTIVATIONAND D REDUCT D ION INRESPIRATION)NVOLVEMENTOFRE F DOX ACTIVETYROSINE3CI ENCE n 1IAN * 3HI 7 0RESSLER - (OGANSON # -ILLS $ "ABCOCK '4 AND&ERGUSON -ILLER3 !SPARTATE IN 2HODO BACTERSPHAEROIDES CYTOCHROME C OXIDASEISNOTREQUIREDFOR PROTON PUMPING OR MANGANESE BINDING "IOCHEMISTRY  n 3ALMEEN) 2IMAI,AND D "ABCOCK'4 K  2AMANSPECTRAOF HEME A CYTOCHROMEOXIDASE LIGAND D COMPLEXES AND D ALKALINE DENATURED D OXIDASE"IOCHEMISTRYn 3HAPLEIGH *0 (OSLER *0 4ECKLENBURG --* +IM9 "ABCOCK '4 'ENNIS2"AND&ERGUSON -ILLER3 $ElNITIONOF THECATALYTICSITESOFCYTOCHROMEOXIDASE4HESPECIlCLIGANDS OFHEME A ANDTHEBINUCLEARMETALCENTER0ROC.ATL!CAD3CI 53! n D "ABCOCK'4 K  0ROTONAND HY H DROGENATOM 4OMMOS#AND CURRENTS INPHOTOSYNTHETICWATEROXIDATION"IOCHIM "IOPHYS !CTA n 4OMMOS# (OGANSON#7 $I6ALENTIN. ,YD , AKIS 3IMANTIRIS, D "ABCOCK K '4 $ORLET0 7ESTPHAL+ #HU(! -C#RACKEN*AND  -ANGANESEANDTYROSYLRADICALFUNCTIONINPHOTOSYN THETICOXYGENEVOLUTION#URR/PIN#HEM"IOLn 4WEEDLE -& 7ILSON ,* 'ARCIA )NIGUEZ , "ABCOCK '4 AND 0ALMER' %LECTRONICSTATEOFHEMEINCYTOCHROMEOXI DASE)))4HEMAGNETICSUSCEPTIBILITYOF BEEF HEARTCYTOCHROME OXIDASE AND SOME OF ITS DERIVATIVES FR F OM n + $IRECT EVIDENCE FORANANT F I FERROMA F GNETICALLYCOUPLED D &E))) #U)) PAIR*"IOL #HEM n 6AROTSIS#AND D "ABCOCK'4 K  !PPEARANCEOFT F HE i &E)6/ VIBRATION FROM A FERRYL OXO INTERMEDIATE IN THE CYTOCHROME OXIDASEDIOXYGENREACTION"IOCHEMISTRYn 6AROTSIS# 7OODRUFF7(AND"ABCOCK'4 4IME RESOLVED RESONANCE 2AMAN DETECTION OF i &E / INANEARLYINTERMEDI ATE INTHEREDUCT D IONOF DIOXYGEN BYCYTOCHROMEOXIDASE*!M #HEM3OCn  6AROTSIS# 7OODRUFF7(AN F D "ABCOCK'4 K  $IRECTDE TECTIONOFF A DIOXYGENADDUCTO D F CYTOCHROME A INTHEMIXED VALENCECYTOCHROMEOXIDASEDIOXYGENREACTION*"IOL#HEM n 6AROTSIS# :HANG9 !PPELMAN%(AND"ABCOCK'4 2ESO LUTIONOFTHEREACTIONSEQUENCEDURINGTHEREDUCTIONOF/ BY CYTOCHROME OXIDASE0ROC.ATL!CAD3CI53!n 7ARD" #A D LLAHAN0- 9OUNG2 "ABCOCK'4AN K D #HANG#+  2ED SHIFTS F INTHEOPTICALSPECTRAOFF PORPHYRIN3CHIFFS

 BASESUPONPROTONATION*!M#HEM3OCn 9ERKES #4 AND "ABCOCK '4  0HOTOSYSTEM )) OXIDATION 9 OFCHARGEDELECTRONDONORS3URFACECHARGEEFFECTS"IOCHIM "IOPHYS!CTAn 9 9OCUM#& 9ERKES#4 "LANKENSHI P2% 3HARP 22AND"ABCOCK Y INHIBITORSENSITIVITY TY AND ORRGANIZATION '4 3TOICHIOMETRY OFF MANGANESEASSOCIATEDD WITH TH PHOTOSYNTH T ETICOXYGENEVOLUTION 0ROC.ATL !CAD 3CI 53!n :OUNI! 7I 7 TT (4 +ERN * &ROMME 0 +RAUSS . 3AENGER 7 AND D /RTH 0  #RYSTALSTRUCTUREOFF 0HOTOSYSTEM))FROM F 3YNECHOCOCCUS ELONGATUS AT  ¯ RESOLUTION .ATURE  n

#HARLES&9OCUM

$EPARTMENTS OF -OLECULAR #ELLULAR AND $EVELOP MENTAL "IOLOGYAND D #HEMISTRY 5NIVERSITY OFF -ICHIGAN !NN !RB R OR -)  53! 2OBERT%"LANKENSHIP $EPARTMENTOF#HEMISTRYAND"IOCHEMISTRY !RIZONA3TATE5NIVERSITY 4EMPE !: 53! 3HELAGH&ERGUSON -ILLER $EPARTMENTOFF "IOCHEMISTRY -ICHIGAN3TATE5NIVERSITY %AST,ANSING -) 53!

!UTHORFORCORRESPONDENCE EMAILCYOCUM UMICHEDU D

$IBQUFS
 *OUSPEVDUJPO
UP
1IPUPTZTUFN
** ,JNJZVLJ
4BUPI

%FQBSUNFOU
PG
#JPMPHZ
0LB Z Z ZBNB
6OJWFSTJU UZ
0LBZBNB

+BQB Z O

5IPNBT + 8ZES[ZOTLJ

1IPUPCJPFOFSHFUJDT
(SPVQ
3FTFBSDI
4DIPPM
PG
#JPMPHJDBM
4DJFODFT
5IF
"VTUSBMJBO
/BUJPOBM
6OJWFSTJUZ UZ
$BOCFSSB
"$5

"VTUSBMJB

(PWJOEKFF %FQBSUNFOU
PG
#JPDIFNJTUSZ SZ
BOE
PG
1MBOU
#JPMPHZ
BOE
$FOUFS
PG
#JPQI Z Z ZTJDT
BOE
$PNQVUBUJPOBM

64" #JPMPHZ
6OJWFSTJUZ
PG
*MMJOPJT
BU
6SCBOB$IBNQBJHO
6SCBOB
*-


4VNNBSZ  *

%JTDPWFSZ
PG
0YZHFO
BOE
0 1SPEVDUJPO CZ
1MBOUT  ** $PODFQUVBM
%FWFMPQNFOU
PG 1IPUPTZTUFN **  ***
0 1SPEVDUJPO ‡ 1IFOPNFOPMPHZ   *7

*TPMBUJPO
PG
UIF
$IFNJDBM
&OUJUZ‡4USVDUVSBM 0SHBOJ[BUJPO
PG
1IPUPTZTUFN
**   "
$PSF
$PNQMFY  #
5IF 5PUBM
1JDUVSF   7

'VODUJPOBM 4JUFT ‡ $BUBMZUJD
3PMF
PG 1IPUPTZTUFN **  7 "
1SJNBSZ 1IPUPDIFNJTUSZ   #
$IBSHF
4UBCJMJ[BUJPO   $
8BUFS
0YJEBUJPO   7*

'VUVSF
1FSTQFDUJWFT   "
4USVDUVSF
BOE
#JPEZOBNJDT  #
0SJHJO
&WPMVUJPO
BOE
"SUJàDJBM
0 1SPEVDJOH 4ZTUFNT   "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 4HISCHAPTERBRIEmYTRACESSOMEOFF THEEARLYSTUDIESAND KEYlNDINGSWHI W CH HAVELEDTOOURCURRENTPERCEPT D ION ANDUNDERSTANDINGOF0HOTOSYSTEM)) THEWATERPLASTOQUINONEOXIDOREDUCTASEINOXYGENICPHOTOSYNTHESIS 3TARTINGWITHTHEDISCOVERY OFOX F YGENAND D THEIDEAOFF TWOPHOTOSYSTEMS THEPROGRESSIVE IDENTIlCATIONOFT F HE F IONALASPECTSOFF 0HOTOSYSTEM))AREOUTLINEDD AND RELATEDD TOTHECORRESPONDINGCHAP UNIQUESTRUCTURAL AND FUNCT TERSINTHEBOOK4HEAIMISTOINTEGRATETHEDETAILEDDESCRIPTIONSINTHEVARIOUSCHAPTERSINTHECONTEXTOFTHE F IONOFF 0HOTOSYSTEM))ASAWH W OLE4HECHAPTERENDSWITHABRIEFF PERSPECTIVE FORT F HE FUTURE F STRUCTUREAND FUNCT D APPLICATIONOFF 0HOTOSYSTEM))RESEARCH STUDYAND

!UTHORS FORCORRESPON F DENCE EMAILKIMIYUKI CCOKAY A AMA UACJ  PTOMWYDRZYNSKI ANUEDUA D U

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



,JNJZVLJ 4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

*
%JTDPWFSZ
PG
0YZHFO
BOE 0 1SPEVDUJPO CZ
1MBOUT 4HE ABILITY OFF PLANTS TO GENERATE OXYGEN WAS lRST DESCRIBEDBY *OSEPH0RIESTLEY  ANDPUB LISHED INHIS BOOK K ENTITLED %XPERIMENTSAN % D /BSERVA TIONSON$IFFERENT+INDSOF!IR )NTHISSTUDY R 0RIESTLEYOBSERVEDTHEPHENOMENONINWHICHMINT PLANTSREVEALEDA@POWERTORESTORET D HEAIRWHI W CH HAD BEENINJ N URED BYTHE BURNINGOFCAN F DLES4HIS@SPECIAL POWEROFPLANTSWASDESCRIBEDINTHESCIENTIlCTERMS OFF THOSEDAY A SASTHEABILITY TOCHANGE@PHLOGISTICATED D AIR )N RETROSPECT THIS AIR INTO @DEPHLOGISTICATED OBSERVATION WAS THE lRST SCIENTIlC RECOGNITION OF THEFUNCT F IONING INPLANTSOFF THEWATER RPLASTOQUINONE D IE 0HOTOSYSTEM))03)) TH T EFOCUS F OXIDO REDUCTASE OFTHISBOOK(OWEVER THE PRECISECHEMICALMECHA NISMAND BIOLOGICAL MACHINERY RESPONSIBLEFORT F HIS PROCESSWERE FAR F BEYOND D THESCIENTIlCUNDERSTANDING ATTHATTIME$URINGTHESAMEPERIOD THEPRESENCEOF A DISTINCTCOMPONENTORELEMENT INTHEATMOSPHERE WAS DISCOVEREDD AND LATERNAMED BY !NTOINE,AURENT ,AVOISIERn AS@OXYGEN,AVOISIER  7 ELM3CHEELE  WH W OHAD D CALLED ALSOSEE +ARL7ILH IT@lREAIR )TWAS LATERRECOGNIZED D THATOXYGENWAS EQUIVALENT TO @DEPHLOGISTICATED AIR AND IT IS NOW WIDELY ACCEPTED D THAT THE BULK K OFF THE LIFE SUSTA F IN INGOXYGEN INTHEATMOSPHERETODAYAROSE FROMT F HE F FUNCTIONINGOF03))SINCE ITSAPPEARANCEDURINGTHE F H EVOLUTIONOF LIFEONEART 4HE REQUIREMENT OF LIGHT CARB R ON DIOXIDE AND WATERINTHEOVERALLPROCESS ASWELLASTHECOUPLING BETWEEN/ PRODUCT D IONAND D ORGANICMATTERSYNTHESIS WERE SUBSEQUENTLY RECOGNIZED THE ENERGETIC MEANINGOFTHISUNIQUEPHYSIOLOGICALPROCESS CALLED @PHOTOSYNTHESIS  WAS ESTABLISHED BY *ULIUS 2OBERT -AYERnSEE-AYER  #HLOROPHY H LL #HL WASIDENTIlEDASTHEMAJORLIGHTABSORBERFOR DRIVINGTHEPROCESS4IMIRIAZEFF  F AND BYTHE BEGINNINGOFF THETH CENTURYOVERALL PHOTOSYNTHESIS WASDESCRIBEDSIMPLYASFOLLOWS !BBREVIATIONS R "2#nBACTERIALREACTIONCENTER#ARnCAROTENE #HL :nREDOXACTIVE#HL OFF 03))#HL nCHLOROPHYLL#YTnCY TOCHROME $ $n 2# CORE SUB U UNITS OF F 03)) ,(#0n LIGHT HARVESTING #HL AB A PROTEIN OF F 03)) /%#n OXYGEN EVOLVING COMPLEX0nPRIMARYELECTRONDONOROF03))0 F HEOnPHEO PHYTIN01nPLASTOQUINONE03))n0HOTOSYSTEM))1! nPRIMARY 01ACCEPTOR1" nSECONDARY01ACCEPTOR2#nREACTIONCENTER 3N nSTATESOFTHEWATEROXIDIZINGCOMPLEXFORN    OR 9$ nREDOX ACTIVETYROSINEON$PROTEIN9: n REDOX ACTIVE TYROSINEON$PROTEIN

KN &2   +  2 } }} }} } m>&+ & 2 &KO

2



W ERE ;#(/=REPRESENTSAUNITOFF ORGANICMATERIAL WH IE SUGAR )NTHI T S FORMU F LATION TH T ESOURCEOF / COULD BEEITHERTHECARBONDIOXIDEORTHEWATER(OWEVER 2OBIN(ILL  QUITEELEGANTLY DEMONSTRATEDD THAT / PRODUCT D IONCOULD BE DECOUPLED FROMORGAN F ICSYN THESISINISOLATEDCHLOROPLASTFRAGMENTSBYUSINGAN INORGANICOXIDANT SUCH ASPOTASSIUM FERR F ICYANIDE AS ATERMINAL ELECTRONACCEPTORFORA F HISTORICALREVIEW SEE'OVINDJEEAND+ROGMANN   **
$PODFQUVBM
%FWFMPQNFOU
PG 1IPUPTZTUFN ** )NTHEEARLYPARTOFTHETH CENTURY 2OBERT%MERSON ANDD 7ILLIAM !RNOLD A B DEMONSTRATEDD THAT R EDTOGET D HERTO SEVERAL HUNDREDD #HLMOLECULESWORK PRODUCE ONE / MOLECULE 4HIS LED D (ANS 'AFFRON ANDD +URT 7OHL  TO PROPOSE THE CONCEPT OF RKASSEMBLYOF THE@PHOTOSYNTHETICUNITASANETWORK #HL MOLECULES WHICH FUNCTIONED TO SUPPLY EXCITA TIONENERGYTOAPHOTOCHEMICALCATALYTICCENTERTHAT POWERS PHOTOSYNTHESIS &OLLOWING THE CONCEPT OF THEPHOTOSYNTHETICUNIT %MERSON AND,EWIS DEMONSTRATEDD THAT FFAR RED LIGHT NM GIVES A POORER QUANTUM YIELDD OF / PRODUCT D ION THAN RED LIGHT4HIS PHENOMENON WAS CALLED THE @RED DROP (OWEVER THEDECLINEINTHEQUANTUM YIELD D COULD BE R ED BYAN@AC AVOIDED IFF SUPPLEMENTARY LIGHTABSORB CESSORYPIGMENTOFSHORTERWAVELENGTHWASADDED TOTHEFAR RE F D LIGHT4HISPHENOMENON ISKNOWNAS K THE@%MERSONENHANCEMENTEFFECT F %MERSONETAL %MERSONAND2ABINOWITCH 'OVINDJEE ANDD 2ABINOWITCH  -YERS ANDD &RENCH  AND LEDD TOTHECONCEPTFORT F HEEXISTENCEOFTWO F LIGHT REACTIONS AND TWO PIGMENT SYSTEMS IN OXYGENIC PHOTOSYNTHESIS4HE IMPLICATION THAT THE TWO LIGHT REACTIONSANDD TWOPIGMENTSYSTEMSACTTOGETHERINA SERIESWASPROPOSEDBY2OBIN(ILLAND&AY"ENDALL  AS PART OF A WORKING HY H POTHESIS TO EXPLAIN THE ROLE OF F CYTOCHROMES #YT IN PHOTOSYNTHETIC ELECTRONTRANSPORTFORAREVIEW SEE$UYSENS   Y GTH 3UBSEQUENTLY THEINVOLVEMENTOFASHORT WAVELEN REACTIONANDD A LONG WAVELENGTH REACTIONINOXYGENIC PHOTOSYNTHESISWASDEMONSTRATEDBYLIGHT INDUCED ABSORBANCE CHANGES AND ELECTRON TRANSPORT STUDIES +OK $UYSENSETAL ,OSADAETAL 

$IBQUFS

*OUSPEVDUJPO
UP
14 **



&IIG4HE: SCHEMEOFOX & F YGENICPHOTOSYNTHESIS FORE F LECTRONTRANSFER F FROMWATERTON F ICOTINAMIDEADENINEDINUCLEOTIDEPHOSPHATE .!$0 MODIlEDFROM&IGOF'OVINDJEE &ROMLEFTTORIGHT-N STANDSFOR-NCLUSTER9Z FORTYROSINE ONTHE$PROTEIN0 FOR THEPRIMARYELECTRONDONOROF03))0HEO FORPHEOPHYTIN1! FORTHElRSTPLASTOQUINONE ELECTRONACCEPTOROF03))1" FORTHESECOND PLASTOQUINONEELECTRONACCEPTOROF03))01 FORTHEPLASTOQUINONEPOOL#YTB, AND#YTB( FORLOWANDHIGHPOTENTIALCYTOCHROME B #YT B F FORCYTOCHROME F F B F COMPLEX#YT F FORCYTOCHROME F F &E3 FOR2IESKEIRONSULFUR0# FORPLASTOCYANIN0 FORTHEPRIMARY ELECTRON DONORFOR03) F ! FORAMONOMER# F HL ! FORV F ITAMIN+ &X & &! AND D &" FOR F IRONSULFURCENTERS@X @!AN F D @" RESPECTIVELY AND &D FOR F FERRE F DOXIN!LSOSHOWNARETHEAPPROXIMATEHALF T F IMES FORMOSTO F F THEREACTIONS THEEXISTENCEOFF THE@1 CYCLE THECYCLIC D 03) AND THEPOSSIBLESITESFORCA F LCIUM CHLORIDEAND BICARB R ONATE IONSIN03))3EE#OLOR0LATE &IG REACTIONAROUND

7ITTETAL  4HUSTHE HYPOT H HESISOFF (ILLAND "ENDALL CALLEDTHE@ZIGZAGMODELOR@: SCHEME WAS U STANTIATED D AND THENAMES@0HOTO EXPERIMENTALLYSUB SYSTEM)LIGHTREACTION AND D @0HOTOSYSTEM))LIGHT REACTION  WERE PROPOSED $UYSENS ET AL   !CURRENTPICTUREOFT F HE: SCHEME IS ILLUSTRATED IN &IG (ERE 03)) IS SHOWN AS THE FUNCT F IONAL UNIT T TRANSFERRINGELECTRONSFROMWATERTOTHEPLASTOQUINONE 01 ACCEPTOR THATINTURN DONATESELECTRONSVIATHE #YT B F COMPLEXTO0HOTOSYSTEM)4HUS THE03)) F FUNCTIONALUNITOPERATINGIN / EVOLVINGORGANISMS ISCALLED D THE@WATER RPLASTOQUINONEOXIDO REDUCTASE D &OREARLIERDETAILED DISCUSSIONSOFF OVERALLOXYGENIC PHOTOSYNTHESIS SEE/RTAND9OCUM AND+E  ***
0 1SPEVDUJPO ‡ 1IFOPNFOPMPHZ 4HEOXIDATIONOFWATERTOMOLECULAROXYGENREQUIRES THE EXTRACTION OF FFOUR ELECTRONS AND FFOUR PROTONS (S FROMTWO F (/MOLECULES&IG 5SINGALGAL SUSPENSIONS 0IERRE *OLIOT ET AL  PROVIDED DRAMATICEXPERIMENTALEVIDENCETOSHOWTHAT/ IS D THROUGH A CYCLICAL PERIOD FOUR F REACTION RELEASED 4HESE RESEARCHERS OBSERVED THAT FOR DARK ADAPTED ALGAE EXCITED BY A TRAIN OFF SINGLE TURNOVER LIGHT mASHES VERY LITTLEORNO / ISPRODUCE D D AFTERT F HE ST AND D NDDmASHESINTHESEQUENCEWHILETHEMAXIMUM / ISPRODUCE D D AFTERT F HERDD mASH4HEREAFTER PROGRES

D OCCURAFTERT F HE SIVELYSMALLERPEAKSINTHE/ YIELD TH ANDD TH mASHES FOLLOWINGABASICPERIODICITYOF F FOUR UNT IL THEOSCILLATIONS DAMPOUTTOASTEADY STATE LEVEL AFTERT F HETHIRDD ORTHEFOURT F H CYCLE&IGURE TOP O SHOWSATYPICALTRACEOF/ mASHYIELDMEASUREMENTS F FROMASP INACHTHYLAKOIDSAMP D LEWHIL W E&IG BOTTOM LEFT E SHOWSAPLOTOFF THEDATA4HEPERIODICITYOF FOUR F CANREADILYBEEXPLAINEDBYTHECHEMISTRYOFWATER D ONTHERDDmASH OXIDATION BUTTHEMAXIMAL / YIELD ANDD THE DAMPEDD OSCILLATIONSINDICATES AMUCH HIGHER LEVELOFCOMPLEXITYINTHEMECHANISMFORAREVIEW SEE7HITMARSHANDD 'OVINDJEE   "ASEDD ON THE RESULTS OFF *OLIOT ET AL  AND +OK ET AL  DEVELOPED AN THEIR OWN "ESSEL + ELEGANTKINETICMODEL TOACCOUNTFORT F HE/ mASH YIELD PATTERN BY03)))NTHISMODEL ASPECIAL OXIDIZABLE COMPONENTISPROPOSEDTOCYCLETHROUGHASERIESOFlVE INTERMEDIATESTATES TERMEDD THE3N STATES WH W EREN F INGWITH WATERTOPRODUCE D    OR BEFOREREACT /%ACH3N STATEDIFFERSFROMTHE PREVIOUS 3N  STATE BYTHEACCUMULATIONOFF ONEOXIDIZINGEQUIVALENTUNTIL 3 ISREACHED ATW D WHICH POINT / ISRELEASEDD AND THE 3 STATEISREGENERATED4OACCOUNTFORTHEPEAKYIELD AFTERT F HE RDDmASHANDTHEDAMPINGINTHEOSCILLATIONS THEFO F LLOWINGADDITIONALASSUMPTIONSWEREMADE FO F R AMACROSCOPICPOPULATIONOF/ EVOLVINGCENTERSI THE 3 STATE ISSTABL A E INTHEDARK R  II THE 3 AND 3 STATES EVENTUALLY DEACTIVATETOTHE3 STATEIFF NOTADVANCED BY ANOTHERINCOMINGPHOTONASINDICATEDBYTHEDASHED I  III UPONREACHING ARROWS IN&IG BOTTOMRIGHT



,JNJZVLJ 4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

&IIG  4OP & O 4Y 4 PICALTRACESOF / mASHYIELDMEASUREMENTSFROMASPINACHTHYLAKOIDSAMPLEASAFUNCTIONOFmASHNUMBER F " "OTTOMLEFT  " /XYGEN YIELDD ASA FUNCT F IONOF mASHNUMBERSSHOWINGTHEPERIODICITY OF FOURAN F D THE lRSTMAXIMUMATTHE RDDmASH*OLIOTETAL   " "OTTOMRIGHT  " ! SIMPLIlED3 STATEMODELWHERETHESUBSCRIPTSUNDER@3REmECT THEPOSITIVECHARGESANDTHE@3STATESWITHPRIMES INDICATETHESTATESFORMEDAFTERALIGHTREACTIONCF+OKETAL  &IGUREMODIlEDFROM7YDRZYNSKI 

THE3 STATE / ISRELEASEDTHROUGHANINSTANTANEOUS DARK RK REACTION IV AFTER F mASH ACTIVATION THERE IS A lNITE TIME BEFORE F AN 3N STATE CAN BE ADVANCED BY ANOTHERINCOMINGPHOTONASINDICATEDBYTHE 3N A 3|N A 3N TRANSITIONSIN&IG BOTTOMRIGHT I AND V THERE ISACERTAINSMALL PROBABILITY FORA F DOUBL U E HITIE WHEREAN/ EVOLVINGCENTERADVANCESTWO D INGA GIVEN LIGHTmASHA DOUBL U EHITTHUS 3 STATESDUR DEPENDSONTHEmASH DURAT D ION ORA MISS IE WH W ERE AN / EVOLVING CENTER FAILS TO ADVANCE TO THE NEXT HIGHER3N STATE DUR D INGA GIVENmASH TOOCCURAFTE F R 7 TH THESEASSUMPTIONS THE ANACTIVATINGLIGHT mASH7I MODELCANREADILYEXPLAINTHEOBSERVED/ mASHYIELD PATTERN4HIS INSIGHTFU F L MODEL IS ILLUSTRATED IN&IG  BOTTOMRIGH G T AND HASGUIDED D THERESEARCH ONTHE OPERATIONOFTHEELECTRONTRANSFERPATHWAYSANDTHE F  PRODUCT D IONIN03))FORMORET F THAN MECHANISMOF/ YEARS&LASH INDUCE D D /RELEASEAND D OTHEROSCILLATORY PROCESSES IN THE 03)) ARE DISCUSSED IN DETAIL BY W ETHEIMPLICATIONSOFF THE 3HINKAREV#HAPTER WHIL

OSCILLATORYREACTIONSINTHEWATEROXIDATIONCHEMISTRY AREDISCUSSED BY (ILLIERANDD -ESSINGER#HAPTER  !HISTORICALACCOUNTOFF THE DISCOVERYOFF THEPERIOD F FOURmASH PATTERNIN/ PRODUCTIONISGIVENBY*OLIOT   *7
*TPMBUJPO
PG
UIF
$IFNJDBM
&OUJUZ‡ 4USVDUVSBM 0SHBOJ[BUJPO
PG
1IPUPTZTUFN
** " $PSF $PNQMFY 7ILLSTÊTTERAND3TO D LL  PROVIDEDD THECHEMICAL IDENTIlCATIONOFF #HL ANDD 3MITH  SHOWEDD THE ASSOCIATIONOF#HLWITHPROTEINSINVIVO4HUS VARIOUS D WERE ATTEMPTED FFOR THE DIRECT ISOLATION PROCEDURES OF F THE FUNCT F IONAL PIGMENT PROTEIN COMPLEXES FROM F PHOTOSYNTHETICTISSUESORFROMISOLATEDCHLOROPLASTS IN ORDER TO IDENTIFY THE MINIMUM UNIT CAPABLE OF

$IBQUFS

*OUSPEVDUJPO
UP
14 ** PHOTOSYNTHESIS4HOMASETAL  !REMARK R ABLE A ROACHWASMADE ACHIEVEMENTINTHISEXPERIMENTALAPP BY +EITH"OARDMANAND*AN!N D DERSON  WH W O WEREABLETOSEPARATEDIGITONIN SOLUBILI U ZEDSP D INACH CHLOROPLASTSINTOTWOFRACTIONSTHATWEREEACHENRICHED INONEOFT F HETWO0HOTOSYSTEMSACCORDINGTOTHE: F ION AND D 03)) HEAVIER SCHEME IE 03) LIGHTERFRACT F FRACTION )NTHISISOLATIONPROCEDURE 03))ACTIVITY WASASSOCIATED D WITHTHEMEMBRANEFRACT F IONENRICHED IN#HL B WHICHISNOWKNOWNTOCONTAINTHEGRANA OFCHLOROPLASTTHYLAKOIDMEMBRANESSEE!NDERSON   2EFINED PREPARATIONS OF F THIS MEMBRANE F ION EG THESO CALLED FRACT D ""9PARTICLES"ERTHOLD ET AL  RETAIN FULL / EVOLVING ACTIVITY AND HAVE BEENUSED D EXTENSIVELY INTHEANALYSISOF03)) F F ION STRUCTUREAND FUNCT 5PON THE INTRODUCTION OF POLYACRYLAMIDE GEL ELECTROPHORESIS 0!'% UNDER PARTIALLY DENATURING CONDITIONS IE THE SO CALLED D GREEN GELS /GAWA ETAL 4HORNBERETAL /GAWA  A SPECIAL PIGMENT PROTEINCOMPLEXOFF THE03))COULD BE ISOLATED D THAT HADNOP D HOTOCHEMICALACTIVITY4HIS PIGMENT PROTEINCOMPLEXBECAMEKNOWNASTHE @LIGHT HARVESTING#HL AB PROTEINOR,(#0 4HORNBERAND (IGHKIN 4HORNBER  THAT FUNCT F IONS AS THEDISTALANTENNAOF03))INGREENPLANTS#URRENT INFORMAT F IONONTHESTRUCTUREAND FUNCT F IONOFT F HEDISTAL AND D EXTRINSICANTENNACOMPLEXESOFT F HE03)) FROM F A VARIETY OF OXYGENIC PHOTOSYNTHETIC ORGANISMS IS SUMMARIZED BY 'REENAND'ANTT D #HAPTER  $URINGTHEEARLYS AVARIETYOFMET F HODSUSING NON IONIC DETERGENTS EG SUGAR BASED DETERGENTS WERE FOUN F D TO BE EFFECT F IVE IN ISOLATING PIGMENT PROTEINCOMPLEXESWITH 03))ACTIVITY FROMA F DIVERSE RANGE OF CYANOBACTERIA GREEN ALGAE AND HIGHER PLANTS 3ATOH AND D "UTLER 3ATOH    4HROUGHTHESESTUDIES THE @03 ))CORECOMPLEXWAS lRMLY ESTABLISHED AS A DElNITIVE CHEMICAL ENTITY F D TOCONSISTOF F 4HEEARLY CORECOMPLEXESWEREFOUN D #0 MAJ A ORPOLYPEPTIDECOMPONENTS IE #0AND WHICH ARE THE #HL A CONTAINING PIGMENT PROTEINS W THATCONSTITUTETHEPROXIMAL ORCOREANTENNAOF03)) F FOR F DETAILS SEE#HAPTER %ATON 2YEAND D 0UTMAN %VANS  THE $ AND $ PROTEINS WHICH CONSTITUTE THECATALYTICCORE FOR F DETAILS SEE#HAPTER .IXON D THE#YT B _ AND ` SUB U UNITS WHI W CH ETAL AND ISPOSSIBLYINVOLVEDASASIDEPATHELECTRONCARRIER FOR F DETAILS SEE#HAPTER 4HORNTONETAL#HAPTER A ORSUB U UNITS  &ALLERETAL )NADDITIONTOTHEMAJ RElNEDPREPARATIONSWEREFOUNDTOCONTAINSEVERAL LOWMOLECULARWEIGHTCOMPONENTSASWELL +ASHINO

 ETAL #HAPTER 4HORNTONETAL  $URING THE EARLY INVESTIGATIONS ISOLATED CORE Y OFT F THEMAJ A ORPOLYPEPT Y IDE COMPLEXESCONSISTINGMAINLY D NOTEXHIBITANY/ EVOLVINGACTIVITY COMPONENTSDID (OWEVER LATERSTUDIES4ANGAND3ATOH  SHOWED THATCORECOMPLEXESWHI W CHRETAINANADDITIONAL MAJ A OR POLYPEPTIDECORRESPONDINGTOTHEK$AEXTRINSIC PROTEIN OR PSB/ GENE PRODUCT ARE CAPABLE OF PRODUC D ING/ 4HUS THEWATER RPLASTOQUINONEOXIDO REDUCTASEWAS D IDENTIlEDD ASTHEMINIMALCORECOMPL M EX PLUSTHEK$AEXTRINSIC PROTEIN 4HE  K$A EXTRINSIC PROTEIN WAS lRST ISOLATED BY +UWABARA AND -URATA  FFROM SPINACH CHLOROPLASTFRAGMENTSANDCOULDBERELEASEDFROMTHE INVERTEDD THYLAKOIDD MEMBRANESBYVARIOUSSALTWASH ANDD CHAOTROPIC TREATMENTS ¯KERLUND AND *ANSSON 9AMAMOTOETAL  4HELOSSOFTHEPROTEIN Y HE CORRELATEDD WITHALOSSINTHE / EVOLVINGACTIVITY4 lRSTSUCCESSFU F L RECONSTITUTIONASSAYSTOANALYZETHE ROLEOFPROTEINSBOUNDTOTHELUMINALSURFACEWERE ACHIEVED BY ¯KERLAND )NADDITIONTOTHE K$A EXTRINSIC PROTEIN TWO OTHER EXTRINSIC PROTEINS IE THE PSB0 AND PSB1GENEPRODUCTSORTHESO CALLED K$AANDD  K$AEXTRINSICPROTEINS RESPECTIVELY WERE IDENTIlED INOPTIMIZINGTHE/ EVOLVINGACTIVITY INTHESESTUDIESWITHHIGHERPLANTS&URTHERDETAILSON F IONOFT F HEOXYGENENHANCING THESTRUCTUREAND FUNCT EXTRINSICPROTEINSAREPRESENTED BY"RICKERAND"URNAP D #HAPTER  #5IF 5PUBM U 1JDUVSF S )NSUB U SEQUENTINVESTIGATIONSOF03)) MAN F NYADDITIONAL SMALL MEMBRANE SPANNINGPOLYPEPTIDESHAVEBEEN IDENTIlED THOUGH MANY OF THEIR FUNCTIONS STILL F DETAILS SEE#HAPTER REMAINTO BE DETERMINED FOR K HAT03))CONTAINS 4HORNTONETAL )T ISNOWKNOWNT ASMANYASDIFFERENTPROTEINCOMPONENTSINVIVO ALTHOUGH SOMEOFF THESEPROTEINCOMPONENTSMAY A BE RESTRICTEDD ONLYTOCYANOBACTERIALSPECIESWHIL W EOTHERS ONLYTOEUKARYOTICSPECIES#RYOELECTRONAND8 RAY F ILITATEDD THE CRYSTALLOGRAPHY HAVERECENTLY GREATLY FAC STRUCTURALELUCIDATIONOFF 03)) FROMT F HE LEVELOFF THE INVIVOMACROSCOPICORGANIZATIONOFTHEPOLYPEPTIDE COMPONENTSTOTH T EHIGHH RESOLUTIONMOLECULARSTRUCTURE OF F THE ANTENNA PIGMENTS AND ELECTRON TRANSFER F COFACTORS(ANKAMERETAL :OUNIETAL  +AMIYA AND D 3HEN  "IESIADKA ET AL  &ERREIRAETAL  4HECURRENTOVERALL STATICVIEW OF03))IS PRESENTEDINSEVERALCHAPTERSOFTHISBOOK 3TRUCTURAL ANALYSISBY ELECTRONCRYO MICROSCOPY OF



,JNJZVLJ 4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

CYANOBACTERIALAND HIGHERPLANT03))ISSUMMARIZED BY (ANKAMER ET AL #HAPTER   THE lRST 8 RAY F 03)) COMPLEXES FROM F CRYSTALLOGRAPHIC ANALYSIS OF 4HERMOSYNECHOCOCCUS ELONGATUS A THERMOPHILIC CYANOBACTERIUMISOLATEDBY3AKAE+ATOHFROMTHE "EPPU HOTSPRING IN*APAN9AMAOKAETAL  INCOMBINATIONWITHVARIOUSBIOPHYS H ICALANALYSES IS SUMMARIZEDBY7ITT#HAPTER THECRYSTALLIZATION AND D THE $ STRUCTURE AT  ¯ RESOLUTION OF 03)) F 4 VULCANUS AREDISCUSSEDBY3HEN 4 COMPLEXES FROM AND +AMIYA #HAPTER   AND THE RElNED HIGHER RESOLUTION  ¯ STRUCTURE AND ITS IMPLICATIONS FOR THE03))COMPLEXES FROM F 4ELONGATUS AREPRESENTED BY"ARBERAND)WATA#HAPTER )NADDITION THE IMMEDIATECHEMICALENVIRONMENTAND D THECONTRIBU TIONOFT F HEPROTEINBACKBONEAND D SIDECHAINSTOTHE VARIOUSFUNCTIONALSITESIN03)) ASDETERMINEDBYTHE POWERFU F LTOOL OF&OUR F IERTRANSFORM F INFRARE F D &4)2 DIFFERENCESPECTROSCOPY ARE F DISCUSSED BY.OGUCHI AND"ERTHOMIEU#HAPTER &INALLY INFORMATION D THROUGH SPACE INTERACTIONS ON THE ORIENTATION AND AMONGTH T EVARIOUSELECTRONTRANSFERCO F F FACTORS IN03)) ASDETERMINEDBYELECTRONPARAMAGNETICRESONANCE %02 SPECTROSCOPY Y ARE SUMMARIZED BY "ITTL AND +AWAMORI #HAPTER  7
'VODUJPOBM
4JUFT ‡ $BUBMZUJD
3PMF
PG 7 1IPUPTZTUFN
** "
1S 1SJNBSZ
1IPUPDIFNJTUSZ J 4HEESTABLISHMENTOFF THECORECOMPLEXASACHEMICAL L INDETAILTH T ESTRUCTURE ENTITYMADEITPOSSIBLETOANALYZE ANDFUNCTIONOF03))!LONGSTANDINGQUESTIONHAD BEEN ON WHI W CH 03)) PROTEIN SUB U UNIT IS THE SITE OF PRIMARY CHARGE SEPARATION LOCATED 3INCE ISOLATED CORECOMPLEXESCANCONTAINUP U TO#HL PER03)) D REACTIONCENTER2# PIGMENTS UNIT BOTH ANTENNAAND AREPRESENT)NEARLY STUDIES SEVERAL LINESOFF EVIDENCE SEEMED TO SUGGEST THAT #0 WAS THE SITE OF THE PRIMARY Y CHARRGESEPARATION.AKATANIETAL FORA REVIEW SEE3ATOH  AND D SOATTTHATTIME ITWASOFTEN F REFERREDTOASTHE@0APO PROTEINFORAREVIEW SEE 3ATOH  (OWEVER ITWASALSOSUGGESTEDTHATTHE $AND$PROTE D INSWERETH T ESITEOFF THEPRIMARYC Y HARG R E SEPARATION SINCETHESEPROTEINSWEREFOUNDTOHAVE SOMESEQUENCEHOMOLOGYWITHTHE,AND-SUBUNITS F HEPURPLE BACTERIAL REACTIONCENTER"2# -ICHEL OFT AND$EISENHOFER 4REBST  3UBSEQUENTLY /SAMU.ANBAAND+IMIYUKI3ATOH  SUCCEEDED

INISOLATINGASUB U CORECOMPLEXCONSISTING OFT F HE$ AND$PROTEINS THE _ AND ` SUBUNITSOF#YT B AND D HISCOMPLEX HADD AMINIMAL THE PSB) GENEPRODUCT4 PIGMENTCOMPOSITIONANDD CONTAINEDD  OR #HL A PHEOPHYTIN0HEO A ANDD  ` CAROTENES` #ARS  F D TOBEPHOTOCHEMICALLY 3INCETHISCOMPLEXWASFOUN ACTIVEINTHEPRIMARYCHARGESEPARATION$ANIELIUS ETAL /KAMURAETAL 4AKAHASHIETAL F HESTRUCTURAL AND  THESTAGEWASSETTOFOCUSONT F HE$AND$PROTE D INSASTHE MOLECULARANALYSISOFT 2#SUBUNITSFORREVIEWS SEE%VANS 3ATOH  #HAPTER .IXONETAL  I IGMENTIN03)) ORIGINALLY L LAB A ELED 4HEPHOTOACTIVEP AS #HL A)) WAS lRST IDENTIlED IN DIGITONIN TREATED D D DIFFERENCE F SPINACH THYLAKOIDS BY THE LIGHT INDUCE R ANCECHANGESAT SPECTRUMWITHMAXIMUMABSORB ANDNM$ÚRINGETAL #HAPTER 2ENGER 7 TT 4HESEABSORB R ANCE ANDD (OLZWARTH#HAPTER 7I CHANGESWERE INITIALLYTAKENTOINDICATETHAT ITWASA @SENSITIZER BUTSOONTHEREAFTERWEREINTERPRETEDTO BEDUETOT D HEPHOTO OXIDATIONOFF ASPECIAL #HL MOL ECULEINTHE03))2#SERVINGASTHEPRIMARYELECTRON DONOR(ENCE #HL A)) BECAMEGENERALLYREFERREDTO AS 0 &LOYD O D ET AL   )N COMPARISON WITH THE"2# 0WASSOMETIMESCONSIDEREDD TO BEA @SPECIALPAIRVAN-IEGHEMETAL  7ITHTHE ADVANCEMENT IN BIOPHYSICAL METHODS OFF ANALYSIS ANDD $CRYSTALLOGRAPHIC DETERMINATIONSOFT F HE03)) CORE:OUNIETAL +AMIYAAND3HEN  &ERREIRAETAL #HAPTER 7I 7 TT#HAPTER R ERANDD )WATA 3HENANDD +AMIYA#HAPTER "ARB 0 IS NOW INTERPRETED AS A SPECIALLY ORGANIZED MULTIMERIC PIGMENT COMPLEX #HAPTER  2ENGER F THE UNIQUE PROPERTIES ANDD (OLZWARTH THAT CONFERS NEEDEDTO PROMOTETHEMAIN PATHELECTRONTRANSFER FFROM WATER TO PLASTOQUINONE 01 AS WELL AS THE F D THEREDOX ACTIVE SIDEPATH ELECTRONTRANSFERAROUN #HL#HL: #YT B AND ` #ARTHATISINVOLVEDIN THEDISSIPATIONOFEXCESSOX F IDIZING POWER#HAPTER  &ALLERETAL 4HUS LIGHTENERGY INITIALLYTRAPPED IN THE DISTAL #HAPTER  'REEN AND 'ANTT ANDOR THE PROXIMAL #HAPTER  %ATON 2Y 2 E ANDD 0UTMAN %VANS ANTENNACOMPONENTS IS DIRECTEDD TOTHECORE 2# WHERE IT ACTIVATES THE PRIMARY ELECTRON DONOR 0FOR F DETAILS SEE#HAPTER "ARTERETAL 3INCE 0 ISTHESTRONGEST KNOWNOX K IDANTIN BIOLOGY IT CANBEHIGHLYDESTRUCTIVETO03))IFITISNOTRAPIDLY DEACTIVATEDD THROUGH THE / EVOLVING REACTIONS4HIS PHOTO DESTRUCTION WAS INITIALLY RECOGNIZEDD WHEN IT WAS DISCOVERED THAT THE $ PROTEIN WHICH BINDS 0 IS THE MOST RAPIDLY TURNING OVER PROTEIN IN

$IBQUFS

*OUSPEVDUJPO
UP
14 ** 03)) %LLIS  !SARESULT OXYGENICORGANISMS Q HAVEDEVELOPEDVARIOUSMECHANISMSTO QUENCHEXCESS ENERGY DISCUSSED IN #HAPTER  0OGSON ET AL PARTICULARLYUNDERCONDITIONSOFF STRESS AND TOREPAIR DAMAGED 03)) COMPONENTS DISCUSSED IN #HAPTER D !RO   #HOWAND #
$IBSH S F
4UBCJMJ[BUJPO 4HE BEAUTYOFP F HOTOSYNTHETIC2#S ISTHATONCETHE PRIMARYCHARGESEPARATIONTAKESPLACE ITISRAPIDLY STABILIZED BY SECONDARYELECTRONTRANSFERW F WHICH CON SIDERABLYREDUCEST D HEPROBABILITY FOR F BACKREACT K ION (ENCE THE QUANTUM YIELD FOR THE PRIMARY CHARGE T ETIC2#SAPPROACHESUNITY TY)N SEPARATIONINPHOTOSYNTH 03)) AFTEREXC F ITATION THEELECTRON INITIALLY LOCALIZED ONTHEPRIMARYACCEPTOR0HEO+LIMOVETAL  LOCATED INTHE0COMPLEX ISRAPIDLY TRANSFERRE F D TOTHElRSTMETASTABLEACCEPTORTERMED 1!4HISAC CEPTORWASDETERMINEDTOBEASPECIALLYBOUND01 D ONITS DIFFERENCEA F ABSORPTIONSPECTRUM MOLECULE BASED 3TIEHL AND D 7ITT  %ARLIER $UYSENSAND3WEERS D  HAD PROPOSED THE EXISTENCE OF @1 ON THE 03))ACCEPTORSIDETHATACTEDASAQUENC D HEROFF #HL A mUORESCENCEWHI W CHWAS LATERIDENTIlED D AS1! FOR F DETAILS SEE0APAGEORGIOUAND'OVINDJEE   1! SERVESONLYASASINGLEELECTRONCARRIERAND D TRANSFERS F THEELECTRONTOASECOND BOUND01MO D LECULETERMED 1" )NCONTRASTTO1! 1" CANACCEPTTWOELECTRONS 5PON BEING DOUBLY U REDUCE D D 1" BECOMESPROTONATED AND D THERESULTINGPLASTOQUINOL ISRELEASED FROMT F HE BINDINGSITEANDISREPLACEDBYANOTHERMOLECULEFROM THE01POOLWITHINTHEMEMBRANE4HUS 1" ACTSAS A TWO ELECTRONGATEREVEALINGBINARYOSCILLATIONSIN THEELECTRONTRANSFERONTHEACCEPTORSIDEOF03)) 4HE FUNCT F IONING OFATWO E F LECTRONGATEWAS lRST DIS COVERED INTHE03))BY"OUGES "OCQUET  AND BY6ELTHUYSAND!MESZ ANDLATERCONlRMED INTHE"2# 6ERMEGLIO   #LOSELY ASSOCIATED D WITH THE 1! AND 1" ELECTRON Q F ACCEPTORSISANON HEMEIRONION!UNIQUEROLEO BICARB R ONATEATTHEACCEPTORSIDEOF03)) PART F ICULARLY ONTH T ETWO ELECTRONGATE WAS DISCOVERED BY'OVINDJEE ETAL VAN2ENSENETAL #HAPTER VAN2ENSENAND D +LIMOV AND D MAAY INVOLVETHENON HEME IRON$ETAILSONTHESTRUCTUREAND FUNCT F IONOF THEQUINONE IRONACCEPTORCOMPLEXAREPROVIDEDBY 0ETROULEASAND#RO D F #HAPTER  FTS /NTHEDONORSIDEOF03)) F THEOXIDATIONOFF WATER TOMOLECULAROXYGENISCATALYZEDTHROUGHTHESTRONG OXIDIZINGPOWERGENERATED BY 0!SDISCUSSED

 ABOVE ONE / MOLECULE ISRELEASEDD AFTEREVERY F F FOUR TURNOVERSOF04HE QUESTIONHADBEENASTOHOW Q THEOXIDIZING POWERISTRANSFERRE F D FROM0 F  TOTHE OXYGEN EVOLVING COMPLEX /%#  )NITIALLY IT WAS DETERMINEDTH D ATTHE lRSTELECTRON DONORTO 0 WAS LOCATEDD ONTHE$ $HETERODIMERBYSELECTIVE IODO LAB A ELINGOFF THEPOLYPEPTIDESINCORECOMPLEXES4AKA HASHIETAL  4HEISOTOPICLABELINGEXPERIMENTS F IONING OFF THE TYROSINE RADICAL ESTABLISHEDD THE FUNCT OBSERVEDD ASTHE%02SIGNAL))F VF ONTHE DONORSIDE OF03))"ARRYAND"ABCOCK  3UBSEQUENTLY $EBUSETAL  PROVIDEDD THElRST DIRECTMOLECULAR BIOLOGICAL EVIDENCETHATTHElRSTDONORTO0 IS TYROSINE RESIDUETERMED9: ONTHE$PROTEIN IN 3Y 3 NECHOCY CYSTIS SP0###HAPTER $INERAND "RITT#HAPTER $EBUS (OWEVER 03))WASKNOWN K TOCONTAINANOTHERREDOX ACTIVETYROSINETERMED9$ LOCATED INAHOMOLOGOUSPOSITIONTO9: ONTHE$ PROTEIN)NCONTRASTTO9: WHI W CH IS DIRECTLY INVOLVED INTHE / EVOLVINGREACTIONS 9$DOESNOTPARTICIPATE INTHEMAINPATH OFE F LECTRONTRANSFER F FROMWATERTO F 01(OWEVER IT DOES INTERACT INTHE DARK RK REDOXEQU Q I LIBRIUMOFTHEEARLY3STATES$ETAILSONTHESTRUCTURE F IONOFT F HEREDOX ACTIVETYROSINES IN03))ARE AND FUNCT DISCUSSED BY$INERANDD "RITT #HAPTER  $
8BUFS
0YJEBUJPO /NE OF THE LARGELY UNRESOLVED PROBLEMS IN 03)) RESEARCH ISTHEMOLECULARMECHANISMBY WHI W CH/ ISPRODUCE D D 2ENGER  5PONPROMULGATIONOF THE3 STATEHYPOTHESISINTHEEARLYS THEQUEST BEGANTO IDENTIFYTHECHEMICAL NATUREOFT F HE@CHARGE ACCUMULATING INTERMEDIATE3INCE-NCANTAKEONA NUMBER OF STABLE OXIDATION STATES AND WAS LOCALIZED IN 03)) #HENIAE ANDD -ARTIN  IT WAS LONG SUSPECTEDD THAT -N IS DIRECTLY INVOLVED IN THE / EVOLVING REACTIONS %ARLY %02 STUDIES REVEALED AN INVERSERELATIONSHIP BETWEENTHE/ EVOLVINGACTIVITY ANDD THEAPPEARANCEOFF THEROOM TEMPERATURE  LINE %02SIGNALCHARACTERISTICOFAQUEOUS-N IONS AFTER TREATMENTSWHI W CH INACTIVATEDD THE / EVOLVINGACTIVITY BYRELEASING BOUNDD -N ,OZIERETAL "LANKEN SHIPAND3AUER  )NADDITION THEREWEREEARLY INDICATIONS FROMNUC F LEARMAGNETICRESONANCE .-2 PROTON RELAXATION RATE MEASUREMENTS THAT BOUND -NCHANGESOXIDATIONSTATEDURINGTHE/ EVOLVING REACTIONS 7YD 7 RZYNSKI ET AL  3HARP  7 DRZYNSKI  (OWEVER lRST DIRECTMEASURE 7Y MENTSOFTHEINTACT BOUND-N IN03))COULDNOTBE F HE LOW TEMPERATUREEG MADEUNTILTHEDISCOVERY OFT



,JNJZVLJ 4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

^+ 3 STATE-NMULTI LINE%02SIGNAL $ISMUKES AND3IDERER  ANDTHElRSTAPPLICATIONSOF-N R ETAL 8 RAY A ABSORPTIONSPECTROSCOPY 8!3 +IRBY A B  7 7ITHTHEDISCOVERYOFTHELOW TEMPERATURE 3 STATE -NMULTI LINE%02SIGNAL AND LATEROFT F HE-N%02 SIGNALARISING FROMT F HE3 STATE ¯HRLINGETAL  -ESSINGERETAL  THEFUNCTIONAL-NCOULDBE PROBED INSITU3TARTING INTHESAND D CONTINUING UNTIL THEPRESENT AVASTLIBRARYOF%02MEASUREMENTS F HAVEACCUMULATEDUNDERCONDITIONSWHICHVARIOUSLY PERTURB R ED D THE-NCENTER)T BECAMECLEARVERYEARLY THATSEVERAL-NIONS INTERACTEDMAGNET D ICALLYTOGIVE RISETOTHESE%02SIGNALS THOUGHITWASCONTROVERSIAL D OR-NIONS #HAPTER ASTOWH W ETHERITINVOLVED ¯HRLING ET AL 4HE $ CRYSTAL STRUCTURES OF 03)) CLEARLYSHOWACLUSTEROF-NIONSATTHECATALYTIC SITE #HAPTER 7I 7 TT#HAPTER 3HENAND D +AMIYA #HAPTER "ARB R ERAND D )WATA BUTDONOTREVEALTHE OXIDATION STATES NOR HOW THE  -N IONSINTERACT WITH ONEANOTHER !NOTHER TECHNIQUE THAT PROVIDES INFORMAT F ION ON THE IMMEDIATE CHEMICAL ENV N IRONMENT AROUND THE FUNCT F IONAL -N AS WELL AS ON THE POSSIBLE -N OXIDATIONSTATESINNON CRYSTALLINESAMPLESOF03)) F IS-N8 RAYABSORPTIONSPECTROSCOPY8!3 ,IKE THE%02 STUDIESUSING THISTECHNIQUEBEGANINTHE SAND D CONTINUETODAY)NCONJ N UNCTIONWITH-N %02 MEASUREMENTS THE -N 8!3 MEASUREMENTS PROVIDESUPPORTAND D VERIlCATIONOFF THE INTERPRETATIONS OF F THE $ CRYSTALLOGRAPHIC STRUCTURE OF F THE -N FUNCTIONAL SITE $ETAILS ONTHE RESULTS FROMTHE -N F 03)) ARE SUMMARIZED BY 8!3 MEASUREMENTS OF 9 HANDRA#HAPTER  9AC #RUCIALTOTHEFUNCTIONALORGANIZATIONOFTHE-N F F FACTORS SITE ASWELL ASOFF THEOTHERELECTRONTRANSFERCO IS THE ROLE OF PROTEIN ENVIRONMENT IN PARTICULAR AS TO WHICH AMINO ACID RESIDUES PROVIDE LIGANDS )NFORMAT F IONONTHISASPECTHASBEENOBTAINED D THROUGH THEUSEOFS F ITE DIRECTED D MUTANTSOF03))PROTE F INS IN CONJUNCTIONWITHTHEVARIOUSTYPESOFSPECTROSCOPIC ANALYSIS IE &4)2 %02 8!3  )T APPEARS THAT MOSTOFF THE LIGANDSTOTHE-NCLUSTERAREPROVIDED BY THE $ PROTEIN ALTHOUGH THE RECENT RElNED $ CRYSTALLOGRAPHICSTRUCTUREOF03))INDICATESTHATONE LIGAND ISPROVIDED BYTHE#0PROTEIN#HAPTER "ARBERAND)WATA (OWEVER ITISIMPORTANTTOPOINT OUTTHATNOTALLLIGANDSTOTHEFOUR-NIONSINTHECLUSTER HAVEASYETBEENACCOUNTED FFOR4HE LATESTINFORMAT F ION ONTHEPROTEINENVIRONMENTOFTHECATALYTIC-NCLUSTER ISPRESENTEDBY $EBUS #HAPTER 

$ESPITETHEWEALTH OFF STRUCTURAL INFORMAT F IONONTHE -NCLUSTER VERYLITTLEISSTILLKNOWNABOUTTHEEXACT MECHANISMBY WHICHMOLECULAROXYGENISFORMED 7ITH FOUR-N F IONS INVOLVEDD ATTHECATALYTICSITEAS WELL AS ONE OR MORE REDOX ACTIVE AMINO ACIDD RESIDUES EG 9: AND D POSSIBLY A HISTIDINE ANY N NUMBER OF REACTIONPATHSMAYTAKEPLACE)NADDITION CALCIUM AND CHLORIDE COFACTORS ARE KNOWN TO BE INTIMATELY INVOLVED #HAPTER  VAN 'ORK R OM ANDD 9OCUM  )MPORTANT QUESTIONS FFOR UNDERSTANDING THE CORRECT REACTIONPATHAREI ATWHICHSTEPINTHECATALYTIC SEQUENCE DOTHETWOSUB U STRATEWATERMOLECULES BIND AND II HOWDOESTHE/ / BOND FORM)NT F HEPASTIT HASBEENEXPERIMENTALLYDIFlCULTTOGETINFORMATION F HEDIFlCULTY ONTHESEQUESTIONS MAINLY BECAUSEOFT TODISTINGUISHTHESUB U STRATEWATERFROMT F HESOLVENT WATER(OWEVER THESUBSTRATEWATERCANBE PROBED BY OXYGEN ISOTOPE EXCHANGE MEASUREMENTS USING MASSSPECTROMETRY%ARLY / ISOTOPE LAB A ELINGSTUDIES ESTABLISHEDTHATWATERISTHEULTIMATESOURCEOFTHE/ PRODUCE D D DUR D ING PHOTOSYNTHESIS2UB U ENETAL   )N OTHER OXYGEN ISOTOPE EXCHANGE MEASUREMENTS 2ADMERAND/LLINGER  "ADERETAL  ASIMILARCONCLUSIONWASMADETHOUGH THE BINDING OFT F HETWOSUB U STRATEWATERMOLECULESCOULD D NOT BE DETERMINED)NMORERECENTMEASUREMENTS HOWEVER THE KINETIC RESOLUTION OF F THE ISOTOPIC EXCHANGE WAS GREATLY IMPROVED IN WHI W CH THE BINDING OF F THE TWO SUBSTRATES COULD BE SEPARATELY DISTINGUISHED -ESSINIGER ET AL  (ILLIER ET AL  4HE RESULTS FFROM THIS EXPERIMENTAL APPROACH REVEALED F RTHE lRSTTIMETHAT ONE SUBSTRATE WATER MOLECULE FO IS BOUNDD TOTHECATALYTICSITETHROUGHOUTTHE3 STATE CYCLEWHIL W ETHESECONDD SU UBSTRATEWATERMOLECULE IS BOUNDINATLEASTTHE 3 AND3 STATES ANDTHATTHE / / BOND FORMS F D INGTHE LASTSTEPOFF THEREACTION DUR D 7YDRZYNSKI   4HESE SEQUENCE (ILLIER AND OBSERVATIONSTHEREFOREPLACELIMITSONTHETYPESOF REACTIONPATHSTHATMAY A BEPOSSIBLEINTHEFORMAT F ION D INGPHOTOSYNTHESIS(ILLIERAND7Y D DRZYNS D KI OF / DUR  !DETAILEDSUMMARYOFTHEMECHANISMFORTHE F BY03)) IS GIVENBY(ILLIERAND OXIDATIONOFWATER -ESSINGER#HAPTER  7*
'VUVSF
1FSTQFDUJWFT "
4USVDUVSF
BOE
#JPEZOBNJDT 3UCCESSINTHE8 RAYCRYSTALLOGRAPHICANALYSIS:OUNI ETAL +AMIYAANDD 3HEN &ERREIRAETAL

$IBQUFS

*OUSPEVDUJPO
UP
14 **  HAS HADAGREAT D IMPACTONWH W ATWECURRENTLY D FUNCTION OF KNOW ABOUTTHE MOLECULAR STRUCTURE AND 03))(OWEVER HIGHERRESOLUTIONSTRUCTURESWILL BE F F LLYUNDERSTAND FU D THEMOLECULAR NEEDED INTHEFUTURETO DETAILSOFCATALYTICCOMPONENTSANDTODETERMINETHE EXACTMECHANISMOFWATEROX F IDATION)NPARTICULAR F F FROM HIGHERPLANTS CRYSTALLOGRAPHICANALYSISOF03)) ANDMUTATEDCYANOBACTERIAWILLBENEEDED /FEQUA F L IMPORTANCETOTHESTRUCTURAL ANALYSIS ISAN F F FUNCT ION UNDERSTANDINGOFF THE BIODYNAMICSOF03)) OFWHICHMUCHLESSISCURRENTLYKNOWN&OREXAMPLE IMPORTANTCLUESONTHEMECHANISMOFWATEROX F IDATION F DERSTANDINGTHEPHOTO ASSEM CANBEGLEANED FROMUN BLYPROCESSOFTHECATALYTIC-NCLUSTER4HECURRENT STATUSOF KNOW K LEDGEONTHISASPECT IS DISCUSSED BY $ISMUKESETAL #HAPTER ,IKEWISE THE DAMAGE REPAIRCYCLEHYPOTHESISPROPOSEDBY+YLEETAL HASNOTYETBEENSUB U STANTIATED A D LTHOUGH CONSIDERABLE EVIDENCE HASACCUMULATED FORT F HE LATEST DETAILS SEE #HAPTER #HOWAND!RO /THERASPECTSOF03)) BIODYANMICSINCLUDETRANSCRIPTIONAL AND D TRANSLATIONAL REGULATION IN THE BIOSYNTHESIS AND HOMEOSTASIS OF 03)) COMPONENTS #HAPTER  9AMAGUCHI ET AL D THETRANSPORTAND D POST TRANSLATIONALPROCESSING AND EVENTS #HAPTER 4HEGAND3 D HI INCLUDING EG THE CARBOXY TERMINAL PROCESSING OF $ PRECURSOR PROTEIN 9AMAMOTOAND3ATO D H  )NALL FUTURE F STUDIES INTHESEASPECTSOFF 03)) THEAPPLICATIONOF COMPREHENSIVEMOLECULARBIOLOGICALTECHNIQUESWILL BEESSENTIAL #
0SJH J JO
&WPMVUJPO
BOE
"SUJGJDJBM
0 1SPEVDJOH 4 4ZTUFNT 4RYINGTOUNDERSTANDTHEORIGINANDEVOLUTIONOF03)) THATMAY A HAVETAKENPLACEn BILLIONYEARSAGOISNOT F L INTERESTTOBIOLOGISTS BUTISA ONLYAMATTEROFGENERA USEFULAPPROACHTOUNDERSTANDTHEUNIQUESTRUCTURAL Q F IONALPROPERTIESOF03))AST F HEY EXISTTODAY AY AND FUNCT (OWEVER ALTHOUGH THEREARESIGNIlCANTVARIATIONSIN THELIGHT HARVESTINGCOMPONENTS#HAPTER 'REEN AND D 'ANTT AND IN THE MINOR PROTEIN COMPONENTS #HAPTER  4HORNTON ET AL OF F 03)) AMONG THE CYANOBACTERIA ALGAEANDHIGHERPLANTS THEREDOESNT D THE SEEMTOBEMUCHVARIATION INTHE03))2#AND WATEROXIDIZINGCATALYT L ICSITEITSELF ˆ TH T EORGANIZATION ANDOPERATIONOFTHECATALYTICSITEAPPEARSTOBETHE SAMEINALL ORGANISMSSTUDIED D THUSFAR4 F HISUNUSUAL ASPECTOF03)) F SINCEMOSTENZYMESSHOWVARIATIONS CHARACTERISTICOFDIVERGENTORCONVERGENTEVOLUTION IS TOUCHEDUPON D BYY $ISMUK U ESAND D "LANKENSHIP#HAPTER

  $ISMUKESANDD "LANKENSHIP .EVERTHELESS THE AUTHORSOFTHIS#HAPTERDOPROMOTESOMEINTERESTING R ONATEIONSMAY A HAVE ARGUMENTSTOSUGGESTTHATBICARB U STRATEFORT F THEPRODUCT D IONOF SERVEDD ASANIMMEDIATESUB / INTHEDISTANTPAST INLIGHTOFTHEFACTTHATTHE#/ LEVEL OFF THEATMOSPHEREATTHATTIMEWASCONSIDERABLY HIGHERTHANIT ISTODAYANDD THElNDINGTHATTHERE ISAN Q A ARENTBICARBONATEREQUIREMENTFORTHEREACTIONSON APP T EDONORSIDEOF0 TH F 3TEMLER #HAPTER VAN 2ENSENANDD +IMOV 4HE LIKELIHOODD OFT F THIS HYPOT H THESIS MAYBEASCERTAINEDINTHEFUTUREBYSTUDIESOFANCIENT REPRESENTATIVESOFC F YANOBACTERIAPRESENTTODAY A EG F 'LEOBACTERORFROMSTROMATOLITES AND R SPECIESFROM OFCYANOBACTERIALIVINGINEXTREMEENVIRONMENTSEG F ICANSODA LAKES 3TILL !RTH T ROSPIRA STRAINSLIVING IN!FR OTH T ERAPPROACHESTOTRYTOUNDERSTANDD THEPLACEOF03)) F F ISTO MAKE MECHANISTIC AND INTHE EVOLUTION OF LIFE STRUCTURALCOMPARISONSBETWEEN03))ANDD CYTOCHROME H C OXIDASE W WHICH CATALYZES THE REVERSE REACTION OF REDUCING / TO WATER FOR DETAILS SEE #HAPTER  "RUDVIG ANDD 7IKSTRÚM ANDD TOMODIFY NATURAL BACTE RIALREACTIONCENTERSTOHAVEUNIQUE03)) Q PROPERTIES THROUGHPROTEINENGINEERINGFORDETAILS SEE#HAPTER  +ÈLMÈN ETAL  !LTHOUGH THERE IS CONSIDERABLE RESEARCH YET TO BE DONE IN ORDERTO UNDERSTAND ALL OFTHE INTRICACIES OF F 03)) THERE IS NOW ENOUGH KNOW K LEDGE OF F THE STRUCTURE AND FUNCT F ION AS DISCUSSED IN THIS BOOK TOMIMIC03))REACTIONSFORFUTUREEFlCIENTENERGY CONVERSION PROCESSES THROUGH PROTEIN ENGINEERING FOR F DETAILS SEE#HAPTER 'IBNEYANDD 4OMMOS ANDBIO INORGANICCHEMISTRYFORDETAILS SEE#HAPTER F F YOUNG SCIENTISTS FOR  -AGNUSONETAL 4HEREFORE INTERESTED IN UNDERSTANDING ONE OFF .ATURES MOST UNIQUEENZYMES THEFUTURERESEARCHOF03))REMAINS ANEXCITING CHALLENGE "DLOPXMFEHNFOUT 7E THANK *OEL &REEMAN FOR HIS UNLIMITED HELP IN 7 PREPARING THISCHAPTERANDD 2UMANA4AAYYAB FOR F HER HELP INDRAWING&IG 3FGFSFODFT ¯HRLING +! 0ETERSON 3 AND 3TYRING 3  !N OSCILLATING MANGANESEELECTRONPARAMAGNETICRESONANCESIGNALFROMTHE 3 STATE OFF THE OXYGEN EVOLVING COMPLEX IN 0HOTOSYSTEM )) "IOCHEMISTRY n



,JNJZVLJ 4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

!NDERSON*- #HANGING CONCEPTSABOUTTHEDISTRIBUTION OF0HOTOSYSTEMS)AND))BETWEENGRANA APPRESSEDANDSTROMA EXPOSEDTHYLAKOIDMEMBRANES0HOTOSYNTH2ESn ¯KERLUND(% 0ARTIALRECONSTITUTIONOFTHEPHOTOSYNTHETIC WATERSPLITTINGININSIDEOUTTHYLAKOIDVESICLES)N!KOYUNOGLOU 'ED 0ROCEEDINGS TH )NTERNATIONAL#ONGRESSON0HOTOSYN THESIS 6OL  PP n "ALABAN )NTERNATIONAL 3CIENCE 3ERVICE 0HILADELPHIA ¯KERLUND(%AND*ANSSON# ,OCALIZATIONOFA AND A -RPOLYPEPTIDETOTHELUMINALSIDEOFF THETHYLAKOID MEMBRANE&%"3,ETTn "ADER +0 4HIBAULT 0 AND 3CHMID '(  ! STUDY ON OXYGENEVOLUTIONANDONTHE3 STATEDISTRIBUTIONINTHYLAKOID PREPARATIONSOFTHElLAMENTOUSBLUE GREENALGA /SCILLATORIA / CHALYBEA:.ATURFORSCH C n "ARRY "!AND D "ABCOCKK '4 4Y 4 ROSINERADICALSAREINVOLVED INTHEPHOTOSYNTHETICOXYGEN EVOLVINGSYSTEM0ROC.ATL!CAD 3CI 53!n "ERTH T OLD$! "A D ABCOCKK '4ANDD9OCUM*& !HIGHLY Y RESOLVED D OXYGEN EVOLVING 0HOTOSYSTEM )) PREPARATION FROM SPINACH THYLAKOIDMEMBRANES&%"3,ETTn "IESIADKA* "ERNHARD, +ERN* )RRGANG+ $AND:OUNI! #RYSTAL STRUCTURE OF CYANOBACTERIAL 0HOTOSYSTEM )) AT  ¯ RESOLUTION!CLOSERLOOKATT K HE-N CLUSTER0HYS# H HEM#HEM 0HYSn "LANKENSHIP2%ANDD 3AUER+ -ANGANESEINPHOTOSYNTH T ETIC OXYGENEVOLUTION)%LECTRONPARAMAGNETICRESONANCESTUDY OFF THEENVIRONMENTOFMAN F GANESEINTRIS WASHEDD CHLOROPLASTS "IOCHIM"IOPHYS!CTAn "OARDMAN.+AND!NDERSON*- )SOLATIONFROMSPINACH CHLOROPLASTS OF PARTICLES CONTAINING DIFFERENT PROPORTIONS OF CHLOROPHYLL A ANDCHLOROPHYLL B ANDTHEIRPOSSIBLEROLEINTHE LIGHTREACTIONSOFP F HOTOSYNTHESIS.ATUREn F BETWEEN THE TWO "OUGES "OCQUET "  %LECTRON TRANSFER PHOTOSYSTEMS INSPINACHCHLOROPLASTS"IOCHIM"IOPHYS!CTA  n #HENIAE'-AND D -ARTIN)&  3TUDIESONTHEFUNCT F IONOF MANGANESE IN PHOTOSYNTHESIS "ROOKHAVEN 3YMPOSIUM IN "IOLOGYn $ANIELIUS26 3ATOH+ VAN+AN0*- 0LIJTER** .UIJS!-AND VAN'ORKOM(* 4HE PRIMARYREACTIONOF0HOTOSYSTEM )) INTHE$ $ CYTOCHROME BCOMPLEX&%"3,ETT n $EBUS2* "ARRY "! 3ITHOLE) "ABCOCK'4AN K D -C)NTOSH ,  $IRECTED D MUTAGENESIS INDICATESTHATTHEDONORTO 0 IN 0HOTOSYSTEM)) IS TYROSINE  OFF THE $ POLYPEPTIDE "IOCHEMISTRYn $ISMUKES'#AND3IDERER9 )NTERMEDIATESOFAPOLYNUCLEAR MANGANESECLUSTERINVOLVEDINTHEPHOTOSYNTHETICOXIDATIONOF WATER0ROC.ATL!CAD3CI53!n $ÚRING' 3TIEHL((AND7 D 7ITT(4  !SECONDD CHLOROPHY H LL REACTIONINTH T EELECTRONTRANSPORTCHAINOFF PHOTOSYNTH T ESIS ˆ REG ISTERED BY THEREPETITIVEEXCITATIONTECHNIQUE:.ATURFORSC F H Bn $UYSENS,.-  4HE DISCOVERY OFT F HETWOPHOTOSYSTEMS! PERSONALACCOUNT0HOTOSYNTH2ESn $UYSENS ,.- AND 3WEERS (%  -ECHANISM OF TWO PHOTOCHEMICAL REACTIONS IN ALGAE AS STUDIED BY MEANS OF mUORESCENCE )N *APANESE 3OCIETY OF 0LANT 0HYSIOLOGISTS ED 3TUDIESON-ICROALGAEANDD 0HOTOSYNTHETIC"ACTERIA PP n5NIV4OKYO0RESS 4OKYO

$UY U SENS,.- !MESZ*AND+AMP"- D  4WOPHOTOCHEMICAL SYSTEMSINPHOTOSYNTHESIS.ATUREn %LLIS2* 0ROTEINSYNTHESISBYISOLATEDCHLOROPLASTS"IOCHIM "IOPHYS!CTAn %MERSON2AND!RNOLD7A !SEPARATIONOFTHEREACTIONS INPHOTOSYNTHESIS BYMEANSOF INTERMITTENTLIGHT*'EN0HYS H IOL  n %MERSON2AND D !RNOLD7 D B 4HEPHOTOCHEMICAL REACTION INPHOTOSYNTHESIS*'EN0HYSIOL n %MERSON2AND D ,EWIS#-  4HE DEPENDENCEOFT F HEQUANTUM YIELDOF#HLORELLA PHOTOSYNTHESISONTHEWAVELENGTHOFLIGHT !M*"OTn %MERSON 2 AND 2ABINOWITCH %  2ED DROP AND THE ROLE OF AUXILIARY PIGMENTS IN PHOTOSYNTHESIS 0LANT 0HYSIOL  n %MERSON 2 #HALMERS 26 AND D #EDERSTRAND D #.  3OME F FACTORS INmUENCING THE LONG WAVELIMITOFP F HOTOSYNTHESIS0ROC .ATL!CAD 3CI 53!n %VANS -#7  0LANT REACTION CENTRE DElNED .ATURE n &ERREIRA+. )VERSON4- -AGHLAOUI+ "ARBER*AND)WATA3  !RCHITECTURE OF THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn &LOYD2! # D HANCE"AND$E6AU D LT$ ,OWTEMPERATURE PHOTO INDUCE D D REACTIONS IN GREEN LEAVES AND D CHLOROPLASTS "IOCHIM"IOPHYS!CTAn 'AFFRON F ( AND 7OHL +  :UR4HEORIE DER!SSIMILATION F   .ATURWISSENSCHAFTEN  'OVINDJEE #HLOROPHYLL A mUORESCENCE!BITOFBASICSAND HISTORY)N0APAGEORGIOU'#AND'OVINDJEEEDS #HLOROPHYLL A &LUORESCENCE ! 3IGNATURE OF 0HOTOSYNTHESIS PP n 3PRINGER $ORDRECHT 'OVINDJEE AND D +ROGMANN $  $ISCOVERIES IN OXYGENIC PHOTOSYNTHESISn !PERSPECTIVE0HOTOSYNTH 2ES  n 'OVINDJEEAND D 2ABINOWITCH%  4WO FORMSO F F HLOROPHYLL A FC WITH DISTINCT PHOTOCHEMICAL FUNCT F IONS 3CIENCE  n  'OVINDJEE 0ULLES-0* 'OVINDJEE2 VAN'ORKOM(*AND$UYSENS ,.-  )NHIBITION OF THE REOXIDATION OF THE SECONDARY ELECTRONACCEPTOROF0HOTOSYSTEM))BYBICARBONATEDEPLETION "IOCHIM"IOPHYS!CTAn (ANKAMER " -ORRIS %0 AND D "ARRBER *  2EVEALING THE STRUCTUREOFF THEOXYGEN EVOLVINGCOREDIMMEROFF 0HOTOSYSTEM)) BY CRYOELECTRONCRYSTALLOGRAPHY.AT3TRUCT" Y IOLn (ILL 2 /XYGENEVOLUTIONBY ISOLATED D CHLOROPLASTS.ATURE n (ILL 2 AND "ENDALL &  &UNCTION OF THE CYTOCHROME COMPONENTS IN CHLOROPLASTS ! WORKING HYPOTHESIS .ATURE  n (ILLIER7 AND7Y D DRZYNSKI 4  4HE AFlNITIES FFOR THE TWO SUB U STRATEWATERBINDING SITESINTHE/ EVOLVINGCOMPLEXOF D ING 3 STATE TURNOVER 0HOTOSYSTEM)) VARY INDEPENDENTLY DUR "IOCHEMISTRY n (ILLIER7AND D 7YDRZYNSKI4  /XYGENLIGAND D EXCHANGEAT METALSITES ˆ IMPLICATIONSFORTHE / EVOLVINGMECHANISMOF 0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn (ILLIER7 -ESSINGER*AND7YDRZYNSKI4 +INETICRESOLUTION AND TEMPERATURE DEPENDENCE OF THE (/(/ EXCHANGE F HETWOSUB U STRATEWATERMOLECULESINTHE3 STATE OF RATES FORT THE PHOTOSYNTHETIC WATER OXIDATION COMPLEX "IOCHEMISTRY

$IBQUFS

*OUSPEVDUJPO
UP
14 ** n *OLIOT0 0ERIOD FOUROSCILLATIONSOFTHEmASH INDUCEDOXYGEN FORMATIONINPHOTOSYNTHESIS0HOTOSYNTH2ESn *OLIOT0 "ARBIERI'AND#HABAUD2 5NNOUVEAUXMODELE DESCENTRESPHOTOCHIMIQUESDUSYSTÒME))0HOTOCHEM0HOTO BIOL  n +AMIYA . AND D 3HEN *2  #RYSTAL STRUCTURE OFF OXYGEN EVOLVING 0HOTOSYSTEM))FROM F 4 ERMOSY 4H S NECHOCOCCUSVULCANUS AT ¯ RESOLUTION0ROC.ATL!CAD3CI53!n U ER7- #ARROLL*! 7ANG 1 7HITMARSH * 3ATOH +ASHINO9 ,AUB +AND0AKRASI(" 0ROTEOMICANALYSISOFAHIGHLYACTIVE 3 SP0## 0HOTOSYSTEM))PREPARATIONFROMTHE 3YNECHOCYSTIS REVEALSTHEPRESENCEOFNOVELPOLYPEPTIDES"IOCHEMISTRY  n +E "  0HOTOSYNTHESIS 0HOTOBIOCHEMISTRY ANDD 0HOTO BIOPHYSICS+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT R *! 'OODIN$" 7YD 7 DRZYNSKI4 2OBERTSON!3ANDD +LEIN-0 +IRBY A 3TATEOFF MANGANESEINTHEPHOTOSYNTHETICAPPARATUS D 8 RAAYAB A SORPTIONlNESTRUCTURESTUDIESONCHLOROPLAST %XTENDED ANDDI — OXO BRIDGEDDIMANGANESEMODELCOMPOUNDS*!M #HEM3OCn +IRBY*! 'OODIN$" 7YDRZYNSKI4 2OBERTSON!3AND+LEIN-0 B 3TATEOFMANGANESEINTHEPHOTOSYNTHETICAPPARATUS 8 RAYABSORPTIONEDGESTUDIESOFF MANGANESE INPHOTOSYNTHETIC MEMBRANES* !M #HEM3OCn +LIMOV 66 +LEVANIKK !6 3HUVALOV 6! ANDD +RASNOVSKY !!  2EDUCT D IONOFP F HEOPHYTIN INTHEPRIMARY LIGHTREACTION OFF 0HOTOSYSTEM))&%"3,ETTn +OK" ,IGHT INDUCEDABSORPTIONCHANGESINPHOTOSYNTHETIC ORGANISMS))!SPLIT BEAMDIFFERENCESPECTROPHOTOMETER0LANT 0HYSIOLn +OK" &ORBUSH"AND-C'LOIN- #OOPERATIONOFCHARGES INPHOTOSYNTHETIC/ EVOLUTION  0HOTOCHEM 0HOTOBIOL  n +UWAB A ARA4AND D -URATA.  0URIlCATIONANDD CHARACTERIZATION OFF  KILODALTON PROTEIN OF F SPINACH CHLOROPLASTS "IOCHIM "IOPHYS!CTAn +YLE $* /HAD ) AND !RNTZEN #*  -EMBRANE PROTEIN DAMAGEANDREPAIR SELECTIVELOSSOFAQUININEPROTEINFUNCTION IN CHLOROPLAST MEMBRANE 0ROC .ATL !CAD 3CI 53!  n ,AVOISIER!, 4RAITÏÏLÏMENTAIREDECHIMIE#HEZ#UCHET LIBRARIE 2UEET(ÙTEL3ERPENTE !CADEMIE2OY O AL DES3CIENCES 0ARIS ,OSADA- 7HATLEY&2ANDD !RNON$  3EPARATIONOFTWO F LIGHTREACTIONSINNON CYCLICPHOSPHORYLATIONOF GREENPLANTS .ATUREn ,OZIER2 "AGINSKY-AND"UTLER7, )NHIBITIONOFELECTRON TRANSPORTINCHLOROPLASTSBYCHAOTROPHICAGENTSANDTHEUSEOF MANGANESEASANELECTRONDONORTO0HOTOSYSTEM))0HOTOCHEM 0HOTOBIOL  n -AY A ER*2 $IEORG R ANISCHE"EWEGUNG IN IHREM:USSAMEN H NHAG MITDEM3TOFFWEC F HSEL%IN"EITRAG ZUR.ATURK RKUNDE6ERLAG DER #$RECHSLERSCHEN"UCHHANDLUNG (EILBRONN -ESSINGER* "ADGER-ANDD 7YDDRZYNSKI4 $ETECTIONOFF ONE SLOWLYEXCHANGINGSUBSTRATE WATERMOLECULEINTHE3 STATEOF 0HOTOSYSTEM))0ROC.ATL!CAD3CI53!n -ESSINGER * .UGENT *(! AND %VANS -#7  $ETECTION ONAN%02MULTILINESIGNALFORTHE 3s STATEIN0HOTOSYSTEM)) "IOCHEMISTRYn -ICHEL (AND D $EISENHOFER* F  8 2AY A DIFFRACT F IONSTUDIESON

 CRYSTALLINEBACTERIALPHOTOSYNTHETICREACTIONCENTER!PROGRESS REPORT AND CONCLUSIONS ON THE STRUCTURE OF 0HOTOSYSTEM)) REACTION CENTERS )N 3TAEHELIN ,! AND !RNTZEN #* EDS %NCYCLOPEDIAOF0LANT0HYSIOLOGY.EW3ERIES0HOTOSYNTHESIS 6 6OL))) PPn3PRINGER 6ERLAG "ERLI N -YERS*AND D &RENCH #3  %VIDENCESFROMACT F IONSPECTRA F FORASPEC IlCPARTICIPATIONOFC F HLOROPHYLL B INPHOTOSYNTHESIS *'EN0HYSIOLn D !RNTZEN#*L )DENTITYOFF THE .AKATANI ( +E" $OLAN%AND 0HOTOSYSTEM))REACTIONCENTERPOLYPEPTIDE"IOCHIM"IOPHYS !CTAn .ANBA/AND3ATOH+ )SOLATIONOFA0HOTOSYSTEM))REACTION CENTERCONSISTINGOF$ AND$ POLYPEPTIDESANDCYTOCHROME B 0ROC.ATL!CAD3CI 53!n /GAWA 4  0HYSICAL SEPARATION OF F CHLOROPHYLL PROTEIN COMPLEXES0HOTOSYNTH2ESn /GAWA4 /BATA&AND3 D HIBATA+ 4WOPIGMENTPROTEINS IN SPINACHCHLOROPLASTS"IOCHIM"IOPHYS!CTAn /KAMURA-9 3ATOH H + )SSACSON2!AND &EHER' %VIDENCE OF THE PRIMARY CHARGE SEPARATION IN THE $$ COMPLEX OF 0HOTOSYSTEM))FROMSPINACH%02OFTHETRIPLETSTATE)N"IGGINS *ED 0ROGRESSIN0HOTOSYNTHESIS2ESEARCH6OL PPn -ARTINUS.IJHOFF "OSTON /RT$2AND9OCUM#& D EDS  /XYGENIC0HOTOSYNTH T ESIS4HE U SHERS $ORDRECHT ,IGHT2EACTIONS+LUWER!CADEMIC0UBLI 0APAGEORGIOU ' AND D 'OVINDJEE EDS  #HLOROPHYLL A &LUORESCENCE ! 3IGNATURE OFF 0HOTOSYNTHESIS 3PRINGER $ORDRECHT 0RIESTLEY *  /BSERVATIONS ON DIFFERENT KINDS OF AIR 0HIL 4RANS23OC,ONDONn 2ADMER2AND/LLINGER/ )SOTOPICCOMPOSITIONOFTHE PHOTOSYNTHETIC/ mASHYIELDSINTHEPRESENCEOF(/ AND (#/n &%"3,ETTn 2ADMER2AND D /LLINGER/ $OTHE HIGHEROXIDATIONSTATES OFF THEPHOTOSYNTHETIC/ EVOLVINGSYSTEMCONTAIN BOUND D (/ &%"3,ETTn 2ENGER'  !PPARATUSAND D MECHANISMOFP F HOTOSYNTHETIC OXYGEN EVOLUTION ! PERSONAL PERSPECTIVE 0HOTOSYNTH 2ES n 2UBEN 3 2ANDALL - +AMEN -$ AND (YDE **  (EAVY 2 // ASTRACERINSTUDYOF0HOTOSYNTHESIS*!M#HEM3OC  n 3ATOH +  0ROTEIN PIGMENTS AND D 0HOTOSYSTEM)) REACTION CENTER 0HOTOCHEM0HOTOBIOL  n 3ATOH H + 2EALITY TY OF 0 CHLOROPHYLL PROTEIN )DENTIlCATION OFF THESITEOFF PRIMARYPHOTOCHEMISTRY INOXYGENICPHOTOSYN THESIS0HYSIOL0LANTn 3ATOH+ 4HEIDENTIlCATIONOFTHE0HOTOSYSTEM))REACTION CENTER!PERSONALSTORY0HOTOSYNTH2ESn 3ATOH+AND"UTLER7, ,OWTEMPERATURESPECTRALPROP ERTIESOFSU F UBCHLOROPLAST FRACT F IONSPURIlED FROMS F PINACH0LANT 0HYSIOLn D FEU2UEE F T 3CHEELE#7 4RAITECHIMIQUE DELAIRET DU (ÙTEL 3ERPENTE !CADEMIE2OY O ALEDES3CIENCES 0ARIS 3HARP22 0ROTON.-2RELAXATIONDUETOT D THEPHOTOSYNTH T ETIC OXYGENEVOLVINGCENTER)N0ECORARO6,ED -ANGANESE2EDOX %NZYMES PPn6#(0UBLISHERS)NC .EW9ORK R 3MITH %,  3OLUTIONS OF CHLOROPHYLL PROTEIN COMPOUNDS PHYLLOCHLORINS EXTRACTED FROM SPINACH 3CIENCE  n  3TEMLER!*  4HEBICARB R ONATEEFFECT OX F YGENEVOLUTION AND



,JNJZVLJ 4BUPI
5IPNBT
+
8ZES[ZOTLJ
BOE
(PWJOEKFF

THESHADOWOFF /TTO7ARRBURG0HOTOSYNTH 2ESn 3TIEHL((AND7ITT(4 $IEKURZEZEITIGENULTRAVIOLETTEN $IFFERENZSPEKTREN BEI DER 0HOTOSYNTHESE : .ATURFORSCHG B  4 4AKAHASHI9 4AKAHASHI-AND3ATOH+ )DENTIlCATIONOF THESITEOF IODIDEPHOTOOXIDATION INTHE0HOTOSYSTEM))REACTION CENTERCOMPLEX&%"3,ETTn 4 KAHASHI9 (ANSSON ½ -ATHIS0AND3ATOH+ 0RIMARY 4A RADICAL PAIR IN THE 0HOTOSYSTEM )) REACTION CENTER "IOCHIM "IOPHYS!CTAn 4ANG 8 3 AND 3ATOH +  4HE OXYGEN EVOLVING 0HOTO 4 SYSTEM))CORECOMPLEX&%"3,ETTn 4HOMAS*" "LAAUW/(AND$UYSENS,.- /NTHERELATION BETWEENSIZEANDPHOTOCHEMICALACTIVITYOFFRAGMENTSOFSPINACH GRANA"IOCHIM "IOPHYS!CTAn 4HORNBER*0 #HLOROPHYLL PROTEINS,IGHT HARVESTING AND REACTIONCENTERCOMPONENTSOFF PLANTS!NN2EV0LANT0HYSIOL n 4HORNBER *0 AND (IGHKIN (2  #OMPOSITION OFF THE PHOTOSYNTHETICAPPARATUSOFNORMALBARLEYLEAVESANDAMUTANT LACKINGCHLOROPHYLLB%UR*"IOCHEMn 4HORNBER*0 3MITH#!AND"AILEY*, 0ARTIALCHARACTERIZA TION OF TWO CHLOROPHYLL PROTEIN COMPLEXES ISOLATED FROM SPINACH BEETCHLOROPLASTS"IOCHEM*PnP 4IMIRIAZEFFF #!  3UR LACTION DE LA LUMIERE DANS LA DECOMPOSITION DE LACIDE CARBONIQUE PAR LA GRANULE DE CHLOROPHYLLE)NTERNATIONAL"OTANICAL#ONGRESS &IRENZE )TALY Y -AY AY PP   4 4REBST! 4HETOPOLOGYOFTHEPLASTOQUINONEANDHERBICIDE BINDINGPEPTIDESOF0HOTOSYSTEM))INTHETHYLAKOIDMEMBRANE :.ATURFORSCHCn 6AN -IEGHEM &*% 3ATOH + AND 2UTHERFORD !7  6 ! CHLOROPHY H LL TILTEDD   RELATIVE TO THE MEMBRANE IN THE 0HOTOSYSTEM))REACTIONCENTRE"IOCHIM "IOPHYS!CTA n VAN2ENSEN**3 8U#ANDD 'OVINDJEE  2OLEOF BICARB R ONATE IN0HOTOSYSTEM)) THEWATER PLASTOQUINONEOXIDO REDUCTASEO D F PLANTPHOTOSYNTHESIS0HYSIOL0LANTn 6 6ELTHUYS"2AND!MESZ* #HARGEACCUMULATIONATTHE

REDUC D ING SIDEOFS F YSTEMOFP F HOTOSYNTHESIS"IOCHIM"IOPHYS !CTAn 6ERMEGLIO !  4HE TWO ELECTRON GATE IN PHOTOSYNTHETIC BACTERIA0HOTOSYNTH2ESn 7HITMARSH*AND'OVINDJEE 0HOTOSYSTEM))%NCYCLOPEDIA OFF ,IFE3C F IENCES-C-ILLAN0UBLI U SHERS,TD .ATURE0U D UBLISHING 'ROUP ,ONDON 5+HTTPWWWELSNET 7ILLSTÊTTER2AND3TO D LL !  5NTERSUCHUNGENàB à ER#HLORO PHYLL*USTUS3PRINGER "ERLIN%NGLISHTRANSLATION BY 3CHERTZ D -ERZ!2 3CIENCE0RINTING0RESS ,ANCASTER 0ENN &-AND SYLVANIA  7 7ITT(4 4 -àLLER!AND2UMBERG" %XPERIMENTALEVIDENCE FORTHEMECHANISMOFPHOTOSYNTHESIS.ATUREn 7 7YDRZYNSKI4 4HEROLEOFMANGANESEINPHOTOSYNTHETIC OXYGENEVOLUTION0H$THESIS5NIVERSITYOFF )LLINOISAT5RB R ANA #HAMPAIGN )LLINOIS 7YDRZYNSKI 4  %ARLY INDICATIONSFORMAN F GANESEOXIDATION STATE CHANGES DUR D ING PHOTOSYNTHETIC OXYGEN PRODUCT D ION ! PERSONAL ACCOUNT0HOTOSYNTH2ESn 7 7YDRZYNSKI4 :UMBULYADIS. 3CHMIDT0' 'UTOWSKY(3AND 'OVINDJEE 0ROTONRELAXATIONANDCHARGEACCUMULATION DURING OXYGEN EVOLUTION IN PHOTOSYNTHESIS 0ROC .ATL!CAD D 3CI 53!n 9AMAMOTO9AND3ATO 9 D H +  4HECARB R OXY TERMINALPROCESSING OFF PRECURSOR$PROTEINOFF 0HOTOSYSTEM))REACTIONCENTERBY A D #TP! )N!RGYROUDI !KOY O UNOGLOU NUCLEAR ENCODEDPROTEASE *( AND D 3ENGER ( EDS 4HE #HLOROPLAST &ROM -OLECULAR Y PP n +LUWER !CADEMIC "IOLOGY TO "IOTECHNOLOGY 0UBLISHERS $ORDRECHT 9AMAMOTO9 $OI- 4AMURA.AND.ISHIMURA- 2ELEASE 9 OFPOLYPEPTIDESFROMHIGHLYACTIVE/ EVOLVING0HOTOSYSTEM  PREPARATIONBY4RISTREATMENT&%"3,ETTn 9 9AMAO KA4 3ATOH+AND D +ATOH 3 0HOTOSYNTHETICACTIVITIES OFF A THERMOPHILIC BLUE GREEN ALGA 0LANT #ELL 0HYSIOL  n :OUNI! 7I 7 TT (4 +ERN * &ROMME 0 +RAUSS . 3AENGER 7 AND D /RTH 0  #RYSTAL STRUCTUREOFF 0HOTOSYSTEM))FROM F 3YNECHOCOCCUS ELONGATUS AT  ¯ RESOLUTION .ATURE  n

$IBQUFS
 %JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT #FWFSMFZ
3
(SFFO

#PUBOZZ %FQBSUNFOU
6OJWFSTJUUZ PG
#SJUJTI
$PMVNCJB
7BODPVWFS
#$
75
;
$BOBEB

&MJTBCFUI
(BOUU $FMM
#JPMPHZ
BOE
.PMFDVMBS
(FOFUJDT
6OJWFSTJUZ
PG
.BSZMBOE
$PMMFHF
1BSL
.%

6 64"

4VNNBSZ   * *OUSPEVDUJPO  ** 1IZDPCJMJQSPUFJOT
BOE 1IZDPCJMJTPNFT  " 1IZDPCJMJQSPUFJO 5 5ZQFT
  
1IZDPCJMJQSPUFJO $IBSBDUFSJTUJDT
JO $ZBOPCBDUFSJB
BOE
3FE
"MHBF   $SZQUPQIZUF 1IZDPCJMJQSPUFJOT    1SPDIMPSPQIZUF 1IZDPCJMJQSPUFJOT  # 1IZDPCJMJTPNFT
   4USVDUVSF    "DDMJNBUJPO
UP -JHIU
BOE /VUSJFOU 4UBUVT   "TTPDJBUJPO
XJUI 1IPUPTZTUFN ** &OFSHZ %JTUSJCVUJPO
BOE %JTTJQBUJPO   *** 1SPDIMPSPQIZUF "OUFOOBT
BOE
UIF *TJ" 1SPUFJOT  *7

5IF
-)$ 4VQFSGBNJMZ
PG $IMPSPQMBTUT   " 0WFSWJFX  #
5IF
$IMPSPQIZMM BC 'BNJMZ
PG
)JHIFS
1MBOUT
BOE
(SFFO
"MHBF   5IF $IMPSPQIZMM BC  1SPUFJO $PNQMFYFT    5ISFF%JNFOTJPOBM 4USVDUVSF
PG
-)$**    .PMFDVMBS #JPMPHJDBM "QQSPBDIFT /BUJWF
BOE 3FDPNCJOBOU $PNQMFYFT   0SHBOJ[BUJPO
PG $IMPSPQIZMM BC  "OUFOOBT JO
UIF 5IZMBLPJE .FNCSBOF    &OFSHZ 5SBOTGFS
5 4UVEJFT
PO $IMPSPQIZMM BC  1SPUFJOT  $
"OUFOOBT
PG
"MHBF
XJUI
$IMPSPQIZMM D   
5IF $IMPSPQIZMM BD  1SPUFJOT    5IZMBLPJE 0SHBOJ[BUJPO
BOE &OFSHZ 5SBOTGFS 5   
5IF
1FSJEJOJO$IMPSPQIZMM B 1SPUFJO
PG %JOPáBHFMMBUFT   %
1IPUPQSPUFDUJPO
BOE
UIF
-)$ 'BNJMZ   "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 4HE DISTALANDD EXTRINSIC LIGHT HARVESTINGANTENNASOFF 0HOTOSYSTEM)) 03)) PROVIDETHECAPABILITYTOMATCH ELECTRONmOWTHROUGH0HOTOSYSTEM)THUSALLOWINGFORREGULATEDRESPONSESTOENVIRONMENTALCHANGES)NTHIS F HESEANTENNACOMPLEXESAND DISCUSSWH W ATISKNOWNA K ABOUT CHAPTERWEPROVIDEACONCISEUP TO DATEDESCRIPTIONOFT THEIRFUNCT F ION4HECYANOBACTERIAAND D RED ALGAL03))ANTENNASAREPHYCO H BILIPROTEINSORGANIZED INTOCOMPLEX MEMBRANE EXTRINSICSTRUCTURESCALLEDPHYCOBILISOMES!SMALLGROUPOFCYANOBACTERIALACKPHYCOBILISOMES D USETHEMEMBRANE INTRINSICPROCHLOROPHYTECHLOROPHYLL #HL AB PROTEINS03))OFEU AND INSTEAD F KARYOTES IS

!UTHORFORCORRESPON F DENCE EMAIL BRGREEN INTERCHANGEUB U CCA

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU

PRIMARILYSERVED BYMEMBERSOFT F HE LIGHT HARVESTINGCOMPLEX,(# SUPERFAM F ILYWHI W CH HAS BECOMEWIDELY DIVERSIlEDINT D O #HL AB AND #HL AC ANTENNAS!SMEMBERSOFTHE,(#SUPERFAMILY CRYPTOPHYTEALGAE ALSO HAVENOVEL PHYCOBILINSINTHETHYLAKOID LUMEN AND DINOmAGELLATEALGAE HAVEAUNIQUEPERIDININ #HL A PROTEIN A ORPLANT,(#AREOPENINGUPANEWERAIN !TOMICRESOLUTIONSTRUCTURESOFF THESETWOANTENNASANDD THEMAJ UNDERSTANDINGENERGYTRANSFER *
*OUSPEVDUJPO ,IGHT HARVESTING ANTENNAS ARE THE PIGMENT PROTEIN COMPLEXES THAT SERVE TO ABSORB LIGHT ENERGY AND TRANSFER F ITTOTHEPHOTOSYNTHETICREACTIONCENTERSOF 0HOTOSYSTEM ) 03) AND D 0HOTOSYSTEM)) 03))  4HECOREANTENNASARETHEANTENNASTHATBINDONLY CHLOROPHYLL #HL A AND D AREMOSTINTIMATELYASSOCI ATED D WITHTHEPHOTOSYSTEMS03) HASACOMBI M NED D CORE ANTENNA REACTIONCENTER WHEREABOUTADDITIONAL #HL A MOLECULESAREBOUND D TOTTHESAMETWOMEMB M RANE INTRINSIC PROTEINS 0SA! AND D 0SA" AS THE REACTION CENTERCHROMOPHORES!NINTERESTINGEXCEPTIONISTHE 03)COREANTENNAOF !CARY R OCHLORIS WITH #HL D PER#HL A (UETAL  )NCONTRAST 03))HAS TWOSEPARATE#HL A PROTEINS #0AND#0 THAT BIND D ONLY  AND D  #HL A RESPECTIVELY 4ABLE   4HECORE#HL A ANTENNASARE DISCUSSED IN#HAPTER %ATON 2YEAND0UTNAM %VANS  F HATTHE03))COREANTENNASBIND D ONLYONE 4HE FACTT THIRD D THENUMBEROF #HLSASTHE03)COREMEANSTHAT 03)) REQUIRES ADDITIONAL LIGHT HARVESTING CAPACITY INORDERTOMAINTAIN LINEARELECTRON mOW4HEDISTAL D EXTRINSIC ANTENNAS PROVIDE THAT CAPACITY 4HE AND @DISTALANTENNACOMPLEXESAREDElNEDASTHOSETHAT AREMEMB M RANE BOUND BUTNOTPARTOFT F HECOREANTENNA D THE@EXTRINSICANTENNASARETHOSETHATAREATTACHED AND TOTHESURFACEOFTHETHYLAKOIDMEMBRANE'REENAND $URNFOR F D D   "OTH KINDS OF F ANTENNA ARE REGU LATEDQUA D LITATIVELYANDQUANT D ITATIVELYINRESPONSETO PHYSIOLOGICALSTATUSANDENVIRONMENTALSIGNALS4HIS PROVIDESTHE mEXIBILITYREQUIRED FORACC F LIMATIONTO !BBREVIATIONS R " 0% 2 0%nPHYCOERYTHRINOFREDALGAE"AN GIOPHYCEAN 2HODOPHYCEAN  #HLn CHLOROPHYLL #0 #0 #0nMINORINTERNAL #HL AB A ANTENNASTHATCONNECTTHE EXTERNAL ANTENNASWITH THE03))CORE#0 #0nANTENNASOFT F HE03)) CORECOMPLEX�nFUCOXANT F HIN #HL A AC PROTEIN)SI!n#HL A PROTEININDUCEDBY&ELIMITATION#0| ,(#nMEMBEROFLIGHT HARVESTING COMPLEXSUPERFAM F ILY,(#))nMAJ A OR,(#ANTENNAOF 03))INHIGHERPLANTS0"3nPHYCOBILISOME0CBnPROCHLOROPHYTE #HL AB  PROTEIN 03) 03))n 0HOTOSYSTEM ) 0HOTOSYSTEM)) 0SA! 0SA"n03)REACTIONCENTER COREANTENNAPROTEINS2# 2#nREACTIONCENTERSOF03) 03))20 (0,#nREVERSED PHASE HIGHPERFORMANCE F LIQUIDD CHROMATOGRAPHY3$3 0!'%nSO H DIUM DODECYLSULFATE PO F LYACRYLAMIDEGEL ELECTROPHORESIS

DIFFERENTLIGHT TEMPERATUREANDNUTRIENTREGIMES AS WELLASTOSHORT TERMCHANGESINTHEREDOXSTATEOF THEELECTRONTRANSPORTCHAIN )N SPITE OF THE GREAT VARIETY OF LIGHT HARVESTING A ORTYPESOF LIGHT ABSORBI R NG ANTENNAS ONLY THREEMAJ CHROMOPHORES ANDD ONLY FFOUR PROTEIN FAM F ILIES ARE EMPLOYEDINALLTHEANTENNASOF03))4ABLE /PEN D HYCOBILINSAREATTACHEDD TO CHAINTETRAPYRROLESCALLEDP MEMB M ERSOFF THEPHYCO H BILIPROTEINFAM F ILYT L THEYAREGEN ERALLYFOUNDINCOMPLEXMEMBRANE EXTRINSICSTRUC TURESCALLEDD PHYCOBILISOMES0"3 3EVERAL GROUPS F BACTERIA LACK0"3S K BUT HAVEAUNIQUEMEM OFCYANO BRANE BOUND#HL A AB ANTENNAUSINGPROTEINSRELATEDTO THECOREANTENNA4HECHLOROPLASTSOFF PHOTOSYNTHETIC F HE LIGHT HARVESTING EUKARYOTESEMPLOYMEMBERSOFT COMPLEX,(# SUPERFAMILY WHICHBIND#HL B OR C INADDITIONTO#HL A AND HAVETHREETRANS MEMBRANE HELICES-EMBERSOFT F HISWELL STUDIED FAM F ILYTRANSFE F R ENERGYTOEITHERORBOTHPHOTOSYSTEMS ANDHAVEAN IMPORTANTROLEINPHOTOPROTECTIONASWELLASLIGHT GATHERING 4HE PERIDININ #HL A ANTENNA IS UNIQUE Q TODINOmAGELLATESBECAUSETHECAROTENOIDPERIDININ RATHERTHAN#HLPLAY A STHEMAJ A ORROLE INLIGHT HARVEST ING ITIS BEINGEXTENSIVELYSTUDIEDD ASAMODELSYSTEM INENERGYTRANSFER)LAGANETAL   -ANY N ASPECTS OF LIGHT HARVESTING ANTENNAS WERE EXTENSIVE I LYCOVERE L D INARECENTBOOK INTHI T SSAMESERIES 'REENAND0ARSON  ANDININDIVIDUALCHAPTERS INTWOOTHERBOOKS&RANKK ETAL ,ARK RKUMETAL  4HISCHAPTERISRESTRICTEDD TOTHEANTENNASOF 03)) ANDOURAIMISTO PROVIDEACONCISEINTRODUCTION ANDD UPDATEOFMATER F IAL INTHEABOVEVOLUMES **
1IZDPCJMJQSPUFJOT
BOE 1IZDPCJMJTPNFT " 1IZDPCJMJQSPUFJO
5 Z 5ZQFT 0HYCO H BILIPROTEINSARETHEPREDOMINANTANTENNAPIG MENTSIN CYANOBACTERIA RHODOPHYTES GLAUCOPHYTES AND INMANY N CRYPTOPHYTES4HEYAREWATERSOLUBL U E D LL BREAKAGE PIGMENTSTHATAREREADILYRELEASEDUPONCE ANDPRODUCEVIBRANTBLUE ORORANGETORED COLORED Y HECHROMOPHORESARE LINEAR SOLUTIONS#HEMICALLY T

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT



4 BLE ,IGHT HARVESTINGANTENNASOFF 0HOTOSYSTEM)) 4A &AMILY #ORECOMPLEX #OREANTENNAS#0 #0 0ROCHLOROPHYTE#HL AB  PROTEINS

#HROMOPHORES

7HERE FOUN F D

/RGANIZATION,OCATION

#HL A ` CAROTENE

!LLOXYGENICORGANISMS

0ARTOF03))CORE

#HLA B

/NLYINGENERAOFCYANOBACTERIA PROCHLOROPHYTES

4HYLAKOIDMEMBRANE

0HYCOBILIPROTEIN

0HYCOBILINS

#YANOBACTERIA GLAUCOPHYTES RHODO PHYTES CRYPTOPHYTES

0HYCOBILISOMES STROMAL SIDEOFT F HYLAKOIDD MEMBRANE EXCEPTCRYPTOPHYTES

,(#LIGHT HARVESTING COMPLEX SUPERFAMILY

#HLA PLUSONEORMOREOF B C C CVARIETYOF CAROTENOIDS

#HLOROPLASTS EXCEPTGLAUCOPHYTES NONE RHODOPHYTES03)ONLY

4HYLAKOIDMEMBRANE

0ERIDININ #HL A PROTEIN

#HLA PERIDININ

$INOmAGELLATESONLY

4 4RIMERSINTH HYLAKOIDLUMEN

TETRAPYRROLESTHATARECOVALENTLY BOUND D TOTHEAPOPRO TEIN BYTHIOETHERLINKAGESTOONEORTWORINGS RINGS !$ -AC#OLLAND'UARD &RIAR -IMUROAND +IKUC K HI 3CHEER  4HEEFFECT F IVENESSOF PHYCO H BILIPROTEINSASANTENNASWASPROVENINTH T ES BYELEGANTACTIONSPECTRADONELARGELYBY(AXOAND F LLY ILLUSTRATED INACOM CO WORK R ERSTHEY AREBEAUTIFU PILATION BY(AXO  )T ISNOWWELLESTABLISHED THAT THE ENERGY ABSORBED BY THE ANTENNA PIGMENTS ISTRANSFERRE F D BETWEENTHEMAND lNALLYTOREACTION CENTERS BY THE &ÚRSTER TYPE EXCHANGE MECHANISM -IMUROAND+IKUCHI 0ARSONAND.AGARAJAN  4HIS MECHANISM REQUIRES AN OVERLAP OFF THE WAVELENGTH FUNCT F IONS AND D OCCURS EFlCIENTLY WH W EN THEDONATINGANDACCEPTINGCHROMOPHORESAREWITHIN ABOUT¯ANDNOMORETHAN¯ APART-AXIMAL TRANSFER F EFlCIENCY IS ASSURED BY CLOSE PACKING OF CHROMOPHORESONTHEPHYCOBILIPROTEINSATONELEVEL F HERED BYTHECLOSEASSOCIATIONOFP F HYCOBILI AND FURT PROTEINSTOONEANOTHER 
1IZDPCJMJQSPUFJO
$IBSBDUFSJTUJDT
JO $ZBOP $  CBDUFSJ SJB
BOE 3FE "MHB M F !LLOPHYCOCYANIN AND PHYCOCYANIN ARE THE TWO PRINCIPAL TYPESOFP F HYCO H BILIPROTEINSTHATOCCURINALL CYANOBACTERIA GLAUCOPHYTES AND RED ALGAE WHICH HAVE PHYCOBILISOMES AS LIGHT HARVESTING ANTENNAS &IGS  !  0HYCOERYT H HRIN IS THE THIRD D PRINCIPAL PHYCOBILIPROTEINTYPE7ITHINCELLSTHESEPIGMENTS EXISTASAGGREGATESOFT F HE _ AND ` SUB U UNITS!N _ AND ` COMBINATIONISOFTENRE F F FERRE D TOASA@MONO MERANDLARGERONESAS@TRIMERSOR@HEXAMERS BUT THETERMINOLOGY CANBERATHERARCANE FORT F HEGENERAL

&IG G  $IAGRAMOFAGENERICPHYCOBILISOMELINKEDTHROUGHTHE ,#- CORETOMEMBRANELINKER TOADIMERIC03))COMPLEX%ACH 03))MONOMERHASA 03))REACTIONCENTER 2# AND #0AND #0COREANTENNASNOTSHOWN 4HEOUTERPHYCOERYTHRIN 0% DARK R GRAY A ISCONNECTED D VIAPHYCOCYANIN 0# LIGHTGRAY A TOTHE ALLOPHYCOCYANINCORE !0# WHI W TECIRCLES WITH LINKERPOLYPEP TIDES SMALL OVALS ASSURING CORRECT PHYCOBILIPROTEIN STACKING %XCITATIONENERGYTRANSFERFROMPHYCOERYTHRINTO #HLISSEENBY THEDECREASEINmUORESCENCEMAXIMAOFPARTICIPATINGPIGMENTS

READER ANDD THUS QUOTES ARE USED )N SOLUTION PHY COBILIPROTEINSTENDTOBEMORESTABLEASAGGREGATES F _` ORAS_`  4HEMOSTSTABLEARETHE OF LARGE AGGREGATES OF F PHYCOERYT H HRINS DESIGNATEDD AS " BANGIOPHYCEANAND2 RHODOPHYCEAN WHICHOFTEN U UNITS CONSISTOFF ACOMBINATIONOFF _ ` AND a SUB WITH ATOTAL MOLECULARMASSOFF ABOUT  K$A !LLOPHYCOCYANINS HAVE THE FEWEST CHROMOPHORES WITH PER@HEXAMEREACHPER_ AND ` SUB U UNIT H BILINCHROMOPHORES ANDD THEYONLYHAVEPHYCOCYANO 0HYCOCYANOBILINISALSOTHECOMMONCHROMOPHORE F HYCOCYANINS BUTMORECHROMOPHORESPER_ OFP



#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU

&IG G  ,OCALIZATIONOFP F HYCOBILIPROTEINS!(EMISPHERICALLY SHAPED PHYCOBILISOMESARROWS ATTACHED D TOTHESTROMALSIDEOFF THYLA KOIDS INA REDALGA0ORPHYRIDIUMCRUENTUM  4HELOCALIZATION ISTHESAME INCYANOBACTERIAALTHOUGH THEPHYCOBILISOMESAREUSUALLY HEMIDISCOIDALLY SHAPED")NCRYPTOPHYTES #HROOMONAS THEPHYCOBILIPROTEINSARECONCENTRATED INTHETHYLAKOID LUMEN ARROWS INAN APPARENTLYHIGHLYORDEREDMANNER

D  PER ` SUB U UNIT ARE BOUND D THAN INAL SUB U UNITAND LOPHYCOCYANINS3OMEPHYCOCYANINAPOPROTEINSMAY D OTTHERCHROMOPHORES IE PHYCOBILIVIOLINAS ALSOBIND IN PHYCOERYT H HROCYANIN OR PHYCOERYT H HROBILIN ASIN 2 PHYCOCYANIN 0HYCOERYRTHRINSBINDMANYPHYCO ERYTHROBILINCHROMOPHORES AND D OFTENA F LSOPHYCOURO BILINS WITH ATOTAL OFF ORMOREPER@HEXAMER _ ` a 4OOLEAND!LLNUTT  "ECAUSEOFTHEIR HIGH CHROMOGENICITY Y INDIVIDUA D L PHYCOBILIPROTEINS HAVE FOUN F D USESAS mUORESCENTCYTOLOGICAL MARK R ERS INANIMALCELLANALYSIS!LSO 0"3SOFSOMEREDALGAE AREHIGHLYUSEFU F L FOR F BIOMEDICALCYTOLOGY SINCETHEY R NGAND mUORESCING CONTAINSPECTRALLYDISTINCTABSORBI CHROMOPHORESANDALARGENUMBEROFCHROMOPHORES 4OOLEAND D !LLNUTT  

&ROMTHECRYSTALSTRUCTURESOFF THEPRINCIPAL TYPES OF F PHYCOBILIPROTEINS ONE CAN SEE THAT THEY FOLLOW F LDING PATTERN A TYPICAL @GLOBIN FO F LD THE SAME FO SEESUMMARYIN-IMUROANDD +IKUC K HI  4HE ARRANGEMENTOFTHE _ AND ` SUBUNITS IS SUCHTHAT @MONOMERSFORMA@TR F IMERAROUNDD ACENTRALCAVITY WITH THE CHROMOPHORES ARRANGEDD ON THE OUTER RIM OFTHE@TRIMER EXCEPTINALLOPHYCOCYANINWHERETHE CHROMOPHORESONTH T E` SUB U UNITFACE F INTOTH T ECAVITY TY!N H BILIPROTEINSEQUENCES EXTENSIVECOMPARISONOFF PHYCO SUGGESTSTHATTHEYAREDERIVEDFROMTWOEVOLUTIONARY LINESTHATORIGINATED FROMACOMMONANCESTOR F !PTET AL A 3EVERALRESIDUESAREO D F ITICAL IMPORTANCE FCR FORCHROMOPHOREATTACHMENTSANDFORFOLDINGOFTHE PROTEINS0RESENTINALMOSTALLPHYCOBILIPROTEINSISTHE

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT #YSATRESIDUE D OR WHI W CH ISACHROMOP H HOREBIND INGSITEIN _ AND ` SUBUNITS !RGAND!SPWHICH F INTAINING TH T EPROPERCHROMOP H HORE AREIMPORTANTFORMA ORIENTATION AND D 'LY OR WHI W CH ISNECESSARY F FORTHEPROPERCHARGEINTERACTIONBETWEENHELICESTHAT PROBABLY A INTERACTWITH T LINKERPOLYPEPT Y IDES!DDITIONAL CHROMOPHORE BINDINGRESIDUES D INPHYCOERYT H HRINSAND PHYCOCYANINSARE#YSOR !PTETAL A -IMUROAND D +IKUC K HI   
$SZ SZQUPQIZUF
1IZDPCJMJQSPUFJO Z T #RYPTOPHYTE H PHYCO H BILIPROTEINSARESIMILARTOTHOSEIN CYANOBACTERIAANDRHODOPHYTESINHAVINGPHYCOERY THRIN ANDP D HYCOCYANOBILIN TYPECHROMOPHORESBUT WITHAVARIETYOFUN F IQUE BILIN ISOMERSINCLUDING   DIHYDROBILIVERDIN MESOBILIVERDIN BILIN  AND BI LINn SOMEOFW F WHICHARERELATED D TO PHYTOCHROME BILIN'LAZERAND7E D DEMAYER 7EDEMAYERET AL  !NYONESPECIESHASONLYPHYCOCYANINOR PHYCOERYTHRIN BUTNEVERBOTH AND D NOALLOPHYCOCYA NINHASBEENREPORTED(OWEVER TH T EPHYCO H BILIPROTEINS AREORGANIZEDAS_` _` @HETERODIMERS7ILKET AL  THE _ AND _ SUB U UNITSARENOTIDENTICAL H LAKOID LUMEN&IG 4HEYARETIGHTLYPACKED INTHETHY " RATHERTHANORGANIZEDINTOCOMPLEXPHYCOBILI SOMES ON THE STROMAL SIDE &IG ! 'ANTT ET AL D 'IBBS  4HECLOSEPACKING ,UDWIGAND PRESUMABLYORIENTSTHECHROMOPHORESFORMAXIMAL ENERGYTRANSFER/NEREASONFORTHISSTRUCTURALDIF F F FERENCE ISTHATTHE _ SUB U UNITSARENOTCLOSELYRELATED TO THE _ SUBUNITS OF CYANOBACTERIA OR RED ALGAE THEY APPEARTO HAVE BEENRECRUITED FROMSOMEOT F HER SOURCE POSSIBLY FFROM A LINKER PROTEIN !PT ET AL A2EUTERETAL  (OWEVER THE ` SUBUNITS OFCR F YPTOP Y HYTEPHYCOCYANINORPHYCOERYTH THRINDOSHARE Q LATEDNESSWITH THOSEOFT F HEIR SUB U STANTIALSEQUENCERE CYANOBACTERIALRHODOPHYTE EQUIVALENTS !PT ET AL F HE0%PHYCOBILIPROTEIN A 4HESTRUCTUREOFT HETERODIMER HAS BEEN SOLVED TO ¯ RESOLUTION BY8 RAYCRYSTALLOGRAPHY7ILKETAL  ANDIS DISCUSSED INSOMEDETAIL IN-AC0HERSONAND D (ILLER  %VOLUTIONARILYTHECRYPTOPHYTEC H HLOROPLASTIS BELIEVEDTOHAVEORIGINATEDBYTHE SECONDARYACQUISI TIONOFARE F D ALGAL CHLOROPLAST WITH PARTIALSUPPORT F FROMAS IMILARITY BETWEENTHE `SUB U UNITS 4HEFUNCTIONALCONTRIBUTIONOFPHYCOBILIPROTEINS ASANTENNAPIGMENTS INCRYPTOPHYTES ISASWELLAC CEPTED D AS INCYANOBACTERIA AND D RHODOPHYTES H -OST CRYPTOPHYTESARECERTAINLYCAPABLEOFPHOTOAUTOTRO PHIC GROWTH BUT ITISNOTCLEARTOWH W ATEXTENTTHEY



ARECAPABLEOFF MIXO OREVEN HETEROTROPHICGROWTH ESPECIALLY SINCE NON PHOTOSYNTHETIC CRYPTOPHYTES ARE BY NO MEANS RARE #HROOMONAS SP GROWN UN DER PHOTOAUTOTROPHIC GROWTH CONDITIONS EXHIBITED AN INCREASE IN #HL ANDPHYCOCYANINCONTENTUNDER LOWLIGHT TYPICAL OFAP F HOTOTROPH BUTWASALSOABLE TOUTILIZEGLYCEROL ASCARB R ONSOURCE'ANTT   5PON EXCITING PHYCOERYTHRIN "RUCE ET AL  F FOUN D mUORESCENCE EMISSION AT BOTH  NM AND NM AT+ INWH W OLECELLS4HIS PLUSAVERY LOWPHYCOERYTHRINEMISSIONSUGGESTSTHATENERGYIS TRANSFERRE F D TO BOTH REACTION CENTERS7HEN ,ICHTLÏ ETAL  CHARACTERIZEDD THEPHOTOSYSTEMPARTICLES FROM #RY RYPTOMONAS THEYESTABLISHEDTHATPHY H COERY THRINWASASSOCIATEDD WITH AFUNCT F IONALLY ACTIVE03)) PARTICLE BUTNOTWITH03)-ACPHERSONANDD (ILLER  REVIEWEDSPECTROSCOPICSTUDIESANDCONCLUDED THATTHEPHYCOBILINSPREFERENT F IALLY TRANSFERENER F GY TO 03))RATHERTHAN03) BUTWH W ETHERITGOESVIATHE#HL A COMPLEX IS NOT CLEAR -ORE STUDIES COMBINING AC BIOCHEMICAL FRACT F IONATIONWITH SPECTROSCOPY US Y ING A BROADERSELECTIONOFF CRYPTOPHYTESPEC H IES ARECLEARLY IN ORDER  1SPD 1 IMPSPQIZUF 1IZDPCJMJQSPUF J J T JO 0HYCOERYTHRIN HAS BEEN FOUN F D IN SEVERAL LOWLIGHT STRAINSOF 0ROCHLOROCOCCUSMARINUS ONEOFF THE@GREEN CYANOBACTERIA OR PROCHLOROPHYTES THAT HAVE A #HL A ANTENNAAND DONOT HAVE0"3S(ESSETAL  AB 4INGETAL 0ARTENSKYAN K D 'ARCZAREK   4HEPHYCOERYTHRINHASPHYCOERYTHROBILINANDPHYCOU ROBILINAND HASTHREEAPOPROTEINTYPES_ ` AND a IE ITISCOMPOSITIONALLYSIMILARTOTHAT IN HIGHERRED ALGAE'ENESFORPHYCOCYANINANDALLOPHYCOCYANIN F EITHER STRAIN $U ARE ABSENT FFROM THE GENOMES OF F FRESNEETA L 0ARTENSKYAN K D 'ARCZAREK   "ECAUSE THESE BASAL PHYCOBILIPROTEINS ARE ABSENT ITISNOTSURPRISING THAT0"3S HAVENOT BEEN FOUN F D 0RESUMABLY THE PHYCOERYT H HRIN CONTAINING STRAINS COULDBENElTUNDERLOWLIGHTBYBEINGABLETOHARVEST THEBLUEAND GREEN LIGHTABSORBI R NGREGIONS PROVIDED THATTHEENERGYABSORB R ED IS FUNCT F IONALLYTRANSFERRE F D TOAREACTIONCENTER(OWEVER THESPECTRALOVERLAP OF F PHYCOERYTHRIN WITH #HL A IS POOR ANDD THUS ANY N F LD BEQUITEINEFlCIENT%VENTHOUGHTHE TRANSFERWOU SPECTRALOVERLAP WITH#HL B ISSLIGHTLYBETTER ACTION SPECTRAL MEASUREMENTSAND FRACT F IONATIONSTUDIESARE F HYCOERYT H HRIN REQUIREDD TOASSESSTHECONTRIBUTIONOFP TOPHOTOSYNTHESIS

 #
1IZDPCJMJTPNFT J  4USVDUVSF S 4HE PHYCOBILISOME0"3 CANBEVIEWEDASANEX QUISITELYENGINEERED D ANTENNATHATACTSASANENERGY TRANSFER F FUNNE F L TO03))&IG 4HECLOSEPACKINGOF @TRIMERSAND@HEXAMERSOFVARIOUSTYPESOFCHROMO PHORESALLOWS FORT F HEIRINTERACTIONWITH ONLYTHEMOST MINIMAL LOSSOFF ENERGY7HETH T ERINSIMPLEORCOMPLEX 0"3 THECHROMOPHORESAREORIENTEDFORMAXIMALAB SORPTIONAND D TRANSFERO F FENER F GY FROM F HIGHERTO LOWER ENERGYSTATESAND BETWEENCHROMOPHORESRECENTLY REVIEWEDBY-IMUROAND+IKUCHI 4OOLEAND !LLNUTT  3PECIlCLINKERPOLYPEPTIDESARE IN VOLVED IN FAC F ILITATINGANOPTIMIZEDP D HYCO H BILIPROTEIN TO PHYCOBILIPROTEIN ARRANGEMENT RESULTING IN THE 0"34HE ROLE TYPE AND LOCATION OF F ROD AND CORE LINKER POLYPEPTIDES THOUGH FASC F INATING IS BEYOND THEGENERALLYINTERESTEDREADERBUTAREREVIEWEDBY D +IKUC K HI  AND !PTETAL A -IMUROAND D !LLNUTT  )NCRYPTOPHYTEST H HEPHYC H O 4OOLEAND BILIPROTEINSARENOTORGANIZEDIN0"3SBUTAREPACKED INTOTHETHYLAKOID LUMEN&IG"  H BILISOME TYPE IS A SIMPLE 4HE SIMPLEST PHYCO ROD WITH ALLOPHYCOCYANIN AS A BASE ON THE THYLA KOID D MEMBRANE AND D A PHYCOCYANIN ROD D EXTENDING DISTALLY INTO THE STROMA EG 'LOEOBACTER  -ORE TYPICAL ARE 0"3S IN MANY CYANOBACTERIA WHERE A MULTIMERIC ALLOPHYCOCYANIN CENTER IS SURROUNDED BYPHYCOCYAN H INRODS0HYCOERYT H HRINMAY BEADDED DISTALLYINCYANOBACTERIATHATADAPTTOREDORGREEN LIGHT APHENOMENONREFERRE F D DTOASCOMP LEMENTARY CHROMATIC ADAPTATION SUMMARIZED IN AN EXCELLENT RECENTREVIEWBY'ROSSMANETAL !LTHOUGH THEMOSTCOMPLEX0"3SOCCURINRED D ALGAE THEY ARE VARIATIONSONTHESAMETHEMEREPRESENTED BYTHEGE NERIC0"3MODELIN&IG3PECTRALLY THEREDALGAL PHYCOBILIPROTEINSARESIMILARTOTH T OSEINCYANOBACTERIA 'ANTT ET AL  -IMURO AND D +IKUC K HI   %NERGY TRANSFER BETWEEN PHYCOBILIPROTEINS OCCURS WITHINPICOSECONDS AND D TRANSFERTO F #HL OCCURSWITHIN NANOSECONDSASHASBEEN DETERMINED INWH W OLECELLS ANDINISOLATED0"3S-IMUROAND+IKUCHI   !TIGHTENERGETICCOUPLING ISVERIlABLE BY THEVERY LOW mUORESCENCEEMISSIONFROMT F HE INDIVIDUA D LPHY H COBILIPROTEINTYPES  "DDMJN J BUJP U O
UP U -JH J IU
BOE /VUSJF S OU 4UB U UVT !CCLIMATIONOFRE F D ALGAETOCHANGES IN LIGHT INTENSITY AND LIGHTQUALITYWERERECENTLYREVIEWED BY'ANTTET

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU AL   )T SHOULD BENOTEDD THAT COMPLEMENTARY CHROMATIC@ADAPTATIONTOREDORGREENLIGHT ASSTUD IED INCYANOBACTERIASUCHAS &REMY M ELLA EXCELLENTLY REVIEWED BY'ROSSMANETAL  HASNEVERBEEN CREDIBLY PROVEN IN ANY RED ALGA -ORE COMMONLY F IALLY NITROGENDEl NUTRIENTLIMITATIONEFFECTS ESPEC CIENCYACCOUNT FORC F HANGES INTHEPHYCO H BILIPROTEIN RATIOSINTHEREDALGAE )NCYANOBACTERIA 3Y 3 NECHOCY CYSTIS SP0##HAS BECOMEAMODELSYSTEMFOR F DETERMININGTHEGLOBAL CHANGESOFNUTRIENTSTRESSANDLIGHTSTRESS7ITHMOST OFT F HE GENESBEING AVAILABLE FO F R MICROARRAY A ANALYSIS (IHARAETAL  FO F LLOWEDD THECHANGESFROM F LOW LIGHTTOHIGHLIGHTTO—MOLPHOTONSMn SECn  F D THAT03)ANDD 03)) GENESWERE 4HEY GENERALLY FOUN DOWN REGULATED D BUT THE RESPONSE OFF 0"3 RELATED GENES VARIED 7HEREAS THE ALLOPHYCOCYANIN GENES WERE INITIALLY DOWNREGULATED T D HEY RECOVERED UN D LIKE PHYCOCYAN H INGENES4HEYSUGGESTTH T ATPHYCOCYAN H INAS THEPERIPHERALPHYCOBILIPROTEINISMORESTRICTLYREGU LATEDD THANALLOPHYCOCYANIN&URTHERMORE THE DOWN H BILIPROTEIN ANDD 03) AND 03)) REGULATION OFF PHYCO TRANSCRIPTSWASNOTEDTORESULTFROMTHEIMP M AIRMENTOF THEINORGANICCARB R ONUPTAKEMECHANISM7ANG ETAL  !NOTHERANALYSISONTHESAMECYANOBACTERIUM EXAMINEDTHERESPONSETOIRONDElCIENCY3INGHET AL  0"3 GENETRANSCRIPTSDECLINEDD WHENCELLS W WERETRANS F FERRE D TOTTHEDElCIENTCONDITIONS BUTAPP A AR ENTLYTHEPHYCOBILIPROTEINAND #HL CONTENTREMAINED ATA LEVEL SUFlCIENTTOCONTINUE INTHEPHOTOSYNTHETIC STATE!LTHOUGHSUCHANALYSESPROVIDEAGLOBALVIEW FOLLOW UPPHYSIOLOGICALANDBIOCHEMICALSTUDIESARE F H SPECIES&OREXAMPLE INEXAMINING NEEDED FOREAC THEEFFECTO F F NITRATEANDD SULFUR F LIMITATIONONPHYC H O BILIPROTEINSYNTHESISIN3YNECHOCYSTIS 2ICHAUDETAL  DID D NOTOBTAINTHEEXPECTED0"3 D DEGRADATION F PREVIOUS STUDIES ON ANDD CHLOROSIS PREDICTED FROM 3 3YNECHOCOCCUS  #LEARLYDIFFERENCESINREGULATION OR F HEIRRESULTS SIGNALING ARESUGGESTED FROMT 
"TTPDJBUJPO
XJUI
1IPUPTZ TZTUFN
**
&OFSSHZ %JTUS J SJCVUJUJPO
BOE %JTT J JQBU J UJPO 4HAT0"3ARESTRUCTURALLYANDFUNCTIONALLYASSOCIATED WITH03)) H INRH R ODOPHYTESANDD CYANOBACTERIAHASBEEN DEMONSTRATED BYISOLATIONOFF 0"303))PARTICLESWITH T 03))ACTIVITYASSHOWNBY/ EVOLUTION ANDD REDUC TIONOFE F LECTRONACCEPTORS'ANTTETAL    F R !LTHOUGH ATTHISTIMETHEREISNO DIRECTEVIDENCE FO ADIRECTPHYSICALCOUPLINGOF0"3STO03) ASVERIlED BY ISOLATIONOF FUNCT F IONAL COMPLEXES IT ISCERTAINLYA POSSIBILITY3TATE)ANDD STATE))CHANGEMEASUREMENTS

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT AND D THESUGGESTIONTHAT0"3SAREMOBILEWHIL W E03)) UNITSARENOT HAVEBEENINTERPRETEDASEVIDENCEFORA 0"3 03)ASSOCIATION*OSHUAAN H D -ULLINEAUX   F BETWEEN03))AND03)WOU D LD BE !SHUTTLINGOF0"3 ANATTRACTIVEMECHANISMTOAVOIDPHOTOINHIBITION 0HOTOINHIBITION AND ITS ATTENDANT PHOTODAMAGE W ENTHEENERGYOF LIGHTABSORB R ED BY AREAPROBLEMWH THEANTENNASYSTEMSCANNOTBEUTILIZEDOROTHERWISE DISSIPATED #HAPTERS  0OGSONETAL #HOWAND !RO /NEMIGHTEXPECT RED D ALGAEAND CYANOBACTERIA W WHICHHAVELOWLIGHTCOMPENSATIONPOINTS TOBEMORE SENSITIVETOPHOTOINHIBITION&URTH T ERMORE TH T EY DONOT HAVEA HIGHLYVARIED D CAROTENOIDCOMPOS D ITIONNORDO THEYHAVEANOBVIOUSXANTHOPHYLLCYCLE WHICHCOULD PLAY A AROLE INMINIMIZING DAMAGEFROMOVER EXC F ITA TION/NTHECONTRARY RRHODOPHYTESAN H D CYANOBACTERIA HAVEBEENSHOWNTOBERATHERRESISTANTTO PHOTODAM AGE FROM F HIGH LIGHTAND D TOHAVERAPID D RECOVERY TIMES F HIGH LIGHTEXPOSURE 'ANTTETAL  #LASSIC AFTER MANIFESTATIONS OF PHOTOINHIBITION SUCH AS THAT IN HIGHERPLANTSINVOLVING LIGHT DEPENDENTPHOSPHORYLA TIONOFF 03)) HAVENOTBEENCLEARLY DEMONSTRATED IN PHYCOBILISOME CONTAININGORGANISMS"IGGINSETAL -C#ONNELLETAL 'ANTTETAL   )NGREENPLANTSANEXCITATIONTRANSFERCONNECT F ION BETWEEN03))AND03)STATECHANGE CANBEBROUGHT ABOUT BY PHOSPHORYLATION OF F ,(#)) POLYPEPTIDES AND D THEIRMIGRATIONTO03) #HAPTER #HOWAND !RO 7ITHTHEAPPARENTLACKOF,(#))POLYPEPTIDES INRED D ALGAEAND D THEIRCOMPLETEABSENCE INCYANO BACTERIA THISMECHANISMOFPREVENT F INGDAMAGE FROM F OVER EXCITATIONOF2#ISNOTLIKELY3TATECHANGES INGREENPLANTS ASMEASURED BY ADECREASE IN03)) mUORESCENCE OCCURRELATIVELYSLOWLY WITH DURAT D IONS OF MINUTES )N ALGAE WITH 0"3S THE APPARENT STATE F GNITUDE FASTER AN F D THEIN CHANGESAREORDERSOFMA CREASEOF03) F mUORESCENCEAPP A EARSTO BEINDEPENDENT OFTHEQUENCHINGIN03))mUORESCENCE"RUCEETAL  $ELPHINETAL   7HATALTERNATIVESEXIST IN0"3 CONTAININGORGAN ISMS TO DISSIPATE ENERGY AND PREVENT PHOTOINHIBI TION IFF THEENERGY ISNOTREDISTRIBUTED D ATTHELEVEL OF REACTIONCENTERS$ISSIPATIONTHROUGH mUORESCENCE OR THERMAL CONVERSION ARE RECOGNIZED ALTERNATIVES BUTDIRECTEVIDENCE ISSTILL LACKING&ROMWH W OLECELL mUORESCENCESTUDIES INCYANOBACTERIA 3ARCINA ETAL  FAVORTHEHYPOTHESISTHATOVER EXCITATIONFROM EXCESS0"3ENERGY R YTO2#ISPREVENTED BYY THEPHYSICAL DETACHMENTOFF THE0"3AND D THEIRMOVEMENTTOAND ATTACHMENT TO 2#7ITH THEIR mUORESCENCE ASSAY USINGWH W OLECELLS THEY HAVEEXAMINED D ANUMBEROF



F HEMAS CYANOBACTERIAANDD THEYCHARACTERIZEALL OFT HAVINGMOBILE0"3S!CCORDINGTOTHEIRINTERPRETA TION THE2#CENTERSARESTATIONARY BUTTHE 0"3SARE MOBILEANDD THEIRMOBILITYISLIMITED BYTHEIRSIZE)F THISISTRUE WHYISITPOSSIBLETOISOLATEACTIVE0"3 03))PARTICLES BUTNOT0"3 03)PARTICLES F IMENTSWASPERFORME F D !COMPELLINGSETOFEXPER THATCRITICALLYADDRESSEDTHESTATE)AND))TRANSITIONS IN THE UNICELLULAR REDD ALGA 2HODELLA VIOLACEA AND ELUCIDATEDD CRITICAL FACTORST F THATRESOLVEAPP A ARENTINCON SISTENCIES$ELPHINETAL  4HESEEXPERIMENTS SHOWEDD THATINHIBITIONOFPROTE F INPHOSPHORYLATION DID NOT INmUENCE THE STATE TRANSITIONS THUS SUPPORTING PREVIOUSlNDINGS"IGGINSETAL  4HEYFURTHER DEMONSTRATEDD THATTHE03))QUENCHED mUORESCENCE STATERESULTS FROMT F HE 6P(ACROSSTHEMEMBRANEAND NOT FROM THE REDUCTION OF THE PLASTOQUINONE Q POOL %VENVERY LOWINTENSITIESOF GREENLIGHTSTATE))LIGHT ABSORBEDONLYBY0"3ANTENNAOF03)) WERESUF F lCIENTFORINDUCINGTHE 6P(4HE03)) mUORESCENCE QUENCHING WAS RELAXED D UPON UNCOUPLING THE 6P( GRADIENTAND BYACTIVATIONOFF !40ASE HENCE ITWAS PROPOSEDTHATTHEQUENCHINGRELAXATIONISRELATEDTO THEUTILIZATIONOFF THE 6P( BY!40SYNTHASE $ELPHIN ETAL  4HECOLLECTIVERESULTSMAKECLEARTHAT SUBSTANTIALDIFFERENCESEXISTBETWEENREDALGAEAND GREENPLANTS INTHEDISTRIBUTIONOFEXC F ITATIONENERGY Y 2ECENTLY -C#ONNELL ET AL  CAME UP WITH A COMBINED MODEL OF REGULATED EXCITATION ENERGY DISTRIBUTIONINCYANOBACTERIAWHI W CHENCOMPASSESAN F IOUSRESULTSAND INTERPRETATIONS EXPLANATION FORVAR 4HEIRMODELALLOWSASIGNIlCANTPORTIONOFEXCITATION F D FROM0"3S F DIRECTLY TOTHE ENERGY TO BETRANSFERRE REACTIONCENTERCHROMOPHORESOFF 03))$ISTRIBUTION TO03)WOULDONLYREQUIREASMALLMOVEMENTOF03) WITH REGARDD TOTHE0"3 03))COMPLEX7HEREASTHE OBSERVATIONSBY$ELPHINWERENOT DISCUSSED BY-C #ONNELLETAL ITSEEMSQUITEPROBABLETHAT CHANGESINTH T E 6P(MAY A BE INVOLVED INBRINGINGAB A OUT CONFORMAT F IONAL CHANGESOFF MEMBRANEPROTEINSAND HENCESOMEENERGYREDISTRIBUTION )F !CARY R OCHLORIS A #HL D CONTAININGPROKARYOTE D THATTENDSTO FORMECTOSYM F BIOTICRELATIONSHIPS -U RAKAMI ET AL  REPRESENTS A PRIMITIVE STATE IT IS AN INTERESTING EXAMPLE FFROM AN EVOLUTIONARY STANDPO D INT(UETAL  FOUN F D THATPHYCOCYAN H IN ANDALLOPHYCOCYANINAREPRESENTINTHISORGANISMBUT ITDOESNOT HAVECYANOBACTERIAL TYPE0"3STRUCTURE 5PONFRACT F IONATING !CARYOCHLORIS THY H LAKOIDS (UET AL FOUNDALLOPHYCOCYANINANDPHYCOCYANIN ASSOCIATEDD WITH THE03))PARTICLES BUTNOTWITH03)

 PARTICLES SUGGESTINGSTRUCTURAL ATTACHMENTTO03)) REACTIONCENTERSONLY3TUDIESTHUSFARHAVESHOWN R RELATIVELY LOW@PHYCOBILISOMEDENSITIESIN !CARYO CHLORIS BUTTHISCOULD BEDIFFERENT F INFREE F LIVINGCELLS W WHEREDEPENDENCEONPHOTOSYNTHESISISGREATER ***
1SPDIMPSPQIZUF
"OUFOOBT
BOE
UIF
*TJ" 1SPUFJOT )NMOSTCYANOBACTERIA 03))ISDEPENDENTON0"3SFOR THEACQUISITIONOFF ABSORB R ED LIGHTENERGY ASEXP Y LAINED D GENERAOF ABOVE(OWEVER THEREARETHREEUNRELATED CYANOBACTERIA 0ROCHLOROCOCCUS 0ROCHLORON AND 0ROCHLO R R RIX THAT HAVE LOSTTHEIR0"3SREVIEWED ROTH IN-ATTHIJSETAL 4INGETAL  )NSTEAD D T THEYEMPLOYMEMBRANE INTRINSIC#HL AB  ANT A ENNAS!LL THE PROCHLOROPHYTE#HL AB A ANTENNAPROTEINS 0CBS ARE RELATED D TO EACH OTHER AND D ARE PART OF F THE SAME PROTEINFAMILYASTHE #0AND #0APOPROTEINSOF THE03))CORE,AROCHEETAL VANDER3TAAY A ETAL REVIEWED IN'REEN  4HERE ISNO SEQUENCERELATEDNESSBETWEENTHESE#HL Q AB PROTEINS AND D THE#HL AB A PROTEINSOF HIGHERPLANTSAND GREEN D !CARYOCHLORIS 3ECTION ALGAE4HE #HL D CONTAINING ))! HAS#HL D BINDINGANTENNAPROTEINSTHATBELONG D D TOTHESAMEGENEFAM F ILY PRO Y BABLY ACQUIRED BY LATERAL GENETRANSFER F #HENETAL   %XTENSIVEBIOCHEMICALSTUDIESONTHE#HL AB  AN TENNAOF 0ROCHLO R R RIX VAN DER3TAAY ROTH A ETAL  AND D THE HIGH LIGHT ADAPTED D -EDSTRAINOF 0ROCHLO R ROCOCCUS 'ARCZAREKETAL  SUGGESTEDTHATIT WASASSOCIATED D PRIMARILYWITH03))(OWEVER OTHER EVIDENCEPOINTED D TOA03)ASSOCIATION 0OSTETAL  )TWASRECENTLYDISCOVEREDTHATINTHEMODERATE LOW LIGHT ADAPTED D -)4STRAINOF 0ROCHLO R R ROCOC

CUS THE0CB SUB U UNITSAREASSOCIATED D WITH 03))UNDER NORMAL GROWTH CONDITIONS 2ATHER THAN ENCIRCLING THE03)) DIMER THE0CBS FORM MEM F BERED D ARCSON EITHERSIDE "IBBYETAL A !SIMILARORGANI ZATIONHASBEENFOUNDINTHETHIRDPROCHLOROPHYTE 0ROCHLO R R DIDEMNI "IBBY ETAL B &IG  RON 4HIS ISACOMPLETELYNOVELORGANIZATION FORANY03)) F ANTENNA)NBOTHCASES SPECTROSCOPICANALYSISSUP PORTSTHETRANSFERO F F EXCITATIONENERGY FROMT F HE#HL AB ANTENNATO03)) (OWEVER INTHEVERYLOWLIGHT ADAPTED33STRAIN A PROTEINSWERE OF 0ROCHLOROCOCCUS 0CB#HL AB F FOUN D TO FORMAR F INGAROUND D THE03)TRIMER"IBBYET AL A )NTHECASEOF 0ROCHLOROCOCCUS -)4 THE0CB SUB U UNITSFOUN F D AROUND THE03))DIMERARE

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU ENCODED BYTHE PCB! B GENE WHI W CH ISCONSTITUTIVELY EXP X RESSED4HE03)03))DILEMMAWASSOLVEDWHENIT WAS DISCOVEREDD THATUNDERCONDITIONSOFF &E LIMITATION THE PCB" GENEISALSOEXPRESSEDD AND THE0CB" #HL AB PROTEINSFORMAN MEMBEREDRINGAROUNDTHE A 03)TRIMER HELPINGTOCOMPENSATEFORA F DECREASE IN THETOTALNUMBEROF03)UN F ITS"IBBYETAL A  4HE0CBSTHATASSOCIATEWITH03))DONOTSEEMTOBE INDUCE D D BY &E DEPRIVATION "IBBYETAL A 4HE LOCATIONOFF THE #HL A AB ANTENNA PROTEINSTHUSAPPEARS TO DEPEND ON THE SPECIES AND ON THE ENVIRONMENT F D W ERE IT IS FOUN WH 0ROCHLOROCOCCUS PREDOMINATES INTHETROPICALAND SUBTROPICALOCEANS WHICHTENDTOBEPOORINNITROGEN AND IRON4INGETAL  4HEREISNORED LIGHTAND LIMITEDD GREEN LIGHTATDEPTHSOFF nMW WHERE LOW LIGHT ADAPTED0ROCHLOROCOCCUS STRAINSPREDOMI NATE#ONSISTENTWITHTHEANTENNAROLE THENUMBER OF PCB GENES WITHIN THE 0ROCHLOROCOCCUS GENUS IS RELATEDTOTHELIGHTENVIRONMENTOFTHEECOTYPETHERE AREEIGHTGENESINTHEVERY LOW LIGHT ADAPTEDD STRAIN T EMODERATELY L LOW LIGHT ADAPTEDD STRAIN 33 TWO INTH -)4 AND ONLY ONE IN THE HIGH LIGHT ADAPTED STRAIN -%$ ,A2OCHE ET AL  'ARCZAREKK ET AL    4HESIGNIlCANCEOFTHEIRONCONNECTIONISTHATIRON ISOFTENT F HE LIMITING NUTRIENT INOCEANWATERS4ING F F CYANOBACTERIATO&ELIMITED ETAL  4RANSFERO CONDITIONSCAUSESDECREASEDSYNTHESISOF0"3SAND REACTIONCENTERS AND INDUCEST D HESYNTHESISOFA# F HL A BINDINGPROTEINCALLEDD )SI! "URNAPETAL  3ANDSTRÚMETAL  ANOTHERMEMBEROFTHECORE F ILY 'REEN   )SI! FFORMS A RING COMPLEX FAM OFMONOMERSAROUN F D 03) "IBBYETAL B "OEKEMAETAL  CLOSELYRESEMBLINGTHERINGOF D IN 0ROCHLO R R ROCOCCUS  0CB MONOMERSAROUND03) 4HISADDITIONSUB U STANTIALLY INCREASES ITSANTENNASIZE ANDTHEREBYCOMPENSATESFORTHEDECREASEDNUMBER F "IBBYETAL A"OEKEMAETAL OF03)CENTERS  4HE FACTT F HAT)SI! ALSOCALLED#0| APPEARS D TOBEASSOCIATEDONLYWITH03)ISPUZZLINGINVIEW F L LINESOFEV F IDENCETHATSUGGEST ITFUNCT F IONS OFSEVERA F LIGHTENERGY IE PHOTO TOPROTECT03))FROMEXCESS OXIDATIVEDAMAGE0ARKETAL )VANOVETAL  3ANDSTRÚM ET AL    (OWEVER IT HASRECENTLYBEENSHOWNTHATUNDERPROLONGED IRON DEPRIVATION THEREISANEXCESSOFFREE)SI! WHICH COULD FUNCT F ION TO SHIELDD 03)) FFROM EXCESS LIGHT 9EREMENKOETAL  

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT



&IG G 0ROCHLORON 03))DIMERCENTER WITHlVE PROCHLOROPHYTE#HL AB A 0CB MONOMERSINANARCABOVE ANDlVEMONOMERSBELOW ,IGHTGRAYSQUARES #HL A OR#HL B /THERATOMSBELONGTOTRANS MEMBRANEHELICES3CALEBAR  ¯#OURTESYOF*"ARBER 3EE#OLOR 0LATE &IG

*7
5IF
-)$
4VQFSGBNJMZ
PG
$IMPSPQMBTUT "
0WFSWJFX 4HEPREDOMINANTLIGHT HARVESTINGANTENNASOFEU F UKARY OTESCHLOROPLASTS AREMEMBERSOFT F HE LIGHT HARVEST INGCOMPLES,(# SUPERFAMILY4HE #HL A AB BINDING PROTEINSOFT F HE@GREEN LINE PLANTSAND GREENALGAE HAVE BEENTHEMOSTEXTENSIVELYSTUDIED PART D ICULARLY THOSEMEMBERSTHATAREASSOCIATEDWITH03))'REEN AND D $URNFOR F D D  0AULSEN  "ASSI ET AL D FFARR F I  VAN  *ANSSON  "ASSIAND#A !MERONGEN AND $EKKER  ! LARGE GROUP OF ECOLOGICALLY IMPORTANT ALGAE HAVE #HL AC BINDING PROTEINSTHATARECLEARLYRELATED D TOTHE#HL AB A PROTEINS OFTHEGREENLINE BUTITISNOTCLEARWHETHERANYOF THESE #HL PROTEINS ARE SPECIlCALLY ASSOCIATED D WITH 03)) ORWH W ETHERTHEYALL TRANSFEREXC F ITATIONENERGY TOBOTHPHOTOSYSTEMS-EMBERSOFTHEFAMILYINRED ALGAEBIND D ONLY#HL A AND D AREASSOCIATED D ONLY WITH 03) SOTHEYWILLNOTBEDISCUSSED FURT F HERBUTSEE 'ANTTETAL  4HISSECTIONWILLTHEREFORECON

CENTRATEONTHE#HL AB  ANTENNASOFHIGHERPLANTSAND GREENALGAE SINCE ITISTHIS GROUPWH W OSERELATIONSHIP TO03 )) IS BESTUNDERSTOOD -EMBERSOFTHISFAMILYAREALSOINVOLVEDINENERGY DISSIPATIONANDP D HOTOPROTECTION #HAPTER 0OGSON ETAL 4HEANTENNAPROTEINS PARTICULARLY ,(#)) ARE ABLETODISSIPATEENERGYDIRECTLYBYmUORESCENCEBUT ALSO BY THERMAL DISSIPATION WITH THE ASSISTANCE OF H LLS)N HIGHER CAROTENOIDS PARTICULARLYTHE XANTHOPHY PLANTS THE0SB3PROTEINPLAYSANIMPORTANTROLEIN THISENERGY DISSIPATION #HAPTER 0OGSONETAL  ! GROUP OFF PROTEINS CALLEDD THE %LIPS ORIGINALLY @EARLYLIGHT INDUCEDPROTEINS PLAYSAROLEINPHOTO PROTECTION)N FACT T F HE ,(#SUPERFAM F ILY MAY A HAVE D HOTOPROTECTIVEPROTEINSDEALINGWITH ORIGINATEDASP EXCESSEXCITATIONENERGY ANDONLYLATERDIVERSIlEDSO F IONTOABSORB R LIGHTINLOW LIGHT SOMEMEMBERS FUNCT K  'REEN ENVIRONMENTS 'REEN ANDD 0ICHERSKY AND+àHLBRANDT  

 #
5IF $IMPSPQIZMM
BC
'BNJMZ
PG
)JHIFS
1MBOUT  BOE (SFFO
"MH M BF

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU H LINEAGESDIVERGED 'REENANDD $URN ANDD STREPTOPHYTE FFORD 3TAUBERETAL %LRADAND'ROSSMAN -INAGAWAANDD 4AKAHASHI  


5IF
$IMPSPQIZMM
BC  1SPUFJO
1 $PNQMFYFT 
5ISFF%JNFOTJPOBM
J 4USVDUVSF
PG
-)$* U * 4HE#HL AB A PROTEINSOFF 03))ARE LISTED IN4ABLE ASREVIEWED D EARLIERBY'REENAND D $URNFOR F D  D "ASSIETAL  4HENATIVEFORMOF ,(#)) THE MAJ A ORCOMPLEX ISATRIMER)NTHETHYLAKOIDD MEM BRANE THEREAREANUMBEROFF TRIMERSPER03)) SOME CLOSELYANDSOMEMORELOOSELYASSOCIATEDWITH03)) COREDIMERS7HENTHE HIGHERPLANT,(#))ISDISSOCI ATED T D HREEPOLYPEPTIDESCANBERESOLVED BY SODIUM DODECYLSULFATE POLYACRYLAMIDEGELELECTROPHORESIS 3$3 0!'% OR REVERSED PHASE HIGH PERFORMANCE F LIQUID CHROMATOGRAPHY H 20 (0,#  ,HCB AND ,HCBAREVERYSIMILARINSEQUENCE ANDAREPRESENT INAPPROXIMATELY RATIO,HCB ISSHORTER AND ISA MINORCOMPONENT!NALYS L ISOFF PURIlED D ,(#))TRIMERS SHOWEDn#HLSPOLYPEPTIDEANDA#HL AB A RATIO OFF n%ACHMONOMERALSO BINDSTWOMOLECULES D ASU UB STOICHIOMETRIC OF LUTEIN ONEOFF NEOXANTHINAND AMOUNT OFF VIOLAXANTHINREVIEWEDIN-OROSINOTTOET AL   #0 AND D #0 CONTAIN ONE POLYPEPTIDE EACH ,HCB AND D ,HCBRESPECTIVELY ANDARECONSIDERED @CORE#HL AB PROTEINS BEING SITUATED BETWEEN,(#)) TRIMERSAND03))CORE D SEE BELOW 4HECOMPONENT #0,HCB ISALSOCONSIDEREDTOBEPARTOFTHE @COREOF03)) F BUTITSROLEHASNOTBEEN DETERMINED %VEN W WHEN ISOLATED BY THE SAME METHODS AS THE ,(#))TRIMER THEMINORCOMPLEXES#0AND#0 F HLSPERPOLYPEPTIDEAND HAVE HIGHER CONTAINFEWER# #HL AB RATIOSTHAN,(#))TRIMERS4ABLE )NAD DITION THEYAPPEARTOBINDONLYONELUTEINANDONE OTHER XANTHOPHYLL PER POLYPEPTIDE )T IS NOT CLEAR WHETHERTHISDIFFERENCE F INPIGMENTBINDINGIS DUETO D LOWERSTABILITYOFMONOMERICCOMPLEXESCOMPAREDTO TRIMERICONES ORWH W ETHERTHESEPROTEINSSIMPLY HAVE F FEWER BINDINGSITES#0 IS DIFlCULTTOWORK RK WITH ASITISRATHERUNSTABLERECONSTITUTED#0BINDS #HLSAND D CAROTENOIDS 0AGANOETAL   F BETWEENGREENALGAE 4HEREARESOMEDIFFERENCES ANDHIGHERPLANTS #HLAMYDOMONASREINHARDTII HAS #0AND#0 D HOMOLOGS BUTLACKSA#0 HOMOLOG 4ERAMOTOETAL %LRAD D AND 'ROSSMAN  -INAGAWAAND4AKAHASHI  &URTHERMORE THE MANY N CLOSELY RELATEDPO D LYPEPTIDESTHATMAKEUPTHE MAJ A OR,(#))CANNOTBEASSIGNED D TO,HCB ,HCBAND ,HCBCATEGORIESTHISSUGGESTSTHATTHEDIVERSIlCATION Y IDES HAPPENED D AFTERT F THECHLOROPHYTE OFF ,(#))POLYPEPT

4HESTRUCTUREOF PURIlED PEA ,(#)) CONSISTINGOF T TRIMERSCONTAININGTHE,HCBAND,HCBPROTEINS WAS lRSTSOLVEDTO¯RESOLUTIONBY ELECTRONCRYSTAL LOGRAPHY H +àHLBRANDT ET AL   )T SHOWEDD THAT EACHPOLYPEPTIDECHAINHADTHREEMEMBRANE SPANNING HELICES WITH TH ASMALL AMPHIPATHI T CALPHA HELIXATTH T E# TERMINUS(ELIX$IN&IG 4HE lRST" ANDD THIRD ! HELICES WHICHSHARESUBSTANTIALSEQUENCESIMILARITY F LDD AXISANDD CONNECTED BY ARERELATED BY ALOCAL TWO FO RECIPROCAL CROSS BRIDGESLINKING!RGONONE HELIXAND 'LUONTHEOTHER4WOCAROTENOIDMOLECULESASSIGNED TOLUTEIN WERE LOCATED INTHEGROOVESONEITHERSIDEOF THEPAIRED HELICES CROSS BRACINGTHEM!TTHIS LEVEL OFRESOLUTION EACHCAROTENOIDHEAD GROUPAPPEARED TOBEINCLOSEPROXIMITY TOOR#HLS ANDD TOBEPRO TECTED BYONEOFF THE LOOPSCONSERVED INALLMEMBERS A FAMILY+àHLBRANDTETAL 'REEN OFTHE #HL AB ANDD +àHLBRANDT  4HE  #HLS WERE ARRAY A ED INTWO LAYERSCORRESPONDINGTOTHETWOGALACTOLIPID LEAmETSOFTHEMEMBRANE!TTHISRESOLUTION ITWAS NOTPOSSIBLETODISTINGUISH BETWEEN#HL A ANDD #HL B SINCETHEY DIFFERON F LY BYAMETHY H L ORFORMY F LATONE POSITION&IG "ASEDONINTERMOLECULARDISTANCES  MOLECULESWERETENTATIVELY ASSIGNEDD TO#HL A ANDD  TO#HL B +àHLBRANDTETAL   4HESTRUCTUREOFTHESPINACH,(#))TRIMERHASRE CENTLYBEENDETERMINEDAT¯ RESOLUTIONBY 8 RAY A CRYSTALLOGRAPHY US H INGANOVELCRYSTALLIZATIONMETHOD ,IUETAL  4HENEWSTRUCTURECONlRMSTHAT THE PROTEIN HAS THREE MEMBRANE SPANNING HELICES CROSS BRACED BY TWO LUTEIN MOLECULES &IGS   ANDCOLOR0LATE8 )NADDITIONTOTHESMALLHELIX$ ONTHE LUMENALSURFACE ANOT F HERSMALL HELIX % IS IN THECORRESPONDINGPOSITIONONTHEOTHERSIDEOFT F HE TWO FOLDAXISRELATINGTHElRSTANDTHIRDHELICES!S SUGGESTED FROMRECONST F ITUTIONANDD MUTAGENESISEX PERIMENTS2OGLANDD +àHLBRANDT #ROCEETAL A ANEOXANTHINISBETWEENTHECROSSEDHELICES " ! ANDD THEMIDDLE HELIX # &IG !LL THREEOF THESECAROTENOIDSARECLOSEENOUGH TO#HLSTOTRANSFE F R ENERGYTOTHEM4HEFOURTHCAROTENOID BINDINGSITEISA W CH ISOCCUPIED BY CREVICEBETWEENMONOMERUNITS WHI VIOLAXANTHI T NANDD OTTHERXANTH T OPHY H LL CYCLECAROTENOIDS &IG 4HERELATIVELYEXPOSEDPOSITIONOFTHISCAROT ENOIDD MAAY ACCOUNTFORT F THESUB U STOICHIOMETRICAMOUNTS

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT



4ABLE #HLOROPHYLL AB A ANTENNASOFP F LANTAND GREENALGAL0HOTOSYSTEM)) #HL PROTEINCOMPLEX PREPARATION

0OLYPEPTIDES

#HL APOLY PEPTIDE

#HL BPOLY PEPTIDE

#AROTENOIDS

2EF F

,(#))CRYSTAL

,HCB ,HCB





,UT .X  8

A

,(#))CRYSTAL

,HCB ,HCB





,UT

B

,(#))BIOCHEMICAL

,HCB ,HCB ,HCB

n   

n   

,UT .X 6X ,UT .X 6X ,UT  .X  6X ,UT .X 6X

C D E F

#0 BIOCHEMICAL

,HCB





,UT .X  6X ,UT .X 6X

G H

#0BIOCHEMICAL

,HCB





8 .X 6X ,UT .X 6X

I H

#0 BIOCHEMICAL

,HCB





,UT 6X

I

,UT LUTEIN .X NEOXANTHIN6X VIOLAXANTHIN8 XANTHOPHYLL CYCLECAROTENOID A ,IUETAL  B +àHLBRANDT ETAL   C .USSBERGERETAL  D 2OGL ANDD +àHLBRANDT   E 2EMELLIETAL  F 0ETERMANETAL  G "ASSI ET AL   H $ASANDD &RANK  I "ASSIETAL  

&IG G  3TEREOGRAMOF,(#))MONOMER NUMBEREDACCORDINGTO,IUETAL  4ABLE "LACK #HL AGREY #HL BLIGHTGREY CAROT ENOIDS RIBBONS _ HELICES#OURTESYOF72#HANGAND:&,IU 

FFOUND INMOSTPURIlEDCOMP D LEXES 4ABLE  W CHCAN 4HENEWSTRUCTUREHAS#HL MOLECULESWHI BECLEARLYIDENTIlEDAS  #HL A AND  #HL B 4HREE OFF THEPORPHYRINRINGSTENTATIVELYASSIGNEDTO# D HL A OR B INTHESTRUCTURE HAVE BEENREASSIGNED AN D D TWO NEW #HLS HAVE BEEN RESOLVED BOUND BETWEEN MONOMER UNITS IN THE TRIMER &IG  "ECAUSE A LARGE NUMBER OF F STUDIES SINCE  HAVE USED D THE #HLNUMBERINGOFTHE+àHLBRANDTETAL PA PER THE#HL DESIGNATIONSOF BOTHMODELSARELISTED

IN4ABLE-OSTOFT F HE FORM F MYL GROUPSOF #HL B ARE ( BONDED D INCONTRASTTOTHEMETHY H LGROUPSOFF #HL A WHICHAPPEARTOBEINHYDROPHOBICENVIRONMENTS BUT ITISNOT KNOWNW K WHETHERTHESESMALL DIFFERENCES F AREENOUGH TO DETERMINE BINDINGSPECIlCITY4HERE ARE EXTENSIVE HYDROPHOBIC INTERACTIONS BETWEEN MONOMERUNITS INVOLVING NOTONLYTHEPROTEIN THE XANTHOPHY H LL CYCLECAROTENOID D AND SEVERAL#HLS BUT ALSOTHESINGLE PHOSPHATIDYLGLYCEROLMOLECULETHAT ISANINTEGRALPARTOFF THESTRUCTUREAND IS KNOWNTO K



#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU

&IG G  #HLOROPHYLL STRUCTURES ! #HL A WITHMODIlCATIONSGIVING#HL DAND D #HL B CIRCLED 4HE PHYTYLSIDECHAINISNOTTOSCALE" #HL C 2  #(#( AND C 2  #(#( !NEWNON POLARFORMISESTERIlEDWITH MONOGALACTOSYLDIACYLGLYCEROLCIRCLE 2 ATTHE POSITION'ARRIDOETAL  

BE ESSENTIAL FFOR TRIMERIZATION (OBE ET AL  .USSBERGERETAL    .PMFDVMBS #JP J MPHJDB J M "QQSPBDIFT /BUJ U WF BOE 3FDPNCJO J BOU
$PNQMFYFT )N THE DECADE BETWEEN THE PUBLI U CATION OFF THE TWO ,(#))STRUCTUREMODELS THEREWEREA LARGENUMBER OFSTUDIESAIMEDATDETERMININGTHEPOSITIONSOFTHE PIGMENTSMOREPRECISELY4 Y HEYWEREMADEPOSSIBLE BYTHE DISCOVERYTHAT ,(#))COULD BERECONSTITUTED F FROMPURIlEDPIGMENTSANDPOLYPEPTIDESSYNTHESIZE D IN %COLI I AND D THATTHERECONSTITUTEDP D IGMENT PROTEIN D CRYSTAL STRUCTURESAND D SPECTROSCOPIC COMPLEXES HAD PROPERTIESALMOSTINDISTINGUISHABLEFROMTHENATIVE COMPLEXES (OBEETAL 0ETERMANETAL  REVIEWED BY0AULSEN  !SARESULT ITWASPOS SIBLETOMAKESITE DIRECTEDANDDELETIONMUTANTS AND TODETECTAMOTIFF REQUIRED FO F R TRIMERIZATIONOF,(#)) F (OBEETAL  4HE #0PROTEIN,HCB HAS ALSOBEENUSEDEXTENSIVELYINRENATURATIONSTUDIES PROBABLY DUETO D ITSRELATIVESTABILITYASAMONOMER 0ASCAL ETAL  

)N GENERAL RECOMBINANT #HL PROTEIN COMPLEXES BINDD ASMUCH#HLASNATIVECOMPLEXES ISOLATED FROM F DETERGENT FRACTIONATED MEMBRANES (OWEVER THE PIGMENTRATIOSOFF THERECOMBINANTCOMPLEXES DEPEND STRONGLY ON THE MIXTURE OF F PIGMENTS PRESENTEDD TO THERENATURINGPROTEIN-EYERAND7ILHELM  0AULSEN  &OREXAMPLE IT ISPOSSIBLETORECON STITUTE ,(#))ACTUALLYTHE,HCBPROTEIN WITH LITTLE ORNO #HL A +LEIMAETAL  AND #0CANFOLD F HL AB RATIOS'IUFFRAE F T CORRECTLYWITHAVARIETY OF# AL  4HERE IS ALSO CONSIDERABLE mEXIBILITY IN CAROTENOID BINDING #ROCE ET AL B (OBE ET AL   /NEOFT F HElRSTSTUDIESSHOWEDD THATSITEBA ONHELIX$WASPROBABLYA#HL A BINDINGSITE2OGL ANDD +àHLBRANDT   THIS WAS CONlRMED IN THE STRUCTURE ,IUETAL  (OWEVER ANUMBER OFSTUDIESSUGGESTEDTHATTHISSITEANDSEVERALOTHERS COULD BIND BOTH #HLS A AND B AND GENERALLY BOUNDD A F HEMINVITRO "ASSI ETAL 2EMELLI MIXTUREOFT ETAL 9ANGETAL  4HISWASDISPUTEDON SPECTROSCOPIC GROUNDS BY 2OGLETAL  ANDD NO EVIDENCE FORM F IXEDD SITESWAS FOUN F D INTHENEW,(#))

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT



&IG G  3TEREOGRAMOF ,(#))TRIMERVIEWEDFROMSTROMALSIDE SHOWINGPOSITIONSOF#HLS ANDCAROTENOIDS$ARK'REY #HL ALIGHTGREY #HL B RIBBONS _ HELICES#OURTESYOF72#HANGAND:&,IU 3EE#OLOR0LATE &IG 4ABLE  ,ABELSAND POSITIONSOF PIGMENTSIN CRYSTAL STRUCTURESOF,(#)) + +àHLBRANDTETAL 

,IUETAL 

2ESIDUEBOUND (ELIX HELIXNUMBER

#HL A

#HL A 

'LU (ELIX! 

#HL A

#HL A 

!SN (ELIX!

#HL A

#HL A 

'LN (ELIX! 

#HL A

#HL A 

'LU (ELIX"

#HL A

#HL A 

(IS (ELIX" 

#HL A

#HL A 



(ELIX " 

#HL A

#HL B 

'LN (ELIX# (/

#HL B

#HL B 

# ! LOOP(/

#HL B

#HL A 



(ELIX!

#HL B

#HL A 

(IS (ELIX$

#HL B

#HL B 

'LU (ELIX#

#HL B

#HL B 



(ELIX# 



#HL B 

4 4YR



#HL B 

6 L " # LOOP 6A P

STRUCTURE,IUETAL  4HESITE DIRECTED D MUTANTS DIDCORRECTLYSUPPORTTHEBINDINGOF#HL B IN SITES B B AND BB AND D THEIRINTERACTIONWITH NEOXANTHIN INTHENEIGHBORH R OOD D OF(E F LIX#2EMELLI ETAL #ROCEETAL A  F HEOTHER#HL PROTEINSARECONCERNED D IT 3O FARAST

SEEMSTO BEGENERALLYAGREEDD THATTHEREARE HIGHLY CONSERVED#HLBINDINGSITESINALLLIGHT HARVESTING F ILY Y INCLUDING THE MEMBERS OFF THE ,(# SUPERFAM REDD ALGAL PROTEINSTHAT BINDD ONLY#HL A ANDD AREAS SOCIATEDONLYWITH03)'RABOWSKIETAL  )N ,(#)) FOURO F F THESESITESCORRESPONDD TO A A

 A A A A AND A A WH W ERETHE#HLS ARELIGATEDTHROUGHTHECENTRAL-G BYSIDE CHAINS D ! lRSTAND D THIRD D TRANS MEMBRANE ON(ELICES"AND HELICES 4HEOTHERSITESARE B B AND B B W WHICHINVOLVEA'LUANDA'LNON(ELIX# SITE A A ATTHEEND D OF(E F LIX"AND D SITE B NOW A ONTHESMALL LUMENAL (ELIX$4HISWOULDD ACCOUNT VERYNICELYFORTHE#HL A AND#HL B OF NATIVE AND D DIESONRECONSTITUTED RECONSTITUTED#0(OWEVER STU #0SUGGESTED D THATSITES AA BA B B AND BB HADMIXEDOCCUPANCY INTHAT COMPLEX"ASSIETAL 3IMONETTOETAL  D SIMILARSTUDIES PROPOSED D THATTHEADDITIONAL #HL AND OF#0WASINASITECORRESPONDINGTO B A #ROCE ET AL   7HETHER THE MIXED BINDING SITESARE FOUN F D INVIVO AND HOWCLOSELYTHEMINOR COMPLEXESMIRRORTHESTRUCTUREOF,(#))AREOPEN QUESTIONS 
0SH S BOJ[BUJPO
PG
$IMPSPQIZMM
BC M "OUFOOBT
JO 5I 5IZMBLPJE
I .FNCSBOF U F5 UI 7E NOW HAVE A GENERAL IDEA OF HOW THE #HL AB 7 A ANTENNAS ARE ORGANIZED D AROUND THE 03)) CORE IN THE THY H LAKOID D MEMBRANE LARGELYBASED D ON ELECTRON MICROSCOPY OF NEGATIVE STAINED PREPARATIONS WITH ADVANCED IMAGE PROCESSING "OEKEMAETAL  A B .IELDD ETAL A BREVIEWED BYVAN !MERONGENAND$EKKER $EKKERAND"OEKEMA  )TWASAIDED BYTHE,(#))STRUCTURE +àHL BRANDTETA D L  WHI W CH H WASUSED TOMODEL TH T EMINOR Q RELATEDNESS COMPLEXES BECAUSE OF THEIR SEQUENCE 'REENAND D 0ICHERSKY  Y AND D MORERECENTLY BYY THE 8 RAYCRYSTALLOGRAPHICSTRUCTURESOFF CYANOBACTERIAL 03))CORES:OUNIETAL +AMIYAAND3HEN &ERREIRAETAL #HAPTERSn &IGURE SHOWS THE 03)) CORE DIMER OUTLINED IN THE CENTER SURROUNDED BY THE THREE MINOR COMPLEXES #0 #0AND D #04HEPOSITIONOFF #0ISBASED D ONITS CROSS LINKINGTO#0(ARRERETAL  SELECTIVE EXTRACTION"OEKEMAETAL B ANDITSABSENCEIN D S ,HCB ANTISENSELINE 9AKUS K HEVSKAET AN !RABIDOPSI AL  4HEPOSITIONOF#0WAS F DEDUCE D D ONTHE BASISOFCROSS LINKINGANDSELECTIVEEXTRACTION(ARRER ETAL -IAOETAL "OEKEMAETAL B  #0ISPLACEDNEXTTO#0 D BYELIMINATION AND BECAUSE ,HCB ANTISENSEPLANTSAREDElCIENTIN BOTHOFF THEM!NDERSSONETAL   -OST03))PREPARATIONSTHATRETAINTHEANTENNAS BINDTWOTYPESOF ,(#))TRIMERS WHICHHAVEBEEN D D @- FORSTRON F GLY AND D MODERATELY BOUND NAMED@3AN "OEKEMA ET AL A  0ARTICLES WITH ADDITIONAL

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU @, LOOSE TRIMERS ASSOCIATEDD WITH THEM ARE ALSO F FOUNDPRESUMABLYTHESECORRESPONDTO,(#))UNITS THATMOVE INTHEPLANEOFT F HEMEMBRANE DUR D INGSTATE TRANSITIONSANDD TRANSFEREXC F ITATIONENERGYTO03)4HE IMPLICATIONS OF 03)) SUPER COMPLEX ORGANIZATION FFOR ENERGY TRANSFER F ARE DISCUSSED IN DETAIL BY VAN !MERONGENANDD $EKKER  !NTISENSE PLANTS HAVE PROVIDED SOME SURPRISES !RABIDOPSI D S PLANTSCARRYING ANANTI ,HCB CONSTRUCT WERE SEVERELY DEPLETED IN BOTH THE MAJ A OR ,(#)) POLYPEPTIDES BUTHADNODECREASEINPHOTOSYNTHETIC K F,(#)) F ACTIVITY ORIN GRANASTACKING DESPITETHE LACKO U ANETAL   TRIMERS !NDERSSONETAL 2UB 7HATTHEYDIDHAVEWASANEXCESSOF,HCB#0 W CH FORME WHI F D TRIMERS WITH A SMALL AMOUNT OF F THE MINOR,HCBPROTEIN ANDD ASSOCIATEDD WITH 03))CORES INAMANNERALMOSTINDISTINGUISHABLEFROMTHE,(#)) TRIMERS IN &IG 4HIS DEMONSTRATES YET AGAIN THE F HE,(#SUPERFAM F ILY 'REEN  ADAPTABILITYOFT ATLEASTUNDERCONTROLLEDGROWTHCONDITIONS(OWEVER NEITHERTHESEPLANTSNORANTISENSELINES FOROT F HER#HL A PROTEINSPRODUCE AB D DASMANYSEE D DSASTHEWILD TYPE !RABIDOP O SIS UNDERlELDCONDITIONS!NDERSSONETAL 'ANETAG ETAL   
&OFSHZ S 5SBOTGFS
5 5S 4UVEJFT
PO
$IMPSPQIZMMM BC
1SS 1SPUFJOT 1 0ROGRESSIVEIMPROVEMENTSINISOLATIONOFNATIVECOM PLEXESANDD RECONSTITUTIONOF DElNEDCOMP D LEXESHAVE PERMITTED FASTERAN F D BETTERENERGYTRANSFERSTU F DIES REVIEWEDBYVAN!MERONGENAND $EKKER )N VIEWOFF THENEW,(#))STRUCTURE ,IUETAL  F mECTIONWILL BENEEDED INORDERTO SOMETIME FORRE INTERPRET BIOPHYSICAL RESULTS IN ITS LIGHT 7E HAVE THEREFORE F DECIDEDD NOTTOATTEMPTTOCOVERTHEWORK R F HELASTFEWYEARS F BUTTO DIRECTTHEREADERSATTEN OFT TIONTOSOMERELEVANTPAPERS FOREXAMPLE#ROCEET AL  'RADINARUETAL  -AETAL 0ALACIOSETAL 2OGL ETAL  /L SZOWKAETAL AND.OVODEREZHKINETAL  2EADERSSHOULD D ALSOCONSULTRECENT ISSUESOFF *OURNAL OF0HYS F ICAL #HEMISTRY " "IOCHEMISTRY *OURNALOF "IOLOGICAL#HEMISTRY *OURNALOF-OLECULAR"IOLOGY F HELATEST INSIGHTS ANDD 0HOTOSYNTHESIS2ESEARCH FORT $
"OUFOOBT
PG
"MH M BF
XJUI
$IMPSPQIZMM Z D 
5IF
$IMPSPQIZMM
BD
1SPUFJO  T !LLTHEALGAETHAT HAVETHE #HLS C C C ORC RATHER THAN #HL B ASACCESSORYPIGMENTAREBELIEVEDD TOHAVE

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT

&IG G  03)) CORE DIMER OUTLINED mANKED BY MINOR #HL AB A COMPLEXES#0 #0 AND#0 STRONGLYBOUND 3 AND D MODERATELYSTRONGLY BOUND - ,(#))TRIMERS)MAGE F FROME LECTRONMICROSCOPY AND IMAGEPROCESSING OFF PURIlED03)) D PARTICLESFROM F !RABIDO DOPSISTH T ALIANA 9AKUS K HEVSKAETAL  AND D EARLIERWORK RKCITEDD THEREIN COURTESYOF%"OE F KEMA 

ORIGINATED BY SECONDARY ENDOSYMBIOSISINVOLVINGA NON PHOTOSYNTHETICEUKARYOTE HOSTAND D A RED D ALGAL INTRACELLULARSYMBIONT'IBBS #AVALIER 3MITH -AC&ADDEN 'REENETAL  !LL THATREMAINSOFT F HEENDOSYMBIONTARETHEPLASTIDS PLUSMANYNUCLEARGENESTHATWERETRANSFERREDFROM THEENDOSYMBIONTNUCLEUSTOTHE HOSTNUCLEUSDUR D ING ESTABLI A SHMENTOFT F HEENDOSYMBI M OTICRELATIONSHIP4HE ALGAEWITH#HL C INCLUDETHEHETEROKONTS HAPTOPHYTES CRYPTOPHYTESAND DINOmAGELLATES4HESEARENOTSMALL EXOTICGROUPS ASONEMIGHTTHINK FROMREA F DINGMOST PLANTPHYSIOLOGYTEXTBOOKS)NFACT THEYDOMINATE THEMARINEENVIRONMENTAND D ACCOUNT FORASMUC F H AS D ONAGLOBAL SCALE &IELD D ETAL  OFF THE#/ lXED  4HESEALGAEHAVEMEMBRANE INTRINSICLIGHT HARVESTINGANTENNASTHATBIND D ONEOFT F HE#HLS C &IG " AS WELL AS #HL A AND D A VARIETY OFF CAROTENOIDS 'REENAND$URNFORD #ARONETAL  4HEIR POLYPEPTIDESAREMEMBERSOFF THE,(#SUPERFAM F ILY Y IE THEYARERELATED D TOTHE#HL AB PROTEINSOFT F HE @GREENLINEANDTHE03) ASSOCIATEDANTENNASOFRED W CH BIND D ONLY #HL A $URNFOR F D ETAL  ALGAE WHI $EANEETAL    PROTEINCOMPLEXESINHETEROKONTSAND 4HE #HL AC HAPTOPHYTESAREFREQUENT F LYREFERRE F D TOAS FUCOXAN F

THIN #HL AC A COMPLEXES�S BECAUSE FUCOXANT F HIN ISTHEPREDOMINANTCAROTENOID4HEHAPTOPHYTESARE DISTINGUISHED BY ASUB U STANTIAL FRACT F IONOFF  ACYLOXY DERIVATIVESOF FUCOXANT F HIN-INORAMOUNTSOFOT F HER CAROTENOIDS SUCH AS VAUCHERIAXANTHINAND HETEROXAN THIN MAY A BE SPECIlC TO CERTAIN HETEROKONT GENERA



#RYPTOPHYTES H HAVE ALLOXANTHIN ANDD CROCOXANTHIN IN THEIR ,(#S AND DINOmAGELLATES HAVE PERIDININ (ILLER  )NADDITION THE#HL AC A PROTEINSBIND THE XANTHOPHY H LLS INVOLVED IN NON PHOTOCHEMICAL QUENCHING VIOLAXANTHIN ANDORR DIADINOXANTHIN AND THEIRDE EPOXIDIZED DERIVATIVES ZEAXANTHIN ANTHER AXANTHINAND DIADINOXANTHIN 6 6ALUESFOR #HL A AC RATIOSOFPURIlED#HL PROTEIN COMPLEXES ARE GENERALLY n HIGHER THAN THE #HL AB RATIOINPLANTS(ILLER #ARONETAL  A -ACPHERSON AND (ILLER   4HE NUMBER OF D MOLECULES PER POLYPEPTIDE VARIES WITH CAROTENOID THESPECIESANDD THE PREPARATIONMETHOD BUTAPPEARS TO BE EQUAL TO OR GREATER THAN THE NUMBER OF #HLS PER POLYPEPTIDE ! � FROM F ,AMINARIA PURIlED BY ISO ELECTRIC FOCUS F ING HAD D  #HL A  #HL C AND  FUCOXANTHIN0ASCALETAL  2ESONANCE2A MANSPECTROSCOPY INDICATEDD THATAT LEASTSOMEOFF THE #HL A WERE BOUND D AT POSITIONS ANALOGOUS TO THOSE Q OF,(#))'IVENTHELACKOFSEQUENCECONSERVATION BETWEEN#HL AB A ANDD #HL AC PROTEINSINTHEREGION OFT F HESECOND HELIX(ELIX# THE#HL C COULD BEAS SOCIATEDWITHTHISHELIXSIMILARLYTO#HL BIN,(#)) POSSIBLY BOUNDD TOCONSERVED 'LNAND' D LURESIDUES D 3OMEOFF THECAROTENOIDSCOULD BEARRANGEDD AROUND THISHELIXASIN,(#))TRIMERS4HETWOMAJOR �S F HE DIATOM #Y # CLOTELLA CAN BEISOLATEDD ASTRIMERS OFT "àCHEL  SOBYANALOGYWITH TOBACCO,(#)) THERE COULD BE ROOM FFOR ADDITIONAL CAROTENOIDS AND #HLSBETWEENTHEMONOMERUNITS 5IZMBLPJE Z 0SSHBOJ[BUJPO
BOE &OFSHZ S 5SBOTGFS 5 0HY H LOGENETICANALYSISSHOWSTHATTHE#HL AB A BRANCH OFTHEFAMILYDIVERGEDFROMTHELINELEADINGTOTHE #HL AC A PROTEINS BEFORE F THE FFORMER SEPARATED INTO 03)) ANDD 03) ASSOCIATEDD COMPLEXES 'REEN AND $URNFORD $URNFORDETAL  4HISMEANS D HL PROTEINSANALOGOUS THATWECANNOTEXPECTTOlND# TOTHEMINOR#HL AB A PROTEINCOMPLEXES IE #0 #0AND#04HEREISNOEVIDENCEFORSPECIlCAS SOCIATIONOFF ANNYOFF THE#HL AC  PROTEINSWITH03)) IN F FACTT HINSECTIONS IMMUNO LABELEDD WITH ANANTI � ANTIBODYSHOWEDTHATTHE�SWEREUNIFORMLYDIS TRIBUTED INAPPRESSEDD ANDNON APPRESSE D D THYLAKOIDS ,ICHTLÏETAL A B0YSZNIAKAN K D' D IBBS   4HEREARENOGRANASTACKSINTHESEALGAEASTHEREARE INHIGHERPLANTS -OST OF F THE #HL AC  GENES THAT HAVE BEEN SE QQUENCED APPEAR TO BE MEMBERS OF FAMILIES WITH A A AND NUMEROUS ALMOST IDENTICAL SEQUENCES "HAY

 'ROSSMAN !PTETAL B$E-ARTINOET Q AL  (OWEVER SOMEOFTHE#HL A AC SEQUENCES R AND D THE OFF THE DIATOMS #YCLOTELLA AND 4HALASSIOSIRA RAPHIDOPHYTE H (ETEROSIGMA AREMORE DIVERGENT AND BRANCH WITHTHE REDALGALANDCRYPTOPHYTE03) A D S SOCIATED D ,(#SONPHYLOGENETICTREES $URNFOR F D ETAL %PPARD D AND 2HIEL %PPARD D ETAL  'REEN !RMBRUSTETAL  4HISSUGGESTS THAT THERE MAY A BE MEMBERS OF F THE FAM F ILY THAT ARE PREFERENT F IALLY ASSOCIATED D WITH 03)4HERE IS SOME SUGGESTIVEBIOCHEMICALEVIDENCEFORASSOCIATIONOF �SWITH03) "àCHELAND D 7ILHELM "ATHKE ETAL "àCHEL   3PECTROSCOPICDATASUGGESTSTHATMOSTOFTHE�S TRANSFERENER F GYTOBOTH PHOTOSYSTEMS-ACPHERSON D (ILLER  2ESEARCHONENERGYTRANSFERUPTO F AND THEBEGINNINGOFWASREVIEWEDBY-AC0HERSON AND D (ILLER  AND D EARLIERWORK RKWASREVIEWED BY (ILLER WITH ANEMPHASISONCAROTENOIDS 
5IF
1FSJEJOJO$IMPSPQIZMM
B
1SPUFJO
PG %JOPGMBHFMMBUF G T )N ADDITION TO THE #HL A AC PROTEINS TWO GROUPS OF H ALGAE HAVE UNIQUE EXTRINSIC ANTENNA CHROMOPHYTE COMPLEXES4HECRYPTOPHYTESHAVEPHYCOBILIPROTEINS WITH UNIQUE _ SUB U UNITS WHI W CH ARE NOT ORGANIZED INTOPHYCO H BILISOMESAND D AREPACKED INTOTHETHY H LA KOIDLUMEN&IG"3ECTION))!(ILLER  -ACPHERSONAND D (ILLER  4HE DINOmAGELLATES HAVEACOMPLETELYNOVELANTENNA THE PERIDININ #HL A PROTEIN ORGANIZEDASTRIMERSINTHETHYLAKOIDLUMEN .EITHERTHEPROTEINSEQUENCENORITSFO F LD ISRELATED TOANYTHINGELSEINTHEDATABANKS)TS DISTINGUISHING CHARACTERISTIC IS THATITBINDS MORE CAROTENOID D THAN #HL4HEMAJ A ORFORM F HASPERIDINSTO#HL A AND D THE MINOR@HIGHSALT FORM F HASPERIDININSTO#HL A 3HARPLESETAL  4HEPROTEINFORMSABOAT LIKE STRUCTUREWITH THALPHA HELICESASTH T ERIBSFOURPER F IDININS D AROUND EACH #HL A (OFMANNETA F L  ARECLUSTERED (ILLER -ACPHERSONAND(ILLER   4HE AVAILABILITY OFF THE HIGH RESOLUTION STRUCTURE ¯ OFTHEMAJORFORMTRIMERANDTHEFACTTHATTHE CAROTENOIDSPLAYAVERYPROMINENTROLEINLIGHT HAR VESTING MEANSTHATTHISPROTEINISAMODELSYSTEM FO F R D CAROTENOID #HL IN STUDYINGCAROTENOID PROTEINAND TERACTIONS #HL #HL ENERGYTRANSFERISALSOIMPORTANT BOTHWITHINAND BETWEENMONOMERS3PECTROSCOPIC STUDIES UP TO  WERE REVIEWED BY -ACPHERSON AND(ILLER 2ECENTSTUDIESHAVEEXTENDEDTHE

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU F )LAGANETAL ANALYSISTOTHEMINOR@HIGH SALT FORM  ANDTOADETAILEDMODELOFTHECAROTENOIDENERGY A ORFORM F 3HIMAETAL  (OW STATESINTHEMAJ EVER THERE ISNO DElNITIVEEVIDENCEFORASSOC F IATION OFTHETRIMERWITHEITHERPHOTOSYSTEM-ACPHERSON ANDD (ILLER   %
1IPUPQSPUFDUJPO
BOE
UIF
-)$
'BNJMZ M !SWELL ASHARNESSINGLIGHTENERGY PHOTOSYNTHETICOR GANISMSNEEDMECHANISMSTOCOPEWITHITSDELETERIOUS F DERTOAVOID DAMAGE FROM F F FREERA DICALS EFFECTS)NOR ANDD ACTIVE OXYGEN SPECIES EXCESS ENERGY MUST BE DISSIPATEDINONEOFTHREEWAYSBYINCREASEDELECTRON mOWIFF ELECTRONACCEPTORSAREAVAILABLE AS mUORES CENCEFROMT F HEANTENNACOMPLEXES ORBYTHERMAL DE ACTIVATION WHICHISREFERREDTOASNON PHOTOCHEMICAL F I -OROSINOTTOET QUENCHING "ASSIANDD #AFFARR AL  +RAUSEAND*A D HNS  (OLTETAL  REVIEWEDIN#HAPTER 0OGSONETAL 8ANTHOPHYLL CAROTENOIDS ARE ESSENTIAL FFOR NON PHOTOCHEMICAL QUENCHING AND INGREENPLANTSANDGREENA D LGAETHEY APPEARTOBEBOUNDTO03)) ASSOCIATEDMEMBERSOF THE,(#FAM F ILY)NT Y HE GREENLINE THEXANTHOPHYLL VIOLAXANTHINISDE EPOXIDIZEDD TO ZEAXANTHININTWO STEPSVIAANTHERAXANTHIN APROCESSACTIVATEDBYTHE DECREASE INLUMENALP(RESULTING FROM F INCREASEDD ELEC TRONmOWDOWNTHEPHOTOSYNTHETICELECTRONTRANSPORT CHAIN)NCONTRAST MOSTHETEROKONTALGAEHAVETHECA ROTENOID DIADINOXANTHIN WHI W CHUNDERGOESAONE STEP DE EPOXIDATIONTO DIATOXANTHIN ,OHRAND7 D 7ILLHELM  4HEINCREASEINDE EPOXIDIZEDXANTHOPHYLLS LEADSTOANINCREASE IN THERMAL DISSIPATIONMEDIATED BYTHECAROTENOIDS)N HIGHERPLANTS THEFOUR F HELIX 0SB3PROTEINHASBEENSHOWNTOPLAYACRUCIALROLEIN NON PHOTOCHEMICALQUENCHING ,IETAL   "ERGANTINOETAL  4HERE ISALSOEVIDENCE THATCONFORMATIONALCHANGESINTHE#HL AB A PROTEINS THEMSELVES ASWELLASTHEMIGRATIONOFF AFRACT F IONOF THE ,(#)) POPULATION BETWEEN GRANA AND D STROMA AREALSOINVOLVEDINNON PHOTOCHEMICALQUENCHING +RAUSEAND*A D HNS   4HEMARINEENVIRONMENTINHABI A TED BY DIATOMSTHE BEST STUDIEDOFTHE #HL AC A ALGAE ISCHARACTERIZEDBY LARG R EANDD OFTENRAP F ID mUCTUATIONS INLIGHTINTENSITY T DUE D TOWATERMOVEMENTSASWELLASCLOUDD COVER!CCLIMA TIONTOHIGHLIGHTISFASTERTHANINPLANTS,AVAUDETAL A  ANDD THENON PHOTOCHEMICALQUENCHING IS PROPORTIONAL TOTHEAMOUNTOF DIATOXANTHIN PRODUCE D D ,AVAUDETAL A )TDOESNOTINVOLVEMOVEMENT OF# F HL PROTEINSINTHEPLANEOFT F HEMEMBRANE SINCE

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT THERE ISNO LATERAL SEGREGATIONIN#HL AC  ALGAE"OTH VIOLAXANTHINANDDIADINOXANTHINCYCLESCANOPERATE D 7ILHELM  %XPOSURE ATTHESAMETIME ,OHRAND TOHIGH LIGHT INTENSITYOVERAPERIOD D OFMORET F HANA F FEWHOURSCAUSESANINCREASEINTHEXANTHOPHYLLPOOL N PHOTOINHIBITIONAPPEARSTO BERAPIDLY SIZE BUTANY D  #HLORORESPIRATION REVERSIBLE4INGAND/WENS  MAYINCREASETHEPROTONGRADIENTINTHEDARK4ING D /WENS *AKOBETAL  $IJKMANAN K D AND + +ROON  BUTCOULDD ALSO BEPROVIDINGARUN OFF PATHWAYTO / INTHELIGHT ,AVAUDETAL B  )N HIGHERPLANTSAND GREENALGAE THEREARESEVERAL F ILYTHATBIND LITTLEOR MEMBERSOFF THE,(#SUPERFAM NO#HLANDAPPEARTOFUNCTIONONLYINPHOTOPROTEC TION 'REEN AND D +àHLBRANDT  -ONTANÏ AND +LOPPSTECH (EDDAD D AND! D DAMSKA  4HE THREE HELIX %LIPSANDTWO HELIX 3EPSAREINDUCEDIN D ARE BELIEVED D TOPLAY AY AROLE IN RESPONSETO HIGH LIGHT AND PHOTOPROTECTION(EDDAD D AND !DAMSKA (UTIN ETAL  (OWEVER NOGENESENCODING0SB3OR F HESEPROTEINS HAVEBEEN FOUN F D INTHEGENOME ANY N OFT 4 ALASSIOSIRAPSEUDONAN D A !RMBRUST OFF THE DIATOM 4H ETAL  4HISSUGGESTSTHATALTHOUGHTHEXANTHO PHYLL CYCLESPLAY A AN IMPORTANTROLEINTHE#HL AC  LINE THE MOLECULAR MECHANISMS OFF NON PHOTOCHEMICAL QUENCHINGARENOTTHESAMEASINTHE#HL AB LINE "DLOPXMFEHNFOUT 7EWOULDLIKETOTHANK$RS7ENRUI#HANG :HENFENG 7 ,IU %GBERT "OEKEMA AND *AMES "ARBER FOR THEIR GENEROUSGIFTSO F F lGURES 3FGFSFODFT !NDERSSON* 7ALTERS2' (ORTON0AND*ANSSON3 !NTI SENSEINHIBITIONOFTHEPHOTOSYNTHETICANTENNAPROTEINS#0 AND#0)MP D LICATIONSFORT F HEMECHANISMOFPROTECT F IVEENERGY DISSIPATION0LANT#ELL n !NDERSSON* 7ENTWORTH- 7ALTERS2' (OWARD#! 2U D UBAN!6 D  !BSENCEOFT F THE,HCBANDD ,HCB (ORTON0AND*ANSSON3 PROTEINS OFF THE LIGHT HARVESTING COMPLEX OFF 0HOTOSYSTEM)) EFFECTS ON PHOTOSYNTHESIS GRANA STACKING AND lTNESS 0LANT *n !PT+% #OLLIER*,AND'ROSSMAN!2A %VOLUTIONOFTHE PHYCOBILIPROTEINS*-OL"IOLn !PT+% #LENDENNEN3+ 0OWERS$!AND'ROSSMAN!2 D B 4HEGENE FAM F ILYENCODINGTHEFUCOXANT F HINCHLOROPHYLL PROTEINS F FROMT HE BROWNALGA -ACROC CYSTISP PYRIF IFERRA-OL 'EN'EN n !RMBRUST%6 "ERGES*! "OWLER# 'REEN"2 -ARTINEZ$ 0UT NAM.( :HOU3 !LLEN!% !PT+% "ECHNER- "RZEZINSKI



-! #HAAL"+ #HIOVITTI! $AVIS!+ $EMAREST-3 $ETTER*# 5 (ILDEBRAND- 'LAVINA4 4 'OODSTEIN$ (ADI-: (ELLSTEN5 *ENKINS"$ *URKA* +APITONOV66 +RÚGER. ,AU779 ,ANE 47 ,ARIMER&7 ,IPPMEIER*# ,UCAS3 -EDINA- -ONTSANT ! /BORNIK- 0ARKER-3 0ALENIK" 0AZOUR'* 2ICHARDSON 0- 2YNEARSON4! 3AITO-! 3CHWARTZ$# 4HAMATRAKOLN + 6ALENTIN+ 6ARDI! 7ILK 7 ERSON&0AND D 2OKHSAR$3 L PSEUDONAN D A %COLOGY Y 4HE GENOMEOFF THE DIATOM 4HALASSIOSIRA EVOLUTION AND D METABOLISM3CIENCEn "ASSI 2AND#A D FFARR F I 3  ,HCPROTEINSAND D THEREGULATION OFPHOTOSYNTHETICLIGHTHARVESTINGFUNCTIONBYXANTHOPHYLLS 0HOTOSYNTH2ESn "ASSI2 3ANDONË$AND#ROCE2 .OVELASPECTSOFCHLORO PHYLL A AB BINDINGPROTEINS0HYSIOL0LANTn "ASSI 2 #ROCE2 #UGINI $AND D 3ANDONË$ -UTATIONAL ANALYSISOFA F HIGHERPLANTANTENNAPROTEINPROVIDESIDENTIlCATION OFF CHROMOPHORES BOUND INTOMULTIPLESITES0ROC.ATL!CAD D 3CI 53! n "ATHKE, 2HI 2 EL % +RUMBEIN7%AND D -ARQUARDT* "IO CHEMICALANDIMMUNOCHEMICALINVESTIGATIONSONTHELIGHT HAR VESTINGSYSTEMOFTHECRYPTOPHYTE 2HODOMONAS SP%VIDENCE FORA0HOTOSYSTEM)SPECIlCANTENNA0LANT"IOLn "ERGANTINO% 3EGALLA! "RUNETTA! 4EARDO% 2IGONI& 'IACOM ETTI '-AND D 3ZAABÛ) ,IHGT AND D P( DEPENDENTSTRUCTURAL CHANGES INTHE0SB3SUB U UNITOFF 0HOTOSYSTEM))0ROC.ATL!CAD 3CI 53!n "HAY A A $ AND D 'ROSSMAN !2  #HARACTERIZATION OF GENE CLUSTERSENCODING THE FUCOXANT F HINCHLOROPHYLLPROTEINSOFT F HE DIATOM 0HAEODACTYLUM TRICORNUTUM .UCLEIC!CIDS 2ES  n "IBBY43 .IELD* 0ARTENSKY&AND"ARBER*A !NTENNA RINGAROUND0HOTOSYSTEM).ATURE "IBBY43 .IELDD *AND "ARRBER* B ! 0HOTOSYSTEM)) LIKE PROTEIN INDUCE D D UNDER IRON STRESS FFORMS AN ANTENNA RING AROUND D THE 0HOTOSYSTEM ) TRIMER IN CYANOBACTERIA .ATURE n "IBBY 43 -ARY ) .IELDD * 0ARTENSKY&AND D "ARRBER* A ,OW LIGHT ADAPTED 0ROCHLOROCOCCUS SPECIESPOSSESSSPECIlC ANTENNAEFOREACHPHOTOSYSTEM.ATUREn "IBBY43 .IELD* #HEN- ,ARKUM!7$AND"ARBER*B 3TRUCTUREOFA0HOTOSYSTEM))SUPERCOMPLEXISOLATEDFROM 0RO R CHLORON R DIDEMNI RETAINING ITSCHLOROPHY H LL AB A LIGHT HARVESTING SYSTEM0ROC.ATL !CAD D 3CI 53!n "IGGINS* #AMPBELL#,AND D "RUCE$  -ECHANISMOFF THE LIGHT STATE TRANSITION IN PHOTOSYNTHESIS ))!NALYSIS OF F PHOS PHORYLATED D POLYPEPT Y IDESINTH T ERED D ALGA 0ORPHYRIDIUMCRUENTUM D  "IOCHIM"IOPHYS!CTAn "OEKEMA%* (ANKAMER" "ALD$ +RUIP* .IELD* "OONSTRA!& "ARBER*AND2ÚGNER- 3UPRAMOLECULARSTRUCTUREOFTHE 0HOTOSYSTEM))COMPLEXFROMGREENPLANTSANDCYANOBACTERIA 0ROC.ATL !CAD 3CI 53!n "OEKEMA%* VAN2OON( #ALKOEN& "ASSI 2AND D $EKKER*0 A -ULTIPLETYPESOFASSOC F IATIONOFF 0HOTOSYSTEM))AND ITS LIGHT HARVESTINGANTENNAINPARTIALLYSOLUBILI U ZED D 0HOTOSYSTEM)) MEMBRANES"IOCHEMISTRY  n "OEKEMA %* * VAN 2OON ( VAN "REEMEN *&, AND $EKKER *0 B 3UPRAMOLECULARORGANIZATIONOF0HOTOSYSTEM))ANDITS LIGHT HARVESTINGANTENNAINPARTIALLYSOLUBILIZED0HOTOSYSTEM)) MEMBRANES%UR*"IOCHEMn "OEKEMA %* VAN "REEMEN *&, VAN 2OON ( AND D $EKKER *0  #ONFORMAT F IONALCHANGESIN 0HOTOSYSTEM))SUPERCOM

 PLEXESUPONREMOVALOFF EXTRINSICSUB U UNITS"IOCHEMISTRY  n "OEKEMA%* (IFNEY! 9AKUSHEVSKA!% 0IOTROWSKI- +EEGSTRA 7 7 "ERRY3 -ICHEL+ 0 0ISTORIUS%+AND+RUIP* ! GIANTCHLOROPHYLL PROTEINCOMPLEXINDUCEDBYIRONDElCIENCY INCYANOBACTERIA.ATUREn "RUCE$ "IGGINS* 3TEINER4ANDD 4HEWALT- %XCITATION ENERGY TRANSFER F INTHECRYPTOPHYTES&LUORESCENCEEXCITATION SPECTRAAND PICOSECONDD TIME RESOLVEDD EMISSIONSPECTRAOF INTACT ALGAEAT+0HOTOCHEM0HOTOBIOL n "àCHEL# &UCOXANTHIN CHLOROPHYLLPROTEINSINDIATOMS ANDK$ASUBUNITSASSEMBLEINTODIFFERENTOLIGOMERICSTATES "IOCHEMISTRYn "àCHEL#AND7ILHELM# )SOLATIONANDCHARACTERIZATION OFF A0HOTOSYSTEM) ASSOCIATEDD ANTENNA ,(#) ANDD A0HOTO SYSTEM) CORECOMPLEX FROMT F HECHLOROPHYLL C CONTAININGALGA 0LEUROCHLORIS MEIRINGENSIS 8ANTHOPHYCEAE * 0HOTOCHEM 0HOTOBIOL "n "URNAP2, 4ROY O AN4ANDD 3HERMAN,! 4HE HIGHLY Y ABUNDANT CHLOROPHYLL PROTEIN COMPLEX OF IRON DElCIENT 3YNECHOCOC 3

CUS SP0###0| ISENCODEDBYTHE ISI!GENE0LANT 0HYSIOLn #ARON, $OUADY$ DE-ARTINO!AND1UINET- ,IGHT HARVESTING IN BROWN ALGAE #AHIERS DE "IOLOGIE -ARINE  n TH 4 -EMB M RANE HEREDITY T ANDD EARLYY CHLOROPLAST #AVALIER 3MITH EVOLUTION4RENDS0LANT 3CIn #HEN- (ILLER2' (OWE#*ANDD ,ARRKUM!7$ 5NIQUE ORIGIN AND LATERAL TRANSFER OF PROKARYOTIC CHLOROPHYLL B AND CHLOROPHYLL D D LIGHT HARVESTING SYSTEMS -OL "IOL %VOL  n #ROCE2 2EMELLI2 6AROTTO# "RETON*AND"ASSI2A 4HE F HEMAJ A ORLIGHTHARVESTINGCOMPLEX NEOXANTHIN BINDINGSITEOFT ,(#)) FROM F HIGHERPLANTS&%"3,ETTn #ROCE2 7EISS3ANDD "ASSI 2B #AROTENOID BINDINGSITES OFF THEMAJ A ORLIGHT HARVESTINGCOMPLEX))OF HIGHERPLANTS*"IOL #HEMn #ROCE 2 #ANINO ' 2OS & AND "ASSI 2  #HROMOPHORE ORGANIZATIONINTHEHIGHER PLANT0HOTOSYSTEM))ANTENNAPROTEINS #0"IOCHEMISTRY n #ROCE2 -ULLER-' "ASSI2AND(OLZWARTH!2 #HLO ROPHY H LL B TOCHLOROPHY H LL A ENERGYTRANSFER F KINETICSINTHE#0 ANTENNA COMPLEX ! COMPARATIVE FEMTOSECON F D ABSORPTION STUDY BETWEEN NATIVE ANDD RECONSTITUTEDD PROTEINS "IOPHYS * n $AS3+AND D &RANK (!  0IGMENTCOMPOSITIONS SPECTRAL PROPERTIES AND ENERGY TRANSFER EFlCIENCIES BETWEEN THE XANTHOPHYLLS AND CHLOROPHYLLS IN THE MAJOR AND MINOR PIG MENT PROTEINCOMPLEXESOF0HOTOSYSTEM))"IOCHEMISTRY  n $EANE*! &RAUNHOLZ- 3U6 -AIER5 ' -ARTIN7 $URNFOR F D D -C&ADDEN')  %VIDENCEFORNUC F LEOMORPHTO $'AND HOSTNUCLEUS GENETRANSFER, F IGHT HARVESTING COMPLEXPROTEINS FFROM CRYPTOMONADS ANDD CHLORARACHNIOPHYTES 0ROTIST  n $ELPHIN % $UVAL * # %TIENNE! , AND +IRILOVSKY $  6P( DEPENDENT 0HOTOSYSTEM)) mUORESCENCE QUENCHING IN D DUCEDBYSATURATING MULTITURNOVERPULSESINREDALGAE0LAN T 0HYSIOLn $E -ARTINO ! $OUADY $ 1UINET 3ZELY - 2OUSSEAU " #RÏPINEAU& !PT+ANDD #ARON,  4HE LIGHT HARVESTING

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU ANTENNAOFBROWNALGAE(IGHLYHOMOLOGUSPROTEINSENCODED BYAMULTIGENEFAMILY%UR*"IOCHEMn $IJKMAN.!AND+ROON"-! )NDICATIONSFORCHLORORESPI RATIONINRELATIONTOLIGHTREGIMEINTHEMARINEDIATOM 4HALAS SIOSIRA R WEISSmOGII * 0HOTOCHEM0HOTOBIOL"n $UFRESNE! 3A F LANOUB U AT- 0ARTENSKY & !RTIGUENAVE& !XMANN U RAT3 'ALPERIN-9 +OONIN%6 ,E'ALL & )- "ARB R E6 $UP -AKAROVA+3 /STROWSKI- /ZTAS3 2OBERT# 2OGOZIN)" 3CANLAN$* * 4ANDEAU DE-ARSAC. 7EISSENBACH * 7I 7 NCKER 0 7OLF9) 0 F AND (ESS 72  'ENOME SEQUENCE OFF THE CYANOBACTERIUM 0ROCHLOROCOCCUS MARINUS 33 A NEARLY MINIMALOXYPHOTOTROPHICGENOME0ROC.ATL!CAD3CI n $URNFORD $' $EANE *! 4AN 3 -C&ADDEN ') 'ANTT % AND 'REEN"2 !PHYLOGENETICASSESSMENTOFF THEEUKARYOTIC LIGHT HARVESTING ANTENNAPROTEINS WITH IMPLICATIONS FORP F LASTID EVOLUTION*-OL %VOLn %LRAD D $AND'ROSSMAN!2 D  !GENOMES EYEVIEWOFF THE LIGHT HARVESTING POLYPEPTIDESOF #HL # AMYD M DOMONASREINHARD R TII #URR'ENETn %PPARD-AND2HIEL% 4HEGENESENCODINGLIGHT HARVESTING SUBUNITSOF#YCLOTELLA # CRYPTICA "ACILLARIOPHYCEAE CONSTITUTE A COMPLEX AND HETEROGENEOUS FAMILY -OL 'EN 'ENET  n %PPARD D - +RUMBEIN7% VON(AESELER!AND D 2HIEL %  #HARACTERIZATIONOF FCPAN F D FCP TWOA F DDITIONAL GENESEN CODING LIGHT HARVESTING PROTEINSOF #Y # CLOTELLACRY RYPTICA "ACIL LARIOPHYCEAE ANDP D HYLOGENETICANALYSISOFF THISCOMPLEXGENE FAMILY0LANT"IOLn &ERREIRA+. )VERSON4- -AGHLAOUI+ "ARBER*AND)WATA3  !RCHITECTURE OF THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn &IELD#" "E D HRENFE F LD-* 2AN D DERSON*4AND D &ALKOWSKI 0  0RIMARY PRODUCT D IONOFF THEBIOSPHERE)NTEGRATINGTERRESTRIAL AND OCEANICCOMPONENTS3CIENCEn &RANK K (! 9OUNG!* "RITTON ' AND D #OGDELL 2* EDS  0HOTOCHEMISTRY OFF #AROTENOIDS +LUWER!CADEMIC 0UBLI U SH ERS $ORDRECHT 'ANETAG5 +àHLHEIM# !NDERSSON*AND*ANSSON3 )S EACHLIGHT HARVESTINGCOMPLEXPROTEINIMPORTANTFORPLANTlT NESS0LANT0HYSIOLn 'ANTT %  0HOTOSYNTHETIC CRYPTOPHYTES H )N #OX %2 ED 0HYTOmAGELLATES PP n %LSEVIER R.ORTH (OLLAND D !M STERDAM 'ANTT% %DWAR D DS-2AND D 0ROVASOLI , #HLOROPLASTSTRUCTURE OFF THE#RYPTOPHYCEAE%VIDENCEFORP F HYCOBILIPROTEINSWITHIN INTRATHYLAKOIDALSPACES*#ELL"IOL n 'ANTT% #LEMENT -ETRAL*$AND#HERESKIN"- 0HOTO SYSTEM)) PHYCOBILISOMECOMPLEXPREPARATIONS)N0ACKER, AND'LAZER!.EDS -ETHODSIN%NZYMOLOGY#YANOBACTERIA 6 L  n!CADEMIC0RESS 3AN$IEGO 6O 'ANTT % 'RABOWSKI " AND D #UNNINGHAM &8  !NTENNA SYSTEMSOFF RED ALGAEPHYCOBILISOMESWITH 0HOTOSYSTEM))AND CHLOROPHYLL COMPLEXESWITH 0HOTOSYSTEM))N'REEN"2AND Y THESIS 0ARSON77EDS ,IGHT (ARVESTING!NTENNASIN0HOTOSYNT PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT 'ARCZAREK, VANDER3TAAY'7- 4HOMAS*#AND0ARTENSKY&  )SOLATIONANDCHARACTERIZATIONOF0HOTOSYSTEM)FROM R R ROCOCCUS  TWOSTRAINSOFTHEMARINEOXYCHLOROBACTERIUM0ROCHLO 0HOTOSYNTH 2ES  n 'ARCZAREK K , (ESS72 (OLTZENDORFF* VAN F DER3TAAY A '7-AND

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT 0ARTENSKY&  -ULTIPLICATIONOFANTENNA F GENESASAMAJ A OR ADAPTATIONTOLOWLIGHTINAMARINEPROKARYOTE0ROC.ATL!CAD 3CIn 'ARCZAREK , VAN DER 3TAAY '7- (ESS72 ,E 'ALL & AND 0ARTENSKY& %XPRESSIONANDPHYLOGENYOFTHEMULTIPLE ANTENNA GENESOFT F HE LOW LIGHTADAPTEDD STRAIN 0ROCHLO R R ROCOC

CUS MARINUS 33 /XYPHOTOBACTERIA 0LANT-OL "IOL n 'ARRIDO*, /TERO* -AESTRO-!AND:APATA- D  4HEMAIN F %MILIANAHUXLEYI E 0RYMNESIOPHY NONPOLARCHLOROPHYLL C FROM CEAE ISACHLOROPHYLLC MONOGALACTOSYLDIACYLGLYCERIDEESTER ! MASSSPECTROMETRYSTUDY*0HYCOLn 'IBBS30 4HECHLOROPLASTSOFSOMEALGALGROUPSMAYHAVE EVOLVEDFROMENDOSYMBIOTICEUKARYOTICALGAE!NN.9!CAD 3CI n F HELLI' 3ANDONË$ #ROCE2 #UGINI $ 'ARLASCHI 'IUFFRA% :UCC D INGS2#  !NALYSISOFF SOMEOPTICAL &- "ASSI2AND*ENN PROPERTIESOFF ANATIVEANDD RECONSTITUTEDD 0HOTOSYSTEM))ANTENNA COMPLEX #0ˆPIGMENT BINDING SITES CAN BE OCCUPIED BY CHLOROPHYLL A ORCHLOROPHYLL B ANDDETERMINESPECTRALFORMS "IOCHEMISTRY n 'LAZER!.AND7EDEMAYER'* #RYPTOMONADBILIPROTEINSˆ ANEVOLUTIONARYPERSPECTIVE0HOTOSYNTH2ES  n 'RAB A OWSKI " #UNNINGHAM&8*RAND'ANTT% D  #HLOROPHY H LL AND D CAROTENOID BINDING INASIMPLEREDD ALGAL LIGHT HARVESTING COMPLEXCROSSESPHYLOGENETICLINES0ROC.ATL !CAD3C D I 53! n 'RADINARU## VAN'RONDELLE2ANDD VAN!MERONGEN(  3ELECTIVEINTERACTIONBETWEENXANTHOPHYLLSANDCHLOROPHYLLS IN,(#))PROBEDBYFEMTOSECONDTRANSIENTABSORPTIONSPEC TROSCOPY*0HYS#HEM"n 'REEN"2 7AS@MOLECULAROPPORTUNISMAFACTORINTHE EVOLUTION OF DIFFERENTP F HOTOSYNTHETICLIGHT HARVESTINGPIGMENT SYSTEMS0ROC.ATL!CAD3C D I53!n 'REEN"2 4HEEVOLUTIONOF LIGHT HARVESTINGANTENNAS)N D EDS ,IGHT (ARVESTING !NTENNAS 'REEN"2AND0ARSON77 IN0HOTOSYNTHESIS PPn+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT 'REEN"2AND$URNFORD$' 4HECHLOROPHYLL CAROTENOID PROTEINSOFOXYGENICPHOTOSYNTHESIS!NNU2EV0LANT0HYSIOL 0LANT-OL "IOL  n D +àHLBRANDT7  3EQUENCECONSERVATIONOF 'REEN"2AND LIGHT HARVESTING ANDD STRESS RESPONSEPROTEINS INRELATIONTOTHE F HOTOSYNTH THREE DIMENSIONAL MOLECULARSTRUCTUREOF,(#))0 2ESn 'REEN "2 AND 0ARSON 77 EDS  ,IGHT (ARVESTING !NTENNAS IN 0HOTOSYNTHESIS +LUWER !CADEMIC 0UBLISHERS $ORDRECHT 'REEN"2AND0ICHERSKY% (YPOTHESISFORTHEEVOLUTION OF THREE HELIX #HL A AB AND #HL AC LIGHT HARVESTING ANTENNA PROTEINSFROMTWO F HELIXAND FOUR F HELIXANCESTORS0HOTOSYNTH 2ES  n 'REEN"2 !NDERSON*-ANDD 0ARSON77  0HOTOSYNTHETIC D THEIRLIGHT HARVESTING ANTENNAS)N'REEN"2 MEMBRANESAND AND0ARSON77 D EDS ,IGHT (ARVESTING!NTENNASIN0HOTOSYN THESIS PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT 'ROSSMAN !2 VAN 7ASSENBERGEN ,' AND +EHOE $  %NVIRONMENTALREGULATIONOFPHYCOBILISOMEBIOSYNTHESIS)N 'REEN"2AND0ARSON77EDS ,IGHT (ARVESTING!NTENNAS IN 0HOTOSYNTHESIS PPn+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT



(ARRER2 "ASSI 2 4ESTI-'AND D 3CHÊFER# F  .EAREST NEIGH BOR ANALYSIS OF A 0HOTOSYSTEM)) COMPLEX FROM -ARCHANTIA POLYMORPHA ,LIVERWORT WHICHCONTAINSREACTIONCENTERAND ANTENNAPROTEINS%UR*"IOCHEMn (AXO&4 4HEWAVELENGTHDEPENDENCEOFPHOTOSYNTHESIS AND D THEROLEOFACCESSOR F Y PIGMENTS)N!LLEN-" ED #OM PARATIVE"IOCHEMISTRY OFF 0HOTOREACTIVE3YSTEMS PPn RKAND ,ONDON !CADEMIC0RESS .EW9ORK (EDDAD D -AND !DAMSKA) 4HEEVOLUTIONOF LIGHTSTRESS PROTEINS INPHOTOSYNTHETICORGANISMS#OMP&UNCT'ENOMICS n (ESS72 0ARENTSKY& VANDER3TAAY'7 'ARCIA &ERNANDEZ*- "ÚRNER4AND6AULOT$ #OEXISTENCEOFPHYCOERYTHRIN ANDACHLOROPHYLL A AB ANTENNAEINAMARINEPROKARYOTE0ROC .ATL!CAD 3CI 53!n (IHARA9 +AMEI ! +ANEHISA- +APLAN!AND D )KEUCHI -  $.! . MICROARRAY A ANALYSISOF CYANOBACTERIAL GENE EXPRESSION D ING ACCLIMATIONTO HIGH LIGHT0LANT#ELL n DUR (ILLER2' #AROTENOIDSASCOMPONENTSOFT F HELIGHT HAR VESTINGPROTEINSOFEUKARYOTICALGAE)N&RANK(! 9OUNG!* "RITTON'AND#OGDELL2*EDS 0HOTOCHEMISTRYOF#AROTENOIDS PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT (OBE3 0RYTULLA3 +àHLBRANDT7AND0AULSEN( 4RIMER IZATION AND D CRYSTALLIZATION OF F RECONSTITUTED LIGHT HARVESTING CHLOROPHYLL AB A COMPLEX%-"/*n (OBE3 &ÚRSTER2 +LINGER*AND D 0AULSEN(  . PROXIMAL SEQUENCE MOTIF IN LIGHT HARVESTING CHLOROPHYLL AB A BINDING PROTEIN IS ESSENTIAL FFOR THE TRIMERIZATION OF LIGHT HARVESTING CHLOROPHYLL AB  COMPLEX"IOCHEMISTRYn (OBE3 .IEMEIER( "ENDER!AND0AULSEN( #AROTENOID BINDING SITES IN ,(#))Bˆ2ELATIVE AFlNITIES TOWARDS MAJOR XANTHOPHYLLSOFHIGHERPLANTS%UR*"IOCHEMn (OFMANN% 7RENC F H0- 3HARPLES&0 (ILLER2' 7ELTE7AND $IEDERICHS +  3TRUCTURAL BASIS OF LIGHT HARVESTING BY CAROTENOIDS0ERIDININ CHLOROPHYLL PROTEIN FROM F !MPH M IDINIUM CARTERAE 3CIENCEn (OLT.% &LEMING'2AND D .IYOGI ++ 4OWARD D ANUNDER STANDINGOFTHEMECHANISMOFNONPHOTOCHEMICALQUENCHINGIN GREENPLANTS"IOCHEMISTRYn (U1 -IYASHITA( -IYASHITA( )WASAKI) +URANO. -IYACHI 3 )WAKI-AND)TOH3 !0HOTOSYSTEM)REACTIONCENTER DRIVEN BYCHLOROPHY H LL D INOXYGENICPHOTOSYNTHESIS0ROC.ATL !CAD 3CI 53!n (U1 -ARQUARDT* )WASAKI ) -IYASHITA( +URANO. -ÚRSCHEL % AND D -IYACHI 3  -OLECULAR STRUCTURE LOCALIZATION AND FUNCT F IONOF BILIPROTEINS INTHECHLOROPHYLL AD A DCONTAINING OXYGENIC PHOTOSYNTHETIC PROKARYOTE !CARYOCHLORIS MARINA "IOCHIM"IOPHYS!CTAn (UTIN # .USSAUME , -OISE . -OYA ) +LOPPSTECH + AND (AVAUX- %ARLYLIGHT INDUCEDPROTEINSPROTECT !RABI R DOPSIS FROM DO F PHOTOOXIDATIVESTRESS0ROC.ATL !CAD3C D I53!  n )LAGAN 20 3HIMA 3 -ELKOZERNOV! ,IN 3 "LANKENSHIP 2% 3HARPLES &0 (ILLER 2' "IRGE 22 AND D &RANK (!  3PECTROSCOPIC PROPERTIES OFF THE MAIN FORM F AND HIGH SALT PERIDININ CHLOROPHYLL A PROTEINSFROM!MPHIDINIUMCARTERAE "IOCHEMISTRYn )VANOV!' 0ARK9 ) -ISKIEWICZ% 2AVEN*! (UNER.0!AND ½QUIST' )RONSTRESSRESTRICTSPHOTOSYNTHETICINTERSYSTEM ELECTRONTRANSPORTIN 3YNEC 3 HOCOCCUS SP0## &%"3 ,ETT  n

 *AKOB4 'OSS2AND7 D 7ILHELM# !CTIVATIONOF DIADINOXAN THINDE EPOXIDASEDUETOACHLORORESPIRATORYPROTONGRADIENT INTHEDARKINTHEDIATOM 0HAEODACTYLUMTRICORNUTUM0LANT "IOLOGYn *ANSSON3 !GUIDETOTHE,HCGENESANDTHEIRRELATIVESIN DOPSIS4RENDS0LANT3CI n !RABIDO D -ULLINEAUX#7  0HYCOBILISOMEDIFFUS F ION *OSHUA3AND IS REQUIRED FOR F LIGHT STATE TRANSITIONS IN CYANOBACTERIA 0LANT 0HYSIOLOGY n +AMIYA.AND3HEN* 2 #RYSTALSTRUCTUREOFOXYGEN EVOLV ING 0HOTOSYSTEM)) FROM 4HERMOSYNECHOCOCCUS VULCANUS AT ¯RESOLUTION0ROC.ATL!CAD3CI53!n +LEIMA&* (OBE3 #ALKOEN& 5RBANUS-, 0ETERMAN%*' VAN 'RONDELLE2 0AULSEN(ANDVAN!MERONGEN( $ECREAS ING THECHLOROPHYLL AB RATIOINRECONSTITUTEDD ,(#))3TRUCTURAL AND FUNCT F IONAL CONSEQUENCES" Q IOCHEMISTRY  n +RAUSE '( AND D *AHNS 0  0ULSE AMPLITUDE MODU D LATED CHLOROPHYLL mUOROMETRY AND ISAPPLICATIONINPLANTSCIENCE)N D EDS ,IGHT (ARVESTING !NTENNAS 'REEN"2AND0ARSON77 IN0HOTOSYNTHESIS PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT +àHLBRANDT7 7ANG$.ANDD &UJIYOSHI9 !TOMICMODEL OFPLANTLIGHT HARVESTINGCOMPLEXBYELECTRONCRYSTALLOGRAPHY .ATURE n ,ARK RKUM7$ $OUGLAS3%ANDD 2AVEN*!EDS  0HOTOSYN THESIS IN!LGAE+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT A '7- 0ARTENSKY & $UCRET! !EBERSOLD ,A2OCHE* VANDER3TAAY RKUM!7$AND 2 ,I 2 'OLDEN33 (ILLER2' 7RENCH0-,ARK 'REEN"2 )NDEPENDENTEVOLUTIONOFTHEPROCHLOROPHYTE ANDGREENPLANTCHLOROPHYLL AB LIGHT HARVESTINGPROTEINS0ROC .ATL!CAD3CI53n ,AVAUD* 2OUSSEAU" VAN'ORKOM(*AND%TIENNE! ,A F HEDIADINOXANTHIN POOL SIZEON PHOTOPROTECTION )NmUENCEOFT INTHEMARINEPLANKTONICDIATOM 0HAEODACT D TYLUMTRICORNUTUM 0LANT0HYSIOL n D RKOM(*ANDD %TIENNE! ,B 0HOTOSYSTEM)) ,AVAUD* VAN'OR ELECTRON TRANSFER F CYCLE ANDD CHLORORESPIRATION IN PLANKTONIC DIATOMS0HOTOSYNTH 2ESn ,AVAUD* 2OUSSEAU"AND%TIENNE! , 'ENERALFEATURES OFPHOTOPROTECTIONBYENERGYDISSIPATIONINPLANKTONICDIATOMS "ACILLARIOPHYCEAE *0HYCOLn ,I 80 "J " ÚRK RKMAN / 3HIH # 'ROSSMAN !2 2OSENQUIST - D .IYOGI ++  !PIGMENT BINDINGPROTEIN *ANSSON3AND ESSENTIAL FFOR REGULATION OFF PHOTOSYNTHETIC LIGHT HARVESTING .ATURE n ,I 80 -àLLER -OULÏ 0 'ILMORE!- ANDD .IYOGI ++  0SB3 DEPENDENTENHANCEMENTOFFEEDBACKDE EXCITATIONPRO TECTS0HOTOSYSTEM))FROMPHOTOINHIBITION0ROC .ATL!CAD3CI 53!n ,I80 'ILMORE!- #AFFARRI3 "ASSI2 'OLAN4 +RAMER$AND T ETICLIGHTHARVESTING .IYOGI ++  2EGULATIONOFF PHOTOSYNTH INVOLVESINTRATHYLAKOID LUMENP(SENSING BYTHE0SB3PROTEIN *"IOL #HEMn ,ICHTLÏ# $UVAL*#ANDD ,EMOINE9  #OMPARATIVE BIO CHEMICAL FUNCT F IONALANDD ULTRASTRUCTURAL STUDIESOFF PHOTOSYSTEM PARTICLES FROM A #RYPTOPHYCEA # #RYPTOMONAS RUFESCENCE ISOLATIONOFANACTIVEPHYCOERYTHRINPARTICLE"IOCHIM"IOPHYS !CTAn ,ICHTLÏ # -C+AY 2-, AND 'IBBS 30 A )MMUNOGOLD LOCALIZATION OFF 0HOTOSYSTEM ) ANDD 0HOTOSYSTEM)) LIGHT HAR VESTING COMPLEXES IN CRYPTOMONADD CHLOROPLASTS "IOL #ELL

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU n ,ICHTLÏ# 3PILAR!AND$UVAL*#B )MMUNOGOLDLOCALIZA TIONOFLIGHT HARVESTINGAND0HOTOSYSTEM)COMPLEXESINTHE R 0HAEOPHYCEAE  0ROTOPLASMA THYLAKOIDS OF &UCUS SERRATUS  n ,IU:& 9AN(# 7ANG+" +UANG 49 :HANG*0 'UL,, !N D #HANG72 #RYSTAL STRUCTUREOFF SPINACH MAJ A OR 8-AND LIGHT HARVESTING COMPLEX AT  ¯ RESOLUTION .ATURE  n ,OHR-AND D 7ILHELM#  !LGAE DISPLAYI A NG TH T E DIADINOXANTH T N CYCLEALSOPOSSESSTHEVIOLAXANTHINCYCLE0ROC.ATL!CAD3CI 53!n ,UDWIG-AND'IBBS30 ,OCALIZATIONOFPHYCOERYTHRINAT THELUMENALSURFACEOFTHETHYLAKOIDMEMBRANEIN2HODOMONAS LENS* #ELL "IOLn -A9: (OLT .% ,I 80 .IYOGI ++ AND D &LEMING '2  %VIDENCE FOR F DIRECTCAROTENOID INVOLVEMENTINTHEREGULATION OFF PHOTOSYNTHETICLIGHT HARVESTING0ROC.ATL!CAD3C D I 53!  n -AC#OLL2AND'UARD &RIAR$ 0HYCOBILIPROTEINS#2# 0RESS "OCA2ATON -ACPHERSON!.AND(ILLER2' ,IGHT HARVESTINGSYSTEMS INCHLOROPHYLL C CONTAININGALGAE)N'REEN"2AND0ARSON 77 EDS ,IGHT (ARVESTING!NTENNAS IN 0HOTOSYNTHESIS PP U SHERS $ORDRECHT n+LUWER!CADEMIC0UBLI -ATTHIJS (#0 VAN DER 3TAAY A '7- AND D -UR ,2  0RO CHLOROPHYTES4HE@OTHERCYANOBACTERIA)N"RYANT$! ED 4HE-OLECULAR"IOLOGYOF# F YANOBACTERIA PPn+LUWER !CADEMIC0UBLISHERS $ORDRECHT -C#ONNELL-$ -C#ONNELL2+ 3ERGEJ6AND"RUCE$ 2EGULATIONOFTHEDISTRIBUTIONOFCHLOROPHYLLANDPHYCOBILINS ABSORBED EXCITATION ENERGY IN CYANOBACTERIA ! STRUCTURE BASED D MODEL FORT F HELIGHTSTATETRANSITION0LANT0HYS H IOL n -C&ADDEN') %NDOSYMBIOSISAND D EVOLUTIONOFT F HEPLANT CELL#URR/PINION0LANT"IOL n -C&ADDEN')  0RIMARY AND D SECONDARYENDOSYMBIOSISAND THEORIGINOFPLASTIDS*0HYCOLn -EYER-AND7ILHELM# 2ECONSTITUTIONOFLIGHT HARVEST ING COMPLEXES FROM # #HLORELLA FUSCA #HLOROPHYCEAE AND -ANTONIELLAS QUAMATA 0RASINOPHYCEAE :.ATURFORSCHC n -IAO*( )RRGANG +$ 3ALNIKOW* &RANKE0AND6ATER* D  #LOSEVICINITYOFF ,HCBAND D ,HCB IN0HOTOSYSTEM))ˆ-EM BRANEFRA F GMENTSASVERIlED BY CHEMICALCROSS LINKING%UR* "IOCHEMn -IMURO-AND+IKUCHI( !NTENNASYSTEMSANDENERGY TRANSFERINCYANOPHYTAANDRHODOPHYTA)N'REEN"2AND0ARSON 77EDS ,IGHT (ARVESTING !NTENNASIN0HOTOSYNTHESIS PP n+LUWER!CADEMIC0UBLISHERS $ORDRECHT -INAGAWA * AND D 4AKAHASHI 9  3TRUCTURE FUNCT F ION AND ASSEMBLY OFF 0HOTOSYSTEM))AND ITSLIGHT HARVESTINGPROTEINS 0HOTOSYNTH2ESn -ONTANÏ -( AND D +LOPPSTECH +  4HE FAM F ILY OF LIGHT HARVESTING RELATED D PROTEINS ,(#S %,)0S (,)0S  7 7AS THE HARVESTINGOFLIGHTTHEIRPRIMARYFUNCTION'ENEn -OROSINOTTO 4 #AFFARRI 3 $ALL/STO , AND "ASSI 2  -ECHANISTIC ASPECTS OF THE XANTHOPHYLL DYNAMICS IN HIGHER PLANTTHYLAKOIDS0HYSIOL0LANTn -URAKAMI ! -IYASHITA( )SEKI - !DACHI+AND D -IMURO #HLOROPHYLL D INANEPIPHYTICCYANOBACTERIUMOFRED

$IBQUFS

%JTUBM
BOE
&YUSJOTJD
1IPUPTZTUFN
**
"OUFOOBT ALGAE3CIENCE .IELD* &UNK# AND"ARBER*A 3UPERMOLECULARSTRUCTURE OF0HOTOSYSTEM))ANDLOCATIONOFTHE0SB3PROTEIN0HIL4RANS 2OY3OC,OND"n .IELD* /RLOVA%6 -ORRIS%0 'OWEN" VAN(EEL-AND"ARBER * B $MAPOFF THEPLANT0HOTOSYSTEM))SUPERCOMPLEX OBTAINED BYCRYOELECTRONMICROSCOPYANDD SINGLEPARTICLEANALY SIS.AT 3TRUCT"IOL n .OVODEREZHKIN6) 0ALACIOS -! VAN!MERONGEN ( ANDD VAN F DYNAMICS INTHE,(#)) 'RONDELLE2 %NERGY TRANSFER COMPLEXOFHIGHERPLANTS-ODIlEDREDlELDAPPROACH*0HYS #HEM"n .USSBERGER3 $ORR+ 7ANG$. +àHLBRANDT7 ,IPID PRO TEININTERACTIONSINCRYSTALSOFPLANTLIGHT HARVESTINGCOMPLEX *-OL "IOLn .USSBERGER3 $EKKER*0 +àHLBRANDT7 VAN"OLHUIS"- VAN 'RONDELLE 2 ANDD VAN !MERONGEN (  3PECTROSCOPIC CHARACTERIZATIONOFF THREEDIFFERENTMONOMER F ICFORMSO F F THEMAIN FT CHLOROPHYLL AB BINDING PROTEIN FROMC F HLOROPLASTMEMBRANES "IOCHEMISTRY n /LSZOWKA$ +RAWCZYK3AND-AKSYMIEC7 !STUDYOF MOLECULARINTERACTIONSINLIGHT HARVESTINGCOMPLEXES,(#))B #0 #0AND#0BY3TARKEFFECTSPECTROSCOPY"IOCHIM H "IOPHYS!CTAn 0AGANO! #INQUE'ANDD "ASSI 2  )NVITRORECONSTITUTION OFF THERECOMBINANT0HOTOSYSTEM)) LIGHT HARVESTING COMPLEX #0AND ITSSPECTROSCOPICCHARACTERIZATION*"IOL#HEM n 0ALACIOS -! &RESE 2. 'RADINARU ## VAN 3TOKKUM )(- 0REMVARDHAN,, (ORTON0 0 2UBAN!6 6 VAN'RONDELLE2AND VAN!MERONGEN( 3TARKSPECTROSCOPYOFTHELIGHT HAR VESTINGCOMPLEX))INDIFFERENTOLIGOMERISATIONSTATES"IOCHIM H "IOPHYS!CTAn D .AGARAJ A AN6  /PTICAL SPECTROSCOPY IN 0ARSON77 AND PHOTOSYNTHETICANTENNAS)N'REEN"2AND0ARSON77 D EDS ,IGHT (ARVESTING !NTENNAS IN 0HOTOSYNTHESIS PP n U SHERS $ORDRECHT +LUWER!CADEMIC0UBLI 0ARTENSKY&AND'ARCZAREK, 4HEPHOTOSYNTHETICAPPARATUS OFGREENOXYPHOTOBACTERIA)N,ARKUM7$ $OUGLAS3%AND 2AVEN*!EDS 0HOTOSYNTHESISIN!LGAE PPn+LUWER !CADEMIC0UBLISHERS $ORDRECHT 0ARK9 ) 3ANDSTRÚM3 'USTAFSSON0AND½QUIST' %X PRESSIONOFF THE ISI! GENE ISESSENTIAL FORT F HESURVIVAL OFF THE CYANOBACTERIUM 3YNECHOCOCCUSSP0## BYPROTECTING F LIGHTUNDERIRON LIMITATION-OL 0HOTOSYSTEM)) FROMEXCESS -ICROBIOL n 0ASCAL!! #ARON , 2OUSSEAU " ,APOUGE + $UVAL *# AND 2OBERT" 2ESONANCE2AMANSPECTROSCOPYOFALIGHT HARVESTINGPROTEINSFROMTHEBROWNALGA,AMINARIA SACCHARINA "IOCHEMISTRY  n 0ASCAL ! 'ASTALDELLA- #EOLDO3 "ASSI 2 2OBERT" 0IGMENTCONFORMAT F IONANDP D IGMENT PROTEIN INTERACTIONSINTHE D ,HCBANTENNA PROTEIN&%"3,ETTn RECONSTITUTED 0AULSEN( 0IGMENTLIGATIONTOPROTEINSOFF THEPHOTOSYN THETICAPPARATUSINHIGHERPLANTS0HYSIOL0LANTn & "ORST*# VAN'RONDELLE 0ETERMAN%*' 'RADINARU## #ALKOEN& 2ANDVAN!MERONGEN( 8ANTHOPHYLLSINLIGHT HARVEST INGCOMPLEX))OFHIGHERPLANTS,IGHTHARVESTINGANDTRIPLET Q QUENCHING"IOCHEMISTR Y  n 0OST!& /HAD D ) 7ARNER+-AND "ULLERJ R AHN'3 %NERGY DISTRIBUTION BETWEEN PHOTOSYSTEMS)ANDD )) INTH T EPHOTOSYNTH T ETIC



PROKARYOTE 0ROCHLOROTH T RIXHOLLANDICA INVOLVESACHLOROPHYLL AB  ANTENNA WHICH ASSOCIATES WITH 0HOTOSYSTEM ) "IOCHIM "IOPHYS!CTAn 0YSZNIAK!-AND'IBBS30 )MMUNOCHEMICALLOCALIZATION OF0HOTOSYSTEM)ANDTHEFUCOXANTHIN CHLOROPHYLL A AC LIGHT HARVESTING COMPLEXINTH T E DIATOM 0HAEODACT D TYLUM TR T ICORNUTUM 0ROTOPLASMAn 2EMELLI2 6AROTTO# 3ANDONË$ #ROCE2AND D "ASSI2 #HLOROPHYLL BINDINGTOMONOMERIC LIGHT HARVESTING COMPLEX F HROMOPHORE BINDING RESIDUES*" D IOL !MUTATIONANALYSISOFC #HEMn 2EUTER7 7IEGAND' (UBER2AND4HAN- 3TRUCTURAL ANALYSISAND¯OFORTHORHOMBICCRYSTALSPRESENTSTHEASYM METRYOFTHEALLOPHYCOCYANIN LINKERCOMPLEX !0,# FROM - IGOCLADUS D LAMINOSUS 0ROC .ATL THE PHYCOBILISOMES OF -ASTI !CAD 3CI 53!n 2ICHAUD# :A D BULON' *ODER!AND D 4HOMAS* #  .ITROGEN ORSULFURSTARVAT F ION DIFFERENT F IALLYAFFECTSP F HYCOBILISOMEDEGRA DATIONANDEXPRESS D IONOFF THENBL! GENE IN 3Y 3 NECHOCY CYSTIS STRAIN 0##*"ACTERIOLn 2OGL(AND+àHLBRANDT7 -UTANTTRIMERSOFLIGHT HARVEST INGCOMPLEX))EXHIBITALTEREDPIGMENTCONTENTANDSPECTROSCOPIC F TURES "IOCHEMISTRY  n FEA 2OGL ( 3CHÚDEL 2 ,OKSTEIN( +àHLBRANDT7AND3C D HUB U ERT!  !SSIGNMENTOFF SPECTRAL SUB U STRUCTURESTOPIGMENT BIND ING SITES IN HIGHER PLANT LIGHT HARVESTING COMPLEX ,(# )) "IOCHEMISTRY  n 2OGL ( +àHLBRANDT 7 AND "ARTH !  ,IGHT INDUCED CHANGES IN THE CHEMICAL BOND STRUCTURE OF LIGHT HARVESTING COMPLEX))PROBEDBY&4)2SPECTROSCOPY"IOCHEMISTRY  n 2UBAN!6 7ENTWORTH- 9AKUSHEVSKA!% !NDERSSON* ,EE0* 2 D (ORTON0 +EEGSTRA7 $EKKER*0 "OEKEMA%* *ANSSON3AND  0LANTS LACKINGTHEMAIN LIGHT HARVESTINGCOMPLEXRETAIN 0HOTOSYSTEM))MACRO ORGANIZATION.ATUREn 3ANDSTRÚM3 0ARK9 ) ½QUIST'AND'USTAFSSON0  #0| D F FUNCT IONSASANEXCITATIONENERGY DIS THE ISI! GENEPRODUCT SIPATERINTHECYANOBACTERIUM 3YNECHOCOCCU 3 S SP0## 0HOTOCHEM0HOTOBIOLn 3ANDSTRÚM3)VANOV!' 0ARK9 ) ½QUIST'AND'USTAFSSON0 3

 )RONSTRESSRESPONSESINTHECYANOBACTERIUM3YNECHOCOC CUS SP0##0HYS H IOL 0LANTn 3ARCINA- 4OBIN-*AND D -ULLINEAUX#7 $IFFUS F IONOF PHYCOBILISOMESONTHETHYLAKOIDD MEMBRANESOFT F HECYANOBAC TERIUM 3YNECHOCOCCUS  %FFECTS F OF PHYCOBILISOME SIZE D MEMBRANE LIPIDCOMPOS D ITION*"IOL#HEM TEMPERATURE AND n 3CHEER( 4HEPIGMENTS)N'REEN"2AND0ARSON77 EDS ,IGHT (ARVESTING!NTENNASIN0HOTOSYNTHESIS PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT D (ILLER2' 4WODISTINCT 3HARPLES&0 7RENCH 0- /U+AND F FORMSO F THEPERIDININ CHLOROPHYLL A PROTEIN FRO F M !MPH M IDINIUM D CARTERAE "IOCHIM"IOPHYS!CTAn 3HIMA3 )LAGAN20 'ILLESPIE. 3OMMER"* (ILLER2' 3HAR PLES &0 &RANK K (! AND "IRGE 22  4WO PHOTON AND mUORESCENCE SPECTROSCOPY AND THE EFFECT OF ENVIRONMENT ON THEPHOTOCHEMICALPROPERTIESOFPERIDINININSOLUTIONANDIN THEPERIDININ CHLOROPHYLL PROTEINFROM!MPHIDINIUMCARTERAE *0HYS#HEM!n Q 3IMONETTO2 #RIMI- 3ANDONË$ #ROCE2 #INQUE' "RETON D "ASSI 2  /RIENTATION OFF CHLOROPHYLL TRANSITION * AND

 MOMENTS INTHEHIGHER PLANT LIGHT HARVESTING COMPLEX#0 "IOCHEMISTRY n 3INGH!+ -C)NTYRE,-AND3HERMAN,! -ICROARRAY ANALYSISOFTHEGENOME WIDERESPONSETOIRONDElCIENCYAND IRON RECONSTITUTION IN THE CYANOBACTERIUM 3YNECHOCYSTIS SP 0##0LANT0HYSIOLn 3TAUB U ER%* &INK K ! -ARRKERT# +RUSE/ *OHANNINGMEIER5AND M DOMONASREINHARD R TII (IPPLER-  0ROTEOMICSOF #HLAMYD LIGHT HARVESTING PROTEINS%UKK #ELLn 4ERAMOTO ( /NO4 AND -INAGAWA *  )DENTIlCATION OF 4 ,HCB GENEFAMILYENCODINGTHELIGHT HARVESTINGCHLOROPHYLL AB  PROTEINSOF0HOTOSYSTEM))IN #HLAMYDOMONAS REINHARDTII 0LANT#ELL0HYSIOLn 4ING #3 AND /WENS 4'  0HOTOCHEMICAL AND NONPHO TOCHEMICAL mUORESCENCE QUENCHING PROCESSES IN THE DIATOM 0HAEODACT D TYLUM TR T ICORNUTUM0LANT0HYSIOLn D /WENS4'  4HEEFFECTSO F F EXCESS IRRADIANCE 4ING#3AND ONPHOTOSYNTHESISINTHEMARINE DIATOM 0HAEODACT D TYLUMTRI CORNUTUM0LANT0HYSIOLn 4ING #3 2OCAP ' +ING * AND #HISHOLM 37  0HYCO BILIPROTEIN GENES OF THE MARINE PHOTOSYNTHETIC PROKARYOTE 0ROCHLO R R ROCOCCUS  %VIDENCE FOR RAPID EVOLUTION OF GENETIC HETEROGENEITY-ICROBIOLOGYn 4ING#3 2OCAP' +ING*AND# D HISHOLM37 #YANOBACTE RIAL PHOTOSYNTHESIS INTHEOCEANS4HEORIGINSANDD SIGNIlCANCE OF DIVERGENTLIGHT HARVESTINGSTRATEGIES4RENDS-ICROBIOL n 4 LE#!AND 4OO D !LLNUTT  2ED CR D YPTOMONADD AND GLAUCO CYSTOPHYTEALGALPHYCOBILIPROTEINS)N,ARKUM7$ $OUGLAS 3%AND2AVEN*!EDS 0HOTOSYNTHESISIN!LGAE PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT VAN!MERONGEN ( AND $EKKER *0  ,IGHT HARVESTING IN D EDS ,IGHT 0HOTOSYSTEM)))N'REEN"2AND0ARSON77 (ARVESTING !NTENNASIN 0HOTOSYNTHESIS PPn+LUWER !CADEMIC0UBLI U SHERS $ORDRECHT A '7- "ROUWER! "AARDD 2, VAN-OURIKK &AND VAN DER3TAAY -ATTHIJ I S(#0  3EPARATIONOFPHOTOSYSTEMS)AND))FROM THEOXYCHLOROBACTERIUMPROCHLOROPHYTE 0ROCHLOROTHRIXHOL

#FWFSMFZ 3 (SFFO
BOE
&MJTBCFUI (BOUU LANDICA AND D ASSOCIATIONOFF #HL B BINDING ANTENNAEWITH 03)) "IOCHIM"IOPHYS!CTAn VANDER3TAAY'7- 9URKOVA.AND'REEN"2 4HE AB PROTEINOFTHEPROKARYOTE 0ROCHLO R R RIX ROTH K$ACHLOROPHYLL A HOLLANDICA ISENCODEDBYADIVERGENT PCB GENE0LANT-OL"IOL  n 7 G ( , 0OSTIER ", AND 7AN D "URNAP 2,  !LTERATIONS IN GLOBALPATTERNSOF GENEEXPRESSIONIN 3YNECHOCY CYSTIS SP0## R ONLIMITATIONAND D THE INAC INRESPONSETO INORGANICCARB TIVATIONOF NDH2 A,Y , S2FAM F ILY REGULATOR*"IOL#HEM  n 7 7EDEMAYER' +IDD$AND'LAZER!. #RYPTOPOMONA D BILIPROTEINS "ILIN TYPES AND LOCATION 0HOTOSYNTH 2ES  n 7ILK+% (ARRO K P 3* *ANKOVA, %DLER$ +EENAN' 3HARPLES& D #URMI 0-' %VOLUTIONOFF A LIGHT HAR (ILLER2'AND VESTINGPROTEIN BY ADDITIONOFF NEWSUUBUNITSAND D REARRANGEMENT OFF CONSERVED ELEMENTS #RYSTAL STRUCTURE OF F A CRYPTOPHYTE PHY H COERYTHRINAT¯RESOLUTION0ROC.ATL!CAD3CI53! n 9AKUSHEVSKA!% +EEGSTRA7 "OEKEMA%* $EKKER*0 !NDERSSON 9 * *ANSSON3 2UBAN!6AND(ORTON0 4HESTRUCTUREOF O SIS,OCALIZATIONOFTHE#0AND 0HOTOSYSTEM))IN !RABIDOP #0ANTENNACOMPLEXES"IOCHEMISTRY  n 9 G # +OSEMUND+ #ORNET#AN 9AN D D 0AULSEN( %XCHANGE OFP F IGMENT BINDINGAMINOACIDS IN LIGHT HARVESTING CHLOROPHYLL A PROTEIN"IOCHEMISTRY n AB 9 9EREMEN KO. +OUǣIL2 )HALAINEN*! $(AENE3 VAN/OSTERWIJK . !NDRIZHIYEVSKAYA %' +EEGSTRA 7 $EKKER (, (AGE MANN- "OEKEMA%* -ATTHIJS(#0AND$EKKER*0 3UPRAMOLECULAR ORGANIZATION AND DUAL FUNCTION OF THE )SI! CHLOROPHYLL BINDING PROTEIN IN CYANOBACTERIA "IOCHEMISTRY  n :OUNI! 7ILL 7 ( 4 +ERN * &ROMME 0 +RAUSS . 3AENGER7 AND D /RTH0  #RYSTALSTRUCTUREOFF 0HOTOSYSTEM)) FROM F G AT  ¯ RESOLUTION .ATURE  3 NECHOCOCCUS ELONGATUS 3Y n

$IBQUFS
 5IF
$1
BOE
$1
$PSF
"OUFOOB
$PNQPOFOUT +VMJBO
+
&BUPO3ZF

%FQBSUNFOU
PG
#JPDIFNJTUSZ SZ
6OJWFSTJUUZ PG
0UBHP
10
#PY

%VOFEJO
/FX
;FBMBOE

$JOEZ 1VUOBN&WBOT %FQBSUNFOU
PG
#JPMPHZ
&BTU
$BSPMJOB
6OJWFSTJUZ
(SFFOWJMMF
/$

64"

4VNNBSZ   * *OUSPEVDUJPO  ** 5IF QTC# BOE QTC$ (FOFT   " 0QFSPO
"SSBOHFNFOU   # (FOF
%FMFUJPOT
BOE
*OBDUJWBUJPO  *** 0WFSWJFX
PG
&OFSHZ
5SBOTGFS
BOE $IMPSPQIZMM #JOEJOH  "
.VUBHFOFTJT
4UVEJFT
PG
1VUBUJWF $IMPSPQIZMM#JOEJOH
-JHBOET   
.VUBHFOFTJT
PG
)JTUJEJOF
3FTJEVFT
JO $1   
.VUBHFOFTJT
PG
)JTUJEJOF
3FTJEVFT
JO $1   *7

5IF
)ZESPQIJMJD
%PNBJOT
PG $1
BOE $1   "
.VUBHFOFTJT
PG
)ZESPQIJMJD
%PNBJOT
PG
$1 JO 4ZOFDIPDZTUJ 4 JT TQ
1$$    #
#JPDIFNJDBM
4UVEJFT
%JSFDUFE
BU
-VNFOBM
%PNBJOT
PG
$1   $
5IF
3FNPWBM
PG
UIF
&YUSJOTJD
1SPUFJOT
JO
$1
.VUBOUT   %
.VUBHFOFTJT
PG
-PPQ
&
JO
$1
JO 4ZOFDIPDZTUJ 4 JT TQ
1$$    &
*OUFSBDUJPO
PG
$1
XJUI
0UIFS 1IPUPTZTUFN ** 4VCVOJUT   
*OUFSBDUJPO
PG $1
XJUI
%   
*OUFSBDUJPO
PG $1
XJUI
1TC0   
*OUFSBDUJPO
PG $1
XJUI
1TC7  ' $PWBMFOU
.PEJàDBUJPOT
PG
)ZESPQIJMJD %PNBJOT PG
$1   7 $PODMVTJPOT  7 "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 4HE #0AND#0SU D UBUNITSOFF 0HOTOSYSTEM)) BIND D CHLOROPHY H LL A AND BELONGTOAFAM F ILYO L F BACTERIO CHLOROPHY H LL BINDINGPROTEINSTHATSERVEASCOREANTENNAPOLYPEPTIDESINBOTHANOXYGENICANDOXYGENICPHOTOSYNTHESIS 5NIQUELY Y BOTHOFF THESEPROTEINSPOSSESSEXTENDED HYDROPHILIC DOMAINSTHATCONTRIBUTETOTHEENVIRONMENTOF THEOXYGEN EVOLVINGCOMPLEX3TRUCTURALSTUDIES HAVESHOWNTHATTHESEPOLYPEPTIDESAREASSOCIATEDD WITH BOTH INTRINSICCOMPONENTSOFTHEREACTIONCENTERANDWITHTHEEXTRINSIC PROTEINSTHATENHANCE/ EVOLUTION UNDER PHYSIOLOGICALCONDITIONS4HE BIOCHEMICAL SPECTROSCOPICANDD MOLECULARTECHNIQUESTHATHAVEBEENUSEDD TO DElNETHESE INTERACTIONS ASWELLASTHEFUNCT F IONAL DOMAINSWITHINTHESE PROTEINS ARE PRESENTEDD AND ENCOMPASS STUDIESPERFORMEDONPHOTOSYNTHETICEUKARYOTESANDCYANOBACTERIA

!UTHORFORCORRESPON F DENCE EMAIL JULIANEATON RYE STONEBOWOTAGOACNZ

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D

 *
*OUSPEVDUJPO 4HE CHLOROPHYLL A BINDING PROTEINS #0 CHLORO PHYLL PROTEINWITH ANAPPARENTMOLECULARMASSOFF  K$A AND#0 D CHLOROPHYLL PROTEINWITH ANAPPAR ENTMOLECULARMASSOFK$A AREENCODEDBYTHE Y D SERVEASTHE PSB" AND PSB# GENES RESPECTIVELY AN PROXIMAL ANTENNA OFF 0HOTOSYSTEM)) 03))  "OTH POLYPEPTIDES POSSESS SIX TRANSMEMBRANE HELICES THATAREARRANGEDINPAIRSTOFORMATRIMEROFDIMERS 4HESEMEMBRANE SPANNING DOMAINSPROVIDE LIGANDS F FORTHEANTENNAPIGMENTMOLECULESANDTHISSTRUCTURAL F ND D INTHE . TERMINAL HELICES OFTHE MOTIFIS ALSO FOU 0HOTOSYSTEM) 03) REACTIONCENTERPROTEINS 0SA! AND D 0SA"ASWELLASTHECORRESPONDINGHELICESINTHE HOMODIMERIC REACTION CENTERS FR F OMTHE #HLOROBIA CEAE AND (ELIOBACTERIACEAE !MESZ &EILERAND (AUSKA *ORDANETAL  2ECOGNITIONOF THESTRUCTURALSIMILARITIESBETWEENTHESECHLOROPHY H LL BINDING PROTEINS HASPROVIDED D SUPPORTFORT F HECONCEPT THAT ALL REACTION CENTERS ARE DERIVEDD FROM A COMMON ANCESTOR2HEEETAL 3CHUBERTETAL   4HEPRIMARY SEQUENCESOFF #0AND #0 FROM F ARANGEOFOXYGENICPHOTOSYNTHETICORGANISMS ARE PRESENTED IN &IGS  AND  TOGETHER WITH THEIR DO MAINORGANIZATION"OTH TOPOLOGICAL AND D STRUCTURAL STUDIESHAVEESTABLISHEDTHATTHE. AND# TERMINI AREFOUNDONTHESTROMALSIDEORCYTOSOLICSIDEIN CYANOBACTERIA OFT F THETHYL T AKOID D MEM MBRANE 3AY A REAND 7ROBEL "OERNER  2HEEETAL  :OUNIETAL +AMIYAAND3HEN &ERRIERAETAL  #HAPTERSn )NBOTH POLYPEPTIDESTHESIX HELIX !BBREVIATIONSAP A C% C% nGENEENCODINGTHEPHYCOBILISOMEANCHOR PROTEIN#0nCHLOROPHYLL PROTEIN#0AnCHLOROPHYLL A BINDING PROTEIN#0#0AnCHLOROPHYLL A BINDING PROTEIN#0$ AND D $nREACTIONCENTERCORESUB U UNITS $430nDITHIOBISSUCCI NIMIDYL PROPIONATE%$#n  ETHYL   DIMETHYLAMINOPROPYL CARBODIIMIDE &!#n-ONOCLONALANTIBODYRECOGNIZINGANEP & I TOPEBETWEEN0RO AND3ER IN#0,AND-nBACTERIAL PHOTOSYNTHETICREACTIONCENTERCORESUBUNITS.(3 BIOTINn. H DROXYSUCCINIMIDE BIOTIN /%#n OXYGEN EVOLVING COMPLEX HY ORF RF n CHLOROPLAST OPEN READING FFRAME  0n PRIMARY ELECTRON DONOROFF 0HOTOSYSTEM))0!'%nPOLYACRYLAMIDE GEL ELECTROPHORESIS PET" ANDPET$ D nGENESFORSU F UBUNITSOFF THECY TOCHROME B F COMPLEX 0SA!n 0HOTOSYSTEM) REACTION CENTER CORESUBUNIT0SA"n0HOTOSYSTEM)REACTIONCENTERCORESUBUNIT PSBXnGENESFOR0HOTOSYSTEM))PROTEINSWHEREX ! " # $ ( ) ) + . / 0 1 4 5 60SBXn0HOTOSYSTEM))PROTEINSUBUNITSWHERE 6 6 X" # / . 5 603)n0HOTOSYSTEM)03))n0HOTOSYSTEM)) 1! nPRIMARYPLASTOQUINONEACCEPTOR1" nSECONDARYPLASTOQUI NONEACCEPTOR3$3nSODIUMDODECYL SULFATE3NnSTATEO F F THE F TRN' ' nCHLOROPLAST WATER OXIDIZING COMPLEXFORN    OR GENE FORT2.! ' F LY9$ nREDOXACTIVETYROSINEON$9: nREDOX ACTIVETYROSINEON$

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT BUNDLEFORM F INGTHETRIMER OF F DIMERSMOTIF ISFORME F D BYHELICES)AND)) HELICES)))AND)6ANDHELICES6 D 6) WITH THTTHEHELICESCONNECTEDD VIAlVEHYDROP D HILIC AND LOOPS DENOTEDD AS!n%)N BOTHCASESLOOP%ISLARGE COMPRISING ^ AMINO ACIDS IN #0 AND ^ RESIDUES D IN#04HESEDOMAINSCONTRIBUTETOTHE PROTEINENVIRONMENTOFT F THEOXYGEN EVOLVINGCOMPLEX /%# OFTHEPHOTOSYSTEMANDCORRESPONDTOMASSES OF^AN F D  K$A RESPECTIVELY &IGSANDD   7ITHINTHE03))DIMER#0ISFOUN F D ONTHE$ SIDE AND#0ONTHE$SIDE OFEACHMONOMERIC REACTIONCENTERTHUSPOSITIONING#0ONTHEOUTSIDE F THEDIMERICCOMPLEX4HEORIGINAL ISOLATIONOFT F THESE OFT POLYPEPTIDESFOUNDTHAT#0WASMOREEASILYPURI lED FROM F ISOLATED03))COMP D LEXESWHIL W ETHEREMOVAL OF F #0 REQU Q IREDD TREATMENT WITH ,I#L/ AND HIGH CONCENTRATIONSOFDODECYLMALTOSIDE'HANOTAKISET AL  )NITIAL WORK RK WASPERFORME F D INSPINACHAND THENOMENCLATUREFORTHESEPROTEINSAROSEFROMAPPAR R ENTMOLECULARMASSESBASEDONPOLYACRYLAMIDEGEL ELECTROPHORESIS0!'% 5SING +LOW TEMPERATURE mUORESCENCESPECTROSCOPYONEXCISED BANDSOBTAINED F FROM0!'% THECHARACTERISTIC  NM EMISSION WAS SHOWNTOARISE FROM#0AN F D THEEMISSIONATNM F FROM#0 .AKATANI ETAL  3INCE#0GAVE RISETOTHELONG WAVELENGTHEMISSIONITWASINITIALLY HYPOTHESIZEDD THAT#0CONTAINEDD THE03))REACTION F THE HOMOLOGY CENTER HOWEVER THE RECOGNITION OF BETWEENTHE$AND$POLYPEPTIDESANDTHE,AND - SUB U UNITSOF THE 2HODOPSEU D D DOMONASVIRI D REACTION DIS &IGOPPOSITE  !LIGNMENTOFPROTEINSEQUENCESFORTHECHLO ROPHYLL A BINDING PROTEIN #0 0SB"  #ONSENSUS SEQUENCES F FORGREENPLANTS'0 GREENALGAE'!  CHLOROPHYLL AC CONTAININGORGANISMS!# RED D ALGAE2! AND D CYANO BACTERIA #9 5PPERCASE INDICATESAMINOACIDSCONSERVED INALL SEQUENCESLOWERCASEINDICATESTHEAMINOACIDSMOST FREQUENT F LY PRESENT)NTHECASEOF'0 F LOWERCASEWASONLY USEDATPOS D ITIONS D ASU UBSTITUTION!MAJ A ORITY W EREMORETHANONESEQUENCECARRIED WH CONSENSUSSEQUENCEATALLPOSITIONSISALSOSHOWN#/. 4HE SEQUENCESFOR %UGLENAGRACILI % IS AND 0ROCHLOROTHRIXHOLLANDICA WERE NOTINCORPORATED4HEDOMAINSTRUCTUREOF#0ISINDICATEDBY THESOLIDBLACKLINE WITH)TO6)REPRESENTINGMEMBRANE SPANNING DOMAINSAND!TO% D H DROP HY D HILIC INTERCONNECTINGLOOPS4HE. AND # TERMINIAREEXPOSED D TOTHESTROMALSIDEOFT F HEMEMBRANE OR D %PRO OJECT THECYTOSOLICSIDE INCYANOBACTERIA AND LOOPS! #AND INTOTHE LUMENALCOMPARTMENT!DDITIONAL DOMAINSSHOWNARE  REGIONSCROSS LINKED BY %$#TOTHE 0SB/PROTEIN GREY LINES   REGIONCROSS LINKEDBY $430TOTHE0SB/PROTEIN BLACKROUND DOTS  ANTIGENICDETERMINANTFORTHEMONOCLONALANTIBODY&!# BLACKSQUAREDOTS AND REGIONSSHIELDEDFROM.(3 BIOTIN MODIlCATIONBYTHE0SB/PROTEINGREYDASHEDLINE 4HEBLACKAR ROW INDICATES A LYSYLENDOPEPTIDASE#CLEAVAGESITEWHI W CH PREVENTS RECONSTITUTIONOFF THE0SB/PROTEINAND INHIBITS / EVOLUTION 4HE CALCULATED D MOLECULARMASSOF #0 IS^K$A

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT





+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT CENTERBY4REBST  TOGETHERWITH THEPURIlCATION OFAPHOTOCHEMICALLYACTIVE03))CORECOMPLEXBY .ANBAAND3ATO D H  lRMLYPOSITIONED D THEREAC TIONCENTERONTHE$AND$PROTE D INS-ICHELAND $EISENHOFER   !SPROXIMAL ORCOREANTENNAPIGMENTPROTEINS#0 D #0AREABLETOABSORB R LIGHTDIRECTLYAND D ALSO AND TOBECOUP U LEDTOTHREEDIFFERENTCLASSESOFPERIPHERAL ANTENNASYSTEMS4HESEARETHE PHYCOBILISOMES FOUN F D INTHECYANOBACTERIAAND RED D ALGAE THE CHLOROPHY H LL AC BINDING PROTEINS THAT ARE CHARACTERISTIC OF THE BROWNALGAE AND D THE CHLOROPHYLL AB BINDING PROTEINS D OTHERPLANTS#HAPTER 'REENAND OFF GREENALGAEAND 'ANTT )NADDITIONTHECOMPOSITIONOFTHELUMENAL PROTEINSTHATFORMPARTO F F HEPROTEINENVIRONMENTOF FT THECATALYTICSITE FORWATERSP F LITTINGARE DIFFERENT F IN THESETAXA!LLORGANISMSTHATUTILIZECHLOROPHYLL AB  BINDINGPROTEINS HAVETHE PSB/ PSB0AND 0 PSB1 GENE PRODUCTSW D WHILETHEREMAININGORGANISMS HAVE0SB/ ANDTWOPROTEINSENCODEDBY PSB6 6 ANDPSB5 5 #HAPTER  (OWEVER AFURT F HER K$APROTEINISASSOCIATED WITH03))INTHERED D ALGA #YANIDIUMCALDARIUM D AND HASBEENSHOWNTOBERELATEDTO0SB1%NAMIETAL   /HTA ET AL   3IMILARLY PROTEINS WITH HOMOLOGYTO 0SB0AND D 0SB1AREALSOENCODED INCYANOBACTERIALGENOMESANDMAYBEFUNCTIONALLY ASSOCIATED D WITH THE/%#+ASHINOETAL $E ,AS2IVASETAL 4HORNTONETAL 3UM MERlELD ET AL   3INCE #0 AND #0 MAY HAVESPECIlC INTERACTIONSWITH DIFFERENTANTENNAAN F D &IIGOPPOSITE &  E  !LIGNMENTOFPROTE F INSEQUENCES FORT F HE CHLORO PHYLL A BINDINGPROTEIN#00SB# #ONSENSUSSEQUENCESFOR GREENPLANTS'0 GREENALGAE'!  CHLOROPHYLL AC CON TAININGORGANISMS!# REDALGAE2! ANDCYANOBACTERIA #9 5PPERCASEINDICATESAMINOACIDSCONSERVEDINALLSEQUENCES LOWERCASE INDICATESTHEAMINOACIDSMOST FREQUENT F LYPRESENT4HE POSITIONSOFVAR F IABLEINSERTIONSORDELETIONSARESHOWNWITH THE PLUSSYMBOL  )NTHECASEOF'0 F LOWERCASEWASONLYUSED D AT POSITIONSWH W EREMORETHANONESEQUENCECARR Q IEDD ASUUBSTITUTION ! MAJ A ORITY CONSENSUS SEQUENCE AT ALL POSITIONS IS ALSO SHOWN #/. 4HESEQUENCESFOR%UGLENAGRACILIS AND 0ROCHLOROTHRIX HOLLANDICA WERENOTINCORPORATED4HEDOMAINSTRUCTUREOF#0 ISINDICATEDBYTHESOLIDBLACKLINE WITH)TO6)REPRESENTING MEM BRANE SPANNINGDOMAINSAND!TO%HYDROPHILICINTERCONNECTING LOOPS4HE. AND# TERM D INI AREEXPOSEDD TOTHESTROMALSIDEOF THEMEMBRANE ORTHECYTOSOLICSIDE INCYANOBACTERIA AND LOOPS D % PRO OJECT INTO THE LUMENAL COMPARTMENT4HE BLACK ! # AND ARROW INDICATESA TRYPSINCLEAVAGESITEWHI W CHPREVENTSRECONSTITU TIONOFT F HE0SB/PROTEINAND INHIBITS/ EVOLUTION 3Y 3 NECHOCY CYSTIS SP0##POSSESSESAONEAMINOACIDDELETIONATPOSITION WHICHMUSTBETAKENINTOACCOUNTWHENIDENTIFYINGTHEPOSITION OFMUTATIONSFROMTHISORGANISM4HECALCULATEDMOLECULARMASS OF #0IS^K$A

 LUMENAL PROTEINS THE CONSENSUS SEQUENCES Q FFOR THE DIFFERENT OXYGENIC PHOTOAUTOTROPHS ARE COMPARED IN&IGSANDD )N BOTH PROTEINSTHE LARGESTVARIA TION IS OBSERVEDD WITHIN CHLOROPHY H LL AC CONTAINING ORGANISMSHOWEVER INSPECTIONOFALLSEQUENCESIN &IGSANDD REVEALSTHATCONSIDERABLECONSERVATION IS FOUN F D&REQUENTLYATPOSITIONSWH W ERE DIFFERENCES F AREOBSERVEDTHEALTERNATIVERESIDUES PRESENT POSSESS SIMILAR BIOCHEMICAL PROPERTIES ANDD A SUMMARY OF THECLASSESOFAM F INOACIDS FOUN F D ATTHESEPOSITIONS ISPROVIDEDIN"RICKERAND&RANKEL  )NEARLY LITERATURE#0AND#0AREA D LSOREFERRE F D TOAS #0AAND#0A D "RICKER 4HEYAREALSO REFE F RREDD TO AS 0SB" AND0SB# ANDOCCASIONALLY0 D 3 )) "AND 03)) #"OTHPROTEINSAREABSOLUTELY REQUIRED F FORWATERSP LITTINGANDP D LASTOQUINONEREDUCT D ION BYTHE PHOTOSYSTEM4HISCHAPTERWILLSURVEYTHELITERATURE F ION OF F THESE PERTAINING TO THE STRUCTURE AND FUNCT POLYPEPTIDESWITHH ANEMPHASISONRECENTBIOCHEMICAL ANDMUTAGENESISSTUDIES DIRECTEDATUNDERSTANDING THECONTRIBUTIONSTHESEPOLYPEPTIDESMAKETO03)) ACTIVITY 4HE READER IS ALSO REFERRE F D TO ADDITIONAL REVIEWSTHATPROVIDEIN DEPTHINFORMATIONONTHESE PROTEINS "RICKER"ARB R ERETAL "RICKER ANDD &RANKEL   **
5IF QTC# BOE QTC$ (FOFT "
0QFSPO
"SSBOHFNFOU )NCHLOROPLASTSOFLANDPLANTSTHE PSB" GENEISPRESENT INTH T E PSB" PS B B4 PSB( PET" PET$ B OPERONAND PSB# # IS CO TRANSCRIBEDD WITH T PSB$4HE PSB" B PSB4 P 4 SB( B PET" PET$ T OPERONISUNUSUALINTHATITENCODES PROTEINSFOR BOTH 03))ANDD THECYTOCHROME B F COMPLEXAND IS THUSAMODEL FORT F HESTUDYOF DIFFERENT F IALEXPRESSION OFGENESCODINGFORCOMPONENTSOFTWOCOMPLEXES 4ANAK A AETAL "ARK R AN+OHCHIETAL  7 HOFFF AND (ERRMANN (IRDD ETAL  )N 7EST THISOPERONGENESARECO TRANSCRIBEDFROMASINGLE PROKARYOTIC LIKE PROMOTER ANDD THEN PROCESSED INTO OLIGOCISTRONICANDD MONOCISTRONICM2.!S 7ESTHOFF AND(ERRMANN $IXITETAL  !DDITIONALLY THEGENEENCODING 0SB.IS LOCATEDD ONTHECOMPLI MENTARYSTRAND BETWEEN PSB4 4 ANDPSB( )NOTHERPHOTOSYNTHETICEUKARYOTESTHISGENECLUSTER ALTHOUGH H PRESENT ISORG R ANIZED INVARIEDD ARRANGEMENTS +ELLER ET AL  *OHNSON AND D 3CHMIDT  2EITH AND -UNHOLLAND  (ONG ET AL   F D BY ITSELF W E INCYANOBACTERIA PSB" HAS BEENFOUN WHIL

 ORWITH PSB4 4 -AYESAND"ARBER +ANEKOET AL   !S A CONSEQUENCE THE PENTACISTRONIC F D IN LAND D PLANTSHASBEENSUGGESTED D TO OPERONFOUN H LETICORIGINOFP F LASTIDS$OUGLAS SUPP U ORTTHEMONOPHY  !DDITIONALLY THE PSB" GENEHASBEENUSEDIN PHYLOGENETICSTUDIESWITHCYANOBACTERIAAND D PLANTS 5RB R ACHETAL 3UMMERlELD D ETAL 3OLTIS ETAL   ,IKEWISE THE PSB$ PSB# OPERON FOUN F D IN CYANOBACTERIA AND LAND PLANTS ALSO SUPPORTS A MONOPHYLETICORIGINOFPLASTIDSANDVARIATIONINTHE PSB#REGIONHASBEENUSEDFORPHYLOGENETICSTUDIES # $OUGLAS :IEGENHAGENAND D &LADUNG  D  4HEOPERONISFOUNDIN PLASTIDSOFF RHODOPHYTESAND D AND CHROMOPHYTES BUTNOTINALL GREENALGAE-AID :ETSCHE +OWALLICK -AUL ETAL   )N BARLEY WHERE THE PSB$ PSB# # OPERON IS FOUND AS PART OFF A COMPLEX REGION THAT INCLUDES PSB+ + PSB) ) ORF RF AND TRN' EXPRESSIONRESULTS INMULTIPLE F RENTSIZES UNDERTHE CONTROL OF ATLEAST M2.!S OF DIFFE THREE DIFFERENT F PROMOTERS 3EXTON ET AL A B #HRISTOPHERAND D (OFFER + F IMETAL  )N # IS NOT CO TRANSCRIBED WITH PSB$ IN CONTRAST PSB# #HLAMYD M DOMONASREINHARD R TII AND D TRANSLATIONCONTROL UTILIZES NUCLEAR ENCODED CIS ELEMENTS INTERACTING WITHSEQUENCESINTHEUN TRANSLATED Q | REGIONOFTHE # PSB#M2.!:ERGESETAL   (OWEVER INCYANOBACTERIATHETRANSLATIONAL STARTCODONISUP STREAM OF THE END OF THE PSB$ GENE AND EVIDENCE SUGGESTSTHAT DICISTRONICAND D MONOCISTRONICM2.!S D ALTHOUGH MONOCISTRONIC MESSAGE FO F R ARE OBTAINED PSB#HASNOTBEENOBSERVEDINALLCASES'OLDENAND # 3TEARNS #ARPENTERETAL 'INGRICHETAL D 6ERMAAS  4HE#0SEQUENCES 9UAND PRESENTEDIN&IGAREBASEDONTHECYANOBACTERIAL STARTSITE # (FOF
%FMFUJPOT
BOE
*OBDUJWBUJPO $ELETION OR INTERRUP U TION OFF THE PSB" GENE IN CYA NOBACTERIARESULTEDINTHEVIRTUALLOSSOFTHEMAJOR 03))REACTIONCENTERCOMPONENTSFROMT F HETHYLAKOID MEMB M RANESGIVINGRISETOOBLIGATEPHOTOHETEROTROPHIC STRAINS6ERMAASETAL  %ATON 2YEAND 6ERMAAS  (OWEVER THE$PROTEINHASBEEN DETECTED IN PSB" DELETIONSTRAINS DEMONSTRATINGTHAT $CANBEPRESENTINTHYLAKOIDMEMBRANESTHATLACK ASSEMBLED D 03)) REACTION CENTERS 6ERMAAS ET AL R EAND%ATON 2YE  D  #LARK 3TUDIES UTILIZING DELETION CHEMICAL AND SITE DIRECTEDMUTAGENESISOFPSB# F P #ALSOPOINTTOAROLEFOR

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT #0IN03))ASSEMBLYANDTHE / EVOLVINGPROCESS &OLLOWINGNITROSOGUANIDINEMUTAGENESIS$ZELZKALNS CYSTIS SP0## ANDD "OGORAD L RECOVEREDD A3YNECHOCY MUTANTCONTAININGADELETIONWITHI T NPSB# # 4HIS M MUTANTWASUNABLETOGROWPHOTOAUTOTROPHICALLYORTO T ATIDENTIlEDD '5'ASTTHE EVOLVE/)NEXPERIMENTSTH T E PSB# # GENEOF3YNEC 3 HOCYSTIS I SP0## STARTCODONINTH  #ARPENTERETAL CONSTRUCTEDTWOMUTATIONS WITHI T NTH T E'5'STARTCODONRESULTING INSTRAINSTH T AT LACKEDD #0 IN TH THYLAK A OIDD MEM MBRANES AND FA F ILEDD TO GROW PHOTOAUTOTROPHICALLY %ARLIER 6ERMAAS ET AL  PRODUCE D D AMUTANTIN 3Y 3 NECHOCY CYSTIS SP0## INWHI W CH PSB# # HADBEENINACTIVATEDTHISSTRAIN WASDEVOIDOF/ EVOLVING ACTIVITYANDIMMUNOBLOTS DEMONSTRATEDD THEAB A SENCEOFF #0 ALTH T OUGH H THE03)) D PRESENT ALBEIT CORE PROTEINS$ $AND#0WERE ATSIGNIlCANTLYREDUCEDAMOUNTS3IMILARRESULTSWERE F IN#HLAM L MYDDOMONAS OBSERVED INFOURSEPARATEMUTANTS R HARD REIN R TII WHI W CH FA F ILEDD TOACCUMULATE #0INTHEIR THYLAKOIDS DUE TO IMPAIRED SYNTHESIS OR STABILITY F THE PSB# OF # GENE PRODUCTS 2OCHAIX ET AL   2ÚGNERETAL CONSTRUCTED 3YNECHOCYSTIS SP 0##MUTANTSWHEREEITHERTHEENTIRE PSB$ # OPERONWAS DELETEDD ORASINGLE BASESUB U STITUTIONWAS INTRODUCE D D INTOTHE PSB# # STARTCODON0HOTOSYSTEM)) CORE COMPLEXES WERE ISOLATED FROM BOTH MUTANTS THOUGH THEY ASSEMBLEDD TO APPROXIMATELY  OF F D IN WILD D TYPE (OWEVER THESE CORE THE LEVEL FOUN COMPLEXESWEREABLETOCARRYOUTLIGHT DRIVENELECTRON TRANSFER F FROMT F HE REDOX ACTIVETYROSINE9Z #HAPTER TOTHEPRIMARY PLASTOQU Q INONEACCEPTOR 1! #HAPTER  4HUS DELETIONORINTERRUPTIONOFPSB# # PRODUCES MUTANTS LACKING #0 BUT CONTAINING OTHER CORE 03)) PROTEINS AT REDUCED LEVELS 4HESE MUTANTS DEMONSTRATED AN ABILITY TO CARRY OUT LIMITED LIGHT DRIVENELECTRONTRANSPORTBUTWEREUNABLETOEVOLVE / ORGROWPHOTOAUTOTROPHICALLY Y ***
0WFSWJFX
PG
&OFSHZ
5SBOTGFS
BOE $IMPSPQIZMM
#JOEJOH "IOCHEMICAL STUDIESWITH SPINACH PREPARATIONS HAVE IDENTIlEDAPPROXIMATELYBOUNDCHLOROPHYLL A AND n ` CAROTENES IN BOTH#0ANDD #0"ARB R ATOET AL :HELEVAETAL  -ODELINGOFPUTAT F IVE (ISLIGANDSFORCHLOROPHYLLIN#0HASBEENCARRIED F INACH INCONJ N UNCTIONWITH AN ¯MAPFOR OUT FORSP THE TRANSMEMBRANE REGIONS OFF #0 AND #0 DERIVED FROM CRYO ELECTRON CRYSTALLOGRAPHY AND THE HOMOLOGY BETWEEN THESE PROTEINS ANDD THE SIX

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT . TERMINAL HELICESOF 0SA!AND D 0SA"OF03) F 2H 2 EE ETAL  (ANKAMER ETAL "ARBERET R AL #HAPTER (ANKAMERETAL 4HISWORK INDICATED D THAT(IS  (IS  (IS  (IS AND (IS  IN #0 ARE POTENTIAL CHLOROPHYLL LIGANDS WHEREASTHEOTHER(ISRESIDUESAPPEARE D D TOBETOO FAR F F FROMT THEREMAININGDENSITIESATTRIBUTED D TOCHLOROPHY H LL MOLECULES"ARBERETAL  )NADDITIONSINCETHE NUMBEROFC F HLOROPHYLLSEXCEEDSTHENUMBEROFF (IS RESIDUES D INBOTH #0AND #0OTHERAMINOACIDS AREEXPECTEDTOSERVEINTHISROLE -ORE PRECISE INFORMAT F ION ON THE NUMBER AND POSITION OF F CHLOROPHY H LLS WITHIN #0 AND D #0 HAS BEEN PROVIDED BY THE CRYSTAL STRUCTURES F 4 ERMOSY 4H S NECHOCOCCUSELONGATUS G AND OBTAINED FROM 4HERMOSYNECHOCOCCUS VULCANUS AT  AND D  ¯ RESPECTIVELY:OUNIETAL +AMIYAAND3HEN F  !N INTERESTING OBSERVATION TO EMERGE FROM A COMPARISON BETWEEN THESE TWO CYANOBACTERIAL 03)) STRUCTURES IS THAT AN ADDITIONAL CHLOROPHYLL WASOBSERVED IN#0OF 4 4 VULCANUS AND D #0 BOTH GAINED D AND LOSTACHLOROPHY H LL4HISGAVEATOTALOF CHLOROPHYLLSIN#0ANDIN#0HOWEVER

 THEELECTRONDENSITIESOFT F HEADDITIONALCHLOROPHY H LLS INTHE 4VULCANUS STRUCTUREAREWEAKANDMAYNOT IN FFACT REPRESENT PORPHYRINS * 2 3HEN PERSONAL COMMUNICATION  4HIS INTERPRETATION IS SUPPORTED 4 BY THE MORE RECENT  ¯ 8 RAY STRUCTURE FROM 4 ELONGATU G S &ERREIRA ET AL  0$" 3, 4HE DATA OBTAINED BY &ERREIRA ET AL  IDENTIlED ANDCHLOROPHYLLSBOUNDTO#0AND#0 RESPECTIVELY &IG   7HENEXAMINING#0ANDD #0 ITISCLEARTHAT THE PIGMENTS ARE GENERALLY ORGANIZED IN TWO TIERS NEARTHESTROMALAND LUMENALSIDESOFF THEMEMBRANE AS PREVIOUSLY OBSERVED IN THE CORRESPONDING CORE ANTENNAREGIONOF0SA!AND0SA"3CHUBERTETAL "ARB R ERETAL 6ASILEVETAL  DE 7 D ET AL A  ! MODEL BASEDD ON THE  7EER ¯ STRUCTURE OF 4ELONGATUSBY:OUNIETAL  IS CONSISTENT WITH ENERGY TRANSFER F TO THE REACTION H LLONTHE CENTERPRINCIPALLYBEINGTHROUGHACHLOROPHY STROMALSIDEOFTHEMEMBRANEINBOTHPROTEINSDE 7 D ETAL A 6ASILEVETAL  HADD ALSO 7EER REACHEDD ASIMILARCONCLUSIONUSINGARElNEMENTOF THE 4ELONGATUS STRUCTURETHATYIELDEDTWOADDITIONAL

&IG!SSIGNMENTOFC & F HLOROPHY H LL A MOLECULES INTHECOREANTENNAPROTEINSOF03))4 F HEPORPHYR H INASSIGNMENTSSHOWNAREFROMT F HE 4 ERMOSY S NECHOCOCCUSELONGATU G S BY&ERREIRAAND D CO WORRKERS0$"3, 4HEVIEWIS ¯DATAOBTAINEDFOR03))CRYSTALSFROM 4H PERPENDICULARTOTHEMEMBRANENORMAL0ORPHYRINASSIGNMENTSAND D REPRESENTTHEPHEOPHYTINSASSOCIATED D WITH$AND D $ RESPECTIVELY Y #HLOROPHYLLSANDD ARETHEACCESSORY CHLOROPHYLLSOFF $AND $ RESPECTIVELY4 Y HEFOURC F HLOROPHYLLSCOMPRISING0AREn4HE HISTIDYL LIGANDSFORT F HEANTENNACHLOROPHYLLSOF#0AN F D #0ARESUMMARIZED BELOWWITH THECORRESPONDING CHLOROPHYLLSHOWNIN PARENTHESES#0(IS  (IS  (IS  (IS  (IS  (IS  (IS  (IS   (IS   (IS  (IS  (IS  AND(IS  #0(IS  (IS   (IS  (IS  (IS   (IS  (IS  (IS  (IS  AND(IS  4HE(ISNUMBERINGISTHESAMEASTHATUSEDIN&IGS AND#HLOROPHYLLIN#0ISLIGATEDBY!SN &ORDETAILS ONOTHERPUTATIVENON HISTIDYLLIGANDSANDTHEASSOCIATIONOF ` CAROTENE MOLECULESWITH#0ANDD #0 SEE#HAPTER

 CHLOROPHY H LLS IN #0 ONE ON EACH SIDE OF F THE MEMBRANE ANDAFURTHERCHLOROPHYLLONTHESTROMAL F #0 5SING THE NUMBERING IN &IG  THE SIDE OF PUTATIVE CHLOROPHY H LLS MOST EFlCIENTLY TRANSFERR F ING ENERGYTOTHEREACTIONCENTERAREIN#0AND IN#0WITH WEAKERCONTRIBUTIONSFROMC F HLOROPHYLLS D  IN#0AND D AND IN#0 6ASILEV AND ET AL &ERREIRAETAL   !CONCLUSIONREACHED FROM F LIMITEDCOUP D LING BE TWEENANTENNAAND D REACTIONCENTERPIGMENTSISTHAT THERATEOFENERGYTRANSFERTOTHEREACTIONCENTERIS SLOWWH W ENCOMPARED D TOTHERATEOFC F HARGESEPARATION -OREOVER A PHYS H ICAL SEPARATION BETWEEN CHROMO PHORESWITHANTENNAANDELECTRONTRANSFERFUNCTIONS APPEARS TO BE A GENERAL PROPERTY AS THIS HAS ALSO BEENOBSERVED INBACTERIAL AND D 03)REACTIONCENTERS 6ISSCHERETAL +RAU”ETAL (UETAL 6ASILEVETAL  )NCONTRAST EXCITATION F ITHIN#0AND D #0 ISRAPID D WITH ENERGYTRANSFERW TYPICALLYSUB PICOSECONDTRANSFERRATESBETWEENPIG MENTSWITHINATIERAND D RATESBETWEENTHESTROMAL AND LUMENALPIGMENTSNOTEXCEEDINGAFEWP F ICOSECONDS 'ROOTETAL 6ASILEVETALDE7EERD ETAL A  R ANCE AT  NM THAT IS 4HE CHARACTERISTIC ABSORB ASSOCIATEDWITH#0ARISESFROMASINGLEOSCILLATOR WITHTHESTRENGTHOFAPPROX F IMATELYONECHLOROPHYLL ORIENTED D ATALARGEANGLEWITH THTTHEPLANEOFT F THETH THYLAKOID MEMBRANEVAN$ORSSENETAL A BDE7EERDET AL A B 5SINGTHENUMBERING OF&ERRE F IRAETAL  IN&IG THISSPECIESHAS BEENASSIGNED D TO CHLOROPHYLLBYMODELINGANDISLOCATEDFURTHEST F FROMC HLOROPHYLL 4HEREFORE T F HISWOULD D TEND TO SLOW DOWN ENERGY TRANSFER F TO THE REACTION CENTER ALTHOUGHATROOMTEMPERATURETHISEFFECTISEXPECTED TOBESMALL 6ASILEVETAL  DE7EERD D ETAL B (OWEVER THE DIMERICSTRUCTUREOFF 03))SUG GESTSTHAT#0 #0INTERACTIONSMAYBEIMPORTANT INTHESUPER COMPLEXAND D THEREFOREENER F GY TRANSFE F R BETWEENNEIGHBORING#0COMPLEXESMAY BEPOS SIBLEUTILIZINGANENERGYTRANSFERPATHWAYINVOLVING THELONG WAVELENGTH ABSORBI R NG CHLOROPHYLL)NCYA NOBACTERIAASTRUCTURAL BASED D ROLEFOR#0REGAR F D INGTHEREDISTRIBUTIONOFEXCITATIONENERGYHASBEEN PROPOSED INTHE LIGHTSTATE TRANSITIONRESPONSIBLE FOR F THEBALANCINGOFF ABSORB R ED D EXCITATIONENERGY BETWEEN THETWOPHOTOSYSTEMS!SSUMINGTHATTHETRANSFEROF A SORB R ED BYCHLOROPHYLL IS INDEPENDENTOFF THAT ENERGY R AB ABSORB R ED BYTHEPHYCO H BILISOMES ENERGYABSORB R ED BY CHLOROPHYLLCOULDBETRANSFERREDTO03)VIATHECORE ANTENNAWH W ENTHEABSORPTIONCROSSSECTIONOFF 03) IS

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT INCREASED!LTHOUGHEITHEROFT F HEPROXIMAL ANTENNA PROTEINSCOULDFULlLLTHISFUNCTION THE PRESENCEOF THENMABSORPTION FAVORS#0 F INTHISROLE-C #ONNELLETAL   "
.VUBHFOFTJT
4UVEJFT
PG
1VUBUJWF $IMPSPQIZMM#JOEJOH
-JHBOET !LTHOUGH #0 AND D #0 SHARE  SEQUENCE IDENTITY HY H DROPHOBICITYPLOTSOF BOTH PROTEINSPLACE OR(ISRESIDUESIN #0AND(ISRESIDUES IN#0INMEMBRANE SPANNING REGIONS!SSHOWN IN&IGSAND T D HESERESIDUESARE D F D ONEITHER FOUN SIDEOFTHEMEMBRANEANDARETYPICALLYSPACEDBY ORAMINOACIDS6ERMAASETAL "RICKER   (ISTIDINEIMIDAZOLESWEREINITIALLYIDENTIlED ASLIGANDSTOTHECENTRAL-G OFBACTERIOCHLOROPHYLL IN PURPLE BACTERIA$EISENHOFERETA F L 4RONRUD ET AL  -C$ERMOTT ET AL  AND LIGHT HARVESTINGCOMPLEX))INPLANTS+àHLBRANDTETAL +  4HESEOBSERVATIONS TAKENTOGETHERWITH THE H LLMOLECULESWEREFOUN F D RECOGNITIONTHATCHLOROPHY INLAYERSONEITHERSIDEOFTHYLAKOIDMEMBRANESIN LIGHT HARVESTING COMPLEX )) ANDD THE CORE ANTENNA OF F 03) RESULTED IN THE CONSERVEDD (IS RESIDUES D IN #0 AND #0 BEING INVESTIGATED FOR A SIMILAR ROLE IN03)) %ATON 2Y 2 EANDD 6ERMAAS 3HEN ETAL 3HENANDD 6ERMAAS -ANNAAND 6ERMAAS   4HE INITIAL EXPERIMENTS IN BOTH #0ANDD #0 D HAT REPLACEDD (ISWITH EITHER'LNOR!SNASRESIDUEST MIGHTPROVIDEAXIALLIGANDS WHILE4YRWASCHOSENAS ITWASNOTEXPECTEDD TOCOORDINATETHE-G %ATON 2 2YEAN D 6ERMAAS 3HENETAL 3HENAND 6ERMAAS -ANNAAND6ERMAAS  )NTEREST INGLY Y ITHASNOW BEENSHOWNTHAT4Y 4 RCOORDINATESA H LL IN0SA" WHIL W ETHEEXPECTATIONTHAT(IS CHLOROPHY RESIDUESSERVEASCHLOROPHYLLLIGANDSIN#0AND #0WAS FURT F HERSTRENGTHENED BY THEFACTT F HAT H LLSOUTOFF ARECOORDINATED BY(ISIM CHLOROPHY IDAZOLESINTHECYANOBACTERIAL03)REACTIONCENTER *ORDANETAL   
.VUBHFOFTJT
PG
)JTUJEJOF
3FTJEVFT
JO
$1 4HENUMBEROFF (ISRESIDUES D INTHE lRSTPAIROF HELICES IN#0HASBEENTHESUBJECTOFDEBATE4HEREARE lVECONSERVEDD (ISRESIDUES D BETWEENTHE. TERMINUS ANDD THESTARTOF LOOP"THATCONNECTSHELIX))WITH HELIX ))) )N HELIX )) THE PREDICTED FOLDING MODELS OF#0P F LACE(IS ONTHELUMENAL SIDEOFF THE

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT MEMBRANEAND HENCE(IS ONTHESTROMALSIDE )NHELIX)HYDROPATHYANALYSESPOSITION(IS AND ONTHESTROMAL ORCYTOSOLICSIDE HOWEVER THE. TERMINAL HY H DROPHILIC DOMAINOFT F HEPROTEINCONTAINS A(ISRESIDUEATPOSITION WHICHIFPOSITIONEDINTHE D RETAINTHESPACINGOFF RESIDUES D MEMBRANEWOULD D (IS ONTHELUMENAL SIDE AND PLACE(IS AND 6ERMAAS  WAS THE lRST TO POINT OUT THAT THE D CAN CHARGESONTHESURROUNDING AMINOACIDSWOULD CEL INA HELICAL DOMAINAND D THEREFOREC F HARGEWOULD NOT BE A CONSTRAINTIF(IS  WAS INTHE MEMBRANE SPANNING DOMAIN %XPERIMENTALLY (IS  HAS BEEN D WITH 4YR IN A 03) MINUSAPC% C MINUS SUB U STITUTED STRAIN OF 3YNECHOCYSTIS 3 SP0##WHICHLACKS BOTH03)AND D ASSEMBLEDP D HYCOBILISOMES 3HENAND 6ERMAAS  4HEPHENOTYPEOFF THE(9MUTANT WHERE(IS HASBEENMUTATEDTO4YR WASSIMILARTO THATOBSERVED FORT F HE(9STRAINWITH BOTH MUTANTS EXHIBITINGANAPPROXIMATELY DROP D INACTIVE03)) CENTERS)TSHOULDBENOTEDTHATTHROUGHOUTTHISCHAPTER THESINGLELETTERAMINOACID D CODEISUSED D TO IDENTIFY MUTANTSCARRYINGAMINOACID D SU UBSTITUTIONS 4HE ASSIGNMENT OF PUTATIVE HISTIDYL LIGANDS TO CHLOROPHYLL BASED D ONTHE ¯03))STRUCTUREFROM 4 LONGATUS 0$"3, ISSUMMARIZED INTH 4E T E LEGEND OF&IG!TTHISRESOLUTION(IS ISTHEAXIALLIGANDTO D (IS  CHLOROPHYLL  (IS TOCHLOROPHYLLAND TOCHLOROPHY H LL#HLOROPHY H LL ISLOCATED INTHE MIDDLEOFTHEMEMBRANEANDHASBEENSUGGESTEDTO PARTICIPATEINENERGY TRANSFER F BETWEENTHEUPPERAND LOWERTIERSOFF ANTENNACHLOROPHY H LLS&ERREIRAETAL  4HUS ALTHOUGH(IS ISACHLOROPHYLLLIGAND (IS REMAINSASSOCIATED D WITH THECYTOSOLICSIDEOF THEMEMBRANE %XPERIMENTALLY SUBSTITUTING 4YR AT (IS  (IS  AND D (IS  PRODUCE D D MUTANTS WITH IMPAIRED 03)) ASSEMBLY AND D REDUCE D D RATES OF / EVOLUTION !SPARAGINE WAS INTRODUCED AT THESE POSITIONS AND SIGNIFICANTLY RESTORED / EVOLUTION SUGGESTING THAT !SN WAS ABLE TO ACT AS A CHLOROPHY H LL LIGAND D THEREBY SUPPORTING THE NOTION THAT THESE RESIDUES F HE-G &IG  AREINVOLVED INTHECOORDINATIONOFT 4HEMOSTSTRIKINGRESULT INTHISSERIESOFF MUTAGENESIS EXPERIMENTSWASOBTAINEDWHEN(IS AND(IS  WEREREPLACED BY'LNWHI W CH ISALSOKNOWNTOACTASA K CHLOROPHYLLLIGAND+àHLBRANDTETAL *ORDAN + ETAL  5NIQUELY THE(1MUTANTDISPLAYED AORNM BLUESHIFTO F F THE NM mUORESCENCE EMISSION"ASED D ONTHISOBSERVATIONAND D MODELING STUDIES (IS HASBEENPROPOSEDTOBETHELIGAND TOTHECHLOROPHYLL GIVING RISETOTHE NMABSORP

 TIONOFTHELOW TEMPERATURETRAPIN#03HENAND 6ERMAAS 6ASILEV ET AL  DE7EERD ET AL B (OWEVER CHLOROPHYLL  HASALSO BEEN SUGGESTEDD AS A CANDIDATE FFOR THE LONG WAVELENGTH ABSORBING SPECIES IN #0 AND CHLOROPHYLL  IN #0HAS BEENSUGGESTEDD TOHAVEASIMILARROLE &ER REIRAETAL   4YROSINE ANDD !SN SUBSTITUTIONS HAVE ALSO BEEN INTRODUCE D D ATTHE(ISRESIDUES D INHELICES)))AND)6 D  D IONOFF 4YRAT(IS ANDD (IS DID 4HE INTRODUCT NOT CREATE SEVERE RESTRICTIONS ON PHOTOAUTOTROPHIC GROWTHANDD (IS  IS IN FACTNOTCONSERVE F D INALL ORGANISMSALTHOUGH THEALTERNATIVERESIDUE D IS!SNIN EACHEXAMPLE)NCONTRAST THE(9AND(9 MUTANTS EXHIBITED IMPAIRED / EVOLUTION THAT WAS LARGELYREVERSED INTHE(.STRAIN4AKENTOGETHER THE RESULTS FROMTHE SUBSTITUTIONS AT(IS RESIDUES IN HELICES) )6IMPLY Y THATPUTATIVEPIGMENT PROTEININTER ACTIONSARENOTALL FUNCT F IONALLYEQUIVALENT(OWEVER DOUBLEANDTRIPLEMUTANTSINVOLVING(ISRESIDUESIN DIFFERENT F HELICESASWELLASTHEWITHINASINGLE HELIX WEREGENERALLYADDITIVE3HENETAL 3HENAND 6ERMAAS   3INCEANUMBEROFMUTANTSEX F HIBITED IMPAIREDD AN TENNA FUNCT F IONBASEDD ONTHELIGHT INTENSITIESREQUIRED TOSATURATE / EVOLUTION ASWELLASALTEREDmUORESCENCE DECAY A KINETICS THEHYPOTHESISWASPUTFORWAR F D THAT REPLACING(ISWITH 4YRMAYRESULT INPHEOPHYT H INATION OFCHLOROPHYLLANDIMPAIREDTRAPPINGANDEXCITATION ENERGY TRANSFER F INSUCH MUTANTS 3HENETAL  3HENAND D 6ERMAAS  DE7EERDD ETAL B 4HIS INTERPRETATIONWASSUPPORTEDBYTHE+mUORESCENCE EXCITATIONSPECTRAMONITORING THENMEMISSION )NTHE(ISTO4YRMUTANTS INCREASES INTHENMAND NMABSORPTIONREGIONSWEREOBSERVEDWHICHCO INCIDEWITHABSORPTIONMAXIMAOFP F HEOPHYTIN 3HEN D 6ERMAAS  7HETHER OR NOTCHLOROPHY H LLSARE AND CONVERTEDTOPHEOPHYTINVIATHELOSSOF-G INTHESE F MUTANTSORPHEOPHYTIN ISINCORPORATED INSTEADD OFA H LL INTHEBIOGENESISOFT F HECOMPLEX ISNOT CHLOROPHY KNOWN-OREOVER THEPOSSIBILITYTHATCERTAINMUTANTS ACTUALLY LOSEPIGMENTBINDINGCANNOT BE DISCOUNTED SINCETHEREISASIGNIlCANTDESTABILIZATIONOFF 03)) IN THE (9 ANDD (9 MUTANTSTHATIS NOT OBSERVED INTHE(9 (9ANDD (9STRAINS 4HERE ARE NO (IS RESIDUES D IN HELIX6 HOWEVER THISDOESNOTPRECLUDETHISTRANSMEMBRANEDOMAIN FFROM A ROLE IN PROVIDING AXIAL LIGANDS FFOR ANTENNA PIGMENTS)N 4E 4 LONGATUS EIGHTCHLOROPHY H LLSON0SA! ARECOORDINATEDBY'LN THECARBONYLBACKBONE  ANDD WATER  ANDD TENCHLOROPHYLLSON0SA"ARE

 COORDINATED BY'LN  !SP 4YR AND D WATER  *ORDANETAL  4OINITIALLYINVESTIGATETHE F FUNCT IONOFT F HETHREECONSERVED D (ISRESIDUES D IN HELIX H RESIDUEWASREP D LACED BY'LNYIELDINGTH T ETH T REE 6)EACH MUTANTS(1 (1AND(1%ATON 2YEAND 6ERMAAS  "OTH THTTHE(1AND D (1MUTANTS F IONAL 03 ))CENTERSATSIMILARLEVELSTO ASSEMBL M ED FUNCT THEWILDTYPE3UBSEQUENTLY THEADDITIONALMUTANTS (4 (9 AND D A DOUBL U EMUTANT&,(1 WERECONSTRUCTED D THATWERENOTPHOTOAUTOTROPHIC7U ETAL  4HISSERIESOFEXPERIMENTSISCONSISTENT WITH(IS AND D (IS PERFORM F ING AROLE INCHLO ROPHY H LL BINDINGAND D (IS  HASBEEN IDENTIlED D AS THEAPPARENTLIGANDTOCHLOROPHYLL THEPUTATIVE F ING ENERGY TO THE SPECIES MOST EFlCIENTLY TRANSFERR REACTIONCENTER6ASILEVETAL &ERREIRAETAL  !T(IS 'LNWASUNABLETOREPLACE(ISAND THENUMBEROFF ASSEMBLED03))CENTERS D INTHE(1 MUTANT FE F LLTOAPPROXIMATELYOFT F HEWILD D TYPE ALTHOUGH THE CENTERS PRESENT RETAINED THE CAPACITY F / EVOLUTION %ATON 2Y FOR 2 E AND D 6ERMAAS  (UANGETAL  4HESERESULTSSUGGESTTHAT(IS MAYPERFORMAROLEASSOCIATEDWITHTHESTABILITY Y HEINTRODUCT D IONOFF 0RO , ,YS OFF 03))!DDITIONALLY T D 4YRRESIDUESATT D HISPOSITIONRESULTED INOBLIGATE AND PHOTOHETEROTROPHICSTRAINSAND03))CENTERSDIDNOT ASSEMBLEINTHESECELLS7UETAL (UANGETAL L INTERGENICSUPPRESSORHASBEEN  )NTERESTINGLY AN OBTAINEDTHATRESTORESSIGNIlCANTRATESOF/ EVOLU TION PHOTOAUTOTROPHIC GROWTHAND D ASSEMBLYOFF 03)) TOTHESEMUTANTS(OWEVER THEGENERESPONSIBLEFO F R THESUPPRESSIONHASNOTYETBEENIDENTIlED(UANG ETAL   
.VUBHFOFTJT
PG
)JTUJEJOF
3FTJEVFT
JO
$1 4HE INVESTIGATIONOFF (ISRESIDUES D IN#0HAS BEEN LESSEXTENSIVE(OWEVER 'LNAND4YRSUBSTITUTIONS HAVE BEENINTRODUCE D D ATTHREEPOSITIONSFROM F HELICES ) AND D ))THATCORRESPOND D TO(IS  (IS AND D (IS IN#04HERESULTING#0MUTANTS(1 (1AND D (9WEREPHOTOAUTOTROPHIC WH W EREAS THESTRAINS(1 (9AND D (9WEREOBLIGATE PHOTOHETEROTROPHS -ANNA AND 6ERMAAS   4HESEEXPERIMENTSWERECONDUCTE D D IN 3YNECHOCY CYSTIS SP0##WHI W CH POSSESSESAONEAMINOACID DELE TIONAT POSITIONWHENCOMPAREDTOPLANTSEQUENCES Q SEE&IG 4HERESULTSOBTAINED INTHE(1AND (9CELLSARECONSISTENTWITH(IS ACTINGASA CHLOROPHYLLLIGAND)NTHISSTUDY(IS WASEFFEC TIVELY SUB U STITUTED BY4Y 4 RASWELL AS'LN4HISRESIDUE D

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT W CH ISTH T E PUTATIVE CORRESPONDSTO(IS  IN#0 WHI LIGANDFORTHELOWENERGYCHLOROPHYLLGIVINGRISETO THENMABSORPTIONBAND AN D D SUGGESTSTHATTHESE D ARE NOT FUNCT F IONAL HOMO CORRESPONDING RESIDUES LOGUES&ERREIRAETAL HAVEASSIGNED(IS  F HLOROPHYLL ANDD (IS ASTHE ASTHE LIGAND FORC LIGAND FORC F HLOROPHY H LL  &IG (OWEVER OFF THE THREERESIDUESINVESTIGATEDSUBSTITUTIONSAT(IS  THEPUTATIVE LIGAND FORC F HLOROPHYLL  PRODUCE D D THE MOST INTERESTING RESULTS AS THE (1 MUTANT WAS STILL ABLETO EVOLVE / EVENTHOUGHITWASUNABLETO SUPPORTPHOTOAUTOTROPHIC GROWTH3URPRISINGLY T Y HIS MUTANT EXHIBITED IMPAIREDD OXIDATION OF 1!n BY THE SECONDARY PLASTOQUINONE ACCEPTOR 1" AND SHOWED INCREASEDD CHARGERECOMBINATION BETWEEN1!nANDTHE DONORSIDEOFF 03)) SUGGESTINGANINCREASEDD STEADY STATELEVELOFTHEOXIDIZEDPRIMARYDONOR0 INTHIS STRAIN-ANNAANDD 6ERMAAS  4HEASSIGNMENT OFT F HE HISTIDYL LIGANDSFORT F HE#0CHLOROPHY H LLS IS SUMMARIZEDINTHELEGENDOF&IG *7
5IF
)ZESPQIJMJD
%PNBJOT
PG
$1
BOE $1 "EFORETHESTRUCTURALDATADEMONSTRATEDTHEARRANGE MENTOFF #0AND#0 D INTHETHYLAKOID D MEMBRANE THETOPOLOGYHAD BEENESTABLISHED FOR#0 F BY3AYRE AND7ROBEL "OERNER WHOUTILIZEDANTIPEPTIDE ANTIBODIESAGAINSTTHEEXTRINSIC LOOPS! # $ANDD % D INI!FTER F LIMITEDTRYPS D INHY H DR D O ANDD THE. AND# TERM LYSISANDPEPTIDEMAPPINGOFTHYLAKOIDMEMBRANES THEY CONCLUDEDD THAT#0CONTAINEDD SIXMEMBRANE SPANNINGHELICES THATTHE. AND# TERM D INIWERESTRO MALLYEXPOSEDANDTHATTHEEXTRINSICLOOPS! #AND %WEREEXPOSEDD TOTHE LUMEN4HISRESULTSUPPORTED THEHYPOT H HESISFORT F HE DOMAINORGANIZATIONOF#0 F AND#0BASEDONANEARLIERANALYSISOFHYDROPATHY PLOTS 6ERMAASETAL "RICKER   4HESIMILARITY BETWEENTHECHLOROPHY H LL A BINDING PROTEINSOF03)AND03))ISMOSTPROMINENTINTHE F DIMERS MEMB M RANE SPANNING DOMAINSOFF THETRIMER OF MOTIFW F WHEREASTH T EHY H DROP D HILIC DOMAINSCONNECTINGTH T E TRANSMEMBRANEHELICESHAVEDIVERGEDTOREmECTTHE A INTHEPHOTOSYSTEMS SPECIlCROLESTHESEPROTEINSPLAY 3CHUB U ERTETAL "ARB R ERETAL  )N03)) HYDROPHILIC AND MEMBRANE SPANNING DOMAINS OF #0AND#0ASWE D LLASTHOSEOF$AN F D $ARE SIMILARBETWEENPLANTS ALGAEANDCYANO D BACTERIAAND THE PSB! B GENE FROM THE HIGHER PLANT 0 0OA ANNUA ENCODING $ HAS BEEN SUCCESSFU F LLY EXPRESSED IN

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT THE CYANOBACTERIUM 3YNECHOCYSTIS SP 0##  RESULTINGINFULLYFUNCTIONAL03))COMPLEXES.IXON # GENESFROM ETAL  (OWEVER THE PSB" AND PSB# SPINACH CANNOT SUB U STITUTE FFOR THEIR CYANOBACTERIAL COUNTERPARTS 4O IDENTIFY DOMAINS THAT COULD BE D CYANOBACTERIAL CORE EXCHANGED BETWEENSPINACH AND ANTENNA PROTEINS #ARPENTERETAL  GENERATED CHIMERIC SPINACHCYANOBACTERIAL #0 MUTANTS IN 3Y 3 NECHOCY CYSTIS SP 0##  AND FOUN F D THAT AN OBLIGATEPHOTOHETEROTROPHWASPRODUCE D D WHENMOST OFTHE PSB#GENEWASOFSPINACHORIGIN(OWEVER # TRANSFORMAT F ION WITH A PLASMID D CONTAINING THE |  OF PSB# FFROM THE CYANOBACTERIUM RESTORED PHOTOAUTOTROPHIC GROWTH TO CHIMERIC MUTANTS )N D THAT CONTRASTSIMILARPSB" CHIMERICSTRAINSINDICATED ONLYAMINOACID D RESIDUESTOO D F #0NEED BE OFCYANOBACTERIALORIGIN6ERMAASETAL  4HIS D F LOOP$ REGION INCLUDESLOOPS" #ANDPARTO 4HEAVAILABLESTRUCTURAL DATA FORT F HEARRANGEMENT OF03))SUP U ER COMPLEXESTHATCONTAINBOTH PERIPH ERAL AND D CORE ANTENNA PROTEINS FROM F GREEN PLANTS D CYANOBACTERIA SUGGEST THE POSSIBILITY THAT THE AND EXTRINSICPROTEINSOF03)) THATAREFOUNDASSOCIATED WITHTHE LUMENAL FACEO F F HEPHOTOSYSTEM MAY FT A PLAY A ASTRUCTURALROLE4HUS INADDITIONTOTHEIRABILITYTO ENHANCE / EVOLUTIONATPHYSIOLOGICAL CALCIUM AND A STABILIZE03))IN CHLORIDECONCENTRATIONS THEYMAY AMANNERTHATISSPECIlCTOTHESTRUCTURALCONSTRAINTS IMPOSEDBYTHEDIFFERENTORGANIZATIONOFTHEPERIPHERAL ANTENNAPROTEINS4HEREFORE F INADDITIONTOFUNCT F ION ALLYIMPORTANTINTERACTIONS BETWEENTHECOREANTENNA PROTEINS AND DIFFERENT TYPES OF PERIPHERAL ANTENNA T E LUMENAL DOMAINSOFF #0AND#0MA D AY SYSTEMS TH HAVESPECIlC PROTEIN PROTEININTERACTIONSIN DIFFER F ENTTYPESOFPHOTOSYNTHETICORGANISMS4HEFUNCTION OFF THE HYDROPHILIC DOMAINS HAS BEEN INVESTIGATED THROUG H H OLIGONUCLEOTIDE DIRECTED D MUTAGENESISSTUDIES IN 3YNECHOCYSTIS SP0##ANDBYBIOCHEMICAL INVESTIGATIONSTHATHAVE BEENPERFORME F D INLARGEPART USINGSPINACH "
.VUBHFOFTJT
PG
)ZESPQIJMJD
%PNBJOT
PG $1
JO
4ZOFDIPDZTUJT
TQ
1 4 $$  %XTENSIVEMUTAGENESISSTUDIES SUMMARIZED IN&IG HAVE BEEN DIRECTED D ATUNDERSTANDINGTHEROLESOFT F HE HYDROPHILICDOMAINSJOININGTHEMEMBRANE SPANNING HELICESIN#0AND D THE# TERMINAL REGION(OWEVER THESYSTEMDESIGNED FOR F INTRODUC D INGMUTATIONSINTO THE lRST  AMINO ACIDS HAS ONLY BEEN UTILIZED IN INVESTIGATINGTHEROLEOFF (IS  (IS AND D (IS 

 AS DISCUSSEDD ABOVE 3HEN AND6ERMAAS D   )N CONTRASTLOOPS " %ANDTHE# TERMINUSHAVEBEEN PROBED BYY THEINTRODUCT D IONOFS F HORTDELETIONSDESIGNED TOCREATEAMAP IDENTIFY F ING IMPORTANTREGIONSWHIL W E CIRCUMVENTINGTHEIMPRACTICALITYOFMUTATINGALLOF THERESIDUES D F FOUN D INTHESEDOMAINS%ATON 2Y 2 EAND 6ERMAAS (AAGETAL -ORGANETAL  #LARKEAND%ATON 2YE #LARKEETAL   &OURSTRAINSWERECREATEDD WITH DELETIONSINLOOP" THATEXTENDS FROM!SP TO,YS 4 F HEMUTANTS 6$ , AND 6$ + WHERE THIS NOMENCLATURE INDICATES DELETIONS FFROM!SP  TO ,EU  AND D !SP  AND ,YS  RESPECTIVELY WEREOBLIGATEPHOTOHETEROTROPHSANDONLYAPPEARED TO HAVE LOW LEVELS OF F ASSEMBLED D 03 )) CENTERS )N CONTRAST STRAINSCARRYING DELETIONS INTHEMIDDLEOF LOOP " REMAINED PHOTOAUTOTROPHIC ,OOP $ SPANS HELICES)6AND6 D FROM!R F G TO4HR  AND IT IS H HATTHECORRESPONDINGLOOPS IN0SA!AND NOTEWORTHYT 0SA"CONTAINANEXTRAHELIXTHATISABSENTIN#0!LL DELETIONS INTRODUCE D D INTOLOOP$PRODUCE D D OBLIGATE PHOTOHETEROTROPHICSTRAINSAND ITIS POSSIBLETHATTHESE MUTANTS PREVENTTHECORRECTINSERTIONOF#0INTO R EETAL  )NTERESTINGLY A Y LL THEMEMBRANE #LARK F HEDELETIONSTHAT HAVEBEEN INTRODUCE D D INTOTHE# OFT TERMINALDOMAINREMAINPHOTOAUTOTROPHIC(OWEVER THE 63 4 MUTANTWASFOUN F D TO HAVESLOWED RATES OF 1!n OXIDATION AND THUS EXHIBITED A SIMILAR DISRUP U TION OF ELECTRON mOW BETWEEN 1! AND 1" AS OBSERVED INCHIMERICCYANOBACTERIALSPINACH #0 F HERUNEXPECTED STRAINS 6ERMAASETAL  !FURT PHENOTYPEWASTHEGENERATIONOFPROTEINADDUCTSTHAT WERERECOGNIZED BYA$ SPECIlCANTIBODY #LARK R E 4 ET AL   )N THE  ¯ 03 )) STRUCTURE FROM 4 VULCANUS ANDTHE ¯ STRUCTURE FROM 4ELONGATUS 4 THE# TERMINALAMINOACIDSINTHEVICINITYOF!R F G  TO'LY OFF #0ARECLOSETO'LU TO'LY  OFTHE$%LOOPOFTHE$ PROTEIN4HECURRENTDATA SUGGESTSTHAT DELETIONS PARTICULARLYTHOSECLOSESTTO F CAN FAC F ILITATETHE FORMAT F ION THE# TERMINUSOF#0 OF PROTEIN AGGREGATES INCLUDING $ #LARKE ET AL   )N ADDITION BOTH 03)) STRUCTURES POSITION RESIDUESATT D THE. TERMINUSOFF #0 INCLOSEPROXIMITY TOTHISREGIONOFTHE$%LOOPALTHOUGHANYFUNCTIONAL F T HESE PUTATIVE PROTEIN PROTEIN SIGNIFICANCE OF INTERACTIONSREQU Q IRESEXPERIMENTAL CLARIlCATION 3HORTDELETIONSOFBETWEENTHREEANDlVEAMINO D D INTO LOOP # BY ACIDS HAVE ALSO BEEN INTRODUCE #LARK R E AND D %ATON 2YE   ,OOP # IN #0 IS APPROXIMATELY HALF THE LENGTH OBSERVED FOR THE CORRESPONDING DOMAIN IN 0SA! ANDD 0SA" AND IT



+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT

&IIG 3UMMARY OFMUTAT & F IONSINTRODUCE D D INTO HYDROPHILIC LOOPSOFF #0 IN3YNECHOCY CYSTIS SP0##$ELETIONSARE INDICATED BY SQUAREBRACKETSANDINDIVIDUALAMINOACIDSTHATHAVEBEENMUTATEDAREINDICATEDBYABLACKDOTABOVETHERESIDUE$ELETIONSORPOINT MUTATIONSCREATINGSTRAINSTHATAREUNAFFECTEDORMILDLYAFFECTEDARESHOWNINNORMALFONTMODERATELYAFFECTEDSTRAINSARESHOWNIN ITALICS ANDINTRODUCEDMUTATIONSTHATRESULTEDINOBLIGATEPHOTOHETEROTROPHICSTRAINSAREUNDERLINED4HEUNDERLININGORITALICFONTFOR DELETIONSCONTAININGPOINTMUTATIONSHAVEBEENMODIlEDTOINDICATETHEPHENOTYPEOFTHEPOINTMUTATIONS

ISRESIDUESSHORTERTHANLOOP#IN#0!LLOF THE DELETIONS INTRODUCE D D INTO LOOP # RESULTED IN A SIGNIlCANTDESTABILIZATIONOFF 03))WITH THEDELETION BETWEEN'LY AND0RO PRODUCINGANOBLIGATE PHOTOHETEROTROPHIC STRAIN AND D THE DELETIONS IN THE D D MUTANTS 63 0 AND 69 ' REDUCE THELEVELOF03 ))TO OFTHATFOUNDINTHECON TROL4HETWOREMAINING MUTANTS 6% ! AND 6& . WEREPHOTOAUTOTROPHICEVENTHOUGH THENUMBEROFPHOTOSYSTEMSWASREDUCEDANDTHEY D ANINCREASEDSUSCEPT D IBILITY TOPHOTOINACTI EXHIBITED VATION4HE 6% ! STRAINWASALSOUNABLETO GROWUNDERR CHLORIDE LIMITINGCONDITIONS WHICHSUG GESTSTHATLOOP#MAY A CONTRIBUTETOTHEPOLYPEPTIDE Q IRED FO F R CHLORIDERETENTIONINTHE STRUCTURETHATISREQU PHOTOSYSTEM"RICKERAND&RANKEL   4OEXPLORETHEROLEOF LOOP#WITHINTHELUMENALLY EXPOSED PROTEIN ENVIRONMENT ASSOCIATED D WITH THE /%# BOTH THE 6% ! AND 6& . A SENCEOFF THE0SB/ STRAINSWERECHARACTERIZED INTHEAB

0SB5AND0SB6PROTEINS)NALLCASES03))ACTIVITYWAS F HERDESTABILIZEDD WITHTHE 6% ! MUTANT FURT BEINGMOREIMPAIREDD THANTHE 6& . STRAIN ANDTHEORDEROFIMPORTANCEFORTHEREMOVALOFTHE EXTRINSICPROTEINSWASFOUN F D TO BE 60SB6 * 60SB/ 60SB5)NTHEAVAILABLESTRUCTURALMODELSOFF 03 )) THE0SB6 PROTEINISINCONTACTWITHLOOP%OF#0 THEREFORE F THE SENSITIVITY OFF #0 MUTANTS TO THE F B6CANNOTBEEXPLAINED INSTRUCTURAL REMOVAL OF0S TERMSATTHECURRENTRESOLUTIONOFTHECRYSTALLOGRAPHIC DATA)NTHECASEOF LOOP#TWOMUTANTSHAVEALSOBEEN INTRODUCE D D AT4RP 4HE72STRAINWASUNABLE TO GROW PHOTOAUTOTROPHICALLY WHEREAS THE 73 CELLSEXHIBITEDD AREDUCE D D RATEOF BOTH THPHOTOAUTOTROPHIC GROWTHAND/ D  EVOLUTION-OREOVER ANENHANCEDD RATE OFPHOTOINACTIVATIONWASOBSERVEDINBOTHMUTANTS %LANSKAY A AETAL 7UETAL   4HE DOMAINTHATCLEARLYDISTINGUISHESBOTH#0 AND#0FROMTHEADDITIONALCHLOROPHYLL BINDING PROTEINS THAT POSSESS THE TRIMER OF F DIMERS MOTIF

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT IS LOOP % SEE &IGS  AND D   ,OOP % OF F #0 IS POSITIONEDSUCHTHATALARGEPORTIONISEXPOSEDDIRECTLY TO THE LUMENAL COMPARTMENT 4HE STRUCTURAL DATA INDICATETHATTHIS DOMAINISNOT IN DIRECTCONTACTWITH T THE$ PROTEINANDISTHEREFORENOTEXPECTEDTOPROVIDE F HECATALYTICSITE +AMIYA ANY N LIGANDSTOTHE-NOFT D 3HEN &ERREIRAETAL  4OPROBE FO F R AND THEFUNCTIONOFTHISREGIONlFTEENSCANNINGSEGMENT DELETIONSHAVE BEEN INTRODUCE D DONEOFT F HESE FROM F 'LY TO4HR  DELETED D AMINOACIDSAND IT IS LIKELYTHATTHEOBLIGATEPHOTOHETEROTROPHICPHENOTYPE F L OFF THECORRESPONDINGMUTANTRESULTED FROMASTRUCTURA PERTURB R ATION %ATON 2YE AND D 6ERMAAS   )N &IGTHEREMAININGFOURTEENDELETIONSARESHOWNAND THESEPRODUCE D D MUTANTSCARRYING DELETIONSOF BETWEEN AND D AMINOACIDS%ATON 2YEAND6ERMAAS  D (AAGETAL -ORGANETAL  &IVEOFTHESE RESULTED INOBLIGATEPHOTOHETEROTROPHICSTRAINSWITH IMPAIRED D 03)) ASSEMBLY HOWEVER THIS PHENOTYPE APPEAREDTODEPENDONTHELOCATIONOFTHEDELETION RATHERTHANTHE LENGTH$ELETIONSINTHElRSTAMINO ACIDSOFF THELOOP WERENOTTOLERATED D AND THEMUTANT 6$ 0 CARRYINGADELETIONADJACENTTOTHE START OF F THE LAST TRANSMEMBRANE DOMAIN WAS ALSO AN OBLIGATE PHOTOHETEROTROPH (AAG ET AL   4HE DELETIONSINLOOP%WEREORIGINALLYDESIGNEDTO INCLUDE'LUAND!SPAM D INOACIDSAND D THEOBJECTIVE WASTOESTABLISH IFF SPECIlCRESIDUESW D ITHINADELETION WERERESPONSIBLEFORTHEOBSERVEDPHENOTYPES4HE D RESIDUES D WITHIN THE DELETIONS IN THE NON CHARGED PHOTOAUTOTROPHIC STRAINS WERE EACH SUBSEQUENTLY INVESTIGATED BY 0UTNAM %VANS ET AL A B  )N THIS WORK RK ALL STRAINS OBTAINED D WERE FOUN F D TO BE PHOTOAUTOTROPHIC AND IT WAS SUGGESTED THAT THE PHENOTYPE OF THE ORIGINAL DELETION STRAINS AROSE FFROM THE STRUCTURAL PERTURB R ATION INTRODUCE D D BY THE DELETION 0UTNAM %VANS ET AL A "RICKER AND &RANKEL   0UTNAM %VANSETAL B ALSOMUTATED D CHARGED D RESIDUES D BETWEEN 'LU  ALL CONSERVED AND!RG INLOOP%4HESEEXPERIMENTSPRODUCED INNOCUOUSMUTATIONSATALL POSITIONSEXCEPTTHEBASIC D !RG AND!R D G )N&IG ITCANBESEEN RESIDUES THATPOSITIVERESIDUESARECONSERVEDATTHESE POSITIONS INALL SPECIESEXAMINED!MINOACIDD SU UBSTITUTIONSAT THISLOCATIONCREATED D MUTANTSEXHIBITING AREDUCT D IONIN / EVOLVINGCAPABILITY ANALTERATIONINTHEQUANTUM YIELD FOR F / EVOLUTION A SLOWING IN THE RISE TIME D INGTHE 3 A ;3= A 3 TRANSITION OF / RELEASE DUR FOR A DISCUSSION OF THE 3 STATES SEE #HAPTER  D AN INCREASE IN THE 3 DECAY A LIFET F IME 0UTNAM AND

 %VANS AND "RICKER  0UTNAM %VANS ET AL B  2ELATIVE TO WILD TYPE THE SEVERITY OF THE F LLOWINGTHEINTRODUCT D IONOFMUTAT F IONS PHENOTYPEFO ATTHESESITESWAS22 %%22 '' 2'2')NADDITION 0SB/WASFOUNDTO EXHIBITWEAKENED BINDING INTHE22 %%AND 22 ''STRAINSWHI W CHACCELERATEDD THET FOR F DARKINACTIVATIONOF03))FROMHINWILDTYPETO H INTHE22 %%MUTANTANDD ANINTERMEDIATE T OFHINTHE22 ''STRAIN1IANETAL  )NADDITION DELETIONOFTHE PSB/ GENEINTHE 22 %%MUTANTWAS FOUN F D TO HAVE LITTLEEFFECT F SUGGESTINGTHATTHEMUTATIONSINTHE22 %% STRAIN ANDREMOVALOFTHE 0SB/ PROTEIN AFFECTSIMILAR PARAMETERSASSOCIATEDD WITHTHEREACTIONMECHANISM F / EVOLUTIONANDTHAT!RG AND!RG FORM FOR ONE OF THE BINDING SITES FOR THE 0SB/ POLYPEPTIDE "RICKERANDD &RANKEL   !DDITIONALSITE DIRECTEDD MUTAGENESISEXPERIMENTS TARGETINGBASICAMINOACIDSINLOOP%HAVEREVEALED THATMUTATIONSINTRODUCE D D AT, ,YS ANDD !RG  ALTERTHECHLORIDE REQU Q IREMENT FOR F PHOTOAUTOTROPHIC GROWTH 0UTNAM %VANS AND "RICKER    4HE+'MUTANTFA F ILEDD TOASSEMBLE03))CENTERS UNDER CHLORIDE LIMITING CONDITIONS AND IN NORMAL "' ONLYOF03))WASPRESENTADDITIONALLY THERATEOFF PHOTOINACTIVATIONWASINCREASEDD THREE FO F LD WHEN COMPAREDD TO WILD D TYPE AT HIGH LIGHT INTENSI TIES0UTNAM %VANSAND"RICKER  %ARLIER THE MUTANT 2' HAD BEEN SHOWN TO PREVENT THE AS SEMBLYOF03))UN F DERCHLORIDE LIMITINGCONDITIONS 0UTNAM %VANSAND"RICKER  5SINGARANDOM H THEADDITIONAL MUTANTS2+ MUTAGENESISAPPROACHT 21 23AND27WEREO D BTAINEDD AND THE STRAIN 2$ WAS SUBSEQUENTLY PRODUCED 7U ET AL "RICKERETAL "RICKERANDD &RANKEL  "OTH THE2$AND27MUTANTSWERE D OBLIGATE PHOTOHETEROTROPHS ALTHOUGH 21 CELLS EXHIBITED HIGHERRATESOF / EVOLUTIONTHANTHE2+ F R STRAINSUGGESTINGTHATCHARGEWASNOTTHEONLYFACTO GOVERNINGTHEREQUIREMENTFOR!RGATTHISPOSITION )NTERESTINGLY W Y WHENTHE23MUTANTWASGROWNIN NORMAL "' MEDIUMANDD THENPLACED INCHLORIDE LIMITING CONDITIONS THE / EVOLVING ACTIVITY WAS LOSTATTHESAMERATEASWILDD TYPE Tš  nMIN  (OWEVER WH W ENTHECHLORIDESTEP DOWNWASREVERSED BYTHEADDITIONOFM-CHLORIDE THERECOVERYOF / EVOLVING ACTIVITY WASRETARDED BY APPROXIMATELY F LD IN THE 23 STRAIN WH W EN COMPAREDD WITH SIX FO THECONTROL"RICKERETAL THEREFORESUGGESTED D DEFECT F INTHE THATMUTATIONSAT!RG  INTRODUCEA

 LOWAFlNITY RAPIDLYEXCHANGING CHLORIDE BINDINGSITE DESCRIBEDBY,INDBERGAND!NDREASSON !DDI TIONAL DETAILSOFSTRA F INSCARRYING MUTATIONSAT!RG R  AREDISCUSSED IN"RICKERAND D &RANKEL   #
#JPDIFNJDBM
4UVEJFT
%JSFDUFE
BU
-VNFOBM %PNBJOT
PG
$1 ! WELL ESTABLISHED INTERACTION EXISTS BETWEEN THE 0SB/ PROTEINAND#0AN D D THISISALSO DISCUSSED IN #HAPTER!NEARLYSERIESOFEXPERIMENTSUTILIZING D 03)) MEMBRANES OBTAINED ALKALINE 4RIS WASHED F FROMSP INACH INDUCE D D ACONFORMAT F IONAL CHANGEAS SOCIATEDWITHLOOP%OF#0"RICKERAND&RANKEL D THE0SB/ 0SB0AND  4HISTREATMENTREMOVED 0SB1PROTEINSALONGWITHALL FOURO F F HEMANGANESE FT ASSOCIATEDWITHTHE / EVOLVINGSITETHISLEDTOTHE F ITOPEBETWEEN0RO AND3ER  D EXPOSUREOFANEP THATWASRECOGNIZED BY&!# ASPECIlCMONOCLONAL ANTIBODY "RICKER AND &RANKEL  &RANKEL AND "RICKER  !LSO IN03))MEMBRANESITHAS BEEN SHOWNTHATTHE# TERMINAL PORTIONOF LOOP %CAN BE CROSS LINKEDTOTHE. TERMINALAMINOACIDSOF0SB/ BY  ETHYL   DIMETHYLAMINOPROPYL CARB R ODIIMIDE %$# "RICKERETAL %NAMIETAL /DOM AND"RICKER  4HISREAGENTCROSS LINKSAMINO GROUPSTOCARB R OXYLICACIDSTHATAREINVAN DER7AALS D 3TRITTMATTER   0EPTIDE CONTACT (ACKETT AND MAPPINGOFTHECROSS LINKEDCOMPLEXOBTAINEDWITH %$#REVEALED D THATTHE DOMAIN BETWEEN'LU AND D TOTHE lRSTAMINOACIDS !SP WASCROSS LINKED OF0SB//DOMAND"RICKER   %ARLIERITHAD BEEN DEMONSTRATED D THATTHEREAGENT DITHIOBISSUCCINIMIDYL PROPIONATE $430 ALSO CROSS LINKEDTHESETWOPROTEINSANDTHATTHISINVOLVED THE REGION ,Y , S  TO ,Y , S  ON #0 %NAMI ET AL  "RICKER ET AL  1UEIROLO  "RICKERAND&RANKEL  )NCOMPLIMENTARYSTUD IES IT HAS BEENSHOWNTHATTHEREAGENT. HYDROSUC CINIMIDOBIOTINLABELSAMINOGROUPSON#0WH W EN 0SB/HASBEENREMOVED&RANKELAND"RICKER   D TWO 3UB U SEQUENTPEPTIDE MAPPING STUDIES IDENTIlED BIOTINYLATED DOMAINSTHESEWERE,YS TO,YS  AND,YS TO,YS INLOOP%FROMSPINACH#0 &RANKEL AND D "RICKER  4HE0SB/PROTEINALSO F LYTIC DIGESTION BYTRYPSIN SHIELDS#0 FROMPROTEO ANDLYSOENDOPEPTIDASE#"RICKERAND&RANKEL  (AY A ASHIETAL  4HEEXPERIMENTSWITH LYSOEN DOPEPTIDASE#ALSO FOUN F D THAT/ EVOLVINGACTIVITY ANDTHEABILITYOFTHE0SB/PROTEINTOREBINDTO03)) DECREASED INPARALLEL WITHTHEPROTEOLYTICCLEAVAGE

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT AT,YS INLOOP%(AYASHI ETAL  4HESE RESULTSTHEREFOREINDICATEDTHATTHEREGIONOFLOOP% INTHEVICINITYOFF ,YS WAS IMPORTANTFOR0S F B/ BINDING AND / EVOLVING ACTIVITY 3EIDLER   4HERESULTSOFTHEPROTEINCROSS LINKINGEXPERIMENTS BIOTINYL N ATIONSTUDIESANDD PEPTIDASESTUDIESARESUM MARIZED IN&IG 0OST TRANSLATIONALMODIlCATIONOFSPECIlCAMINO F HE/%#HAS BEENREPORTEDD THAT ACIDS INTHEVICINITYOFT PRODUCE D D ACTIVATEDD CARRBONYL GROUPSCAPABLEOF FORM F

INGCOVALENTADDUCTSWITHAMINES/UELLETTEETAL A ELING OFF THESE GROUPS !NDERSONETAL  ,AB HAS PROVIDEDD EVIDENCE FO F R QU Q INONECOFACTORS F LOCATED ON$ $AND#03INCELABELINGDIDNOTOCCURIN THEPRESENCEOFF THE0SB0ANDD 0SB1PROTEINS ANDD THE ABSENCEOFF 0SB/ANDMANGANESE D F FAVORE D LABELING IT HASBEENSUGGESTEDTHATTHEPUTATIVEQUINONECOFACTORS ARELOCATED INASTERICALLY RESTRICTED DOMAIN7HILE IT HAS BEENPOSTULATEDD THATSUCHMODIlEDD AMINOACIDS ASSOCIATEDWITHTHE / EVOLVINGSITEOF03))COULD A AFUNCT F IONALROLE INWATERSPLITTING TH T ELOCATIONOF PLAY F HASNOTYET BEENMAPPEDONTOANYO D F THESECOFACTORS THELABELEDPROTEINS/UELLETTEETAL !NDERSON ETAL  (OWEVER EVIDENCEFORPOST TRANS F LATIONAL MODIlCATIONOFF !SP  INSPINACH#0RESULTING INTHEGENERATIONOFAN ASPARTYLALDEHYDEHASBEEN OBTAINED !NDERSONETAL   ,OOP! OF F #0 HAS ALSO BEEN SHOWN TO CROSS LINK TO 0SB/ WITH %$# THEREFORE INDICATING THAT ,OOP! ISINVANDER7AALSCONTACTWITHA DOMAIN ON 0SB/ /HTA ET AL  4HESE STUDIES USING SPINACH 03)) ENRICHED MEMBRANES INDICATE THAT !SP OR'LU  IN LOOP!INTERACTWITH0SB/INTHE VICINITYOFF ,YS  ,YS  ,YS ANDOR,YS  )NTRIGUINGLY THEAVAILABLE03))STRUCTURALDATASUP U PORTSTHISCONCLUSIONAND FURT F HERINDICATESTHATEACH 0SB/PROTEIN INTHE DIMERMAY BINDTO,OOP%O D F ONEREACTIONCENTERMONOMERANDLOOPS!AND%OF THEOPPOSITEMONOMER4HISTHEREFORESU F GGESTSTHAT PROTEIN INTERACTIONSBETWEEN LUMENALSUB U UNITSOFT F HE TWOREACTIONCENTERSOFTHEDIMERIC03))HOLOENZYME AREIMPORTANTINOPTIMIZINGTHEPROTEINENVIRONMENT SURROUNDINGTHESITEOFF WATERSPLITTING $
5IF
3FNPWBM
PG
UIF
&YUSJOTJD
1SPUFJOT
JO $1
.VUBOUT 4HE INTERPRETATIONOFT F HEPHENOTYPESOBSERVED INTHE T NHY H DROP D HILIC DOMAINS SEGMENT DELETIONMUTANTSWITHI OF #0 IS DIFlCULT SINCE IT IS HARD TO DISTINGUISH BETWEENAPHENOTYPEARISING FROMT F HEREMOVAL OFAN F

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT IMPORTANTRESIDUEORAMORE D INDIRECTSTRUCTURALCHANGE 0UTNAM %VANSETAL A"RICKERAND&RANKEL F THE LOOP %   &UNCTIONAL COMPLEMENTATION OF OBLIGATE PHOTOHETEROTROPHIC STRAIN 6$ 0 F NDTH D ATRESTORATION USING COMBINATORIAL MUTANTS FOU OFF THE WILD TYPE LENGTH WAS SUFlCIENT TO SUPPORT PHOTOAUTOTROPHICGROWTH TH WHILENONEOFT F THEAMINOACID RESIDUESWITHINTHEDELETIONWEREINDISPENSABLE4ICHY D 6ERMAAS  3EVERALOFT F HEPHOTOAUTOTROPHIC AND D EXHIBIT STRAINSOBTAINED BYTHESEAUTHORS HOWEVERDID AN ALTERED CHLORIDEREQUIREMENTANDTHEREFORERESIDUES Q BETWEEN!SP AND0RO APPEARTOP D LAY AYASIMILAR D TOTHE!RG  ROLE INCHLORIDE BINDINGASASCRIBED MUTANTSEXAMINEDBY"RICKERETAL  !DIFFERENTAPPROAC F HWASTAKEN BY -ORGANETAL  W WHO SELECTED D TWO LOOP % DELETION MUTANTS THATHADREMAINEDPHOTOAUTOTROPHIC4HESEDELETIONS WERE CHOSEN TO ACT AS PROBES FFOR THE EFFECT F OFF THE T ERWISE INNOCUOUS REMOVAL 0SB/ 0SB6OR0SB5 ONOTH MUTATIONSWITHINLOOP%4HESESTUDIESTARGETEDBOTH THEDOMAINBETWEEN'LU AND!SP T D HAT HAD BEEN SHOWN TO CROSS LINK K TO . TERMINUS OF F 0SB/ /DOMAND"RICKER  ANDTHEEPITOPERECOGNIZED BY &!# "RICKER AND D &RANKEL  &RANKEL AND "RICKER  4HE TWO DELETION MUTANTS SELECTED WERE 62 6 WHICH IS LOCATED WITHIN THE &!# EPITOPE AND 6' 4 WHI & W CH IS AT THE # TERMINAL ENDD OFT F HE LOOP %DOMAINTHATCROSS LINKS TOTHE. TERMINUSOF0SB/ 4HE REMOVAL OF F 0SB/ 0SB5 OR 0SB6 IN THE 6' 4 STRAINRESULTED INOFF 03))CEN TERSASSEMBLINGANDNONEOFTHESEMUTANTSREMAINED PHOTOAUTOTROPHIC 4HUS REMOVAL OF F THE EXTRINSIC PROTEINS HAD D ANADDITIONALEFFECTTOT F HATINTRODUCE D D BYDELETIONOF'LY TO4HR )NCONTRAST THE 62 6 60SB/ AND 62 6 60SB5 STRAINSREMAINED PHOTOAUTOTROPHICAND D THUS BEHAVED IN A SIMILAR FASHION TO THE 22 %% MUTANT INDICATING THATREMOVAL OFF 0SB/HASASIMILAREFFECT F ON/ EVOLUTIONTOTHECREATIONOFT F HE22 %% AND 62 6 STRAINS%ATON 2YEAND6ERMAAS  0UTNAM %VAN ET AL B -ORGAN ET AL  )THASALSOBEENSHOWNTHATTHE 6! $ MUTANT AS WELL AS THE 62 6 22 %% AND 22 '' STRAINS ALL EXHIBIT WEAKENED BINDING OF F 0SB/ 'LEITER ET AL  1IAN ET AL   )NTERESTINGLY THE 62 6 60SB6 MUTANTASSEMBLED D APPROXIMATELYOFT F HECONTROL LEVELOFF 03 ))CENTERS BUTTHESEWEREINACTIVEAND D THE STRAINWASANOBLIGATEPHOTOHETEROTROPH-ORGANET D WITH AL  4HUS ALTHOUGH0SB6 ISASSOCIATED

 LOOP%OFF #0 ITAPPEARSTOPLAYANIMPORTANTROLE INSTABILIZING03))INBOTHTHE 62 6 AND 6' 4 MUTANTS INTHESEEXAMPLES ITISPOS SIBLETHAT0SB6CONFERSSTA F BILIZATIONTHROUGH 0SB5 ASTHIS PROTEINISSEENTOBEINCONTACTWITH0SB/AND 0SB6INTHE ¯ AND¯ RESOLUTIONSTRUCTURES +AMIYAANDD 3HEN &ERREIRAETAL   )THASALSOBEENSHOWNTHATREMOVALOF0SB/ IN WILD D TYPEAN Y D THE22 %%MUTANTINCREASESTHE D OF PHOTOACTIVATION IN HY H DROXYLAMINE QUANTUMYIELD T TREATED03))"URNAPETAL 1IANETAL  )N CONTRAST THEQUANTUM YIELD D OFP F HOTOACTIVATIONINTHE 62 6 MUTANTASWELLASTHE 6! $ STRAINWASCONSIDERABLYREDUCEDWHENCOMPAREDTO D TYPE 'LEITERETAL -ORGANETAL   WILD 7HILETHEEXPERIMENTAL CONDITIONS INTHESESTUDIES ARENOTDIRECTLYCOMPARABLE THERESULTSSUGGESTTHAT D INTHEVICINITY OFF !LA TO6AL  OTHERRESIDUES D PERTURB R EDD AS A RESULT OF F THE TWO OR OTHER RESIDUES DELETIONSINmUENCETHE PHOTOACTIVATIONPROCESS!N IN DEPTH T DISCUSSIONOFF THISPOINTISPROVIDED IN"RICKER ANDD &RANKEL   !SDISCUSSEDEARLIER3ECTION)6! THEINTRODUC TIONOFMUTAT F IONSATCONSERVEDD CHARGEDD AMINOACIDS BETWEEN'LU AND D !SP DEMONSTRATEDD THATONLY L SUBSTITUTIONSAT!RG AND!RG IN 3YNECHOCYSTIS I SP 0##  PRODUCE D D IMPAIREDD PHENOTYPES )N CONTRAST WH W EN OTHERWISE INNOCUOUS MUTATIONS AT 'LU  WERE INVESTIGATED IN THE ABSENCE OF THE EXTRINSICPROTEINSUNEXPECTEDD RESULTSWEREOBTAINED D 60SB6 WERE 4HEMUTANTS%'60SB6AND%1 NOTPHOTOAUTOTROPHICANDASSEMBLEDLESSTHANOF THEWILD TYPE LEVEL OF03)) F HOWEVER THEPHENOTYPES OFT F HE%'AND%1STRA D INSWEREBOTHSIMILARTO WILDTYPE-ORGANETAL  )NCONTRAST MUTANTS D LACKING 0SB6WEREFOUN F D AT!SP AND!SP  TOHAVEPHENOTYPESSIMILARTOTHEPHOTOAUTOTROPHIC 60SB6CONTROLSTRAINEVENTHOUGHCHARGEDRESIDUESAT 2 EAND THESEPOSITIONSAREHIGHLY CONSERVED %ATON 2Y D 4HEREFORE ' F LU APPEARSTOPLAYAN 3HAND  IMPORTANTROLEINTHEASSEMBLYOROPERATIONOF03)) D L OFF 0SB6)TISTHEREFORE F THATIS UNMASKEDUPONREMOVA INTERESTINGTHAT&ERREIRAETAL  HAVE IDENTIlED THEADJACENT0HE AND0HE RESIDUESASPARTOF ACONSERVEDD MOTIF &&%3&06), IN 4 4 ELONGATUS G &IG F WHICH 0HE ANDD 0HE CONTRIBUTETO  FROMW THEHYDROPHOBICENVIRONMENTINTHEVICINITYOFTHE REDOXACTIVE9$ FROMT F HE$PROTEIN4HIS LOCATION Q IVALENTPOSITIONOFT F HEMETAL CORRESPONDSTOTHEEQU CLUSTEROFTHE/%#ON$#HAPTER  !N EQUALLY INTRIGUING OBSERVATION HAS BEEN

 THE DISCOVERY THAT THE %160SB6 6 62 6 60SB6 AND 62 6 60SB/60SB5 STRAINSAREALLUNABLETO GROWPHOTOAUTOTROPHICALLY IN"' MEDIAAT P(BUT HAVETHEIRPHOTOAUTO TROPHIC GROWTH RESTORED WHEN GROWN AT P(  %ATON 2Y 2 E ET AL   )N CONTRAST THE &2 6' 4 AND 6% ! MUTANTS WHI W CH EXHIBIT PHOTOAUTOTROPHIC GROWTH ALBEIT WITH AN D REQUIREMENT FFOR CHLORIDE BECOME OBLIGATE ALTERED PHOTOHETEROTROPHSUP U ONREMOVALOFF 0SB6ATBOTH P( ANDP(#LARKEAND%ATON 2YE   %ATON 2Y 2 EETAL  4HESPECIlCITYOFF THISP( DEPENDENCE POINTSTOAMECHANISMFORT F THEACCLIMATION OF03))ACTIVITYINRESPONSETOTHEEXTERNALP(THAT F IONONTHEOPERATIONOF MAY A PROVIDENOVEL INFORMAT 03)) DUR D INGENVIRONMENTAL CONDITIONSTHATRESULT IN P(VARIATION %
.VUBHFOFTJT
PG
-PPQ
&
JO $1
JO Z 4ZOFDIPDZTUJT
TQ
1$$
 4Z -UTAGENESISTECHNIQUES HAVEALSO BEENUTILIZED D TO STUDYTHEROLEOFTHELARGEEXTRINSICLOOP%OF#0 IN03))ASSEMBLY AND FUNCT F ION+UHNAND6ERMAAS D  PRODUCE D D ASETOF EIGHT DELETION MUTANTS IN 3 3YNECHOCYSTIS SP0##INWHICHnCODONS WEREDELETEDWITHINTHEREGIONOFPSB# FP ENCODING LOOP %4HE DELETEDSEGMENTSSPANNE D D 'LN TO!LA  W WHICHCOMPRISEAPPROXIMATELYOFTHISDOMAIN AND D ARESHOWN IN&IG3IGNIlCANTLY A Y LL DELETION F ILED D TOGROWPHOTOAUTOTROPHICALLYAN L D NONE MUTANTSFA EXHIBITEDDETECTABLE/ EVOLVINGACTIVITY4HISRESULT ISSTRIKING INCOMPARISONTOTHESIMILAREXPERIMENTS WITH PSB" IN W WHICH ONLY A SUB U SET OF F THE DELETIONS PRODUCEDINLOOP %OF#0RESULTEDINSTRAINSTHAT EXHIBITED IMPAIRED D PHOTOAUTOTROPHICGROWTH OR03)) D 6ERMAAS (AAGETAL ACTIVITY%ATON 2YEAND -ORGANETAL  4HEPRESENCEOF#0IN THETHYLAKOIDSOFT F HESEMUTANTSRANGED FROMA F LMOST WILD TYPELEVELS INONESTRAINTONO DETECTABLE#0

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT INSEVERALMUTANTS4HE LEVELSOFF THEOTHER03 ))CORE PROTEINS$ $AND#0WEREALSODECREASEDINALL F BEING MOSTPRONOUNCED FO F R MUTANTS WITH THEEFFECT $4HESEOBSERVATIONSWERESIMILARTOTHOSESEEN IN MUTANTS IN WHI W CHTHE ENTIRE PSB#GENEHADBEEN # DELETEDD ORINACTIVATED/NLYONEOFT F HEEIGHT DELETION STRAINSASSEMBLEDD ANYFUNCT F IONALCENTERSASASSESSED BY03)) SPECIlCHERBICIDE BINDINGSTUDIESEMPLOY ING ;#= DIURON!DDITIONALLY Y mUORESCENCEEMISSION SPECTRASUPPORTEDD THE LACKO K F FUNCT F IONAL ASSEMBLYOF THE03))COMPLEX 'IVENT I THESEVEREPHENOTY TYPEOF THEDELETIONMUTANTS D ISWASUSEDD TOALTERSPECIlC SITE DIRECTEDMUTAGENES AMINO ACID RESIDUES WITHIN LOOP % OF #0 IN AN EFFORTTO F IDENTIFY RESIDUEST D HATARECRITICAL FOR03)) F F FUNCT IONANDD THATMAY INTERACT DIRECTLYWITH THE/ EVOLVINGSITE4HENUMBERINGOFRESIDUESIN#0IS PROBLEMATICBEYONDD THEDISCREPANCY INTRODUCE D D BYY THE DELETIONOFAS F INGLEAMINOACID INTHE 3YNEC 3 HOCYSTIS SP0##SEQUENCE4HEORIGINALPROPOSALFOR THE START CODON BY!LT ET AL  WAS  BASES F THE SUB U SEQUENTLY IDENTIlEDD POSITION UP STREAM OF IN CYANOBACTERIA #HISHOLM AND 7ILLIAMS  'OLDENANDD 3TEARNS #ARPENTERETAL   4HE. TERMINUSOFT F HEPROTEINHASALSO BEENSHOWN TOUNDERGOPOST TRANSLATIONALMODIlCATIONSINCLUDING D HR  ANENDOPROTEOLYTICCLEAVAGEBETWEEN'LU AND4 )KEUCHI ETAL -ICHELETAL  4HEREFORE F INCERTAINCASES4HR IN&IGISREGARDEDASTHESTART OFT F THEPROTEINEG +NOEPmEETAL "ARB R ERETAL F LLOWING DISCUSSIONTHENUMBERING IN  )NTHE FO THEORIGINALPAPERSISGIVENTOGETHERWITHIDENTIlCATION OFT F HEEQUIVALENTRESIDUES D IN&IGSANDD  &IVE AMINO ACID D SU UBSTITUTIONS WITHIN THE LARGE 3 SP0##HAVE EXTRINSICLOOP IN 3YNECHOCYSTIS BEENCONSTRUCTEDD THATSHOWDEFECTSASSOC F IATEDD WITH THE /%# -UTAGENESIS OFF !RG  AND !RG  TO3ERRESULTEDINMUTANTSSHOWINGIMPAIRED03)) ACTIVITY +NOEPmEETAL  4HE23MUTANT SEE!RG  IN &IG  GREW PHOTOAUTOTROPHICALLY

&IG  3UMMARYOFMUTAT & F IONS INTRODUCE D D INTO LOOP%OF#0 F IN 3YNEC 3 HOCYSTIS SP0##$ELETIONSORPOINTMUTATIONSCREATING STRAINSTHATAREUNAFFECTE F D ORMILDLYAFFECTE F D ARESHOWNINNORMAL FONTMO F DERATELYAFFECTE F D STRAINSARESHOWN IN ITALICS AND INTRODUCE D D MUTATIONSTHATRESULTED INOBLIGATEPHOTOHETEROTROPHICSTRAINSAREUNDERLINED

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT AND D ACCUMULATED D NORMAL QUANT Q ITIES OF F #0 $ #0ANDTHE0SB/ PROTEINBUTITEVOLVED/ TOONLY OFF CONTROL RATESATSATURATING LIGHTINTENSITIES )N AGREEMENT CHLOROPHYLL FLUORESCENCE YIELD MEASUREMENTS INDICATEDD THATTHIS MUTANT ASSEMBLED F THE 03)) CENTERS FOUN F D IN APPROXIMATELY  OF THECONTROLSTRAIN4HE23MUTANT SEE!RG  IN &IG  FAILED TO GROW PHOTOAUTOTROPHICALLY AND D NOCAPACITY FOR/ F W EN EXHIBITED  EVOLUTION EXCEPTWH GROWN PHOTOHETEROTROPHICALLY IN MEDIA CONTAINING BOTHGLUCOSEANDDIURON5NDERTHESECONDITIONS/ EVOLVING ACTIVITY TYWASOBSERVED INTH T E23MUTANTAT A LEVEL OFF APPROXIMATELYOFF THECONTROLRATE4HE APPARENTPROTECTIVEEFFECTOFDIURONMAYHAVERESULTED F FROMARE D IONOF DAMAGING OXIDIZING SIDERADICALS DUCT INTH T EPRESENCEOFT F THIS03))INHIBITOR#HUETA H L  7 7UETAL  )SOLATEDTHYLAKOIDMEMBRANESFROM THIS MUTANT ALSO ACCUMULATED D NORMAL AMOUNTS OF 03))COREPROTEINS6ARIABL A ECHLOROPHY H LL mUORESCENCE YIELDMEASUREMENTSFORTHE23MUTANT HOWEVER INDICATED D THATITASSEMBL M ED D ASEVERELYREDUCE D D NUM MBER F ABLETOOXIDIZEWATER R AND D THESECENTERS OF03))CENTERSA SHOWEDANEXTREMESENSITIVITYTOPHOTOINACTIVATION "OTHSTRAINSEXHIBITED D AN INCREASE AND D  FO F LD F FOR23AN D 23 RESPECTIVELY INmUORESCENCE RISETIMESUSINGWATERASANELECTRONDONOR INDICATIVE OFF ASLOWINGOFE F LECTRONTRANSPORT FROMT F HE/%#TO 9Z 4HESE DATACONlRMED D THATTHE23AND D 23 MUTATIONSEACH PRODUCEDADEFECTASSOCIATEDWITHTHE /%#OF03)) F 4HE23MUTANTADDITIONALLYY SHOWED F HERIMPAIRMENTOFF 03))ACTIVITYWH FURT W ENGROWNIN CHLORIDE DEPLETEDMEDIA4HISEFFECTMAYBEEXPLAINED INPARTBYTHE LOSSOFF 0SB63ECTION)6%  "ASED D ONTHE03))MINUSPHENOTYPEOFF THE23 MUTANTNUMBERINGSYSTEMUSEDBY+NOEPmEETAL D THEFACTT F HAT#0 IS INCONTACTWITH$  AND !RG  HAS BEEN PROPOSED D AS A POTENTIAL LIGAND TO THE MANGANESE CLUSTER $EBUS   %ARLIER WORK R %NAMI ETAL  SHOWED D THATTHISRESIDUE D CORRESPONDINGTO!RG  IN&IG WASCLEAVED BY T TRYPSININTHEABSENCEOF0SB/ANDTHUSWASPRESUME D TOBEATORNEARABINDINGSITE FORT F HE0SB/PROTEIN 3ECTION )6%  (OWEVER THE 23 MUTANT IS MORESEVERELYIMPAIREDTHANMUTANTSLACKING0SB/ D THUSTHEPHENOTYPEOFT F HISMUTANTCOULD D NOT BE AND EXPLAINED BY AN ALTERATION IN 0SB/ BINDING ALONE 4HE RECENT STRUCTURAL MODEL FOR 03)) PROVIDED BY &ERREIRAETAL  INDICATESTHATTHE/%#CONTAINS ACUB U ANE-N#A/ CLUSTERLINKED D TOAFOURT F H -NBY A MONO — OXO BRIDGE AND AT  ¯ RESOLUTION THE PUTATIVE LIGANDS FORT F HECLUSTERHAVEBEENASSIGNED

 #HAPTER 3IGNIlCANTLY !RG  &IG ISAS SOCIATEDWITHANON PROTEINLIGANDTOTHEFOURTH-N IONAND ISSUGGESTEDD TO FORMA F HYDROGENBONDD TOTHE D INGTHEREACTIONCYCLE4HEREFORE F SUB U STRATEWATERDUR T THERESULTSOBTAINEDWITHTHE23MUTANT+NOEPmE ET AL  PROVIDE EXPERIMENTAL SUPPORT FFOR THE DIRECTPARTICIPATIONOFF #0INWATERSPLITTING 2OSENBERGETAL CONSTRUCTEDTHREEMUTANTS IN 3YNECHOCY CYSTIS SP0##WH W ERE'LURESIDUES D INTHE LARGEEXTRINSICLOOPWEREALTEREDTO' D LN4HE %1 STRAIN SEE 'LU  IN &IG  EXHIBITED NORMAL PHOTOAUTOTROPHIC GROWTH ANDD ACCUMULATED Q ITIESOFF THE#0 #0 $ANDD 0SB/ NORMAL QUANT PROTEINS(OWEVER THISMUTANTEVOLVED/ TOONLY OFCONTRO F LRATESATSATURATING LIGHTINTENSITIESAND A ECHLOROPHY H LL mUORESCENCEYIELD D MEASUREMENTS VARIABL INDICATED THAT THIS STRAIN ASSEMBLED APPROXIMATELY  OF F THE FUNCT F IONAL 03)) CENTERS FOUN F D IN THE CONTROL 4HE %1 MUTANT SEE 'LU  IN &IG  GREW PHOTOAUTOTROPHICALLY BUT AT A SEVERELY REDUCED RATE "OTH IMMUNOLOGICAL ANALYSIS AND VARIABLECHLOROPHY H LL mUORESCENCEYIELDEXPER D IMENTS INDICATEDTHATTHE%1STRAINASSEMBLEDANORMAL COMPLEMENT OFF 03)) CENTERS (OWEVER LESS THAN ONE HALFF OFF THE CENTERS COULD SUPPORT ELECTRON TRANSFER FROM WATER TO 1! ANDD THE MUTANT EVOLVED / AT  OF F THE RATE FOUN F D IN THE CONTROL "OTH %1 AND D %1 STRAINS EXHIBITEDD AN INCREASED SENSITIVITYTOPHOTOINACTIVATION4HESERESULTSSHOW THATTHEMUTATIONS INTHE%1 ANDD %1 CELLSALSO PRODUCE D D DEFECTSASSOC F IATEDD WITH THTTHE/%#-OREOVER THEMODELBY&ERRIERAETAL BASEDONTHEIR  ¯8 RAY A CRYSTALSTRUCTURESHOWSTHAT'LU OF #0LIGATESTHETHIRDD -N INTHECUB U ANECLUSTERTHIS IS'LU INTHENUMBERINGSYSTEMOF2OSENBERGET AL  ANDD 'LU  IN&IGTHENUMBERING GIVEN F HISRESIDUE FORT D IN&ERRIERAETAL ASSUMESTHE -ETSTARTOF#0TOBERESIDUESUP U STREAMOFTHE D ITHINTHE STARTSHOWN IN&IG 4HUS TWORESIDUESW #0LARGEEXTRINSIC LOOP 'LU AND!RG  D IN &IG APPEARTOBEINVOLVEDINWATERSPLITTING 4HE%1MUTANTSEE'LU IN&IG EXHIBITED THEMOSTSEVEREPHENOTYPEOFF ALLMUTANTSCONSTRUCTED 4HISMUTANTFAILEDTOGROWPHOTOAUTOTROPHICALLYAND EXHIBITED D NO CAPACITY FFOR / EVOLUTION UNDER ANY N CONDITIONS-EASUREMENTSOFT F THEVARIABL A ECHLOROPHY H LL FLUORESCENCE YIELD INDICATED THAT THIS MUTANT ASSEMBL M EDD NOFUNCT F IONAL03))CENTERS)MMUNOLOGICAL ANALYSISOF ISOLATEDD THY H LAKOID D MEMBRANES FROMT F HE %1 STRAIN REVEALED A COMPLETE ABSENCE OF ANY DETECTABLE#0ANDD REDUCE D D LEVELSOF BOTH THE$

 AND D 0SB/ PROTEINS4HE'LUTO'LNMUTATIONINTHE %1 STRAIN APPEARS TO AFFECT THE STABILITY OF THE 03))COMPLEX F ION ON THE ROLE STRUCTUR 4HERE IS LITTLE INFORMAT ALLY OR OTHERWISE OF THE LUMENALLY EXPOSED LOOPS D #IN#04HE 3YNECHOCY CYSTIS SP0## !AND D "OGORAD MUTANT CHARACTERIZED BY $ZELZKALNS AND L WHICHLACKEDANY03))ACTIVITY WASFOUNDTO RESULTFROMA F DELETIONIN PSB# # THATCORRESPONDSTOA DELETIONOFF AMINOACIDSnIN&IG,OOP# APPROXIMATELYSPANSAMINOACIDSn ANDTHUS MORETH T ANHALFF OF,OOP# F ISMISSING INTHI T SMUTANT TOGETH T ERWITH TH ALL OFF TRANSMEM MBRANE HELIX)6AND D ALL BUTTHREEAMINOACIDSOFLOOP$WHICHISLOCATEDONTHE F ITISLIKELYY THATTH T EPHENOTY TYPE CYTOSOLICSIDE4HEREFORE OFF THISMUTANTRESULTED FROMAGROSSPERTUR F RBATIONOF T E NATIVE TH I STRUCTU TURE &
*OUFSBDUJPO
PG
$1
XJUI 0UIFS
1IPUPTZ TZTUFN
**
4VCVOJUUT 
*OUFSBDUJPO
PG
$1
XJUI
% 'IVEN THE INTIMATE ASSOCIATION OFF #0 AND $ H :OUNI ET NOWCONlRMED BY8 RAYCRYSTALLOGRAPHY AL +AMIYA AND 3HEN  &ERREIRA ET AL  #HAPTERS n THERE IS LITTLE INFORMAT F ION BASED D ON BIOCHEMICAL STUDIES THAT DEMONSTRATES A DIRECT INTERACTION OF THESE TWO PROTEINS -ORI AND 9 9AMAMOTO  SHOWED D THAT$ #0AND D #0 WERE LOST DUR D ING4RIS TREATMENT UNDER LOW LIGHT IL LUMINATION +MOL PHOTONSq Mn SECn INSPINACH 03))MEMBRANES4HEYSPECULATED D THATTHISLOSSWAS Q F DONOR SIDEPHOTOINHIBITION SINCE ACONSEQUENCEO ITCOULDBEPREVENTEDBYTHEADDITIONOFEXOGENOUS ELECTRON DONORS TO 03)) .O DEGRADATION PRODUCTS D OFF EITHER$OR#0WERE DIRECTLYOBSERVED)NAN EXPANDED STUDY THIS SAME LOSS OF $ #0 AND #0 WAS OBSERVED BOTH IN 03)) MEMBRANES AND IN ISOLATED D 03)) COMPLEXES SUBJ U ECTED D TO THE SAME TREATMENT-ORIETAL  )NBOTHPREPARATIONS HIGH MOLECULAR MASS POLYPEPTIDES WERE DETECTED BY3$3UREA0!'%AND IMMUNOBLOTTING AND D THESE CROSS REACTEDWITHANTIBODIESAGAINST$ANDANTI BODIES GENERATED D AGAINSTEITHER#0OR#04HUS THELOSSOF#0COU F LDPART D IALLYBEATTRIBUTED D TOTHE F FORMATIONOFACROSS LINKED PRODUCTBETWEEN#0 D $ -ORI ETAL  3IMILARRESULTSWEREOB AND TAINED D WITH TH 4RIS TREATED D 03))MEM MBRANESSUBJ U ECTEDD TO ILLUMINATIONATVERYHIGHLIGHTINTENSITIES +MOL PHOTONSqM Mn SECn 9AMAMOTOAND D !KASAKA  

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT )NTHESEEXPERIMENTSTHREEDEGRADATIONPRODUCTSOF #0WEREOBSERVED ASWELLASHIGHMOLECULARWEIGHT CROSS LINKEDPRO D D DUCTSCONTA INING$)NRECONSTITU TIONEXPERIMENTS THEPRESENCEOFT F HE0SB/PROTEIN APPEAREDTOINHIBITBOTHTHEDEGRADATIONOF#0AND THEAPPEARANCEOFF CROSS LINKEDD PRODUCTS4 D HISSUG GESTSTHATALUMENAL DOMAINON#0 FORMSAT F LEAST ONESITESUSCEPTIBLETOPROTEOLYTICATTACK#URRENTLY THEEXACTSITESOFCROSS F LINKING BETWEEN#0AND $AREUNKNOWN K 'IVENTHEWELL KNOWNROLEOF$INTURNOVEROF THE PHOTOSYSTEM UNDER PHOTOINHIBITORY CONDITIONS REVIEWED IN9AMAMOTO  #HAPTER  #HOW AND!RO THEABOVEDATAALSOINFERAROLEFOR#0 INTHISPROCESSBY VIRTUEOF ITSCLOSEASSOCIATIONWITH $ %ARLY WORK R INDICATEDD THAT $ TURNOVER DUR D ING PHOTOINHIBITIONOCCURREDVIAPROTEOLYTICDEGRADATION !ROETAL 6I 6 RGINETAL  3TUDIESUSING VARIOUSCLASSESOF PROTEASEINHIBITORSDEMONSTRATED THAT ASERINE TYPEPROTEASEWASRESPONSIBLEFORTHE TURNOVER OFF $ 6I 6 RGIN ET AL 6I 6 RGIN ET AL  ANDD THATTHISPROTEASEWASTIGHTLYASSOCIATED WITHTHE03))COMPLEX6IRGINETAL !ROET AL  3OMEEVIDENCEPOINTSTO#0ACTING AS D ING A SERINE PROTEASE CAPABLE OF DEGRADING $ DUR PHOTOINHIBITION 'IACOMETTI ET AL  3ALTER ET AL   5SING SPINACH 03)) CORE PREPARATIONS 3ALTERETAL  DEMONSTRATEDD THAT#0COULD BE COVALENTLYLABELEDWITHTHESERINEPROTEASEINHIBITOR ;#= DIISOPROPYL mUOROPHOSPHATE 0HOTOINACTIVA TION INDUCE D D CLEAVAGEOFF $ W WHICH OCCURRED INTHE REGIONBETWEENTRANSMEMBRANEHELICES$AND%ON ASTROMA EXPOSEDD SITE WASINHIBITED BYTHIS LABELING OF #04HERADIOLABELEDD SERINE IN#0WASNOT IDENTIlEDHOWEVER SINCECLEAVAGEOF$OCCURSIN THEEXPOSEDD REGION THELABELEDD SERINEBYNECESSITY HASTOOCCURONTHESTROMAL SURFACEO F F #0 $EGRADATIONOF$AND$UNDERPHOTOINHIBITORY CONDITIONSHAS BEENREPORTED INISOLATEDD 03))REAC TIONCENTERCOMPLEXESCONTAINING$ $ THE _ AND ` SUBUNITS OF CYTOCHROME B AND THE PSB) ) GENE PRODUCT D BUT LACKING #0 3HIPTON ANDD "ARRBER  )TWASSUGGESTEDD THATDEGRADATIONOFF $OCCURS THROUGHANAUTOPROTEOLYTICMECHANISM4HISARGUES AGAINST#0ACTING ASASERINEPROTEASE(OWEVER THEPRESENCEOFF CONTAMINATINGAMOUNTSOFF #0OR SOMEOTHERPROTEASEWASNOTADDRESSED!DDITIONALLY 6 RGIN ET AL  SHOWEDD THAT ONLY  INHIBI 6I TIONOFF $ DEGRADATIONOCCURRED INTHEPRESENCEOF DIISOPROPYLmUOROPHOSPHATE SUGGESTING THAT OTHER PROTEASESANDORMECHANISMSAPPEARTOEXISTTHATARE

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT INVOLVED INTHEPROCESSOF$TURNOVER F REVIEWED IN 9 9AMAMOTO   
*OUFSBDUJPO
PG
$1
XJUI
1TC0 %ARLY BIOCHEMICAL EVIDENCEBY )SOGAIETAL  SUPPORTED D THE HYPOT H HESIS THAT THE LUMEN EXPOSED LOOPSOF#0PROVIDEDSITESOFINTERACTIONBETWEEN #0ANDCOMPONENTSO D F THE/%#4HESEAUTHORS D THAT #0 COULD BE CLEAVED BY TRYPSIN IN SHOWED SPINACH03))MEMBRANESFOLLOWINGREMOVALOFTHE 0SB/PROTEIN BY WASHING WITH #A#L 4HEEXTENTOF DEGRADATIONOF#0WAS F DIRECTLYCORRELATED D WITH THE EXTENTOFREMOVALOF0SB/ ANDTHEAUTHORSCONCLUDED THAT THESE DATA DEMONSTRATED D A DIRECT INTERACTION BETWEEN0SB/AND D #0(OWEVER THEEXACTSITES OFCLEAVAGEWITHIN#0WERENOTIDENTIlED%NAMI ETAL MAPPED D THETRYPSINCLEAVAGESITESAND SHOWED D THAT!RG !RG  IN&IG LOCATED D ON THE STROMAL SIDE OF THE MEMBRANE WAS CLEAVED IN .A#L WASHEDSP D INACH 03))MEMBRANESTHATLACK K THE 0SB0 AND D 0SB1 EXTRINSIC PROTEINS (OWEVER UP U ON REMOVALOF0SB/TWOADDITIONALRESIDUES !RG  !RG  IN&IG AND D ,YS ,Y , S IN&IG WERECLEAVED/NEOFF THESERESIDUES !RG  D LIES WITHINTHELARGEEXTRINSICLOOP%)NADDITIONTOCLEAV AGEOF#0 C F LEAVAGEAT!RG OFF THE _ SUB U UNITOF CYTOCHROME B WAS ALSO OBSERVED )N RECONSTITU TION EXPERIMENTS THE ABILITY OF 0SB/ TO REBIND TO TRYPSINIZED #A#L WASHED MEMBRANES DECREASED WITH INCREASING TRYPSIN CONCENTRATION INDICATING THAT EITHER A DOMAIN AROUND!RG  WITHIN LOOP % ANDORADOMAINCLOSETO!RG INCYTOCHROME B WASREQUIRED FOR F BINDING)TSHOULD BENOTED D THAT THEFRAGMENT'LY TO!SN CORRESPONDINGTO 'LY TOTHE# TERMINUSIN&IG ISFOUN F D INTHE F HIGHLYUNLIKELYTOBEINVOLVED STROMAAND ISTHEREFORE INTHEBINDINGOF0SB/4HEAUTHORSSPECULATEDTHAT REMOVAL OF F 0SB/ CAUSES A CONFORMAT F IONAL CHANGE WITHIN#0THATALLOWS,YS TOBEACCESSIBLETO TRYPSIN4HESEDATACOLLECTIVELYINDICATETHAT0SB/ IS CLOSELY ASSOCIATED D WITH #04HESE RESULTS ARE ALSOCONSISTENTWITH THECURRENTSTRUCTURAL MODELSOF 03))INWHICH#0APPEARSTOBEINCONTACTWITH THE0SB/ 0SB5AND D 0SB6PROTEINS 
*OUFSBDUJPO
PG
$1
XJUI
1TC7 4HEMUTATIONINTHE23STRAIN THATCORRESPONDSTO AMUTATIONAT!RG IN&IG WASINTRODUCEDINTO THELARGEEXTRINSIC LOOPOFF #0FROM F 3YNECHOCY CYSTIS

 SP0##ANDRESULTEDINASTRAINTHATGREWPHO TOAUTOTROPHICALLYANDEXHIBITEDAPPROXIMATELY F HE/ EVOLVINGACTIVITYOFW F ILD D TYPE+NOEPmE OFT ETAL  (OWEVER 03))ACTIVITYINTHISMUTANT WAS DRAMATICALLY DECREASED WHEN IT WAS GROWN IN MEDIADEPLETEDD OF CHLORIDE9OUNGETAL 3EC TION)6$ 5NDERCHLORIDE LIMITINGCONDITIONS THIS MUTANT FAILED TO GROW PHOTOAUTOTROPHICALLY AND IT EVOLVED / TOONLYAPPROXIMATELYOFF THECONTROL 4HEMUTANTALSOSHOWEDD ANENHANCEDSUSCEPT D IBILITY Y STABILIZATION OF TO PHOTOINACTIVATION !DDITIONALLY F HE/%#WASOBSERVED FORT F HEMUTANT THE 3 STATEOFT UNDERCHLORIDE LIMITINGCONDITIONS 4HEOBSERVEDPHENOTYPEOFTHISMUTANTISSIMILAR TOTHATOBSERVEDD WHEN PSB6 THEGENEENCODINGCYTO 6 6

CHROME C HASBEENDELETED3HENETAL A B  4HE PSB6DELETIONMUTANTDOESNOTGROWPHOTOAU 6

TOTROPHICALLY UNDERCHLORIDE LIMITINGCONDITIONSOR F HATMATTER UNDERCALCIUM LIMITINGCONDITIONS FORT ANDEXHIBITSANENHANCEDSUSCEPTIBILITYTOPHOTOIN ACTIVATIONANDD ASTABILIZATIONOFT F HE3 STATE-ORGAN ETAL 3HENETAL  4OSTUDYTHEPOSSIBLE EFFECTSOFTHEMUTATIONON03))ASSEMBLY THE03)) COMPLEXESOF BOTHMUTANTANDD CONTROLSTRAINSGROWN IN CHLORIDE DEPLETEDD MEDIA WERE ISOLATEDD UTILIZING A SIX RESIDUE (IS TAG LOCATED ON THE | TERMINUS OF F THE #0 PROTEIN "RICKER ET AL    )MMUNOBLOTS OFF THE ISOLATED D 03)) CORE PARTICLES DEMONSTRATEDTHATFORCONTROLAND23CELLSTHERE WASNODETECTABLELOSSOFT F HE#0 #0 $AND 0SB/ PROTEINS4HE03))PARTICLESWEREALSOPROBED FORTHE PRESENCEOF0SB62EDUCEDMINUSOXIDIZED DIFFERENCESPECTRAO F F FCONTRO L 03))PARTICLESSHOWED R ANCEAT ANASYMMETRICPEAKK WITHAMAXIMUMABSORB NMANDA PRONOUNCEDSHOULDERATABOUTNM 4HESEPEAKSARISE FROMC F YTOCHROME B AND 0SB6 RESPECTIVELY L +ASHINOETAL  4HE03))PARTICLES F FROMTHE23MUTANT HOWEVER SHOWEDAHIGHLY SYMMETRICALPEAKK WITHNOAPPARENTSHOULDERAT NMANDD ANABSORPTIONMAXIMUMOFF NM INDI CATINGTHATTHEPARTICLESCONTAINEDLITTLEORNO0SB6 3TAINING OFF CYTOCHROMESUSINGACHEMILUMINESCENT SUB U STRATESHOWEDD NO DETECTABLE0SB6 PRESENTINTHE 03)) PARTICLEPREPARATIONHOWEVER 0SB6WAS PRES ENTINWH W OLECELL EXTRACTSFROMT F HEMUTANTAT LEVELS A ETOTHOSEOBSERVED FORT F HECONTROL INDICAT COMP M ARABL INGTHATTHEPROTEINWASSYNTHESIZEDBUTFAILEDTOBE INCORPORATED INTOTHE03))COMPLEX4HUS ITAPPEARS THATREPLACEMENTOFF !RG !RG  IN&IG BY 3ER PREVENTS THE STRONG ASSOCIATION OF 0SB6 WITH 03)) INDICATING THIS RESIDUE D MAAY FFORM PART OF THE

 BINDING DOMAIN )NSPECTION OF THE  ¯ STRUCTURE F OM 4 FR 4 ELONGATUS SUGGESTS THAT!RG  IN &IG  F HYDROGEN BOND D WITH!SN  FROM0S F B6 MAY A FORMA )NTERESTINGLY AN!SPTO!SNMUTATIONATTHEADJACENT RESIDUE IN 3YNECHOCYSTIS 3 SP0##ALSORESULTSIN AMUTANTSHOWING ANALTERED DEPENDENCYONCHLORIDE #0UTNAM %VANS UNPUBLI U SHED  ' $PWBMFOU
.PEJGJDBUJPOT
PG
)ZESPQIJMJD %PNBJOT
PG
$1 D TANDEMMASSSPECTROM !NDERSONETAL USED ETRY T TOMAPPOST TRANSLATIONAL MODIlCATIONSOCCURRING INTHELARGEEXTRINSICLOOPOF#03PECIlCALLY THESE MODIlCATIONSWEREMAPPED D TOACONSERVED PEPTIDE !07,%0,2WITHINTHE LARGEEXTRINSIC LOOP BETWEEN !LA AND!RG IN&IG7ITHINTHISPEPTIDE THE INDOLE SIDE CHAIN OF4RP  F WAS SHOWNTO BE POST TRANSLATIONALLYMODIlED D SUCH THATITEXHIBITED MASSSHIFTSOF AND4HEMODIlCATION WAS IDENTIlED D AS KYNURENINE AND D THEAND D  MODIlCATIONSWEREIDENTIlED D AS OXINDOLYLALANINEAND HYDROXYINDOLE DERIVATIVES RESPECTIVELY +YNUREN INEAND D THEOXINDOLYLALANINEDERIVATIVESHAVEBEEN H DROXYIN REPORTED INOTHERPROTEINSHOWEVER THEHY DOLEMODIlCATIONAPPEARSTOBEUNIQUE4HEAUTHORS Q SPECULATED D THAT THESE MODIlCATIONS ARE CAUSED BY D INGTH T ECATALYT L IC REACTIVEOXYGENSPECIESGENERATED DUR CYCLEANDORUNDERCONDITIONSINWHICHNORMAL03)) F FUNCT IONHAS BEEN DISRUPTED3INCE BOTH THEMODIlED D UNMODIlED D PEPTIDESWEREOBSERVED D INDICATINGA AND TRANSIENTACCUMULATION THESEMODIlCATIONSMAYACT ASASIGNAL FORTURNOVERO F F THEPHOTOSYSTEM D . TERMINUSOFF #0 ISALSO 4HESTROMALLYEXPOSED POST TRANSLATIONALLYMODIlED. TERMINALSEQUENCE DATAFORSP F INACH#0SHOWSTHATTHE lRSTRESIDUE D OFF THEMATUREPROTEINIS. ACETYL PHOSPHOTHREONINE -ICHELETAL L6ENERETAL  4HETWO. TERMINAL RESIDUESO D F HEPRIMARYTRANSLATIONPRODUCT FT D -ETAND' D LU AREREMOVED D POST TRANSLATIONALLYAND 4HRATPOSITIONISBOTHACETYLATEDANDPHOSPHORY LATED 4HIS PHOSPHORYLATED D 4HR IS ORIENTED D TO THE H LAKOID -ICHEL ETAL L  STROMALSIDEOFF THETHY 4HE CYANOBACTERIAL PSB# # GENE ENCODES A PROTEIN INWHI W CHA4HRRESIDUE D LOCATEDATPOS D ITION ISALSO D 7ILLIAMS L (OWEVER CONSERVED #HISHOLMAND7 PHOSPHORYLATIONOF#0HASNOTBEENDEMONSTRATED INCYANOBACTERIA)N HIGHERPLANTS LIGHTACTIVATESTHE REVERSIBLE REDOX DEPENDENTPHOSPHORYLATIONOFF MANY THYLAKOIDPROTEINSINCLUDINGTHE03))PROTEINS$ $ 0SB( AND D #06ENERETAL  2EVERSIBLE

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT PHOSPHORYLATION OF 03)) PROTEINS PLAYS A ROLE IN THECONTROLOF03))TURNOVER THOUGHTHEEXACTROLE F HOSPHO #0 ISNOTCLEAR)N BOTH SPINACH AND OFP !RABIDO DOPSIS 03)) PROTEINS INCLUDING#0 HAVE BEENSHOWNTOUNDERGORAPIDDEPHOSPHORYLATIONIN RESPONSETOCERTAINSTRESSCONDITIONS SUCH ASELEVATED TEMPERATUREORHIGH LIGHTIRRADIANCE %LICHH ETAL  2OKKAETAL 6ENERETAL  )NSPINACH THIS DEPHOSPHORYLATIONISCATALYZED BY ATHYLAKOID PHOSPHATASE THAT IS ACTIVATED BY THE HEAT INDUCE D D RELEASE OF A MEMBRANE ASSOCIATED PEPTIDYL PROLYL CIS TRANSISOMERASE 4,0 2OKKAETAL  4HE 4,0POLYPEPTIDEACTSASAREGULATORYSUB U UNITOFT F HE PHOSPHATASE$URINGTURNOVEROFTHEPHOTOSYSTEM IT IS KNOWNT K HATTHE$PROTEIN ISNOTSUBJ U ECTTOPROTE OLYSISUNTIL DEPHOSPHORYLATED7HETHERORNOTTHIS ISALSOTRUEFOR#0ANDTHEOTHERPHOSPHORYLATED 03))PROTEINSISUNCLEAR 7 $PODMVTJPOT 7 "OTH#0AND#0REPRESENTSINGLEGENEPRODUCTS WITH MULTIPLEFUNCT F IONSWITHIN03))4HEPRESENCEOF THESIX HELIXTRIMER OF F DIMERSMOTIF ISEVIDENCEFO F R THEIR EVOLUTION FROM AN ANCESTRAL REACTION CENTER AND F THEIRHYDROP D HILIC DOMAINS PARTICULARLY MODIlCATIONOFT THEIRRESPECTIVE LARGELUMENAL DOMAINSCONNECTING HELICES6AND6) ISRELATEDTOESTABLISHINGTHEPROTEIN F HEASSEMBLYAND FUNCT F ION ENVIRONMENTREQUIRED FORT F HECATALYTICSITEFORWATERSP F LITTING4HEAVAILABLE OFT DATAALSO POINTTOCRITICALROLESFORTHESEPROTEINSIN THEASSEMBLYANDD TURNOVEROFT F HEPHOTOSYSTEM!S F 3 )) STRUCTURALSTUDIESRElNETHECURRENTMODEL FOR0 T THERATIONALDESIGNOFEXPERIMENTSTOIDENTIFYESSENTIAL PROTEIN PROTEIN AND PROTEIN COFACTOR F INTERACTIONS WILL PROGRESS4HEPASTTWENTYYEARS HASWITNESSED CONSIDERABLE PROGRESS AND FREQUENTLY UNEXPECTED DEVELOPMENTSREGARDING TH T EROLESOF BOTH TH OFT F THESECORE ANTENNAPOLYPEPTIDES4HE IDENTIlCATIONOFF THE NMmUORESCENCEEMISSIONWITH#0LEDTOAPROPOSAL U UNITCONTAINEDD THE03))REACTIONCENTER THATTHISSUB AND ISOLATEDD REACTIONCENTERCORES LACKING#0 HAVE RESULTEDINCOMP M LEXESABLETOEVOLVE/ "àCHELETAL  (OWEVER / EVOLUTION FROMT F HESE#0 LESS PHOTOSYSTEMSWASNOTSHOWNTOEXHIBITTHECLASSICAL PERIOD OF FOUR/ RELEASEASSOCIATEDWITHTHE 3 STATES OF03)) F "RICKERANDD &RANKEL  )NCONTRAST THE CURRENT IMAGESOFF 03))REVEALTHEPROXIMITYOFF #0 TO$ANDINDICATETHATLOOP%OF#0 PARTICIPATESIN LIGATIONOFT F HE-NCLUSTERATTHEACTIVESITE!SISTHE

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT CASEWITH OTHERAREASOFF 03))RESEARCHTHE IMPENDING DEVELOPMENTOFAHIGH RESOLUTIONSTRUCTUREOF03)) HOLDSTHEKEY FORARAP F ID D ADVANCE INUNDERSTANDING THEBIOLOGYAND FUNCT F IONOF#0AN F D D#0AN DA F FULLAPPRECIATIONOFTHEIRESSENTIAL ROLESINOXYGENIC PHOTOSYNTHESIS "DLOPXMFEHNFOUT 3UPP U ORT FOR THE PREPARATION OF THIS CHAPTER WAS PROVIDED BYA GRANTFROMT F HE.EW:EALAND D -ARSDEN &UND 5// TO **% 2 AND D A .ATIONAL 3CIENCE &OUNDATIONGRANT-#"  TO#0 %4HEAU THORSTHANK"RONW K YN#ARLISLEFORT F HEPREPARATIONOF &IGAND D $R4INA3UMMERlELD FORACR F ITICAL READING OFTHETEXT 3FGFSFODFT !LT* -ORRIS* 7ESTHOFF0AN F D (ERRMAN2' .UCLEOTIDE SEQUENCEOFTHECLUSTEREDGENESFORTHEKDCHLOROPHYLL A APOPROTEINANDTHE@KD LIKEPROTEINOFTHE0HOTOSYSTEM)) REACTIONCENTERINTHESPINACHPLASTIDCHROMOSOME#URR'ENET  n !MESZ*  4HEANTENNA REACTIONCENTERCOMPLEXOF HELIO BACTERIA)N"LANKENSHIP2% -ADIGAN-4AND"AUER#% D EDS !NOXYGENIC 0HOTOSYNTHETIC "ACTERIA PP n +LUWER !CADEMIC0UBLI U SHERS $ORDRECHT !NDERSON," /UELLETTE!*!ANDD "ARRY "! 0ROBINGTHE STRUCTUREOF0HOTOSYSTEM))WITHAMINESANDPHENYLHYDRAZINE *"IOL#HEMn !NDERSON ," -ADERIA - /UELLETTE !*! 0UTNAM %VANS # (IGGINS,! +RICK4 -AC#OSS-* ,IM( 9ATES)))*2AND "ARRY"! 0OSTTRANSLATIONALMODIlCATIONSINTHE#0 SUBUNIT OFF 0HOTOSYSTEM)) 0ROC .ATL !CADD 3CI 53!  n !NDERSON," /UELLETTE!*! %ATON 2Y 2 E* -ADERIA- -AC#OSS F -* 9ATES)))*2ANDD "ARRY"!  %VIDENCEFORAPOST TRANSLATIONALMODIlCATION ASPARTYLALDEHYDE INAPHOTOSYN THETICMEMBRANEPROTEIN*!M#HEM3OCn !RO%- (UNDAL4 #ARLBERG)AND!NDERSSON" )NVITRO STUDIESONLIGHT INDUCEDINHIBITIONOFPHOTOSYSTEM ))AND$ PROTEIN DEGRADATION AT LOW TEMPERATURES "IOCHIM "IOPHYS H !CTA  n !RO% - 6I 6 RGIN)ANDD !NDERSSON" 0HOTOINHIBITIONOF 0HOTOSYSTEM)) )NACTIVATION PROTEIN DAMAGE ANDD TURNOVER "IOCHIM"IOPHYS!CTAn "ARBARTO2 2ACE(, &RISO'AND"ARBER*  #HLOROPHYLL LEVELS IN THE PIGMENT BINDING PROTEINS OF 0HOTOSYSTEM))! STUDYBASEDONTHECHLOROPHYLLTOCYTOCHROMERATIOINDIFFERENT 0HOTOSYSTEM))PREPARATIONS&%"3,ETTn U UNIT "ARB R ER* .IELDD * -ORRIS%0ANDD (ANKAMER"  3UB POSITIONING IN 0HOTOSYSTEM))REVISITED4RENDS"IOCHEM3CI  n "ARB R ER* * -ORRIS%ANDD "àCHEL # 2EVEALING THESTRUCTURE

 OFF THE0HOTOSYSTEM))CHLOROPHYLL BINDINGPROTEINS #0AND #0"IOCHIM"IOPHYS!CTAn "ARKAN! 0ROTEINSENCODEDBYACHLOROPLASTTRANSCRIPTION UNIT ARE EACH TRANSLATED FROM BOTH MONOCISTRONIC AND POLYCISTRONICM2.!S%-"/*n "RICKER4- 4HESTRUCTUREAND FUNCT F IONOF#0A AN F D D#0A  IN 0HOTOSYSTEM))0HOTOSYNTH2ESn "RICKER4-AND D &RANKEL ,+ 5SEOFAMONOC F LONALANTIBODY IN STRUCTURAL INVESTIGATIONS OFF THE  K$A POLYPEPTIDE OF 0HOTOSYSTEM))!RCH"IOCHEM"IOPHYSn "RICKER4-AND&RANKEL,+ 4HESTRUCTUREANDFUNCTION OFTHEK$AEXTRINSICPROTEINOF0HOTOSYSTEM))!CRITICAL REVIEW0HOTOSYNTH2ESn "RICKER4-AND&RANKEL,+ 4HESTRUCTUREANDFUNCTION OFF #0 AND #0 IN 0HOTOSYSTEM)) 0HOTOSYNTH 2ES  n "RICKER4- /DOM72AND D 1UEIROLO#" #LOSEASSOCIATION OFF THE K$AEXTRINSIC PROTEINWITH THEAPOPROTEINOFF #0A IN 0HOTOSYSTEM))&%"3,ETTn "RICKER4- -ORVANT* * -ASRI. 3UTTON(-AND&RANKEL,+  )SOLATIONOFAHIGHLYACTIVE0HOTOSYSTEM))PREPARATION 3 S USINGAHISTIDINE TAGGEDMUTANTOF FROM 3YNECHOCYSTI #0"IOCHIM"IOPHYS!CTAn "RICKER 4- ,OWRANCE * 3UTTON ( AND D &RANKEL ,+  !LTERATIONSOFT F HEOXYGEN EVOLVING APPARATUSINA !RG A 3 MUTANTINTHE#0PROTEINOFF 0HOTOSYSTEM))UNDERNORMALAND LOWCHLORIDECONDITIONS"IOCHEMISTRYn "RICKER4- 9OUNG ! &RANKEL ,+AND D 0UTNAM %VANS#  )NTRODUCTIONOFTHE !RG A 3ERMUTATIONINTHELARGEEXTRINSIC LOOP % OF THE #0 PROTEIN OF 3YNECHOCYSTIS SP 0##  LEADSTOTHELOSSOFCYTOCHROME CBINDINGTO0HOTOSYSTEM)) "IOCHIM"IOPHYS!CTAn "àCHEL # "ARB R ER* !NANYEV' %SHAGHI 3 7ATT2AND D $ISMUKES D OXYGEN #  0HOTOASSEMBLY OFF THEMANGANESECLUSTERAND EVOLUTIONFROMMONOMER F ICAND DIMERIC#0REACTIONCENTER 0HOTOSYSTEM)) COMPLEXES 0ROC .ATL !CAD 3CI 53!  n "URNAP 2, 1IAN - AND 0IERCE #  4HE MANGANESE STABILIZING PROTEIN OF 0HOTOSYSTEM)) MODIlES THE IN VIVO DEACTIVATIONANDPHOTOACTIVATIONKINETICSOFTHE(/OXIDATION COMPLEX IN 3YNECHOCYSTIS 3 SP 0## "IOCHEMISTRY  n #ARPENTER3$ #HARITE* %GGERS"AND D 6ERMAAS7&*  4HE PSB# STARTCODONIN 3Y 3 NECHOCY CYSTIS SP0##&%"3 ,ETT  n #ARPENTER 3$ /HAD D ) AND 6ERMAAS 7&*  !NALYSIS OF CHIMERICSPINACHCYANOBACTERIAL#0MUTANTSOF3YNECHOCYSTI 3 IS SP 0##  4HE CHLOROPHYLL PROTEIN #0 AFFECTS THE WATER SPLITTING SYSTEM OF 0HOTOSYSTEM)) "IOCHIM "IOPHYS !CTA  n #HISHOLM$AND D 7ILLIAMS*'+ .UCLEOTIDESEQUENCEOF PSB# THE GENE ENCODING THE #0  CHLOROPHYLL A BINDING PROTEINOFF 0HOTOSYSTEM)) INTH T ECYANOBACTERIUM U 3Y 3 NECHOCY CYSTIIS 0LANT-OL"IOL n #HRISTOPHER $! AND (OFFER F 0(  $%4 REPRESSES A CHLOROPLASTBLUELIGHT RESPONSIVEPROMOTERINADEVELOPMENTAL AND TISSUE SPECIlC MANNER IN !RABIDOPSIS THALIANA 0LANT * n #HU (! .GUYEN!0 AND $EBUS 2*  !MINO ACID RESI D DUEST HATINmUENCETHE BINDINGOFMANGANESEORCA F LCIUMTO R ELICAL DOMAINSOFF THE$ 0HOTOSYSTEM))4HE LUMENAL INTERH

 POLYPEPTIDE"IOCHEMISTRY n #LARKE3-AND%ATON 2YE** -UTATIONOF0HE INTHE 0HOTOSYSTEM))PROTEIN#0IMPAIRSPHOTOAUTOTROPHICGROWTH ALTERSTHECHLORIDEREQUIREMENT ANDPREVENTSPHOTOSYNTHESISIN 3 S SP THEABSENCEOFEITHER03 )) / OR03 )) 6 IN 3YNECHOCYSTI 0##"IOCHEMISTRY n R E3-AND D %ATON 2 2YE** !MINOACID DELETIONSIN LOOP #LARK #OFF THECHLOROPHYLL A BINDING PROTEIN#0ALTERTHECHLORIDE REQUIREMENTANDORPREVENTTHEASSEMBLY OFF 0HOTOSYSTEM)) 0LANT-OL"IOL n #LARKE3- &UNK# (ENDRY'3 3HAND*! 7YDRZYNSKI4AND %ATON 2YE **  !MINO ACID DELETIONS IN THE CYTOSOLIC DOMAINSOFTHECHLOROPHYLL A BINDINGPROTEIN#0SLOW1!nOXI DATIONANDORPREVENTTHEASSEMBLYOF0HOTOSYSTEM))0LANT -OL "IOL  n $EBUS2* 4HEPOLYPEPTIDESOFF 0HOTOSYSTEM))ANDD THEIR INmUENCEONMANGANO TYROSYL BASEDD OXYGENEVOLUTION)N3IGEL ! AND D 3IGEL (EDS -ETAL )ONS IN"IOLOGICAL3YSTEMS 6OL  PPn-ARCEL $EKKER .EW9ORK R $EISENHOFER * %PP / 3INNING ) AND -ICHEL (  #RYSTALLOGRAPHIC RElNEMENT AT  ¯ RESOLUTION AND RE lNED MODEL OF THE PHOTOSYNTHETIC REACTION CENTER FROM 2HODOP O SEUDOMONAS VIRIDIS*-OL"IOLn $E LAS 2IVAS * "ALSERA - ANDD "ARRBER *  %VOLUTION OF OXYGENICPHOTOSYNTHESIS'ENOME WIDEANALYSISOFF THE/%# EXTRINSIC PROTEINS4RENDS0LANT3CIn DE7EERD D &, VAN 3TOKKUM K )(- VAN!MERONGEN ( $EKKER D DELLE2A 0ATHWAY A S FORENER F GY TRANSFE F R *0ANDVAN'RON IN THE CORE LIGHT HARVESTING COMPLEXES #0 AND #0 OF 0HOTOSYSTEM))"IOPHYS*n DE7EERD&, 0ALACIOS-! !NDRIZHIYEVSKAYA%' $EKKER*0 ANDVAN'RONDELLE2B )DENTIFYINGTHELOWESTELECTRONIC STATESOFF THECHLOROPHYLLSINTHE#0COREANTENNAPROTEINOF 0HOTOSYSTEM))"IOCHEMISTRY n $IXIT2 4RIVEDI 0+ .ATH 0AND3ANE06 D  /RGANIZATION AND D POST TRANSCRIPTIONALPROCESSINGOFT F HE PSB"OPERON FROM F CHLOROPLASTSOF 0OPULUSDE D LTOIDE D S#URR'ENETn $OUGLAS3% #HLOROPLASTORIGINSANDEVOLUTION)N"RYANT $!ED -OLECULAR"IOLOGYOFTHE#YANOBACTERIA PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT $ZELZKALNS6!AND"OGORAD, -OLECULARANALYSISOFA F IVEINPHOTOSYNTHETICOXYGENEVOLUTIONAND ISOLA MUTANTDEFECT TIONOFF ACOMPLEMENTING CLONEBYANOVEL SCREENINGPROCEDURE D %-"/ * n %ATON 2Y 2 E**ANDD 3HANDD *!  -UTATIONS BETWEEN'LY  AND D 4HR  IN LOOP % OFF THE CHLOROPHYLL A BINDING PROTEIN #0CHARACTERIZEDINTHEPRESENCEORABSENCEOFTHEMEM BRANE EXTRINSIC PROTEINS OF THE WATER OXIDIZING COMPLEX OF 0HOTOSYSTEM)) )N 03 0ROCEEDINGS TH )NTERNATIONAL #ONGRESS ON 0HOTOSYNTHESIS 3  #3)2/ 0UBLISHING -ELBOURNE #$ 2/- %ATON 2Y 2 E**AND D 6ERMAAS7&* /LIGONUCLEOTIDE DIRECTED O NGA MUTAGENESISOF PSB" THE GENEENCODING #0 EMPLOYI DELETIONSTRAINOFF THECYANOBACTERIUM 3Y 3 NECHOCY CYSTIS SP0## 0LANT-OL"IOL n %ATON 2YE ** AND6ERMAAS 7&*  #HARACTERIZATION OF A HISTIDINETOGLUTAMINESUBSTITUTIONATRESIDUEIN#0OF 0HOTOSYSTEM)))N-URATA.ED 2ESEARCHIN0HOTOSYNTHESIS 6 6OL) PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT %ATON 2YE ** 3HANDD *! AND .ICOLL 73  P( DEPENDENT PHOTOAUTOTROPHIC GROWTHOFSPEC F IlC0HOTOSYSTEM))MUTANTS

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT LACKING LUMENALEXTRINSICPOLYPEPTIDES IN 3YNECHOCY CYSTIS 0## &%"3,ETTn %LANSKAYA)6 !LLAKHVERDIEV3) "OICHENKO6! +LIMOV6 $EME TER3 4IMOFEEV+.AND3HESTAKOV36 0HOTOCHEMICAL CHARACTERIZATIONOFCYANOBACTERIUM U 3YNECHOCYSTIS 3 SP0## D 0HOTOSYSTEM))PROTEINS"IOCHEMISTRY MUTANTSWITH IMPAIRED -OSCOW n %LICH 4$ %DELMAN- AND D -ATOO!+  $EPHOSPHORYLA TIONOFF 0HOTOSYSTEM))COREPROTEINS ISLIGHT REGULATED INVIVO %-"/*n %NAMI) 3ATOH+AND+ATOH3 #ROSSLINKINGBETWEENTHE K$AEXTRINSICPROTEINANDTHEK$ACHLOROPHYLL CARRYING PROTEINOFTHE03))REACTIONCENTERCORECOMPLEX&%"3,ETT  n %NAMI) +ANEKO- +ITAMURA. +OIKE( 3ONOIKE+ )NOUE9 AND D +ATOH3 4OTAL IMMOBILIZATIONOFT F HEEXTRINSIC K$APROTEININSPINACH H 0HOTOSYSTEM))MEMB M RANEPREPARATIONS D STABILIZATIONOFOX F YGENEVOLUTION 0ROTEINSTOICHIOMETRYAND "IOCHIM"IOPHYS!CTAn %NAMI) 4OHRI! +AMO- /HTA(AND3HEN* 2 )DENTIl CATIONOFDOMAINSONTHEK$ACHLOROPHYLL CARRYINGPROTEIN #0 THATARESHIELDEDFROMTRYPTICATTACKBYBINDINGOFTHE EXTRINSICK$APROTEINWITH0HOTOSYSTEM))COMPLEX"IOCHIM "IOPHYS!CTAn H  %NAMI ) +IKUC K HI 3 &UKU K DA4 /HTA ( AND D 3HEN * 2  "INDING AND FUNCT F IONAL PROPERTIES OF FFOUR EXTRINSIC PROTEINS OFF 0HOTOSYSTEM)) FROMARE F D ALGA #Y # ANIDIUMCALDARIU D M AS STUDIED BY RELEASE RECONSTITUTION EXPERIMENTS "IOCHEMISTRY n %NAMI) 9OSHIHARA3 4OHRI! /KUMURA! /HTA(AND3HEN* 2  #ROSS RECONSTITUTIONOFVARIOUSEXTRINSICPROTEINSAND 0HOTOSYSTEM))COMPLEXESFROMCYANOBACTERIA REDALGAEAND HIGHERPLANTS0LANT#ELL 0HYS H IOLn &EILER5AND D (AUSKA'  4HEREACTIONCENTERFROM F GREEN SULPHURBACTERIA)N"LANKENSHIP2% -ADIGAN-4AND D "AUER #% EDS !NOXYGENIC0HOTOSYNTHETIC"ACTERIA PPn U SHERS $ORDRECHT +LUWER!CADEMIC0UBLI &ERREIRA+. )VERSON4- -AGHLAOUI+ "ARBER*AND)WATA3  !RCHITECTURE OF THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn &RANKEL ,+ AND "RICKER 4-  %PITOPE MAPPING OF THE F #0A  IN MONOCLONAL ANTIBODY &!# ON THE APOPROTEIN OF 0HOTOSYSTEM))&%"3,ETTn &RANKEL,+AND D "RICKER4- )NTERACTIONOFF #0A WITH COMPONENTSINVOLVED D WITH WATEROXIDATION IN0HOTOSYSTEM)) -APPING OFF .(3 BIOTINYL N ATION SITES AND D THE EPITOPE OF F THE MONOCLONALANTIBODY&!#TOTHELARGEEXTRINSICLOOPREGION OF #0A  )N "ALTSCHEFFSKY - ED #URRENT 2ESEARCH IN 0HOTOSYNTHESIS6OL) PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT &RANKEL ,+ AND "RICKER 4-  )NTERACTION OFF #0A  WITH THE MANGANESE STABILIZING PROTEIN OFF 0HOTOSYSTEM)) W CHARESHIELDED FROM F .

. )DENTIlCATIONOF DOMAINSON#0A WHI HYDROX D YSUCCINIMIDE BIOTINYL N ATIONBY Y THEMANGANESE STABILI A ZING PROTEIN"IOCHEMISTRYn 'HANOTAKIS$& DE0AULA*# $EMETRIOU$- "OWLBY.2 0ETERSON * "ABCOCK'4AND9OCUM#& )SOLATIONANDCHARACTERIZA TIONOFTHEK$APROTEINANDTHE$ $ CYTOCHROME B  COMPLEX"IOCHIM"IOPHYS!CTAn 'IACOMETTI '- "ARB R ATO 2 &RISO ' &RIZZO! AND D 2IGONI &  0HOTOSYSTEM)) DEGRADATION PATHWAY A S AFTER F PHOTOIN

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT HIBITION OF ISOLATEDD THYLAKOIDS )N -URATA . ED 2ESEARCH IN 0HOTOSYNTHESIS 6OL )6 PP n +LUWER !CADEMIC 0UBLISHERS $ORDRECHT 'INGRICH *# 'ASPARICH '% 3AUER + AND "RYANT $!  .UCLEOTIDESEQUENCEANDEXPRESSIONOFTHETWOGENESENCODING $PROTEINANDD THESINGLE GENEENCODINGTHE#0PROTEINOF 0HOTOSYSTEM)) INTHECYANOBACTERIUM 3YNECHOCOCCUS SP0## 0HOTOSYNTH2ESn 2 E ** )NOUE 9 AND 'LEITER (- (AAG % 3HEN * 2 %ATON 2Y 6 6ERMAAS7&*  &UNCTIONAL CHARACTERIZATION OF F MUTANT STRAINS OF THE CYANOBACTERIUM 3YNECHOCYSTIS SP 0##  LACKINGSHORTDOMAINSWITHINTHELARGE LUMEN EXPOSEDLOOPOF THECHLOROPHYLLPROTEIN#0IN0HOTOSYSTEM))"IOCHEMISTRY  n 2 E** 3EELIGER!' )NOUE 'LEITER(- (AAG % 3HEN* 2 %ATON 2Y 9 6ERMAAS7&*AND 2ENGER'  )NVOLVEMENTOFF THE#0 9 PROTEIN IN STABILIZATION AND PHOTOACTIVATION OFF A FUNCT F IONAL WATER OXIDIZING COMPLEX INTHECYANOBACTERIUM 3Y 3 NECHOCY CYSTIS SP0##"IOCHEMISTRY n 'OLDEN 33 AND 3TEARNS '7  .UCLEOTIDE SEQUENCE AND TRANSCRIPT ANALYSIS OF THREE 0HOTOSYSTEM)) GENES FROM THE CYANOBACTERIUM 3YNECHOCOCCUS 3 SP 0## 'ENE  n 'ROOT- , &RESE2. DE7EERD&, "ROME D K + 0ETTERSSON ¯ K )(- VAN 'RONDELLE 2 AND 0ERTERMAN %*' VAN 3TOCKKUM F HE#0CORE $EKKER*0 3PECTROSCOPICPROPERTIESOFT ANTENNAPROTEINOFF 0HOTOSYSTEM))"IOPHYS*n (AAG % %ATON 2Y 2 E ** 2ENGER ' ANDD 6ERMAAS 7&*  &UNCTIONALLYIMPORTANTDOMAINSOFTHELARGEHYDROPHILICLOOP OF#0ASPROBEDBYOLIGONUCLEOTIDE DIRECTEDMUTAGENESISIN 3YNECHOCYSTIS SP0##"IOCHEMISTRYn (ACKETT#3AND3TRITTMATTER0 #OVALENTCROSS LINKINGOF F ICLE BOUNDD CYTOCHROME B ANDD .!$( THEACTIVESITESOFVES CYTOCHROME B REDUCTASE D * "IOL #HEMn (ANKAMER " -ORRIS %0 ANDD "ARRBER *  2EVEALING THE STRUCTUREOFF THEOXYGEN EVOLVING CORE DIMEROFF 0HOTOSYSTEM)) BY CRYOELECTRONCRYSTALLOGRAPHY.AT3TRUCT" Y IOL n (AYASHI( &UJIMURA9 -OHANTY03AND-URATA. 4HE ROLEOF#0INTHEEVOLUTIONOFOXYGENANDTHEBINDINGOFTHE EXTRINSIC K$APROTEINTOTHECORECOMPLEXOF0HOTOSYSTEM)) AS DETERMINED BY LIMITED PROTEOLYSIS 0HOTOSYNTH 2ES  n (IRD3- 7E D BBER!. 7IL 7 SON2* $YER4!AND'RA D AY *#  $IFFERENT F IALEXPRESSIONOFF THEPSB"ANDD PSB( GENESENCODING T EK$ACHLOROPHYLL A PROTEINANDD THEK$APHOSPHOPROTEIN TH OFF 0HOTOSYSTEM))DUR D INGCHLOROPLAST DEVELOPMENTINWH W EAT #URR'ENETn (ONG, 3TEVENSON*+ 2OTH7"AND(ALLICK2" %UGLENA GRACILISCHLOROPLASTPSB" PSB4 PSB(ANDPSB.GENECLUSTER 2EGULATION OF PSB" PSB4 PRE M2.! PROCESSING -OL 'EN 'ENETn (U8 $AMJ M ANOVIC! 2ITZ4ANDD 3CHULTEN+ !RCHITECTURE AND D MECHANISM OF F THE LIGHT HARVESTING APPARATUS OF F PURPLE BACTERIA0ROC.ATL !CAD 3CI 53!n (UANG )9 ,Y , NCH$0ANDD %ATON 2 2YE** -UTAGENESISOF HISTIDINEn IN THE 0HOTOSYSTEM)) CHLOROPHYLL A BINDING PROTEIN #0 IN 3YNECHOCYSTIS SP 0##  )N 03 0ROCEEDINGSTH )NTERNATIONAL#ONGRESSON0HOTOSYNTHESIS 3  #3)2/0UBLISHING -ELBOURNE#$ 2/- )KEUCHI- 0LUMLEY&' )NOUE9AND3C D HMIDT'7  0HOS PHORYLATIONOFF 0HOTOSYSTEM))COMPONENTS #0APOPROTEIN

 $ $ ANDTOKILODALTONPROTEININCHLOROPLASTTHY H LAKOIDS OFHIGHERPLANTS0LANT0HYSIOLn )SOGAI9 9AMAMOTO9 AND .ISHIMURA -  !SSOCIATION OF THE  K$A POLYPEPTIDE WITH THE  K$A COMPONENT IN 0HOTOSYSTEM))PARTICLES&%"3,ETTn *OHNSON#(AND D 3CHMIDT'7  4HE PSB" GENECLUSTEROF M DOMONAS REINHARD R TII CHLOROPLAST SEQUENCE Q AND THE #HLAMYD . AND PSB( ( 0LANT -OL "IOL TRANSCRIPTIONAL ANALYSES OF PSB.  n *ORDAN0 &ROMME0 7I 7 TT(4 +LUKAS/ 3AENGER7AND D +RAU” .  4HREE DIMENSIONALSTRUCTUREOFCYANOBACTERIAL0HOTO SYSTEM)AT ¯RESOLUTION.ATUREn +AMIYA . AND 3HEN * 2  #RYSTAL STRUCTURE OF OXYGEN EVOLVING0HOTOSYSTEM))FROM 4HERMOSYNECHOCOCCUSVULCANUS 4 AT ¯RESOLUTION0ROC.ATL!CAD3CI53!n +ANEKO4 3ATO3 +OTANI( 4ANAKA! !SAMIZU% .AKAMURA 9 IYAJI 9 A MA. (IROSAWA- 3UGIURA- 3ASAMOTO3 +IMURA 4 -ATSUNO! -URAKI ! .AKAZAKI. .ARUO+ 4 (OSOUCHI4 4 K 3 3HIMPO 3 4AKEUCHI # 7ADA 4 7ATANABE ! /KUMURA 9 9AMADA- 9ASUDA- 4ABATA3 3EQUENCEANALYSISOF THEGENOMEOFTHEUNICELLULARCYANOBACTERIUM 3YNECHOCYSTIS SP3TRAIN0##))3EQUENCEDETERMINATIONOFTHEENTIRE GENOMEANDASSIGNMENTOFPOTENTIALPROTEIN CODINGREGIONS $.! . 2ES  n +ASHINO 9 ,AUB U ER 7- #ARROLL *! 7ANG 1 7HITMARSH * 3ATOH +AND D 0AKRAS K I(" 0ROTEOMICANALYSISOFF AHIGHLY ACTIVE 0HOTOSYSTEM)) PREPARATION FFROM THE CYANOBACTERIUM CYSTIS SP 0##  REVEALS THE PRESENCE OF F NOVEL 3 NECHOCY 3Y POLYPEPTIDES"IOCHEMISTRYn + +ELLER- 7EIL*(AND.AIR#++ .UCLEOTIDESEQUENCE OF THE PSB" GENE OF %UGLENA GRACILIS 0LANT -OL "IOL  n +IM- 4HUM+% -ORISHIGE$4AND D -ULLET*%  $ETAILED ARCHITECTUREOFT F HEBARLEYCHLOROPLAST PSB$ P

SB# # BLUELIGHT RESPONSIVEPROMOTER*"IOL#HEMn + +NOEP mE. "RICKER4-AND D 0UTNAM %VANS# 3ITE DIRECTED D D  INTHE#0 MUTAGENESISOF BASICARGININERESIDUESAN PROTEINOF0HOTOSYSTEM))AFFECTSOXYGEN EVOLVINGACTIVITY IN 3YNECHOCYSTIS "IOCHEMISTRYn +OHCHI4 9OSHIDA4 +OMANO4AND/HYAMA+ $IVERGENT + M2.!TRANSCRIPTIONINTHECHLOROPLAST PSB" OPERON%-"/ * n + +OWA LLIKK +6  /RIGIN AND D EVOLUTION OF F PLASTIDS FROM F CHLOROPHYLL A  C CONTAINING ALGAE 3UGGESTED ANCESTRAL RELATIONSHIPS TO RED D AND GREEN ALGAL PLASTIDS )N ,EWIN 2! ED /RIGINOFF 0LASTIDS3YMBIOGENESIS 0ROCHLOROPHYTESAND THE/RIGINOF#HLOROPLASTS PPn#HAPMANAND(ALL .EW9ORK +RAU” . 3CHUBERT 7 $ +LUKAS / &ROMME 0 7ITT (4 AND + 3AENGER7 0HOTOSYSTEM)AT¯RESOLUTIONREPRESENTS F JOINTPHOTOSYNTH T ETICREACTIONCENTRE T ElRSTSTRUCTURAL MODEL OFA TH AND D COREANTENNASYSTEM.AT3TRUCT"IOLn + HN-' AND +U D 6ERMAAS7&* $ELETIONMUTATIONSINA LONG HYDROPHILIC LOOP IN THE 0HOTOSYSTEM)) CHLOROPHYLL BINDING PROTEIN#0INTHECYANOBACTERIUM 3YNECHOCY CYSTIS SP0## 0LANT-OL"IOLn + +àHLBRANDT7 7ANG$.AND&UJIYOSHI9 !TOMICMODEL OFPLANTLIGHT HARVESTINGCOMPLEXBYELECTRONCRYSTALLOGRAPHY .ATURE  n ,INDBERG + AND D !NDRÏASSON , %  ! ONE SITE TWO STATE F HE BINDING OFAN F IONS IN 0HOTOSYSTEM))"IOCHEM MODEL FORT

 ISTRYn -AID5AND:ETSCHE+ !KBSMALLSINGLE COPYREGION SEPARATESTHEPLASTID$.!INVERTEDREPEATOFTHEUNICELLULARRED ALGA #YANIDIUMCALDARIUM # 0HYSICALMAPPINGOFTHE)2 mANKING

SB# RP R S 3 REGIONSANDNUCLEOTIDESEQUENCESOFTHE PSB$ P R2.! AND RPL L GENES0LANT-OL "IOLn -ANNA0AND D 6ERMAAS7 -UTATIONALSTUDIESONCONSERVED HISTIDINERESIDUES D INTHECHLOROPHYLL BINDING PROTEIN#0OF 0HOTOSYSTEM))%UR*"IOCHEMn -AUL *% ,ILLY*7 #UI, DE0AMPHILIS#7 -ILLER7 (ARRIS%( AND3TERN$" 4HE #HLAMYDOMONASREINHARDTI # I PLASTID CHROMOSOME)SLANDSOFGENESINASEAOFREPEATS0LANT#ELL n -AYES32AND"ARBER* 0RIMARYSTRUCTUREOFTHE PSB. PSB( P

ET# P

ET! T GENECLUSTEROFF THECYANOBACTERIUM 3Y 3 NECHO CYSTI Y S 0## 0LANT-OL "IOLn -C#ONNELL-$ +OOP2 6ASILEV3ANDD "RUCE$ 2EGULA TIONOFT F HE DISTRIBUTIONOFF CHLOROPHYLLANDD PHYCOBILIN ABSORB R ED EXCITATIONENERGY R INCYANOBACTERIA!STRUCTURE BASEDD MODEL FO F R THELIGHTSTATETRANSITION0LANT0HYSIOLn -C$ERMOTT' 0RINCE3- &REER!! (AWTHORNTHWAITE ,AWLESS !- 0APIZ -: #OGDELL 2* AND )SAACS .7  #RYSTAL STRUCTUREOFANINTEGRALMEMBRANELIGHT HARVESTINGCOMPLEX F FROMP HOTOSYNTHETICBACTERIA.ATUREn -ICHEL (AND D $EISENHOFER* F  2ELEVANCEOFT F HEPHOTOSYN THETICREACTIONCENTERFROM F PURPLEBACTERIATOTHESTRUCTUREOF 0HOTOSYSTEM))"IOCHEMISTRY n -ICHEL ( (UNT$& 3HABANOWITZ*ANDD "ENNETT*  4ANDEM MASSSPECTROMETRYREVEALSTHATTHREE0HOTOSYSTEM))PROTEINS OFSPINACHCHLOROPLASTSCONTAIN . ACETYL . . / PHOSPHOTHREONINE ATTHEIR.( TERMINI*"IOL#HEMn -ORGAN 42 3HAND *! #LARKE 3- AND %ATON 2YE **  3PECIlCREQUIREMENTSFORCYTOC F H HROME C ANDD THEMANGANESE STABILIZINGPROTEIN INPHOTOAUTOTROPHICSTRAINSOF 3Y 3 NECHOCY CYSTIS SP0##WITH MUTATIONSINTHEDOMAIN'LY TO4HR  OF F THE CHLOROPHYLL BINDING PROTEIN #0 "IOCHEMISTRY n -ORI(AND9AMAMOTO9 $ELETIONOFANTENNACHLOROPHYLL A BINDINGPROTEINS#0AND#0BYTRIS TREATMENTOF03)) MEMBRANESINWEAKLIGHT%VIDENCEFORAPHOTO DEGRADATIVE EFFECTONTHE03))COMPONENTSOTHERTHANTHEREACTIONCENTER BINDINGPROTEINS"IOCHIM "IOPHYS!CTAn H -ORI ( 9AMASHITA9 !KASAKA4AND9AMAMOTO D 9  &URTHER CHARACTERIZATION OFF THE LOSS OFF ANTENNA CHLOROPHYLL BINDING PROTEIN#0 FROM0 F HOTOSYSTEM)) DUR D ING DONOR SIDEPHOTO INHIBITION"IOCHIM"IOPHYS!CTAn .AKATANI (9 +E " $OLAN % AND!RNTZEN #*  )DENTITY OF THE 0HOTOSYSTEM)) REACTION CENTER POLYPEPTIDE "IOCHIM "IOPHYS!CTAn .ANBA/AND3ATOH+ )SOLATIONOFA0HOTOSYSTEM))REACTION CENTERCONSISTINGOFF $ AND$ PO D LYPEPTIDESANDCYTOC D HROME B 0ROC.ATL!CAD 3CI 53!n .IXON0* 2ÚGNER-ANDD $INER"! %XPRESSIONOFF AHIGHER PLANT PSB! B GENE IN 3YNECHOCY CYSTIS  YIELDS A FUNCT F IONAL HYBRIDD 0HOTOSYSTEM)) REACTION CENTER COMPLEX 0LANT #ELL n /DOM72AND"RICKER4- )NTERACTIONOF#0A WITHTHE MANGANESE STABILIZINGPROTEINOF0HOTOSYSTEM)))DENTIlCATION OFDOMAINSCROSS LINKEDBY ETHYL  ; DIMETHYLAMINO PROP YL=CARB R ODIIMIDE"IOCHEMISTRYn /HTA( 9OSHIDA. 3ANO- (IRANO- .AKAZATO+ANDD %NAMI )

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT  %VIDENCE FORE F LECTROSTATIC INTERACTIONOFF THELOOP!ON #0  WITH THE EXTRINSIC  K$A PROTEIN )N -ATHIS 0 ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL)) PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT /HTA( 3UZUKI4 5ENO- /KUMURA! 9OSHIHARA3 3HEN* 2 AND D %NAMI )  %XTRINSICPROTEINSOFF 0HOTOSYSTEM))!N INTERMEDIATEMEMBEROFT F HE0SB1PROTEIN FAM F ILY INRED D ALGAL 03))%UR* "IOCHEMn /UELLETTE!*! !NDERSON,"AND D "ARRY "! !MINEBIND INGAND D OXIDATIONATTHECATALYTICSITEFORP F HOTOSYNTHETICWATER OXIDATION0ROC.ATL!CAD3CI53!n 0UTNAM %VANS # AND "RICKER 4-  3ITE DIRECTED MUTA GENESIS OF THE #0A  PROTEIN OF 0HOTOSYSTEM)) !LTERATION OF THE BASIC RESIDUE PAIR  2 TO  ' LEADS TO A DEFECT ASSOCIATED D WITHTHEOXYGEN EVOLVINGCOMPLEX"IOCHEMISTRY  n 0UTNAM %VANS#AND D "RICKER4- 3ITE DIRECTED D MUTAGEN ESISOFT F HE#0PROTEINOFF 0HOTOSYSTEM))!LTERATIONOFF THE BASICRESIDUE D 2 TO 'PREVENTSTHEASSEMBLYOF FUNCT F IONAL 0HOTOSYSTEM)) CENTERS UNDER CHLORIDE LIMITING CONDITIONS "IOCHEMISTRYn 0UTNAM %VANS#AND"RICKER4- 3ITE DIRECTEDMUTAGEN ESIS OFTHE BASIC RESIDUES +T + O 'INTHE#0 PROTEINOF F H AND 0HOTOSYSTEM))ALTERSTHECHLORIDEREQUIREMENT FORGROWT OXYGEN EVOLVING ACTIVITY IN 3YNECHOCY CYSTIS  0LANT -OL "IOL n 0UTNAM %VANS # 7U * AND D "RICKER4- A 3ITE DIRECTED MUTAGENESISOFF THE#0PROTEINOFF 0HOTOSYSTEM))!LTERATION OFCONSERVEDCHARGEDRESIDUESWHICHLIEWITHINLETHALDELETIONS OFTHELARGEEXTRINSICLOOP%0LANT-OL"IOLn 0UTNAM %VANS# "URNAP2 7U* 7HITMARSH*AND"RICKER4B 3ITE DIRECTED MUTAGENESIS OF THE #0  PROTEIN OF F D CHARGED D RESIDUES D IN 0HOTOSYSTEM))!LTERATIONOFCONSERVE THE DOMAIN % 2"IOCHEMISTRY n 1IAN- !L +H + ALDI 3& 0UTNAM %VANS# "RICKER4-AND D "URNAP 2,  0HOTOASSEMBLY M Y OF THE0HOTOSYSTEM)) -N CLUSTERIN SITE DIRECTED D MUTANTS IMPAIRED INTHEBINDINGOFT F HEMANGANESE STABILIZINGPROTEIN"IOCHEMISTRYn 1UEIROLO #  !SSEMBLAGE OF SPINACH 0HOTOSYSTEM)) PROTEINS #0A  AND -30 INTERACTIONS 0H$ $ISSERTATION ,OUISIANA3TATE5NIVERSITY "ATON2OUGE 2EITH -AND D -UNHOLLAND D * ! HIGH RESOLUTIONGENEMAP OFF THECHLOROPLASTGENOMEOFT F HERED D ALGA 0ORP 0 HYRAPURPUREA 0LANT#ELLn 2HEE + ( -ORRIS %0 0 :HELEVA $ (ANKAMER " +àHLBRANDT 7 AND D "ARRBER *  4WO DIMENSIONAL STRUCTURE OF F PLANT 0HOTOSYSTEM))AT ¯ RESOLUTION.ATUREn 2 2HEE+ ( -ORRIS%0 "ARBER*AND+àHLBRANDT7 4HREE

DIMENSIONAL STRUCTURE OF THE PLANT 0HOTOSYSTEM)) REACTION CENTER AT  ¯ RESOLUTION .ATURE  n 2OCHAIX *$ +UCHKA - -AYlELD D 3 3CHIRMER 2AHIRE - 'I RARD "ASCOU*AND D "ENNOUN0  .UCLEARAND D CHLOROPLAST MUTATIONSAFFECTT F HESYNTHESISORSTABILITY OFT F HECHLOROPLAST PSB#GENEPRODUCT D IN #HLAMYD M DOMONASREINHARD R TII%-"/ * n 2ÚGNER - #HISHOLM $! AND $INER "!  3ITE DIRECTED MUTAGENESISOFTHE PSB#GENEOF0HOTOSYSTEM)))SOLATIONAND FUNCTIONALCHARACTERIZATIONOF#0 LESS0HOTOSYSTEM))CORE COMPLEXES"IOCHEMISTRYn 2OKKA! !RO%- (ERRMANN2' !NDERSSON"AND6ENER!6 D  $EPHOSPHORYLATIONOFF 0HOTOSYSTEM))REACTIONCENTER

$IBQUFS

5IF $1
BOE
$1
1SPUFJOT PROTEINSINPLANTPHOTOSYNTHETICMEMBRANESASANIMMEDIATE RESPONSE TO ABRUPT ELEVATION OF TEMPERATURE 0LANT 0HYSIOL n 2OSENBERG # #HRISTIAN * "RICKER 4- AND 0UTNAM %VANS #  3ITE DIRECTEDMUTAGENESISOFGLUTAMATERESIDUESINTHE LARGEEXTRINSICLOOPOFF THE0HOTOSYSTEM))PROTEIN#0AFFECTS F OXYGEN EVOLVING ACTIVITY AND D 03)) ASSEMBLY Y "IOCHEMISTRY  n 3ALTER!( 6IRGI R N) (AGMAN!AND!N D DERSSON"  /NTH T EMO LECULARMECHANISMOF LIGHT INDUCE D D $PROTEINDEGRADATION IN 0HOTOSYSTEM))COREPARTICLES"IOCHEMISTRYn 3AYRE24AND7ROBEL "OERNER%! -OLECULARTOPOLOGY OF THE 0HOTOSYSTEM)) CHLOROPHYLL A BINDING PROTEIN #0 4 4OPOLOGYOFATHYLAKOIDMEMBRANEPROTEIN0HOTOSYNTH2ES  n 3CHUB U ERT7$ +LUKAS/ 3AENGER7 7I 7 TT(4 &ROMME0AND +RAU”.  ! COMMON ANCESTOR FFOR OXYGENIC ANDD AN OXYGENICPHOTOSYNTHETICSYSTEMS!COMPARISONBASEDD ONTHE STRUCTURALMODELOFF 0HOTOSYSTEM)*-OL "IOLn 3EIDLER! 4HEEXTRINSICPOLYPEPTIDESOF0HOTOSYSTEM)) "IOCHIM"IOPHYS!CTAn 3EXTON4" #HRISTOPHER$!AND-ULLET*%A ,IGHT INDUCED SWITCH IN BARLEY PSB$ PSB# # PROMOTER UTILIZATION ! NOVEL MECHANISM REGULATING CHLOROPLAST GENE EXPRESSION %-"/ *  n 3EXTON4" *ONES*4ANDD -ULLET*%B 3EQUENCEANDD TRAN SCRIPTIONALANALYSISOFT F HE BARLEY CT$.!REGIONUPSTREAMOF PSB$ PS

B#ENCODING TRN T +555 RPS TRN T 155' PSB+ PSB) AND TR T N3'#5 #URR'ENETn 3HEN ' AND6ERMAAS7&*  -UTATION OF CHLOROPHYLL LI GANDSINTHECHLOROPHYLL BINDING#0PROTEINASSTUDIEDINA 3YNECHOCYSTIS SP0##0HOTOSYSTEM) LESSBACKGROUND "IOCHEMISTRYn 3HEN ' %ATON 2Y 2 E ** AND6ERMAAS D 7&*  -UTATION OF HISTIDINE RESIDUES D IN #0 LEADS TO DESTABILIZATION OF F THE 0HOTOSYSTEM)) COMPLEX ANDD TO IMPAIRMENT OF LIGHT ENERGY TRANSFER" F IOCHEMISTRYn 3HEN * 2 "URNAP 2, AND )NOUE9 A !N INDEPENDENT ROLE OF CYTOCHROME C  IN CYANOBACTERIAL 0HOTOSYSTEM)) ASREVEALEDBYDOUBLE DELETIONMUTAGENESISOFTHE PSB/AND PSB6 GENESIN 3YNECHOCYSTIS 3 SP0##"IOCHEMISTRY n 3HEN * 2 6ERMAAS7 ANDD )NOUE9 B 4HE ROLE OFF CYTO CHROME C ASSTUDIEDD THROUGHREVERSEGENETICSANDD MUTANT CHARACTERIZATION IN 3Y 3 NECHOCY CYSTIS SP0##*"IOL#HEM  n 3HEN* 2 1IAN- )NOUE9AND"URNAP2, &UNCTIONAL CHARACTERIZATION OF 3YNECHOCYSTIS SP 0##  6PSB 6PP 5 AND 6P 6PSB P 6 MUTANTS REVEALS IMPORTANT ROLES OF CYTOCHROME C  IN CYANOBACTERIAL OXYGEN EVOLUTION "IOCHEMISTRY  n 3HIPTON#!AND D "ARRBER* 0HOTOINDUCE D D DEGRADATIONOFF THE $POLYPEPTIDEIN ISOLATEDD REACTIONCENTERSOFF 0HOTOSYSTEM)) F LYTICPROCESSTRIGGERED BYTHEOXI %VIDENCE FORANAUTOPROTEO DIZINGSIDEOFF THEPHOTOSYSTEM0ROC.ATL !CAD3C D I 53! n 3OLTIS $% 3OLTIS03AND:ANIS-, 0HYLOGENYOFSEED PLANTS BASED ON EVIDENCE FROM EIGHT GENES !M * "OT  n 3UMMERlELD4# D 'ALLOWAY $* ANDD %ATON 2YE **  4HE USE OF PSB" FFOR THE IDENTIlCATION OFF CYANOBACTERIAL LICHEN

 SYMBI M ONTS)N030ROCEEDINGSTHH )NTERNATIONAL#ONGRESS ON 0HOTOSYNTHESIS 3  #3)2/ 0UBLISHING -ELBOURNE #$ 2/- 3UMMERlELD4# 3HAND*! "ENTLEY&+AND%ATON 2YE** Q 0SB13LL IN 3YNECHOCYSTIS SP0##ISREQUIREDFOR 0HOTOSYSTEM)) ACTIVITY IN SPECIlC MUTANTS AND IN NUTRIENT LIMITINGCONDITIONS"IOCHEMISTRYn 4ANAKA- /BOKATA* #HUNWONGSE* 3HINOZAKI+AND D 3UGIURA-  2APIDD SPLICING ANDSTEPW D ISEPROCESSING OFF ATRANSCRIPT FROMTHE PSB" OPERON INTOBACCOCHLOROPLASTS$ETERMINATION OF THE INTRON SITES IN PET" AND PET$ -OL 'EN 'ENET  n 4HORNTON,% /HKAWA( 2OOSE*, +ASHINO9 +EREN.AND 0AKRASI ("  (OMOLOGS OF PLANT 0SB0 AND 0SB1 PRO TEINSARENECESSARY FORRE F GULATIONOFF 0HOTOSYSTEM))ACTIVITY IN THE CYANOBACTERIUM 3YNECHOCY CYSTIS  0LANT #ELL  n 4ICHY-AND6ERMAAS7 D  &UNCTIONALANALYSISOFCOM F BI NATORIALMUTANTSALTERED INACONSERVED D REGION INLOOP%OFT F HE 3 SP0##"IOCHEMISTRY #0PROTEININ 3YNECHOCYSTIS n 4REBST! 4HETOPOLOGYOFTHEPLASTOQUINONEANDHERBICIDE BINDINGPEPTIDESOF0HOTOSYSTEM))INTHETHYLAKOIDMEMBRANE :.ATURFORSCH C n 4RONRUD D $% 3CHMIDT-&AN D D -ATTTHEWS"7 3TRUCTUREAND D F BACTERIOCHLOROPHYLL A PROTEIN FA 8 RAY A AMINOACIDSEQUENCEO F FROM 0ROSTH T ECOCHLORISAESTUARII RElNEDAT ¯ RESOLUTION* -OL"IOL n 5RBACH% 3CANLAN$* $ISTEL$, 7ATERBURY*"AND#HISHOLM37  2APIDDIVERSIlCATIONOFMARINEPICOPHYTOPLANKTONWITH DISSIMILARLIGHT HARVESTINGSTRUCTURESINFERREDFROMSEQUENCES OF 0ROCHLO R R ROCOCCUS AND 3YNECHOCOCCU 3 S #YANOBACTERIA *-OL %VOL  n VAN$ORSSEN2* "RETON* 0LIJTER** 3ATOH +ANDVAN'OR D RKOM(* A 3PECTROSCOPICPROPERTIESOFT F HEREACTIONCENTERAND D OF THEK$APROTEINOFF 0HOTOSYSTEM))"IOCHIM"IOPHYS!CTA  n VAN$ORSSEN2* 0LIJTER** $EKKER*0 DEN/UDEN! !MESZ*AND VAN'ORKOM(*B 3PECTROSCOPICPROPERTIESOFCHLOROPLAST GRANAMEMBRANESANDOFTHECOREOF0HOTOSYSTEM))"IOCHIM "IOPHYS!CTAn D "RUCE$ %X 6ASILEV3 /RTH0 :OUNI ! /WENS4'AND CITED STATEDYNAMICSIN 0HOTOSYSTEM)))NSIGHTSFROMT F HE8 RAY A CRYSTAL STRUCTURE0ROC.ATL!CAD3C D I53!n D )AND !NDERSSON"  0ROTEINPHOSPHORYLA 6ENER!6 /HAD TIONAND D REDOXSENSING INCHLOROPLASTTHYLAKOIDS#URR/PIN 0LANT"IOLn 6ENER!6 (ARMS! 3USSMAN-AND6IERSTRA2$ -ASS SPECTROMETRICRESOLUTIONOFREVERSIBLEPROTEINPHOSPHORYLATION O SISTHALIANA* "IOL INPHOTOSYNTHETICMEMBRANESOF !RABIDOP #HEMn 6ERMAAS7  -OLECULAR BIOLOGICAL APPROACHESTOANALYZE F ION!NN2EV0LANT0HYSIOL 0HOTOSYSTEM))STRUCTUREAND FUNCT 0LANT-OL"IOL n D  3EQUENCING 6ERMAAS7&* 7ILLI 7 AMS*'+AND!RNTZEN#* ANDMODIlCATIONOFF PSB" THEGENEENCODINGTHE#0PROTEIN OF0HOTOSYSTEM)) INTHECYANOBACTERIUM 3YNECHOCYSTIS  0LANT-OL"IOLn 6ERMAAS7&* )KEUCHI-AND)NOUE9 0ROTEINCOMPOSITION OFF THE0HOTOSYSTEM))CORECOMPLEX INGENETICALLYENGINEERED 3 NECHOCY CYSTIS SP0## MUTANTSOFF THECYANOBACTERIUM 3Y

 0HOTOSYNTH 2ESn 6ERMAAS 7&* 3HEN'AND/HAD) #HIMERIC#0MUTANTS 6 OFTHECYANOBACTERIUM 3YNECHOCYSTIS 3 SP0##CARRYING SPINACH SEQUENCES #ONSTRUCTION AND FUNCTION 0HOTOSYNTH 2ES  n 6IRGIN) 'HANOTAKIS$&ANDD !NDERSSON"  ,IGHT INDUCE D D $ PROTEIN DEGRADATIONIN ISOLATEDD 0HOTOSYSTEM))CORECOM PLEXES&%"3,ETTn 6IRGIN ) 3ALTER!( 'HANOTAKIS $& ANDD !NDERSSON "  ,IGHT INDUCE D D$PROTE D IN DEGRADATION ISCATALYZED BYASERINE TYPEPROTEASE&%"3,ETTn 6IRGIN) 3ALTER!( (AGMAN! 6ASS) 3TYRING3AND!NDERSSON" 6  -OLECULARMECHANISMSBEHINDLIGHT INDUCEDINHIBITION OF0HOTOSYSTEM))ELECTRONTRANSPORTANDDEGRADATIONOFREACTION CENTERPOLYPEPTIDES"IOCHIM "IOPHYS!CTAn 6ISSCHER +* "ERGSTÚM ( 3UNDSTRÚM 6 (UNTER #. ANDD VAN F RGY TRANSFE F R 'RONDELLE2 4EMPERATURE DEPENDENCEOFENER F FROMT HELONGWAVELENGTHANTENNA"#HL TOTHEREACTION CENTER IN 2HODOSPIRI D LLUM RUBRUM 2HODO D BACTER SPHAEROIDES D 74AND-MUTANT FROMTO+ STUDIEDBYPICOSECOND ABSORPTION SPECTROSCOPY0HOTOSYNTH2ESn 7 7ESTHOFF0AND(ERRMANN2' #OMPLEX2.!MATURATION INCHLOROPLASTS4HE PSB" OPERONFROMSPINACH%UR*"IOCHEM  n 7U * 0UTNAM %VANS # AND "RICKER 4-  3ITE DIRECTED 7 MUTAGENESISOFF THE#0PROTEINOFF 0HOTOSYSTEM)) 7 IN THE LUMENALLYEXPOSED LOOP#ISREQUIRED FOR0 F HOTOSYSTEM)) ASSEMBLY AND D STABILITY0 Y LANT-OL "IOLn 7U* -ASRI. ,EE7 &RANKEL,+AND"RICKER4- 2ANDOM 7 MUTAGENESISINTHELARGEEXTRINSICLOOP%ANDTRANSMEMBRANE _ HELIX6)OFTHE#0PROTEINOF0HOTOSYSTEM))0LANT-OL "IOL  n 9 9AMAMOTO9  1UALITY CONTROL OFF 0HOTOSYSTEM)) 0LANT

+VMJBO
+
&BUPO3ZF
BOE
$JOEZ
1VUOBN&WBOT #ELL0HYSIOL n 9AMAMOTO9 AND !KASAKA 4  $EGRADATION OF ANTENNA 9 CHLOROPHYLL BINDING PROTEIN #0 DURING PHOTOINHIBITION OF 0HOTOSYSTEM))"IOCHEMISTRYn 9 9OUNG! -C#HARGUE- &RANKEL,+ "RICKER4-AND0UTNAM

%VANS#  !LTERATIONSOFT F HEOXYGEN EVOLVINGAPPARATUS INDUCE D D BYA !RG A 3ERMUTATION INTHE#0 PROTEINOF F 3YNECHOCY CYSTIS SP0##UNDERCHLO 0HOTOSYSTEM)) FROM RIDE LIMITINGCONDITIONS"IOCHEMISTRY n 9 9U*AN D D6ERMAAS7&*  4RANSCRIPT LEVELSAND D SYNTHESISOF 0HOTOSYSTEM)) COMPONENTS IN CYANOBACTERIAL MUTANTS WITH INACTIVATED0HOTOSYSTEM))GENES0LANT#ELLn :ERGES7 'IRARD "ASCOU*AND2OCHAIX* $ 4RANSLATION OFTHECHLOROPLAST PSB#M2.!ISCONTROLLEDBYINTERACTIONS # BETWEENITS | LEADER AND D THE NUCLEAR LOCI 4"# AND 4"# IN #HLAMYD M DOMONASREINHARD R TII-OL #ELL "IOL n :ERGES7 !UCHINCLOSS!( AND D 2OCHAIX * $  -ULTIPLE Q IN THE | LEADER OF THE CHLO TRANSLATIONAL CONTROL SEQUENCES ROPLAST PSB# # M2.! INTERACT WITH NUCLEAR GENE PRODUCTS IN # #HLAMYDOMONASREINHARDTII 'ENETICSn :HELEVA$ 3HARMA* 0ANICO- -ORRIS(2AND"ARBER* )SOLATION AND CHARACTERIZATION OF MONOMERIC AND DIMERIC #0 REACTIONCENTER0HOTOSYSTEM))COMPLEXES*"IOL#HEM  n :IEGENHAGEN " AND D &LADUN D G -  6ARIATION IN THE PSB# GENE REGION OF GYMNOSPERMS AND D ANGIOSPERMS AS DETECTED BY ASINGLERESTRICTIONSITEPOLYMORPHISM4HEOR!PPL'ENET  n :OUNI! 7ITT ( 4 +ERN * &ROMME 0 +RAU” . 3AENGER 7 AND/RTH0 #RYSTALSTRUCTUREOF0HOTOSYSTEM))FROM 3YNECHOCOCCUS ELONGATUS AT  ¯ RESOLUTION .ATURE  n

$IBQUFS
 5IF
%
BOE %
$PSF 1SPUFJOT 1FUFS
+
/JYPO

8PMGTPO
#JPDIFNJTUSZ SZ #VJMEJOH
%JWJTJPO
PG
#JPMPHZ
*NQFSJBM
$PMMF Z HF
-POEPO 4PVUI
,FOTJOHUPO
$BNQVT
-POEPO
48
";
6,

.BSZ 4BSDJOB %FQBSUNFOU
PG
#JPMPHZ
6OJWFSTJU Z UZ
$PMMFHF
-POEPO
%BSXJO
#VJMEJOH (PXFS
4USFFU
-POEPO
8$&
#5
6,

#SVDF " %JOFS $FOUSBM
3FTFBSDI
BOE
%FWFMPQNFOU
&YQFSJNFOUBM
4UBUJPO & &
*
EV
1POU
EF
/FNPVST

$P
*OD
8JMNJO HUPO
%&

64"

4VNNBSZ   *
*OUSPEVDUJPO  **

*EFOUJàDBUJPO
PG
UIF
%
BOE
%
1SPUFJOT   "
5FSN 5 JOPMPHZ   #
-JOLJOH
UIF QTC" (FOF
UP
*UT
(FOF
1SPEVDU
JO
UIF
5IZMBLPJE
.FNCSBOF    5IF 3BQJEMZ5VSOJOH0WFS
L%B
1SPUFJO   5IF L%B )FSCJDJEF#JOEJOH 1SPUFJO    5IF 2##JOEJOH 1SPUFJO   $
5IF
-JOL
#FUXFFO
UIF QTC% (FOF
BOE
*UT
(FOF
1SPEVDU   ***

5IF
1SJNBSZ 4USVDUVSFT
PG
%
BOE
%   *7

*EFOUJàDBUJPO
PG
UIF
%
BOE
%
1SPUFJOT
BT
UIF
1IPUPTZTUFN **
3FBDUJPO $FOUFS 4VCVOJUT     "
*OUFSQSFUBUJPOT
PG
4FRVFODF
4JNJMBSJUJFT     #
*TPMBUJPO
PG 1IPUPTZTUFN **
3FBDUJPO
$FOUFS
$PNQMFYFT   $
'PMEJOH
.PEFMT
PG
UIF
%%
)FUFSPEJNFS   7

.VUBHFOFTJT
PG
UIF
%
BOE
%
1SPUFJOT   7 "
.VUBUJPOT
"GGFDUJOH
UIF
%POPS
4JEF    5ZSPT 5 JOFT :; BOE :%    %)JT
BOE %)JT   
.VUBOUT
"GGFDUJOH
UIF
#JOEJOH
BOE
'VODUJPO
PG
UIF
.BOHBOFTF $MVTUFS   B 5IF -'
.VUBOU
PG 4DFOFEFTNVT
PCMJRVVT   C %"TQ   D
)JT (MV
)JT
"TQ
JO
UIF $BSCPYZM5FSNJOBM
3FHJPO
PG
%   E
5IF $BSCPYZM5FSNJOBM
3FTJEVF
BU "MB  F 0UIFS
3FTJEVFT
"GGFDUJOH
UIF
.BOHBOFTF $MVTUFS   G
.PEFM
GPS
-PDBUJPO
BOE
"TTFNCMZ
PG
UIF
.BOHBOFTF $MVTUFS
#BTFE
PO .VUBHFOFTJT
%BUB    %)JT
BOE %)JT    %)JT
BOE %)JT  

!UTHORFORCORRESPON F DENCE EMAILPNIXON IMPERIALACUK 47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS

#
.VUBUJPOT
"GGFDUJOH
UIF
"DDFQUPS
4JEF    5IF 1IFPQIZUJOT   
5IF
"DDFTTPSZ $IMPSPQIZMMT #" BOE ##    5IF *SPO2VJOPOF $PNQMFY   7* $PODMVEJOH
3FNBSLT   "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ )N #HUAAND'ILLHAM*#ELL"IOLOGYn REPORTEDFORTHElRSTTIMETHEEXISTENCEOFTWOCHLO ROPLAST ENCODED D PROTEINSWITHINTHETHYLAKOID D MEMBRANEOFF THE GREENALGA #HLAMYD M DOMONASREINHARD R TII WHI W CH THEYTERMED D $ AND $ 4HE$AND D $PROTEINSARENOWRECOGNIZEDD ASTHE0HOTOSYSTEM))REACTIONCENTER POLYPEPTIDESWITHAKEYROLEINBINDINGALLOFTHECO FACTORSINVOLVEDINPHOTOSYNTHETICWATEROXIDATION)NTHIS N CHAPTERWESUMMARIZESOMEOFF THE BIOCHEMICALANDD MUTAGENESISDATATHAT HAS BEENINSTRUMENTAL INSHAPING THISVIEWOFF THE $AND D $ PROTEINS *
*OUSPEVDUJPO !LTHOUGH H THE0HOTOSYSTEM))  03)) COMPLEX ˆ IE WATER PLASTOQU Q INONEOXIDOREDUCTASE D ISCOMPOSEDD OF OVERTWENTY lVESUBUNITS THEIMPORTANTLIGHT INDUCED F IONSOCCURWITHI T NAHETERODIMER ELECTRONTRANSFERREACT COMPOSED D OF THE $AND$PO D LYPEPTIDES $$  2ECENT STRUCTURAL STUDIES HAVE NOW IDENTIlED D THE POSITIONSOFF THE#HLAND D 0HEOPIGMENTSWITHIN$$ D IMPORTANTLY THE LOCATION OF F THE -N #A METAL AND CLUSTERWHICHCATALYSESPHOTOSYNTHETICWATEROXIDA TION :OUNI ET AL  +AMIYA AND D 3HEN  "IESIADKAETAL &ERREIRAETAL  0ERH R APS ONEOFTHEMOSTREASSURINGASPECTSOFTHESTRUCTURAL !BBREVIATIONS R "2# n REACTION CENTER FROM F PURPLE NON SUL FFUR PHOTOSYNTHETIC BACTERIA "!"" n 03)) CHLOROPHYLLS IN SIMILAR POSITION TO THE TWO ACCESSORY "#HLS OFF THE "2# "#HL n BACTERIOCHLOROPHYLL"0HEO n BACTERIOPHEOPHYTIN# TER MINALnCARBOXYL TERMINAL#HLnCHLOROPHYLL#HL:$ nPERIPHERAL #HLINTHE03))2# LIGATEDBY$ (IS#HL :$ n PERIPHERAL #HLINTHE03))2# LIGATEDBY$ (IS#YTnCYTOCHROME %.$/2 n ELECTRON NUCLEAR DOUBLE RESONANCE %02 n ELECTRON PARAMAGNETICRESONANCE%3%%-nELECTRONSPIN ECHOENVELOPE D LATION&4)2n&OURIERTRANSFORM F INFRARE F D 0nSPECIAL PAIROF MODU F HEPRIMARY ELECTRON "#HL INTHE"2#0nHISTORICALTERM FORT DONORWITHIN03))0 0!0" n CHLOROPHYLLS IN03))INSIMILARPOSITION F HL OF"2#0 F HEO!0HEO" n PHEOPHYTIN A TOTHESPECIAL PAIROF"# MOLECULESIN03))INSIMILARPOSITIONTOTHE"0HEOMOLECULESOF THE"2# 1! n PRIMARYQUINONEELECTRONACCEPTOR1" n SECONDARY Q QUINONEELECTRONACCEPTOR2#n REACTIONCENTER3 n-NCLUS TERTHATHASACCUMULATEDTWOOFTHEFOUROXIDIZINGEQUIVALENTS F IDATION3$3 0!'%nSODIUMDODECYLSULFATE F NEEDED FORWATEROX POLYACRYLAMIDEGEL ELECTROPHORESIS 9$ n REDOX ACTIVETYROSINE 4 R 9: n REDOX ACTIVETYROSINE $ 4Y 4 R WHI W CH ACTS $ 4Y ASTHE IMMEDIATEOXIDANTOFF THE-NCLUSTER

STUDIES HASBEEN HOWMUCH HAD BEENCORRECTLYPRE DICTEDINADVANCE)NTHISCHAPTER WEDESCRIBETHE BACKGROUNDD THATLEDD TOTHEEMERGENCEOF$AN F D $ U UNITS PLUSTHE ASTHE03))REACTIONCENTER2# SUB KEYMUTAGENESISANDBIOCHEMICALEXPERIMENTSTHAT IDENTIlEDD THE LIKELY BINDINGSITES FORT F HEVARIOUSPIG MENTSANDD REDOX ACTIVECOMPONENTSWITHINTHE$ ANDD $ HETERODIMER ** *EFOUJàDBUJPO PG UIF % BOE % 1SPUFJOT "
5FSN 5 J MPHZ JOP 4HE$ANDD $NOMENCLATUREWASlRSTUSED INSTUD IESAIMEDD AT IDENTIFY F INGTHOSETHY H LAKOIDD PROTEINSTHAT WERESYNTHESIZEDBYTHECHLOROPLASTOFTHEGREENALGA #HLAMYD M DOMONAS REINHARD R TII #HUA AND D 'ILLHAM  #ELLSWEREPULSE LABELEDD WITH;#= ACETATE IN THEPRESENCEOFANINHIBITOROFCYTOPLASMICPROTEIN SYNTHESIS ANDD THE RADIOLABELEDD THYLAKOIDD PROTEINS SEPARATED BY DENATURING POLYACRYLAMIDE GEL ELEC TROPHORESIS4WO BROAD AREAS OF RADIOACTIVITY WERE IDENTIlED BY AUTORADIOGRAPHY ANDD TERMED @DIFFUSE F BAND  $  ANDD @DIFFUSE F BAND $  )NTHESE EXPERIMENTS $ POSSESSEDANAPPARENTMOLECULAR F ABOUT K$AANDD $ APPEARED TOBEK$A MASSOFA !LTHOUGH $ ANDD $ MIGHTHAVEBEENCOMPOSED OFANUMBEROFDIFFERENTCO MIGRATINGPOLYPEPTIDES THENOTATION$ANDD $SOMETIMES $ AND $ HAS BEEN SUB U SEQUENTLY USEDD TO DESCRIBE THE PSB! B AND PSB$ GENE PRODUCTS RESPECTIVELY IN ALL OXYGENIC PHOTOSYNTHETICORGANISMS

$IBQUFS

%
BOE
%
1SPUFJOT #
-JOLJOH
UIF
QTC"
(FOF
UP
*UT
(FOF
1SPEVDU
Z JO
UIF
5IZMBLPJE
.FNCSBOF 5IF 3BQJEMZ5VSOJOH0WFS
L%B
1SPUFJO J %ARLY STUDIES ON THE SYNTHESIS OF F THYLAKOID D MEM BRANE PROTEINS IN ISOLATED D CHLOROPLASTS OF HIGHER PLANTSREVEALEDTHATAMAJORPRODUCTWASAPROTEIN F APPARENT MOLECULAR MASS  K$A "OTTOMLEY OF ET AL  %AGLESHAM AND D %LLIS   'IVEN THAT IT DID NOT SEEM TO CORRESPOND TO AN ABUNDANT POLYPEPTIDE THIS PARTICULAR PROTEIN APPEARED D TO BE D HESISAND DEGRADATIONWITHIN UNDERGOINGRAPIDSYNT THEMEMBRANE ˆ HENCEITSNAME4HEGENEENCODING THISPROTEIN NOWTERMED PSB! WASMAPPED D ONTOTHE F CHLOROPLASTGENOMEBYANUMBEROF DIFFERENTGROUPS ANDTHElRSTSEQUENCES FROMS Q PINACHANDTOBACCO WEREPUBLI U SHED IN:URAWSKI ETAL   
5IF L%B
)FSCJDJEF#JOEJOH 1SPUFJO J ! LARGENUMBEROF HERBI R CIDES BLOCKP K HOTOSYNTHETIC ELECTRONmOWONTHEACCEPTORSIDEOF03))4HROUGH THEUSEOFT F HERADIOLABELED D PHOTOAFlNITY HERBI R CIDE D CO WORRKERSSHOWED AZIDO ;#= ATRAZINE 0lSTERAND T THATA K$APROTEINWASATARGET0lSTERETAL   "Y PROTEOLYTIC lNGERPRINTING THISPARTICULARTAGGED PROTEINAND D THE K$ARAPIDLY TURN L ING OVERPROTEIN IT WASCONCLUDEDTHATTHEYWEREONEANDTHESAME3TEIN BACK K ETAL  #ONlRMATIONTHAT PSB! B ENCODED THEHERBI R CIDE BINDINGPROTEINCAMEWITH THE IDENTI lCATIONOFAMUTATION 3ER'LY INTHE PSB! B GENE PRODUCTO D F FASPONTANEOUSATRAZ INE RESISTANT BIOTYPE T USHY H BRIDU D S (IRSCHBERGAND D -C)NTOSH OF !MARANTH  4HISSAMEMUTATIONHASNOWBEENFOUNDINA R CIDE RESISTANTPHOTOSYNTHETICORGAN VARIETY OF HERBI ISMSFORAREV F IEW SEE/ETTMEIER   5IF 2##JO J EJOH
J 1SPUF 1 J JO 3EVERALLINESOFEVIDENCEFORAREVIEW SEE+YLE  HAVE INDICATED D THATTHEBINDINGSITE FOR F HERBI R CIDESIN 03)) WAS COINCIDENT WITH OR NEAR TO THE BINDING SITEFORTHESECONDARYQUINONEELECTRONACCEPTOR 1" WHICHTAKESPARTINTHEBINARY GATEMECHANISM FORT F HE TRANSMISSIONOFE F LECTRONSINTOTHE PLASTOQU Q INONE POOL "OUGES "OCQUET 6ELTHUYSAND!MESZ  4HESE INCLUDEDD THE INmUENCEOFT F HE1"REDOXSTATEON THEAFlNITYOFF 03)) FOR F HERBI R CIDES 6ELTHUYS  %02STUDIES2UTHERFORDETAL  INHIBITIONOF HERBI R CIDE BINDING USING PLASTOQUINONE ANALOGUES

 6ERMAASETAL /ETTMEIERETAL  AND SLOWINGOF1" REDUCTIONINATRAZINE RESISTANT PLANTS "OWESETAL  4OGETHERTHESE DATA LEDD TOTHE IDEATHAT$ ENCODED BY PSB! WASTHE1" BINDING PROTEINOF03)) ASWELLASATARGETFORSEVERALCLASSES OF HERBI R CIDEFORAREV F IEW SEE/ETTMEIER   $
5IF
-JOL
#FUXFFO
UIF
QTC%
(FOF
BOE
*UT (FOF
1SPEVDU 4HE PSB$ GENEWASLOCATEDlRSTONTHECHLOROPLAST GENOME OF # REINHARD R TII USING A COMBINATION OF D INSTPROTEINSINTHE$REGION ANTIBODIESRAISEDAGA OFDENATURINGGELSANDAN %COLI BASEDTRANSCRIPTION TRANSLATION OF F CLONEDD CHLOROPLAST $.! FRA F GMENTS 2OCHAIX  4HESEQUENCEWASlRSTPUBLI U SHED IN 2OCHAIXETAL  ANDLATERINANAMENDED F FORM %RICKSONETAL  5SINGTHIS GENE PSB$ F CHLO WAS SUB U SEQUENTLY ISOLATED FFROM A VARIETY OF ROPLASTS !LT ET AL  (OLSCHUH ET AL  2ASMUSSENETAL  ANDD CYANOBACTERIAL SOURCES 7ILLI 7 AMSANDD #HISHOLM  4HEIDENTIlCATION OFTHE PSB$ GENEPRODUCTINTHYLAKOIDSWASINITIALLY UNCERTAIN ESPECIALLY FOR F HIGHER PLANTS BECAUSE OF THELACKK OF SPECIlCANTIBODIES&OR #REIN # HARD R TII $ WASIDENTIlEDINDIRECTLYONTHEBASISOFRADIOACTIVE LABELINGOFT F HYLAKOID D PROTEINSANDD CONCLUDEDD TOBEA COMPONENTOFF THE03))CORECOMPLEX$ELEPELAIRE  #ONlRMATIONTHATTHE PSB! AND PSB$ GENE PRODUCTSWERE D INDEEDD COMPONENTSOF THE03))COM PLEXFROM F HIGHERPLANTSCAMEWITHTHEUSEOFF SPECIlC ANTIBODIESRAISEDTOTHEGENEPRODUCTSEXPRESSEDIN % COLI .IXONETAL   ***
5IF
1SJNBSZ
4USVDUVSFT
PG
%
BOE
% $ ISSYNTHESIZEDASAPRECURSORPROTEININHIGHER Y A PLANTS 'REBANIER ET AL  WITH UNUSUALLY CARB R OXYL TERMINAL # TERMINAL EXTENSION -ARDERET AL  7ITHTHEAPPARENTEXCEPTIONOF %UGLENA G GRACI LIS +ARABINETAL  ANDD SOMESPECIESOF DINOmAGELLATES 9AMAMOTOETAL  THEPRESENCE OFA# TERMINALEXTENSIONFOR$APPEARSUBIQUITOUS Q 0ROTEINSEQUENCINGOFF MATURE$ FROMSP F INACH 4AKAHASHI ETAL 94AKAHASHI ETAL  THE # "!$INER PERSONALCOM GREENALGA #REINHARDTII MUNICATION ANDD THECYANOBACTERIUM 3YNECHOCY CYSTIS .IXONETAL A HASCONlRMEDD THAT INALL CASESTHE # TERMINALRESIDUEOFMATURE$IS!LA D RESIDUESAREREMOVE D D D SOTHAT ANDD AMINO ACID

 RESPECTIVELY FROMT F HEIRPRECURSORMOLECULES)NCON TRAST $ISNOT# TERMINALLYPROCESSED94AKAHASHI ETAL   )N SPINACH THE INITIATING . FORMY F LMETHIONINE RESIDUEOFBOTH$AND$ISREMOVEDLEAVING4HR W CH IS ACETYLATED4HESE4HR AT THE . TERMINUS WHI D LSO BEPHOSPHORYLATED INBOTH$AND RESIDUESCANA $ -ICHEL ET AL   4HE PHYSIOLOGICAL SIG NIlCANCEFOR$AN F D $PHOSPHORYLATION INHIGHER PLANTSREMAINSUNCLEARALTHOUGH EVIDENCEPOINTSTO AROLEINREGULATINGTHEIRDEGRADATION+OIVUNIEMI ETAL  $ HASALSOBEENSUGGESTED D TO BE PAL MITOYLATED IN HIGHER PLANTS -ATTOO AND D %DELMAN  BUTTHESITEOFATTACHMENTANDPHYSIOLOGICAL IMPORTANCEREMAINOBSCURE&ORCYANOBACTERIA $ AND D $DONOTAPPEARTO BEPHOSPHORYLATED W D WHEREAS # THEREAREREPORTSTHAT INTHEGREENALGA #REINHARDTII $ BUT NOT $ IS PHOSPHORYLATED $ELEPELAIRE   DE6I 6 TRYETAL  "ANDSASSIGNEDD TO THEPHOSPHORYLATED$ ANDNON PHOSPHORYLATED F FORMS $ OFF $ HAVEBEENRESOLVED BYY 3$3 0!'% CONDUCTE D D INTHEPRESENCEOF  -UREA $ELEPELAIRE   4HEPHOSPHORYLATIONSITEIN$HAS YETTOBEIDENTIlED F D $ DETERMINED 4HEMOLECULARMASSESFOR$AN BYMASSSPECTROMETRY AREAPPROXIMATELYK$A AND D  K$A RESPECTIVELY Y AS PREDICTED FFROM THE GENE SEQUENCES 3HARMA ET AL   (OWEVER UPON3$3 0!'%ANALYSISTHEYUSUALLYMIGRATEWITH APPARENTSIZESOF BETWEENnK$A3ATOH ETAL  DEPENDINGONTHEELECTROPHORETICCONDITIONS MOSTNOTABLYTHECONCENTRATIONOFUREAINTHEGEL)N THEABSENCEOFF UREA $MIGRATESASTWO IMMUNODE TECTABLE BANDS ATAND D  K$A THEFASTERO F F WHICH ISLIKELYTOBEAMORECOMPACTSTRUCTURAL@CONFORMER 4AYL A ORETAL  $MIGRATESBETWEENTHESETWO $BANDS.IXONETAL 3AYREETAL   )NCLUSIONOFUREAATCONCENTRATIONSGREATERTHAND OF CAUSESTH T ETWO$ BANDSTOCOLLAPSETOASINGLEBAND APPARENTSIZE K$A SOTHAT$NOWMIGRATESSLOWER THAN$"EFORETHEAVAILABILITYOFSPECIlCANTISERA THEABERRANTELECTROPHORETICMIGRATIONOFF $AND $ TOGETHERWITHTHEIRPOORSTAINING BY#OOMASSIE BLUE LEDTOMUCHCONFUSIONCONCERNINGTHEIRIDENTIlCATION )TISEVENPOSSIBLETHATTHERADIOLABELED BANDS lRST ASSIGNED D TO$AND $INNON UREACONTAININGPOLY ACRYLAMIDEGELS#HUAAND'ILLHAM  ACTUALLY REPRESENTTH T ETWODIFFERENTE F LECTROPHORETICCONFORMERS F OFF $ .IXON  

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS *7
*EFOUJàDBUJPO
PG
UIF
%
BOE
%
1SPUFJOT BT
UIF
1IPUPTZTUFN
**
3FBDUJPO
$FOUFS 4VCVOJUT "
*OUFSQ S SFUBUJPOT
PG
4FRVFODF
4JNJMBSJUJFT $.!SEQUENCINGSTUDIESREVEALEDD EARLYONTHATTHE PSB! B AND PSB$GENEPRODUCTSSHAREDSOMESEQUENCE SIMILARITYANDD POSSESSED SIMILARHYDROPATHYPROlLES SUGGESTIVEOFF ANEVOLUTIONARYRELATEDNESS 2OCHAIX ET AL   7HILE THE OVERALL SEQUENCE IDENTITY BETWEENTHETWOWASRELATIVELY UNIMPRESSIVE IN # #REIN HARD R TII THEREWEREREGIONSOF HIGH SEQUENCE IDENTITYAPPROACHING2OCHAIXETAL  )N CONCURRENTWORK R ITWASREALIZEDD THATTHE,ANDD F HE2# OF PURPLENON SULFURP F HOTOSYN SUB U UNITSOFT T THETICBACTERIAHEREAFTERCALLEDTHE"2# ALSOSHOWE D SIGNIlCANTREGIONSOFSEQUENCES F IMILARITY TO$AND $7ILLI 7 AMSETAL  9OUVANETAL   )NTHEABSENCEOFDETAILEDSTRUCTURALINFORMATION THE SIGNIlCANCEOFT F HESESIMILARITIESWASUNCLEAR"ASED K BOUT$AND03))ATT D HETIME ONWH W ATWASKNOWNA IT WAS SUGGESTED THAT THE AREAS OF SIMILARITY WERE F IONINQUINONEBINDING (EARSTAND RELATEDD TOAFUNCT 3AUER 2OCHAIXETAL   ! MAJOR STEP IN UNDERSTANDING THE STRUCTURE OF 03))CAMEPARADOXICALLY WITHTHEELUCIDATIONOFT F HE F THE"2# FRO F M 2HODO DOPSEUDOMONASVIRI D DIS DI STRUCTUREOFT $EISENHOFERETAL  4HEPREVIOUSLYOBSERVED F GSEQUENCESIMILARITY BETWEEN$$ AREASOFSTRON U UNITSWERE INREGIONSOFF THE"2# IN ANDD THE,-SUB VOLVEDINBINDINGTHESPECIALPAIROF"#HLMOLECULES D ANDD NOTQUINONEAS lRSTTHOUGHT/THERKEY RESIDUES D INVOLVED IN LIGATING SUCH AS THE FFOUR (IS RESIDUES THENON HEMEIRONONTHEACCEPTORSIDEOFTHE"2# WEREALSO FOUN F D IN$ANDD $#ONSEQUENTLY ITWAS F HESESTUDIESTHAT$ANDD $ QUICKLYREALIZED FROMT WEREMOSTLIKELYTHE2#SUBUNITSOF03))-ICHEL ANDD $EISENHOFER  F FU F LlLLINGROLESANALOGOUSTO ,-OFT F HE"2#/THERKEYRESIDUES D IN,-WERE CONSERVED IN $$ SO THAT FOLDING MODELS COULD BEDRAWN FOR$ F $ INTHEABSENCEOF DIRECTEXPERI MENTAL DATA4REBST "ARB R ER A-ICHELAND $EISENHOFER  !TTHATTIMETHEDOGMAINTHE LITERATURE BASEDD ONAVARIETY TYOF BIOCHEMICALEVIDENCE WASTHAT #0 ENCODED BY PSB" WASTHE03))2# SUBUNIT #AMM AND 'REEN  .AKATANI ET AL DE6I 6 TRYETAL 3ATOH  (OWEVER WITH HINDSIGHT ITISNOWCLEARTHAT$ANDD $WERE EITHER PRESENT IN THE ACTIVE 03)) PREPARATIONS BUT

$IBQUFS

%
BOE
%
1SPUFJOT HAD D ESCAPED DETECTIONORTHAT INAPPROPRIATEASSAYS F FOR03))2#ACTIVITYWEREUSED #
*TPMBUJPO
PG
1IPUPTZTUFN
**
3FBDUJPO $FOUFS $PNQMFYFT 4HE lRST DIRECTEXPERIMENTALSUPP U ORT FORT F HE$$ 2#MODELOF03))CAMEWITHTHEISOLATIONBY.ANBA AND D 3ATOH  FROMSP F INACH OFF THESO CALLED03)) D 2#COMPLEX SOMETIMESREFERRE F D TOASTHE@$$ #YT BCOMPLEX!LTHOUGHTHISPREPARATIONDIDNOT CONTAIN-N LACKED D AFU F LLY FUNCT F IONAL SECONDARYELEC TRONDONOR 9: ANDHADLOSTTHETWO QUINONEELECTRON Q ACCEPTORS 1! AND1" ITSTILLRETAINEDTHEABILITYTO PERFORM F LIGHT INDUCE D D CHARG R ESEPARATIONINDICATIVEOF THE FORMAT F IONOFF THEPRIMARYRADICALPAIR 0 0HEOn $ANIELIUSETAL 94AKAHASHIETAL  !S THECOMPLEX LACKED D #0 THISRESULTSUGGESTED D THAT $AND D $BOUND D 0AND THEREDOX ACTIVE0HEO /THERFEATURESTOSUPP U ORTTHEPRESENCEOF0AND THE0HEOELECTRONACCEPTORWITHINTHISCOMPLEXWERE THEABILITIESTOGENERATEASPIN POLARIZED D CHLOROPHY H LL TRIPLETSTATE2UTHERFORDETAL /KAMURAETAL D WITH CHARGERECOMBINATIONOFT F HE  ASSOCIATED RADICAL PAIRAND D TO PHOTOACCUMULATEEITHERREDUCE D D 0HEOINTHEPRESENCEOFDITHIONITE.ANBAAND3ATOH D CHLOROPHYLL POSSIBLY 0 UPON  OROXIDIZED F ILICOMOLYBDATE"ARB R ERETAL  &OR ADDITIONOFS AREVIEWOFTHEISOLATED03))2#SEE3ATOH  !NALYSISOFF THE03))2#COMPLEX BY3$3 0!'% AND#OOMASS D IE BLUESTAININGREVEALED D TWO BANDSWITH T APPROXIMATESIZESOFK$A PLUSAFURTHERFAINTER STAINING BAND D ATABOUT K$A4HE#0AND D #0 APOPOLYPEPTIDESWEREBOTH UNDETECTABLE .ANBAAND 3ATOH   6ITAL IMMUNOCHEMICAL EXPERIMENTS LATERCONlRMED D THETWO K$ABANDSASTHE$AND $SUB U UNITSAND D THE K$ABAND D ASEITHERAMIXTURE OF$AND$HOMODIMERS3ATOHETAL  ORA $$HETERODIMER-ARDERETAL  !LSOPRESENT WITHINTHISPREPARATIONWERETHELOWMOLECULARMASS _ AND ` SUBUNITSOFCYTOCHROMEB WHICHCOULDBE DETECTEDSPECTROSCOP D ICALLY .ANBAAND3ATO D H  D THE0SB)SUB U UNIT ALSOENCODED BYTHECHLOROPLAST AND GENOME)KEUCHIAND)NOUE A7EBBERETAL D 0SB7SUB U UNITWAS FOUN F D  4HENUCLEAR ENCODED LATERINSOMEPREPARATIONSOFT F HE$$#YT B COM PLEX,ORKOVIƾ ETAL  BUTITSABSENCEINOTHER PREPARATIONSSUGGESTSALOCATIONONTHEPERIPHERYOF THE$$#YT B COMPLEX !LIZADEH ETAL   )MPORTANTCONlRMATIONTHAT$AND$BOUNDTHE #HL AND D 0HEOMOLECULESOFF THE2#CAMEWITH THE

 ISOLATIONANDD ANALYSISOFF A$$COMPLEXDEPLETED OF#YT B AND0SB)4ANGETAL   $$#YT B COMPLEXES HAVE NOW BEEN ISO LATED FROM F # REINHARD # R TII !LIZADEH ET AL  AND 3YNECHOCYSTIS 3 'IORGIETAL  SOIT ISCLEARTHATTHESUB U UNITSTRUCTUREOFF THE03))2#IS CONSERVEDD ACROSSTHEWH W OLERANGEOFF OXYGENICORGAN ISMS2ADIO LABELINGSTUDIES!LIZADEHETAL  AND DETERMINATIONOFF THERATIOOFAM F INOACIDS3ATOH F HERINDICATEDD THATTHElVESUB U UNITSARE  HAVE FURT PRESENTINEQUIMOLARAMOUNTS Q  $
'PMEJOH
.PEFMT
PG
UIF
%%
)FUFSPEJNFS "ASEDD ON HYDROPATHY PLOTS $WASORIGINALLY PRO POSEDD TOCONSISTOFSEVENTRANSMEM F MBRANE HELICES2AO ETAL  RATHERTHANTHElVEPREDICTEDBY-ICHEL F  %XPERIMENTS INVOLVINGTHE ANDD $EISENHOFER BINDINGOFF SITE SPECIlC$ANTIBODIESTOINSIDE OUT AND RIGHT SIDE OUT THYLAKOID VESICLES AND TRYPSIN ACCESSIBILITY TO $ PROVIDEDD EARLY SUPPORT FFOR A lVE TRANSMEMBRANE HELIX MODEL FFOR $ 3AYRE ET AL   )NTHEABSENCEOF DETAILEDD STRUCTURAL INFORMAT F ION ANUMBEROFF COMPUTER BASEDD MODELSWEREDEVELOPED FORTHE$$HETERODIMERUSINGTHESTRUCTUREOFTHE "2#ASATEMPLATE 3VENSSONETAL 2UFmEET AL 3VENSSONETAL 8IONGETAL   )NALLMODELSTHEREARETWOBRANCHES !AND" OF CHLORINSSPANNINGTHEMEMBRANE &IG "ASEDD ON F HE"2# THE#HLS THENOMENCLATURE DEVELOPED FORT ANALOGOUSTOTHESPECIALPAIROF"#HLMOLECULESARE DESIGNATEDINTHISCHAPTERAS0 0! AND 0" THE#HLS ANALOGOUSTOTHEACCESSORY"#HLMOLECULESAREDES IGNATED"! ANDD "" THESE#HLSARETERMED#HL$ AND #HL$ IN#HAPTERSn ANDD THEPHEOPHYTINSARE DESIGNATEDD 0HEO! ANDD 0HEO" -IDWAYBETWEENTHE PRIMARY1! ANDSECONDARY1" QUINONEELECTRON ACCEPTORS ISANON HEME IRONATOM&E 4WOADDI TIONAL CHLOROPHY H LLS#HL :$ AND #HL :$ FOUN F D IN 03)) BUTNOTTHE"2# AREINCLUDEDIN&IG8IONG ETAL   3UPERlCIALLYTHECOMPUTER GENERATEDD MODELSARE VERY SIMILAR ALTHOUGH THERE ARE SIGNIlCANT DIFFER ENCESWITH REGARDD TOTHEAMINO ACID D RESIDUES D LINING THECO FACTOR F BINDINGSITESREVIEWED IN8IONGETAL  .EVERTHELESSTHESEMODELSARECONSISTENTWITH EARLY STRUCTURAL DATA OBTAINED BY %02 SUCH AS THE ORIENTATIONSOFF 0HEO! AND 1! $ORLETETAL  AND DISTANCESESTIMATEDFROMTHESPIN LATTICERELAXATION OFPARAGMAGNETICSPECIES› ¯FOR&E))



1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS

&IIG  .OMENCLATUREANDD MODELOFT & F HETRANSMEMBRANEARRANGEMENTOFF THEPIGMENT QUINONEAND D NON HEMECOFACTORSW F ITHINTHE$$ HETERODIMER BASEDONACLOSEANALOGYTOTHE"2#4HE0 0!0" AND D "!"" #HL MOLECULESWITHIN03))OCCUPY SIMILARBUTNOTIDENTICAL POSITIONSTOTHESPECIAL PAIRANDD ACCESSORY"#HLMOLECULES RESPECTIVELY O Y FT F HE"2#0HEO! AND D 0HEO" AREEQUIVALENTTOTHETWO"0HEO MOLECULESOFTHE"2# 1! ISTHEPRIMARYQUINONEELECTRONACCEPTOR 1" THESECONDARYQUINONEELECTRONACCEPTOR ANDÐENON HEME IRONATOM)NTHE"2# ELECTRONTRANSFERPROCEEDSDOWNTHE@!BRANCHFROM0 0!0" VIA "! TO"0HEO! THEN1! ANDlNALLYTO 1")N03)) Y THEREARETWOEXTRA#HLSTERMED#HL:$ AND#HL:$ LIGATEDBY$ (ISAND$ (IS RESPECTIVELY

#HL :+OULOUGLIOTISETAL  › ¯FORBOTH &E)) 9$s AND D &E)) 9:s +OULOUGLIOTISETAL  AND›¯FOR&E)) 0HEO!n $ELIGIANNAKISAND 2UTHERFORD   4HE CLOSE STRUCTURAL SIMILARITY 2 BETWEENTHEACCEPTORSIDESOFF 03))AND THE"2# SUG GESTED BYTHESEAND D OTHEREARLYSTUDIES HASNOWBEEN CONCLUSIVELYSHOWNINRECENT8 RAYCRYSTALLOGRAPHIC D 3HEN  STUDIES :OUNIETAL +AMIYAAND "IASIADKAETAL &ERREIRAETAL   &IGUREDISPLAYSFOLDINGMODELSFOR$AND$ BASED D ONTHOSEOFF 3VENSSONETAL (IGHLIGHTED ARE KEY RESIDUES PREDICTED FFROM SEQUENCE COM PARISONSTOBEINVOLVEDINBINDINGVARIOUSCOFACTORS WITHIN 03)) 5NFORTUNATE F LY THE LACK K OFF SEQUENCE D ,- INTHEEXTRINSIC SIMILARITY BETWEEN$$AND LOOPS CONNECTING THE TRANSMEMBRANE HELICES HAS LIMITED D THEABILITYOFF THECOMPUTER BASED D STRUCTURAL MODELSTOPROVIDECLEARGUIDANCEASTHESTRUCTUREOF THELUMENALREGIONSANDHENCETHEPOSSIBLESITEOF THE-NCLUSTER 7 7
.VUB HFOFTJT
PG
UIF
%
BOE
%
1SPUFJOT 4WOMODELORGANISMSHAVEBEENWIDELYUSEDFORTHE MUTAGENESISOFF $AND $THECYANOBACTERIUM 3YN ECHOCYSTIS SP0##REVIEWED BY.IXONETAL B6ERMAAS $EBUS$INER AND THE GREENALGA #HL # LAM MYD DOMONASREINHARD RDTII REVIEWED

BY2UFmEAND3AYRE  4HEABILITYOFA$PROTEIN FFROMACHLOROPLASTTOBESUCCESSFU F LLY INTEGRATED INTO ACYANOBACTERIAL 03))COMPLEX WITHOUT LOSSOF/ F  EVOLVINGACTIVITY.IXONETAL  SUGGESTSTHAT THEMECHANISMOFWATEROX F IDATIONBY03))ISHIGHLY CONSERVEDD THROUGHOUTNATURE)NGENERAL MUTAGENESIS EXPERIMENTS HAVE PROVIDED STRONG SUPPORT FOR THE GENERAL ARRANGEMENTOFF PIGMENTSSHOWN IN&IGAND HAVEPROVIDEDD CRUCIAL INFORMAT F IONONTHE LOCATIONOF THEREDOX ACTIVECOMPONENTSWITHIN03))INCLUDING THE -N CLUSTER )N THE FO F LLOWING SECTIONS WE WILL DISCUSSSOMEOFT F HEKEYMUTANTS5NLESSINDICATED OTHERWISE THE NUMBERING OF RESIDUES COMES FROM CYSTIS SEQUENCES&ORFURT F HERDETAILS SEE THE 3YNECHOCY #HAPTERS$INERANDD "RITT ANDD  $EBUS  "
.VUBUJPOT
"GGFDUJOH
UIF
%POPS
4JEF 5Z 5ZSPTJOFT :; BOE
:% 4HE lRST DEMONSTRATION OFF THE POWER ANDD USEFU F L NESSOFMUTAGENESISSTUDIESFOR03))CAMEWITHTHE IDENTIlCATIONOFF THETYROSINEELECTRON DONORS 9: AND 9$)NTHEEARLY LITERATURETHESEELECTRONDONORSARE SOMETIMESCALLED:AND$ RESPECTIVELY:LIESONTHE F HWAY A CONNECTING THE-NCLUSTER ELECTRONTRANSFERPAT W EREAS$ ISONASIDE PATH)NTHEIROXIDIZED TO0 WH STATESTHEYGIVERISETO%02SIGNALSOFSIMILARSHAPE D IGNAL )) BUTSHOWDIFFERENT F KINETICS THESO CALLED3

$IBQUFS

%
BOE
%
1SPUFJOT



&IIG  &OLDING MODELSFORT & F HE$TOP ANDD $BOTTOM SUB U UNITSOF 3Y 3 NECHOCY CYSTIS BASED D ONONESDRAWNBY3VENSSONETAL   %ACHSUB U UNITCONSISTSOF lVE ! % TRANSMEMBRANE HELICESAND D TWO #$AND D $% HELICESTHAT LIEPARALLELTOTHEMEMBRANE!MINO ACID RESIDUESTHATAREDISCUSSEDINTHETEXTAREINDICATEDBYTHEONE LETTERCODE0OSSIBLEAMINO ACIDLIGANDSTO0 0! 0" #HL:$ #HL :$ 0HEO! 0HEO" AND&EAREINDICATED

F ION AND DECAY OXIDATION OF F : GIVES RISE OF FORMAT TO 3IGNAL))VFF VERYFAST ININTACT03))OR 3IGNAL))F FAST F INSAMPLES LACKING A-NCLUSTER"LANKENSHIP K AND 3AUER ETAL   3IGNAL ))S SLOW "ABCOCK  ISANEXTREMELYSTABLESIGNALANDISASSOCIATED WITHOXIDATIONOFF $/NTHE BASISOFF THEHYPERlNE

STRUCTUREOF3 F IGNAL))AND ITSSIMULATIONUSINGMODEL COMPOUNDS TOGETHER WITH OTHER ARGUMENTS EARLY WORK RKSUGGESTEDD THAT:MIGHTBEAPLASTOSEMIQUINONE CATION 01( /-ALLEY ET AL   (OWEVER 03)) COMPLEXES WITH FULLY FUNCTIONAL : AND 1! ONLYCONTAINSUFlCIENTEXTRACTABLEPLASTOQUINONETO

 ACCOUNT FORT F HESINGLE 1! MOLECULEDE6I 6 TRYETAL  ! RESOLUTION TO THE CHEMICAL IDENTITY OF $ F LSERIES AND BYEXTRAPOLATION: CAMEWITH ABEAUTIFU D "ABCOCK INWHI W CH THEY OFF EXPERIMENTSBY"ARRYAND SHOWEDTHAT3IGNAL))S BECAMENARROWERWHEN03 )) INCORPORATED DEUTERATED D TYROSINEBUTNOT DEUTERATED PLASTOQUINONE SO IMPLICATINGTYROSINEASTHEORIGIN OF$"ARRYAND"ABCOCK  !SIMILARANALYSIS D 03)) COMPLEXES CONTAINING DEUTERATED OF ISOLATED TYROSINE LATER CONlRMED D : TO BE ALSO A TYROSINE RESIDUE"OERNERAND"ARRY  2ADIO IODINATION W CH )nISACTIVATEDBY03)) MEDI EXPERIMENTS INWHI ATED D OXIDATIONPRIORTOMODIlCATIONOF PROTEINSIDE CHAINS SUGGESTEDTHAT$AND$CONTAINED:AND$ RESPECTIVELY 94AKAHASHI ETAL )KEUCHI AND )NOUE  &OLDINGMODELSPREDICTED D THEPRESENCE OFTWOSYMMETRICALLYPLACED4YRRESIDUES $ 4YR 4 R INTHEREGIONOF IODINATION )KEUCHI AND $ 4Y AND D )NOUE B94AKAHASHI AND D 3ATOH   -UTATIONOF$ 4YRTO0HEIN 3YNECHOCYSTIS 3  CAUSED LOSSOFT F THE DARK RK STAABLE3IGNAL))ASSOCIATED D WITH T $ $EBUSETAL A6ERMAASETAL  BUTSTILL ALLOWEDASSEMBLYOFAN/ EVOLVING03))COMPLEX -UTATION IN 3YNECHOCY CYSTIS OFT F HESYMMETRICAL TYROSINE IN$TOEITHER0HE$EBUSETAL B -ETZETAL  OR4RP (IS #YSAND-ET.IXON ETAL B ALL PRODUCE D D STRAINSTHATWERE INCAPABL A E OFF PHOTOSYNTHETICWATEROXIDATION!NALYSISOFMA F N GANESE DEPLETEDCORECOMPLEXESISOLATEDFROMTHESE MUTANTS BY %02 AND D OPTICAL SPECTROSCOPY FURT F HER DEMONSTRATED D THATTHE LESIONINELECTRONTRANSFERWAS F ATTHELEVELOFDONOR:-ETZETAL  3OFARNO OTHERRESIDUEAPPEARSTO D BEOXIDIZED D WHENPLACEDD AT THISPOSITION4OGETHERTHESE DATALED D TOTHEATTRIBUTION OF$ 4YRAND$ 4YRASDONORS9: AND 9$ RESPECTIVELY)T Y ISNOWRECOGNIZED D THATWH W ENOXIDIZED D 9: AND D 9$ FORMT F HENEUTRALRADICALSPECIES 9:% AND 9$% ANDNOTTHECATIONICSPECIESSOMETIMESCITEDIN THELITERATUREREVIEWED BY $INER #HAPTER $INNERAND D "RITT   %)JT
BO J E %)JT J !VARIETYOF%02EVIDENCEHASINDICATEDTHAT9:% AND 9$% ARE( BONDED REVIEWED BY Y $INER  %.$/2 4ANGETAL  #AMPBELLETAL  HIGH lELD %02 5N ET AL  AND &4)2 (IENERWADEL ET AL  EXPERIMENTS USINGA$ (ISMUTANT 3 HOCYSTIS  STRONGLY SUPPORT THE PRES OF 3YNEC ENCE OF A ( BOND BETWEEN 9$% AND D $ (IS 4HE SITUATIONREGARDINGTHEROLEOFF $ (ISAPPEARED

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS MORECOMPLICATEDD WITH .PULSED %.$/2NOTFA VORINGADIRECT( BONDBETWEEN9:s ANDD $ (IS #AMPBELL   4HESE EXPERIMENTAL RESULTS ARE CONSISTENT WITH SEVERAL OF F THE COMPUTER GENERATED MODELS WHICHHAVESUGGESTEDTHAT$ (ISMIGHT ( BONDD TOTHEPHENOLICOXYGENOFF 9$% BUTTHAT$ (ISISTOOFARAWAYTO( F BONDD TO9:% REVIEWED BY8IONGETAL   !LL MUTANTSCREATEDD AT$ (IS IN 3Y 3 NECHOCY CYSTIS $INERETAL A.IXONETAL B.IXON AND$INER #HUETAL A$INERAND.IXON # HARD RDTII 2OF F (AY A SETAL  AND #REIN F FEYETA L  AREUNABLETOEVOLVE/ APARTFROM , ,YSAND!RGSUBSTITUTIONSATAPPROXIMATELYOF WILD D TYPERATES #HUETAL A(AY A SETAL   F : ISDRAMATICALLYSLOWEDBYGREATER 4HEOXIDATIONOF9 THAN FOLDINMUTANTSSUCHAS$ (IS'LNAND A S !SP $INERETAL A$INERANDD .IXON (AY ETAL  BUTCANBEPARTIALLYRESTOREDD THROUGHTHE ADDITION OF SMALL ORGANIC BASES SUCH AS IMIDAZOLE (AY A SETAL  3IMILARLY THERATEOFRE RE F D C DU TIONOFF 9:% INMANGANESE DEPLETEDD 03))COMPLEXES THROUGHCHARGERECOMBINATION ISALSOMUCHSLOWER AND LIKEWISECANBEACCELERATEDD THROUGHADDITIONOF SMALL BASES(AYSETAL  4OGETHERTHESEDATA SUPPORTAMODELINWHICH$ (ISACTSASAGENERAL ACIDBASECATALYZING THEPROTONATIONABSTRACTIONOF THEPHENOLICPROTONOF 9: DUR D INGITSPROTON COUPLED REDUCTIONOXIDATION(AYESETAL  5NLESS 9: ISABLETO BEDEPROTONATED D ITSOXIDATION ISBLOCKED 4HEREISSTILL DEBATEASTOWH W ETHER$ (ISFORMS F A DIRECT( BONDD TO 9: ORISLINKEDINDIRECTLYTHROUGH OTHERPROTON CARRIERS3EE #HAPTER $INERANDD "RITT F FORAMORE DETAILEDD ACCOUNT 
.VUBOUT
"GGFDUJOH
UIF
#JOEJOH
BOE
'VODUJPO PG
UIF
.BOHBOFTF
$MVTUFS "ASEDD ON FO F LDINGMODELS FOR$AN F D $ RESIDUES D EXPOSEDD TOTHE LUMEN HAVE BEENSUBJ U ECTEDD TOSITE DIRECTEDMUTAGENESISFORREVIEWS SEE$INER  ANDD $EBUS  4HECHIEFTAR F GETSFORMUTA F GENESIS D BOTH OF WERE CARB R OXYLATES AND HISTIDINE RESIDUES WHICH ARE EXCELLENT CANDIDATES FOR LIGATING METAL IONS3OMEEARLYSUPPORT FORARO F LE FOR( F ISIN LIGATING THECLUSTERCAMEFROMC F HEMICAL MODIlCATIONEXPERI MENTS4AMURAETAL  0ULSED%02EXPERIMENTS HAVESINCEUNAMBIGUOUSLYCONlRMEDD THISPROPOSAL 4ANGETAL  4HEPARTICIPATIONOF HISTIDINEAND CARBOXYLATERESIDUESIN-NBINDINGIN03))HASALSO BEENSUGGESTEDD ONTHEBASISOFNON COMPET F ITIVEINHI

$IBQUFS

%
BOE
%
1SPUFJOT BITIONEXPERIMENTSBETWEENEXOGENOUS-N AND D THE ARTIlCIALELECTRONDONORTO03)) DIPHENYLCARBAZIDE 3EIBERTETAL 0RESTONAND D 3EIBERT   /NEOFF THESURPRISESOFT F HEBRUTE FORCEMUTAGENES F IS APPROACHWASHOWDIFlCULTITWASTOKNOCKOUTTHE F A FUNCT F IONAL -N CLUSTER 0AKRAS K I AND ASSEMBLY OF 6ERMAAS.IXONETAL B )NPARTTHISMAY REmECTTHEABILITYOFALTERNATELIGANDSSUCHASWATER D CHLORIDETOSUB U STITUTE FORAM F ISSINGPROTEINACEOUS AND LIGAND.EVERTHELESS THISOBSERVATIONSTRENGTHENED THESIGNIlCANCEOFRESIDUESATWHICHMUTATIONSPECIl CALLY BLOCKED D -NASSEMBLY Y B
5IF -'
.VUBOU
PG
4DFOFEFTNVT
PCMJRVVT /NEOFT F HElRSTAND D MOSTIMPORTANTMUTANTSAFFECT F INGTHEASSEMBLYOFTHE-NCLUSTERIN03))WASTHE ,&  LOW mUORESCENCE n MUTANT OF 3CENE 3 D DES MUSOBLIQUUS WHI W CH WASGENERATED D THROUGH8 RAY MUTAGENESIS -ETZ AND "ISHOP   /RIGINALLY IDENTIlED INCHLOROPHYLL mUORESCENCE BASED D SCREENS ASLOW mUORESCENT THE,& WASCONSISTENTWITH AN IMPAIRMENTONTHEDONORSIDEOF03))"IOCHEMICAL D THATTHE03))COULD D ACCUMULATEAND STUDIES INDICATED F IVITYACROSSTHEMEMBRANE RETAINELECTRONTRANSFERACT BUTWASUNABLETOASSEMBLEAFUNCTIONAL-NCLUSTER WITHONLYn-NBOUND D TOEACH 03))CENTER-ETZ AND D "ISHOP -ETZETAL  !POLYPEPTIDE OFAPPARENTMOLECULARMASSK$AINTHEWILDTYPE D ASHIFTTOANAPPARENTMO F LECULARMASSOFF  SHOWED K$AIN,& .OOTHERDIFFERENCES F INTHEELECTROPHO RETOGRAMSWEREDETECTED SOTHE K$ASUBUNITWAS D TOBE INVOLVED IN BINDING-N-ETZETAL SUGGESTED  /RIGINALLYTHOUGHTTO BEAMODIlED FORMO F F THE K$AEXTRINSICPROTEININVOLVEDINSTABILIZINGOF THE-NCLUSTER"ISHOP  ORPOSSIBLY $ -ETZ D 3EIBERT  THE K$APOLYPEPTIDE ISNOW AND KNOWNTOBEPRECURSOR$-ETZETAL 4AYLOR ETAL $INERETAL  4HEPHENOTYPEOF ,& RESULTS FROMAS F INGLE BASE DELETIONAND D ACON SEQUENTFRAMESHIFTINTHECODINGREGIONFORR #TTP! THE $PROCESSINGPROTEASE4ROSTETAL  WHI W CH ISNEEDED D TOREMOVETHE# TERMINALEXTENSION4HAT THEINABILITYTOASSEMBLETHE-NCLUSTERWASDUETO A BLOCK IN $ MATURATION RATHER THAN SOME OTHER TARGET FFOR THE PROCESSING PROTEASE WAS CONlRMED THROUGH THE CONSTRUCTION OF THE $ 30 MUTANT OF 3Y 3 NECHOCY CYSTIS WHI W CH WASUNABLETOPROCESS PRECURSOR$ BECAUSEOFAMO F DIlED D CLEAVAGESITE4HE PHENOTYPEOFTHISMUTANTWASESSENTIALLYIDENTICALTO THATOFF ,& .IXONETAL A 

 /VERALLTHE,& MUTANTPROVIDEDTHElRSTMUTAGEN ESISEVIDENCETOINDICATEAROLEFOR$INTHEASSEMBLY F HE-NCLUSTER NOTJUST IN1" BINDING(OWEVER OFT ITSTILLREMAINSUNCLEARTOWH W ATEXTENTTHE # TERMINAL EXTENSIONCAUSESINDIRECTSTRUCTURAL PERTURBATIONSON F THELUMENAL SIDEOF03)) C %"TQ 4HISWASTHElRSTAMINOACIDRESIDUETOBEIDENTI lEDASIMPORTANTFORASSEMBLYOFAFUNCTIONAL-N CLUSTER.IXONANDD $INER  -UTANTSCREATEDD AT THISRESIDUE D INEITHER 3YNEC 3 HOCYSTIS  .IXONAND $INER "OERNERETAL  OR#REINHARDTII # 7HITELEGGEETAL  WERETOVARYING DEGREESABL A E F A RESIDUE D TO ACCUMULATE ACTIVE 03)) 2ETENTION OF THATWASCAPABLEOFLIGATIONTOAMETALALLOWEDTHE ASSEMBLY M OFF ACLUSTERWH W EREASSUB U STITUTIONWITHH ANON LIGANDD SUCH AS!LASHOWEDD NO/ EVOLUTION BUTWAS STILLABLETOASSEMBLEANOTHERWISEFUNCTIONAL03 )) COMPLEXWITHREGARDD TOELECTRONTRANSFER F FROM9 F : TO 1" .IXONANDD $INER  -UTATIONSAT!SP ALSOAFFECTEDTHEABILITYOFDETERGENT SOLUBILIZED03)) COMPLEXES WITH THE-NCLUSTERREMOVED TO D BINDD AND OXIDIZE-N TO-N AT A HIGH AFlNITYSITEWITHIN 03)) $INER AND .IXON  .IXON AND $INER  4HISHIGH AFlNITY SITE IMPLICATED lRSTBY%02 (OGANSONETAL  ISPROBABLY IDENTICALTOTHE BINDINGSITEFORTHElRST-NIONINTHENATURALLIGHT DRIVENPROCESSOF PHOTOACTIVATIONTHATASSEMBLESTHE F FUNCT IONAL-NCLUSTER2ADMERAND# D HENIAE   -ORERECENTEVIDENCETOSUPP U ORTTHEIDEATHAT!SP ACTSASALIGANDD TOTHE-N))) HASCOME FROMPARA F LLEL MODE%02MEASUREMENTS#AMPBELL ETAL   4HE STRUCTURAL MODEL OF 03)) FROM )MPERIAL #OL LEGE,ONDONHASNOWCONlRMEDD AROLEFOR!SP F INLIGATINGONEOFF THE-NIONSOFT F HE INTACTCLUSTER &ERREIRAETAL  3OMEWHATSURPRISINGLYMUTANTS $ !SP6AL ,EUANDD )LEAREABLETOASSEMBLEA F FUNCT IONALCLUSTERINA FRACT F IONOFF THECENTERS DESPITE THE PRESENCE OF A NON LIGAND AT THIS POSITION #HU ET AL A  0RESUMABLY AN ALTERNATIVE LIGAND IS OPERATINGINTHESEMUTANTS D
)JT
(MV
)JT 
"TQ
JO
UIF
$BSS CPYZM5FSNJOBM
3FHJPO
PGG % -UTATIONOFANUM F BEROFRES F IDUES D INTHE# TERMINAL REGIONOFF $SPECIlCALLYCOMPROMISEDD THEABILITYTO ASSEMBLEAFUNCTIONAL-NCLUSTER.IXONETAL A .IXONANDD $INER #HUETAL B )NTHECASE

 OFF SOME BUTNOTALL OFT F HEMUTANTSCREATED D AT $ (IS $ 'LUAND$ !SP/ EVOLUTION IS BLOCKED .IXONETAL B.IXONAND D $INER  #HUETAL B CONSISTENTWITH THEIRROLESAS-N LIGANDS&ERREIRAETAL  4HOSESUBSTITUTIONSTHAT ALLOWASSEMBLY OFA-NC F LUSTERHAVETHEPOTENTIALTO F THEMUTANTSCONSTRUCTED INTHI T S ACTAS LIGANDS-ANYOFT REGIONOF$ SUCHASAT $ (IS ALLOW/ EVOLU TION BUTTHEACTIVITY ISREADILYPHOTOINHIBITED .IXON ETAL B#HUETAL B 4HEMOST DETAILED ANALYSISSOFAROFMUTANTSINTHISREGIONOF$HASCOME F FROMAN%3%%-STU DY OF03))COMP F LEXES ISOLATED F FROMA$ ( IS'LUMUTANTOF 3YNECHOCYSTIS  IN WHI W CHTHE -N CLUSTER ASSEMBLES BUTIS UNABLETO PASS BEYOND D THE 3 STATE $EBUSETAL  3INCE THISRESIDUE D ISNOW KNOWNTO K BEALIGAND D TOTHE-N CLUSTER&ERREIRAETAL  THESERESULTSHIGHLIGHT THEIMPORTANCEOFAPPROPR F IATE LIGATIONFORCONTRO F LLING THEREDOXPROPERTIESOFT F HECLUSTER/F IMP M ORTANCEWAS THElNDINGTHATTHE HIGH AFlNITY-N BINDINGSITE D 03))COMPLEXESIS LARGELY CHARACTERIZED INISOLATED PRESERVED IN# TERMINAL MUTANTSLACKINGA FUNCT F IONAL CLUSTER.IXONETAL A B.IXONAND$INER   4HIS FEATURE F DISTINGUISHESTHE# TERMINAL $MUTANTS F FROMT HOSEAT$ !SP E
5IF
$BSCPYZM5FSNJOBM
3FTJEVF
BU
"MB )N SOME OF THE lRST MUTAGENESIS STUDIES ON $ IN 3YNECHOCY CYSTIS  THEEFFECTO F F DELETING PROGRES SIVELY L LARGERPORTIONSOFF THE# TERMINUSWASEXAMINED .IXON ET AL A  2EMOVAL OF THE # TERMINAL F F F$WAS EXTENSION SOTHATONLY THEMATUREFORMO SYNTHESIZED D DID D NOT BLOCK K ASSEMBLY OF FUNCT F IONAL 03))4HISIMPORTANTlNDING WHICHHOLDSALSOFORTHE GREENALGA #REINHARD R TII ,ERSETAL 3CHRADER AND D *OHANNINGMEIER  SHOWED D THATTHE# TER MINALEXTENSIONWASNOTREQUIREDFORINTEGRATIONOF $INTOTHETHYLAKOID D MEMBRANE APOSSIBILITY THAT HAD BEENSUGGESTED INEARLYSTUDIESON$MATURA TION-ARDERETAL  2EMOVALOFANADDITIONAL D LEAVING $ ,EUASTH T E# TERMINAL RESIDUE D RESIDUE ALLOWED D THEASSEMBLYOFF 03))COMPLEXESCOMPETENT INTHE OXIDATION OF 9: BUTBLOCKEDD THE FO F RMATION OF AN ACTIVE -N CLUSTER $ELETION OF F ADDITIONAL RESI D DUES F HERREDUCE FURT D D THEABILITYTOACCUMULATE03)) CENTERS ALLOFWHICHWEREINACTIVEFOR/ EVOLUTION .IXONETAL A 4HESERESULTSTHEREFORE F HIGH LIGHTED D THEFUNCT F IONAL IMPORTANCEOFF THE# TERMINAL RESIDUEFORTHE-NCLUSTER!NUMBEROFRESIDUES'LY -ET 3ER 6AL 4Y 4 R ,Y , S COULDD SUUBSTITUTE FOR! F LA 

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS WITHOUT BLOCKING TOTALLY THE ASSEMBLY OFF THE -N CLUSTER4HEREFOREITWASSUGGESTEDTHATITWASTHEFREE F FUNCT F IONALITY CARB R OXYL GROUPTHATWAS IMPORTANTFOR .IXONETAL A  F 0UIFS
3FTJEVFT
"GGFDUJOH
UIF
.BOHBOFTF $MVTUFS ! NUMBER OF F $ MUTANTS HAVE BEEN CHARACTERIZED THATRETAINOXYGEN EVOLVINGABILITYBUTSHOWMODI lEDPROPERTIES!MONGSTTHESEAREMUTANTSCREATEDAT $ !SP $ !SPANDD $ 'LU INTHE LUMENAL LOOP CONNECTING HELICES ! ANDD " 4HESE RESIDUES D HADBEENSPECULATEDFORSOMETIMETOBEINVOLVED INBINDING METAL IONS EITHER-NOR#A!ROLEFO F R $ !SPAND$ !SP D IN BINDING#A WASGIVEN IMPETUSWITHTHElNDINGTHATPHOTOAUTOTROPHICGROWTH OFMUTANTSCONSTRUCTE F D ATTHESESITESWASSENSITIVETO T EDEPLETIONOF# TH F A FROMT F THEGROWTH THMEDIUM.IXON AND$INER #HUETAL A -OREDETAILED ANALYSESOFMUTANTSATT F HESEPOSITIONSARECONSISTENT WITH AN IMPORTANTROLEFORT F HESERESIDUES D INSTABILIZ INGINTERMEDIATESFORMEDDURINGTHEASSEMBLYOFTHE -NCLUSTER POSSIBLY THROUGH THEBINDING OFACA F LCIUM ION ALTHOUGH THIS HASYETTOBEPROBED DIRECTLY1IAN ETAL  )NADDITION MUTANTSATTHESERESIDUES SHOWSLOWERPRODUCT D IONOF / INDICATIVEOFAS F LOWER THROUGH THEHIGHER3 STATETRANSITIONS(UNDELT CYCLINGTH ETAL 1IANETAL   "ESIDES$ !SPAND$ !SP T D HEPHOTOAU TOTROPHICGROWTHOFF MUTANTSAT$ 'LUANDD $ !SPINTHE# TERMINALREGIONAREALSOSENSITIVETO THEDEPLETIONOF#A F  .IXONANDD $INER #HU ETAL A ORCHLORIDE .IXONANDD $INER  FROMTHEGROWTHMEDIUM4HESEPHENOTYPESAREALSO N $MUTANTS +EILTYETAL  OBSERVEDD WITHMANY F SUM ANDD MUTANTS LACKINGEXTRINSICPROTEINSOF03)) MARIZEDIN,IAND"URNAP  #A AND CHLORIDE PLAY A AROLE INTHE FUNCT F IONOFT F HE-NCLUSTER SUGGEST ING DIRECTORINDIRECTEFFECTSO F F LLTHESEMUTATIONS FA ONTHEIRNORMALBINDINGWITHIN03))ORINTHECASE F HLORIDE APOSSIBLEROLEASAREPLACEMENT LIGAND OFC TOTHECLUSTER/NE INDIRECTEFFECTO F F FMUTAT INGTHESE AND OTHER RESIDUES SUCH AS$ !RG MIGHTBETO PREVENTCORRECTBINDINGOFF THEEXTRINSICPROTEINSTO THELUMENAL SIDEOF03)) F ,IANDD "URNAP   !NOTHERFEATUREOFMANY$MUTANTS INCLUDING MUTANTSCONSTRUCTEDD ATTHE# TERMINUSOFF $ ASWELL ASTHE $ 'LU'LNMUTANT 6ERMAASETAL  ISTHEENHANCEDSENSITIVITYTOLIGHT INDUCEDINACTIVA TIONOFF / EVOLUTION BYMUTANTCELLS4HEREAREMANY N

$IBQUFS

%
BOE
%
1SPUFJOT POSSIBLECAUSESFORSUC F H APHENOTYPEINCLUDINGTHE DAMAGINGEFFECTSOFRELATIVELYLONG LIVEDHIGHLYOXI DIZING SPECIESWITHIN03)) THEPRODUCT D IONOFF REACTIVE OXYGENSPECIESATTHEMODIlED D -NCLUSTERS #HUET AL B ANDEFFECTSONTHE$REPAIRCYCLE$ALLA #HIESA ET AL  4HUS ENHANCED D SENSITIVITY TO PHOTOINHIBITION ISINITSELFF NOTCOMPELLINGEVIDENCE F FORAROLEFORTHATRESIDUEINBINDING-N&ORMORE DETAILSSEE#HAPTER $EBUS GG
.PEFM
GPS
-PDBUJPO
BOE
"TTFNCM G MZ
PG UIF .BOHBOFTF $MVTUFS
#BTFE
PO
.VUBHFOFTJT %BUB U /NTHEBASISOFT F HEMUTAGENESISDATA $INERETAL A PROPOSED D ASIMPLESTRUCTURAL MODEL FORT F HE LOCATION OF THE -N CLUSTER WITHIN $ &IG   )N D IDENTIlED BY ORDERTO BRING ALL THECRUCIAL RESIDUES MUTAGENESIS INTOACOMPACTCLUSTERTHATCOULD LIGATE THE-NCLUSTER THE# TERMINALREGIONOF$HADTOBE F LDED BACK FO K SOTTHATITWASPRESENTCLOSETO$ !SP 4HE LOCATIONOF!SPON F LYRESIDUESAWAY D F FROM $ 4YR THEOXIDANTOFTHECLUSTER ALSOSUGGESTED THATTHE-NCLUSTERWASLOCATED D CLOSETO9:INDEED ESTIMATES FFROM COMPUTER MODELING STUDIES PLACED

 !SPn¯FROM9: EG 3VENSONETAL  !T THETIMEOFTHEPROPOSALOFTHEMODELIN&IG %02 DATASUGGESTEDD THATTHE-NCLUSTERWASGREATERTHAN D ¯FROM 9: (OGANSONANDD "ABCOCK  AND INOTHERWORK POSSIBLYUPTO¯AWAY5NETAL F  3UCH DISTANCES LEDD TOTHEEXCLUSIONOF!SP ASA LIGAND INEARLYCOMPUTERMODELS3VENSSONET AL  (OWEVERMORERECENT%02ANALYSESHAVE INDICATEDAMUCHSMALLERDISTANCEn ¯ BETWEEN THE-NCLUSTERAND 9: $ORLETETAL 0ELOQUIN ET AL  ,AKSHMI ET AL  4HE PROPOSED LOCATIONOFF THE-NCLUSTERIN&IGALSOAGREESVERY WELLWITHTHESTRUCTURALMODELS DETERMINED BY8 RAY CRYSTALLOGRAPHY+AMIYAAND3HEN "IESIADKA ETAL &ERREIRAETAL   4HEMUTAGENESIS DATAHAVEALSOALLOWEDD AMODEL TOBEPROPOSEDFORTHECOUPLINGOF$PROCESSINGTO THEASSEMBLYOFF THE-NCLUSTER$INERETAL A .IXONETAL A )NTHISMODEL THE lRST-NION IS PROPOSEDTOBINDATAHIGH AFlNITYSITEOFWHICH $ !SPISAN IMPORTANTCOMPONENT)T ISTHIS-N IONTHATISOXIDIZED lRST BY03))IN PHOTOACTIVATION )N ORDER TO COMPLETE THE ASSEMBLY OF THE CLUSTER REMOVAL OFF THE # TERMINALEXTENSIONISREQUIRED!T THE SAME TIME THIS CLEAVAGE REACTION WOULD D REVEAL

&IG 4HEFOLDINGMODELOF$PROPOSEDBY$INERETALA SHOWINGTHELOCATIONOFTHE-NCLUSTER-N WITHIN03))BASEDON & 64HECLUSTER THEANALYSISOFSITE DIRECTEDMUTANTSOF 3YNECHOCYSTIS 4HElVETRANSMEMBRANEHELICESOF$ARELABELEDHEREAS) 6 D TO BECOORDINATED BY$ !SP $ AND BYRESIDUESW D ITHINTHE# TERMINAL REGIONOF$ F INCLUDINGTHE# TERMINUSITSELF WASPROPOSED # !LSOSHOWNARE9Z 9 THEOXIDANTOFF THE-NCLUSTER WHI W CH ISTHOUGHTTO BECLOSETO$ ( ( ASWELL AS0P AND D THE D TO BRINGTHE# TERMINALRESIDUESTOWAR D DS$ !SP$  NON HAEMIRON&E !POSSIBLEAMPHIPHILICHELIX HELIX! WASSUGGESTED D WITH PERMISSION FROM% F LSEVIER3CIENCE 2EPRINTED

 THE CARB R OXY TERMINUS AT $ !LA WHI W CH MIGHT ACT AS A LIGAND TO THE CLUSTER PERHAPS FOLLOWING A F IONAL CHANGE TO BRING THE # SUB U STANTIAL CONFORMAT TERMINUSCLOSETO$ !SP4HE INVOLVEMENTOF THEFREE# TERMINUSINLIGATING-NOR#A EXPLAINS W PROCESSING ISREQUIRED FORASSEM F BLYOFF A NICELYWHY F FUNCT IONAL -NCLUSTER 4HE PRESENCEOFTHE# TERMINALEXTENSIONPROVIDES ASELECTIVEADVANTAGETOCELLSGROWING INMIXED D CUL TURECOMPAREDD TOTHOSEWITHOUT)VLEVAETAL  (OWEVER IT REMAINS UNCLEAR WHAT ADVANTAGE THE EXTENSIONCONVEYS/NEPOSSIBILITY ISTHATREMOVAL OFF THEEXTENSIONISCOUPLED D TOTHESTEPWISEASSEMBLY OFTHEMOSTSTABLEANDACTIVECLUSTER)NITSABSENCE THEREMIGHTBEANINCREASED D CHANCETHATINAPPROPRI ATE LIGANDSAREUSED D SOTHATALTHOUGH ACTIVECLUSTERS MIGHTSTILLASSEMBLE THEREISAGREATERLIKELIHOODOF F FORM INGPOORERFUNCT F IONINGCLUSTERSWITHA GREATER PROPENSITYFORCAUS F ING DAMAGETO03))!LTERNATIVELY THE# TERMINALEXTENSIONMIGHTPLAYAROLEINOPTIMIZ INGTHEASSEMBLY OFF 03)) INTHE03))REPAIRCYCLE 7HILETHEMODEL IN&IGEMPHASIZED D ANEXCLUSIVE ROLEFOR$INBINDINGTHE-NCLUSTER ITWASALWAYS RECOGNIZED D THATOTH T ER03))SUB U UNITSMIGHTPARTICIPATE ASEVENTUALLYREVEALED INTHESTRUCTURAL MODEL FROM F )MPERIAL#OLLEGE&ERREIRAETAL    %)JT BOE %)JT 4HE HISTIDINESTHATCOORDINATETHETWOMEMB M ERSOFT F HE F D INTHE,AND D -SUUBUNITSOFT F HE"2# SPECIAL PAIRFOUN ARECONSERVEDIN03))AT$ (ISAND$ (IS D TOAMODEL INWHI W CHTHESELATTER 4HISNATURALLY LED RESIDUESALSOLIGATEDTHECHLOROPHYLLMOLECULES 0 0!AND 0" RESPECTIVELY &IG INABROADLYSIMILARWAYTO THAT FOUN F D INTHE"2# ALTHOUGHTHEREHAD D TOBESOME STRUCTURAL DISTORTIONTOEXPLAINTHEABSENCEOFF STRONG EXCITONICCOUPLINGBETWEENTHETWO#HLS$URRANTET AL  (OWEVER THEPRESENCEOF0 0! AND 0" IN03)) WASNOT IMMEDIATELYACCEPTED BECAUSEOFT F HESIGNIl CANTREDOXANDSPECTRALDIFFERENCESOBSERVEDBETWEEN 03))AND D THE"2#&ORINSTANCE BECAUSE 0 IS MUCHMOREOXIDIZINGTHANTHEBACTERIAL EQUIVALENT VAN'ORKOMAND3CHELVIS POINTEDOUTTHAT0" ASWELLAS "" MIGHTNOT BERETAINED IN03))TOAVOID D OXIDATIONOF# F HL!LSOMODELSWERESUG UNWANTED GESTEDINWHICH0 0! WASROTATEDFROMPERPENDICULAR TO THE MEMBRANE PLANE TO  DEGREES .OGUCHI ET AL  SO AS TO ACCOMMODATE %02 DATA ON THE ORIENTATIONOFTHECHLOROPHYLLMOLECULECARRYINGTHE 04 STATEVAN-IEGHEMETAL  

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS %ARLY MUTAGENESIS STUDIES INDICATED THAT $ (ISAND$ (ISWEREINDEEDIMPORTANTSINCE REPLACEMENT BY LEUCINE .IXON ET AL B AND TYROSINE 6ERMAAS ET AL  RESPECTIVELY LED TOLOSSOF03))FROMTHEMEMBRANE-ORERECENTLY F MUTATIONS HAVE BEEN MADE AT THESE A NUMBER OF F IONAL 03)) POSITIONSTHATALLOWEDD ASSEMBLYOF FUNCT COMPLEXES4HESEMUTANTSHAVESUBSEQUENTLYPROVED TOBEANINVALUABLERESOURCE INADDRESSING QUESTIONS REGARDINGTHENATUREOF 0$INERETAL  AND THEENERGETICSOFPRIMARYCHARGESEPARATION-ERRY ETAL   )NTHEMOSTDETAILEDD WORRK SO FARONT F HESEMUTANTS $INER ET AL  HAVE APPLIED OPTICAL AND %02 SPECTROSCOPIES TO EXAMINE THE 0 ANDD 04 STATES IN CORE COMPLEXES ISOLATED FFROM MUTANTS OF 3 3YNECHOCYSTIS  CONSTRUCTED ATEITHER $ (IS OR $ (IS -UTATION OFF $ (IS CAUSED BOTH SHIFTS F IN THE 0n0 ABSORB R ANCE DIFFE F R ENCE SPECTRUM AND APPRECIABLE MODULATION OF THE 00REDOXCOUPLE-UTATIONOFF $ (IS HAD LESS DRAMATICEFFECTSONT F HESE PARAMETERS4HE TRIPLET MINUS SINGLET OPTICAL DIFFERENCE SPECTRUM F FOR0WAS INCONTRASTUNCHANGED INALL MUTANTS F SUUPPORT A MODEL IN W WHICH 4HESE RESULTS THEREFORE FOLLOWINGCHARGESEPARATIONTHECHLOROPHYLLCATION USUALLYCALLEDD 0 ISSTABILIZEDPRIMARILYON0 0! LIGATED BY$ (IS ANDD TOA LESSERDEGREE AP PROX  ON 0" LIGATED BY $ (IS  2ESULTS OBTAINEDD WITH THEABOVEMUTANTSANDD OTHERDATA INTHE LITERATUREALSOSUPPORTTHEIDEATHATCHARGESEPARATION ISINITIATEDMAINLYFROMTHESINGLETEXCITEDSTATEOF THE ACCESSORY CHLOROPHYLL "! ANDD THAT THE 0 TRIPLETSTATEISSTABILIZEDD AT LOWTEMPERATUREMAINLY ON "! ANDNOT0 0! .OGUCHIETAL 3ARCINAETAL  !TlRSTSIGHT SUCH ASCHEME FOR03))PR F IMARY PHOTOCHEMISTRYIS INSHARPCONTRASTWITH THAT FORT F HE "2#WHERETHE"#HLSINGLET CATIONANDTRIPLETSTATES ARE LOCATEDD ONTHE SPECIAL PAIROF"# F HLS 0 (OWEVER F HISDOGMASUGGESTSTHATTHERE AMAJ A ORRETHINKINGOFT AREMULTIPLEPATHWAYSFORSTABLECHARGESEPARATIONIN F THE"2#INCLUDING CHARGESEPARATIONFROM" ! NOT JUST 0 VAN "REDERODE ANDD VAN 'RONDELLE  $EKKERANDVAN'RONDELLE  03))DIFFERSFROM THE"2# INTHATTHEEXCITEDD SINGLETSTATESAREMUCH CLOSERINENERGY INTHE FORMERRESU F LTINGINEXTENSIVE DELOCALIZATIONOFTHEEXCITEDSTATEOFTHE2# WITHIN AMULTIMEROFF PIGMENTSRATHERTHAN LOCALIZEDD TOTHE SPECIAL PAIR$URRANTETAL  !TLOWTEMPERATURE + THEEXCITEDSTATEENERGYIN03))ISEXCLUSIVELY LOCALIZEDD ON "! IMPLYINGTHATCHARGESEPARATIONAT

$IBQUFS

%
BOE
%
1SPUFJOT

THISTEMPERATUREMUSTBEOCCURRING FROM" F ! $INER ETAL $INERAND2APPAPORT  

 %)JT BOE %)JT )NITIAL MEASUREMENTSOFF THEPIGMENTSTOICHIOMETRY OF THE$$#YT B COMPLEXISOLATED BY.ANB N AAND3A D TOH SUGGESTEDARATIOOFABOUT#HLPER0HEO MOLECULES&ORTHE"2# THEREARE"#HLMOLECULES D "0HEO(ENCE ITWASANTICIPATED BYANALOGY AND THAT THE PIGMENT STOICHIOMETRY FOR THE $$#YT B COMPLEXWOULD IN FACT F BE#HL0HEO"ARB R ER F LLOWINGSUGGESTIONSTHATTHISWAS B (OWEVER FO SOEG "ARBERETAL !UREDETAL #HANG ETAL 0UEYOETAL  ACONSENSUS HASNOW EMERGED IN FAVORO F F FARAT IOOFF #HLPER0HEO FOR F THEMOSTSTABLEFORMOFTHE03))2#'OUNARISETAL A ASHI ETAL %IJCKELHOFFF AND $EKKER +OBAY  4HUS THE$$#YT B COMPLEXCONTAINS ADDITIONAL#HLSCOMPAREDWITHTHE"2#)MPORTANT CONlRMATIONTHATITWASTHE$$HETERODIMERTHAT BOUND D THESEEXTRA#HLCAMEWITHTHEISOLATIONOFA F $$COMPLEX4ANGETAL   4HE POSSIBLE LIGANDS FFOR THESE TWO @EXTRA #HL MOLECULESFOCUSE F D ONTWOSYMMETRICALLYRELATED D (IS RESIDUES $ (ISAND$ (IS NOTCONSERVED INTHE"2# WHI W CHAREPREDICTED D TOLIEINTHESECOND TRANSMEMBRANE HELIX OF F $ AND D $ RESPECTIVELY -ICHELAND$EISENHOFER  #OMPUTERMODELING STUDIESPLACED D THESE#HLSONTHEEXTERIOROFT F HE$$ HETERODIMERINAPOSITIONTO INTERACTWITHTHENEIGH BORING#0AND#0SUBUNITSANDTOACTPOSSIBLY ASLINKER#HLSCONNECTINGTHEPIGMENTS IN#0AND #0TOTHOSE INVOLVED INPRIMARYPHOTOCHEMISTRY IN$AND$2UFmEETAL  -EASUREMENTSOF F ITHINTHE ISOLATED D 03))2# INDEED ENERGY TRANSFERW D LATIONSOFF PIGMENTS!PERIPHERAL SUGGESTEDTWOPOPU POOL WHICHSHOWSARELATIVELYSLOWRATEOFENERGY T TRANS F FERTO0 3CHELVISETAL  POSSIBLY BOUND TO$ (ISAND D $ (IS AND D AN INNERCOREOF PIGMENTSTHATSHOWSRAPIDEQUILIBRATIONOFEXCITATION ENERGY $URRANTETAL   -UTATION OFF $ (IS TO RESIDUES D THAT COULD POTENTIALLYCOORDINATE#HL#YS -ET !SNAND4HR STILL ALLOWED D PHOTOAUTOTROPHIC GROWTH IN 3YNECHO CYSTIS  WH W EREAS OTHER SUB U STITUTIONS IMPAIRED ACCUMULATIONOF03))TOVARYINGDEGREES0AKRASIAND 6ERMAAS ,INCEAND6ERMAAS 3TEWARTE D T AL  &LUORESCENCESTUDIESONTH T E$ (IS4HR MUTANTSUGGESTEDTHATENERGYTRANSFERINTOTHE03)) 2# WAS SLOWED D CONSISTENT WITH A ROLE IN BINDING

 #HL ,INCEAND6ERMAAS 6AS D ILEVAND"RUCE D  !COMPARISONBY%02 NEAR INFRAREDABSOR BANCEANDD RESONANCE2AMANSPECTROSCOPIESOFF 03)) COMPL M EXESISOLATED FRO F M 3YNECHOCYSTIS I  MUTANTS $ (IS'LNANDITSCOUNTERPART $ (IS'LN 3TEWARTETAL  LEDD TOTHECONCLUSIONTHAT$ (IS ANDD NOT$ (IS WASTHEAXIAL LIGANDD TO #HL: APHOTOOXIDIZABLE#HLMOLECULETHATHADBEEN IDENTIlEDD EARLIERIN03))FROM F HIGHERPLANTSDE0AULA ETAL  4HEPOSSIBLEPHYS H IOLOGICALROLEOF #HL : IN PROTECTIONFORMPHOTOINHIBITIONANDITSMODEOF D ION ARE DESCRIBED IN DETAIL IN OXIDATION ANDD REDUCT #HAPTER &ALLERANDD 2UTHERFOR F D OFF THISVOLUME "ASEDONSYMMETRYARGUMENTS3TEWARTETAL F HERSUGGESTEDD THAT$ (ISALSOACTEDD ASAN FURT AXIAL LIGANDTO# D HL BUTTHATTHIS#HL TERMEDD #HL $ WASREDOX INACTIVE4HE #HL:AND#HL$NOMENCLA TURE ISANEXTENSIONOFT F HATUSEDD TO DESCRIBETHETWO REDOX ACTIVETYROSINESIN03))n9: AND 9$ 4HE 03 )) STRUCTURE DETERMINED TO A RESOLUTION OF  ¯ BY :OUNI AND CO WORKERS CONlRMED THE PRESENCEOFF TWO#HLS TERMEDD #HL :$ AND #HL :$ WITHINTHEPREDICTEDVICINITYOF$ (ISAND$ (IS RESPECTIVELY :OUNIETAL  /NEOFF THE INTERESTING FFEATURES OFF THIS 03)) STRUCTURE WAS THE lNDINGTHAT #YT B WAS LOCATED ONTHE $ SIDE OF THECOMPLEX :OUNI ETAL  4HISMEANTTHAT#YT B SUGGESTED INITIALLYTOBETHE DIRECTREDUCTANTO D F #HL : 4HOMPSONAND"RUDVIG  WASINFACT CLOSERTO#HL$THAN#HL:(ADD #HL:BEENMISAS SIGNEDD SOTHAT$ (ISRATHERTHAN$ (ISWAS THETRUELIGAND4HEPOSSIBILITYTHATTHE#HLBOUND TO $ (IS COULD BE REDOX ACTIVE HAS RECEIVED RECENT EXPERIMENTAL SUPP U ORT FFROM STUDIES OFF $ (IS!SN'LN AND $ (IS'LN MUTANTS OF # # REINHARDTII R 2UFmEETAL 7ANG ETAL   D HL : OBTAINED FROM F 4HE%02SPECTRUMASSIGNEDTO# 03 )) 2#SISOLATEDFROMTHE$ (IS!SNMUTANT WAS BROADENEDCOMPARE D D TOTHEWILD D TYPEANDD THE $ (IS'LNMUTANT WHI W CH BOTH SHOWEDD SIMILAR SPECTRA4HISBROADENINGHASBEENINTERPRETEDINTERMS OFAPOSS F IBLEROTATIONOFF THE#HL 7ANG ETAL   -UTATIONOF BOTH$ (ISANDD $ (ISIN # # R REINHARDTII ALTEREDENERGYTRANSFERINTOTHE03))2# CONSISTENT WITH MODIlCATION TO PERIPHERAL #HLS OF THE2#4HESERESULTSAREINAPPARENTCONTRADICTION TO THE CONCLUSIONS MADE BY 3TEWART ET AL   !POSSIBLERECONCILIATIONOFF THESE DATA HASRECENTLY EMERGED FROMCOMPARAT F IVESTUDIESONTHE IDENTITYOF T THEPHOTOOXIDIZABLEACCESSORY#HLSIN03))FRO M 3YN 3 ECHOCY CYSTIS ANDSP D INACH 4RACEWELLETAL  

 &OR 3YNEC 3 HOCYSTIS  IT APPEARS THAT ONLY #HL : #HL:$ ISPHOTOOXIDIZEDWHEREASINSPINACH AND HENCEPOSSIBLY #REIN # HARD R TII BOTH#HL :AND# D HL $ #HL :$ CAN BEPHOTOOXIDIZED4HISCONTRASTSWITH EARLYSTUDIESWHEREONLYONEOFTHEACCESSORY#HLS WASTHOUGHTTO BEPHOTOOXIDIZABL A E INTHEPLANTSYSTEM DE0AULAETAL  'IVENTHECURRENTUNCERTAINTY ITSEEMSPRUDENTTOADOPTTHENOMENCLATURE#HL :$ AND D #HL :$ TO DESCRIBETHESETWO#HL SPECIESRATHER THAN #HL :AND #HL $ #
.VUBUJPOT
"GGFDUJOH
UIF
"DDFQUPS
4JEF 
5IF 1IFPQIZUJOT )T HAS LONG BEEN ESTABLISHED D THAT 0HEO IS REDUCE D D DURINGPRIMARYCHARGESEPARATIONIN03))+LIMOV ETAL  0IGMENTANALYSISOFT F HE03))2#SUP U PORTED D THE PRESENCEOFF 0HEOPER2#"ASED D ONTHE ANALOGY WITH THE "2# THE TWO 0HEO WOULD BE ON SEPARATE BRANCHESOFF CO FACTORS F INTHE2# WITHTHE 0HEO BOUND D TO$ONTHEACTIVE BRANCH3EQUENCE Q COMPARISONSINDICATEDTHATTHE# KETOGROUPOFEACH 0HEOWOULD BEPOSITIONED D SOTHAT0HEO! COULD FORMA F ( BOND D TORESIDUEO D F F$AN D 0HEO" TORESIDUE D OF$-ICHELAND$EISENHOFER  )NTERESTINGLY THERESIDUEAT$  D ISNOTTOTALLY CONSERVED D WITHIN 03))nFORT F HEMAJ A ORITYOFORGAN F ISMSITISAGLUTAMIC ACID WH W EREAS FOR F 3YNECHOCYSTI 3 IS ITISAGLUTAMINE %ARLYY %.$/2,UBI U TZETAL  RESONANCE2AMAN -OENNE ,OCCOZETAL  AND D &4)2EXPERIMENTS .ABEDRYKETAL  ALLSUGGESTEDTHEPRESENCEOF A( BOND D TOTTHEPHOTOACTIVE0HEO INPLANTS#ONlRMA TIONTHATRESIDUEO D F $PLAYSANIMPORTANTROLE INTHEOPTICALPROPERTIESOFAREDOX ACTIVE0HEOCAME WITHTHEANALYSISOFF 03))2#S ISOLATED FROMMUTANTS F $ 'LN'LU AND D $ 'LN,EU CONSTRUCTED IN 3 3YNECHOCYSTIS  'IORGI ET AL   4HE 1X TRANSITION WAS SHIFTE F D FFROM  NM IN THE WILD TYPETONM INTHE $ 'LN,EUMUTANTANDD TO NMINTHE $ 'LN'LUMUTANT)NPLANTS WHERE RESIDUE$  D ISNATURALLYA'LU THE 1X TRANSITION ISALREADYATNM4HEDEGREEOFT F HERED D SHIFT F IN THEMUTANTSCORRELATESWITHTHEPROBABLESTRENGTHOF THE( BOND D TOTHISRESIDUE AS D IMILARBEHAVIORTOTHAT F FOUN D INTHEEQU Q IVALENTMUTANTSINTHE"2#AT,  "YLINAETAL  3INCEONLYOFTHE0HEOIN THE03))2# ISPHOTOACTIVE.ANBAAND3ATO D H  THESEDATAALSOPROVIDEDCOMPE D LLINGEVIDENCETOSUP U PORTTHEASSUMPTIONTHATITWASTHE$ BOUND0HEO THAT WAS REDOX ACTIVE AND D THAT ELECTRON PROCEEDED

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS PREDOMINANTLY DOWN THE! BRANCH AS IN THE "2# 4HISCONCLUSIONHASBEENRECENTLYREINFORCEDFROM ANALYSISOFF 03))2#S INWHI W CHH THE$ BOUNDD 0HEOWAS REPLACED BYA /( 0HEO DERIVATIVE 'ERMANOETAL  &4)2ANALYSISOFMUTANT 3YNECHOCYSTIS  03))2#S0*.IXONANDD *"RETON UNPUUBLISHED HAVECONlRMEDD THE PRESENCEOFF A( BOND BETWEEN RESIDUEANDTHE# KETOGROUP4HISCONCLUSION ISALSOSUPPORTED BYARECENT HIGH lELDD %02STUDY OF03))2#S F ISOLATED FROM$ ' F LU(IS 'LNAND ,EU MUTANTS OF #REINHARDTI # I $ORLETETAL   /VERALL THESEDATAPROVIDED IMPORTANTEXPERIMENTAL SUPP U ORTTOTHE IDEATHATTHE LOCATIONANDD ORIENTATION OFTHEREDOX ACTIVE0HEOISCONSERVEDBETWEEN03 )) ANDD THE"2# "ESIDESIDENTIFY F INGTHE LOCATIONOFP F IGMENTMOL ECULESWITHIN03))THROUGHTHEEFFECTSOFMUTATION ONTHEOPTICAL REDOXORVIBRATIONAL PROPERTIESOFF THE MOLECULE IT IS POSSIBLETOCREATEMUTANTSINWHI W CHA DIFFERENTTYPEOFPIGMENTMOLECULEISINSERTEDINTOTHE SITE)N"2#S WH W ENAN IMIDAZOLELIGAND ISSUPPLIED INTHECORRECTREGIONOFT F HE"0HEOPOCKET A"#HL ISINSERTEDINSTEAD PRESUMABLYBECAUSETHECENTRAL -G ISSTABILIZED +IRMAIERETAL  "ASEDD ON SEQUENCECOMPARISONS THEANALOGOUSMUTANTS HAVE NOWBEENCONSTRUCTEDIN03 ))4HE$ ,EU(IS MUTANT OF 3YNECHOCY CYSTIS  $! &ORCE ANDD "! $INER UNPUBLI U SHED DATA REPLACESTHEREDOX ACTIVE 0HEOWITHA#HL THE RESULTOFWHICH ISTHATPRIMARY D DD QUANTUM CHARGE SEPARATION OCCURS WITH A REDUCE YIELD-UTANT$ ,EU(ISSTABILIZESINSERTIONOF A#HLFORTHEREDOX INACTIVE0HEO$!&ORCEAND"! $INER UNPUBLI U SHED DATA 4HISLATTERMUTANT ISVERY IMPORTANT ASITHASPROVIDEDD THE lRSTMUTAGENESIS DATA INFAVOROFASTRONGSTRUCTURALSIMILARITYBETWEEN03)) ANDD THE"2#WITH REGARDD TOTHEREDOX INACTIVE0HEO MOLECULE!STRONGSIMILARITY BETWEEN03))ANDD THE "2#INTERMSOFTHEORIENTATION NOTJUSTTHELOCATION OFT F HETWO0HEOMOLECULES ISALSOSUGGESTED FROM F RECENTSPECTROSCOPICEXPERIMENTSONISOLATEDD 03)) 2#SCONTAININGA /( 0HEODERIVATIVE'ERMANO ETAL    
5IF
"DDFTTPSZ SZ
$IMPSPQIZMMT Z #" BOE
## 4HE(ISRESIDUEST D HATACTASAXIAL LIGANDSTOTHEAC CESSORY"#HLSINTHE"2#ARENOTCONSERVEDIN$ ANDD $-ICHEL ANDD $EISENHOFER  F 4HISFEATURE F F ITEDRAMATICDIFFERENCESTO F RAISEDD THEPOSSIBILITYOFQU THE"2#INTHISPARTOF03))4HEIDENTIlCATIONOF F ACCESSORYMOLECULESINTHElRSTSTRUCTURESOF03))

$IBQUFS

%
BOE
%
1SPUFJOT WASVITAL EVIDENCETHATTHISWASNOTTHECASE2H 2 EEET AL  0RESUMABLYTHEREAREALTERNATIVELIGANDS TOTHESE#HL IN03))SUCH ASWATERMOLECULES FO F R WHICHTHEREISPRECEDENCE (OFMANNETA F L  THEPROTEINBACKBONEORPERHAPSADIFFERENTTYPEOF AMINO ACIDD SIDECHAIN)NSPECTIONOFF THE#$ HELICES IN$AND D $ HASIDENTIlED D TWOPOTENTIAL LIGANDS $ !RGAND$ !SN-UTATIONOF$ !RG IN 3YNECHOCY CYSTIS  HAS NOT PROVIDED DElNITIVE EVIDENCECONCERNINGAROLEASA#HL LIGAND D ALTHOUGH THERE WERE CLEAR EFFEC F TS ON 9$ -ANNAETAL   5IF *SPO2VJOPOF $PNQMFY F !VARIETY OFF EARLY BIOPHYSICALEVIDENCESUGGESTEDD A D THE"2#WITHREGARD CLOSEANALOGY BETWEEN03))AND THEACCEPTORSIDEOFTHECOMPLEXREVIEWEDBY$INER ETAL B "OTH CONTAINATIGHTLY BOUND D QUINONE 1! WHI W CHACTSASA ELECTRONCARRIER1!1!n AND Q 1" WHICH IS REDUCED BY 1!n TO A SECOND QUINONE lRST THE SEMIQUINONE ANION 1"n AND D FO F LLOWING A SECOND D TURNOVEROFT F HE2# TOTHEQUINOL&OLLOWING PROTONATIONTHE QUINOL Q 1"( ISRELEASEDFROMTHE 1" BINDINGSITE"OUGES "OCQUET 6ELTH THUYSAND D F &OR FTS !MESZ #HAPTER 0ETROULEASAND#RO THE"2# THEBINDINGSITESFOR1! AND1" ARE LOCATED INTHE-AND D ,SU UBUNITS RESPECTIVELY" Y YANALOGY THE Q IVALENTSITES IN03))WOULD BE IN$AND D $&OR EQU THE"2# THENON HEMEIRONLOCATEDBETWEEN 1! AND 1" ISLIGATED BY (ISRESIDUESP D LUSAGLUTAMATE AT POSITIONINTHE-SUB U UNITOF 2HODO DOPSEUDOMONAS D VIRIDIS $EISENHOFERETAL  )N03))THEREIS D D NOOBVIOUS HOMOLOGUETOTHIS'LURESIDUE)NSTEA &4)2 EXPERIMENTS INDICATE THAT BICARB R ONATE ACTS ASABIDENTATELIGANDTOTHEIRON(IENERWADELAND "ERTHOMIEU  4HE BICARB R ONATE LIGAND D CAN BE DISPLACED D COMPETITIVELY BY A COLLECTION OF LIGANDS W WHICHINCLUDE./ANDCARBOXYLATEANIONS RESULTINGIN F HEREACTION1!n 1" A 1!1"n AND AMODESTSLOWINGOFT D SLOWINGOFF THEREACTION 1!n 1"n A 1!1"( AMARK R ED $INERAND0ETROULEAS 0ETROULEASETAL   D EARLIERBICARB R ONATE DEPLETION 4HESEOBSERVATIONSAND EXPERIMENTS HAVELED D TOTHECONCLUSIONTHATBICARBO R NATEISINVOLVEDINPROTONATIONREACTIONSCOUP U LEDTOTHE F FORMAT IONOF 1"n %ATON 2Y 2 EAND'OV D INDJEE  AND OFF 1"(n VAN2ENSENETAL  REVIEWED IN $INER ET AL B  %XTENSIVE MUTAGENESIS EXPERIMENTS HAVEALSO BEENDIRECTED D AT IDENTIFYING RESIDUEST D HAT MIGHT BE IMPORTANTFOR F BICARB R ONATE BINDING-UTA TION OF$ ,YSAND$ !RG WHICHLIECLOSE F TO$ (ISIMPAIRBICARB R ONATEBINDING $INERET

 AL A 4WOOTHERRESIDUES $ !RGAN D D $ !RG HAVEAWEAKEREFFECTONBICARBONATEBINDING #AOETAL   0HOTOSYSTEM)) DIFFERS F FFROM "2#S IN THAT THE NON HEME IRON IS REDOX ACTIVE &IRST IDENTIlED AS 1 THE IRONSHOWSREVERSIBLEOXIDATIONREDUCT D ION D IONPOTENTIALOFF M6ATP($INER WITH AREDUCT AND0ETROULEAS  4HE(ISRESIDUESTHATLIGATE THEIRON INTHE"2#ARECONSERVED IN$ANDD $AT $ (IS $ (IS $ (ISANDD $ (IS 4HEEFFECTOFMUTATINGTHESERESIDUESIN03))HASNOT BEENFU F LLY EXPLOREDD ALTHOUGH INITIAL REPORTSSUGGEST PERH R APSSURPRISINGLY THATREPLACEMENTOFF $ (IS BY'LN6ERMAASETAL  ANDOF$ (ISBY ,EU .IXONETAL B LEADSTOTHEACCUMULATION OF03))CENTERS F BUTWITHAMUCH LOWERQUANTUMYIELD OF1! REDUCTION #OMPUTERGENERATEDD MODELS FOR03)) F HAVEIDEN TIlEDD A NUMBER OF F $ RESIDUES D THAT COULD LINE THE 1! BINDING POCKET INCLUDING $ !LA 3ER !LA !LAANDD (ISEG 2UFmEETAL   -UTAGENESISSTUDIES HAVENOWCONlRMEDD THEIMPOR TANCEOFTHESERESIDUESFOR03))FUNCTION6ERMAAS D ETAL %RMAKOVA 'ERDESAND6ERMAAS  %RMAKOVA 'ERDESETAL  !NINTERESTINGPROP ERTY OF THESE MUTANTS SUCH AS THE $ 6AL-ET U E MUTANT %RMAKOVA 'ERDES AND !LA4HR DOUBL 6ERMAAS  ANDD THE$ (IS'LNMUTANT 6ER MAASETAL  ISTHEAPPARENTEASEOFLOSSOF1! F FROM ITS BINDINGSITEANDD THEABILITY TORECONSTITUTE Q INONES $ 4RPHASATTRACTEDD AT WITH ARTIlCIAL QU TENTIONBECAUSEITISINANANALOGOUSPOSITIONTORESIDUE - 4RPOF 2PVIRI 2 DIS WHI W CH LIES BETWEEN 1! AND "0HEO! ANDD WHICH ISREQU Q IRED FORRETENT F IONOF 1! #OLEMANAND9OUVAN  )NTHE$ 4RP0HE MUTANT 1!n ISDESTABILIZED CONSISTENTWITHACLOSE LOCATION FORT F HISRESIDUE D 6ERMAAS   !NALYSIS BY %3%%- OF 03)) CENTERS ISOLATED F FROMW ILD D TYPE 3YNECHOCY CYSTIS ANDD MUTANT$ !LA'LY IN W WHICH . HAD BEEN INCORPORATED D HAS PROVIDED OVERWHELMING EXPERIMENTAL SUPPORT FFOR A WEAK ( BOND BETWEEN AN OXYGEN OF 1! AND THEPEPTIDENITROGENOFF $ !LA0ELOQUINETAL  3UCHANINTERACTIONISPREDICTEDFROMSTRUC TURAL MODELS BASEDD ONTHE"2#$INERETAL B  !NOTHER STRONGER( BONDD EXISTS BETWEEN 1!n ANDD AN UNIDENTIlED PEPTIDE NITROGEN (OWEVER EVIDENCE F FORAPRE DICTEDD ( BOND BETWEEN1!n ANDD $ (IS THOUGHTTO LIGATETHENON HEME IRON BASEDD ONSTRUC TURAL HOMOLOGY WITH THE "2#S IS NOT APPARENT IN THE%3%%-SPECTRA0ELOQUINETAL  BUTSEE

 $ELIGIANNAKISETAL  4HEINABILITYTOSEETHIS INTERACTIONDOESNOTNECESSARILYMEANTHATITDOESNOT EXIST AS THE MAGNETIC COUPLING MAY A BE EXTREMELY WEAK K AND MAYDEPEND D ONTHEP(AND D THECOORDINATION OFTHENON HEMEIRON4HUS WHILETHEREISSIGNIlCANT STRUCTURAL CONSERVATION BETWEENTHE 1! BINDINGSITES OFF 03))AND THE"2# THESTRENGTHOFF THE( BONDS TO1!n ISLIKELYTODEPENDONSUBTLEDIFFERENCESINTHE POSITIONOFF THE( BONDING RESIDUESAN D D ONTHE INTERAC TIONOF$ (ISWITHTHENON HEMEIRON 4HE 1" BINDING POCKET WITHIN 03)) IS FORMED F FROMRES IDUESPRESENT D INTRANSMEMBRANE HELICES$ D % OF $ AND THE STROMAL $% LOOP CONNECTING AND THEM &IG  4HE PREDICTED $% LOOP IN $ IS  AMINO ACIDD RESIDUES D LONGER THAN THAT FOUN F D IN THE , SUB U UNIT-UTAGENESISHAS INDICATED D THAT03))CAN TOLERATEDRAMATICCHANGESINTHELENGTHANDSEQUENCE OFF THISLOOPALTHOUGH THEREARE DELETERIOUSEFFECTSON F 1" FUNCT F ION +LESSETAL .IXONETAL   3EQUENCEALIGNMENTSBETWEEN$ANDTHE,SUBUNIT INDICATEONLY OUTOFF RESIDUESTO D BEIDENTICAL INTHE F HETO$ 'LY/FF THECON REGION FROM$ 0 SERVEDRESIDUES RESIDUE$ (ISISTHEEQUIVALEN Q T TO, (ISOF 2PVIRIDIS WHI W CH( BONDSTO 1" AND LIGATESTHENON HEME IRON$ 3ERAPPEARSTOBE HOMOLOGOUSTO, 3EROFTHE"2# SINCEMUTATION OF BOTH GIVESRISETO HERBI R CIDERESISTANCE/ETTMEIER  , 3ERPLAYSAROLEINTHEPROTONATIONOF 1"n TO1"(0ADDOCKETAL  ANDMIGHTBEIN VOLVED INSTABILIZINGTHE BINDING OFF 1"(n INTHE1" SITE,ANCASTERAND D -ICHEL  2EPLACEMENTOF $ 3ERBY'LYREDUCESTHEAFlNITYFOR1" BUTHAS LITTLEEFFECTONT F HEPROTONATIONOFT F HEQUINOL IN03)) 4AOKAAND#RO D F FTS  INDICATINGOTHERPATHWAYS F PROTONATION SUCHASVIA$ (IS WHICHWAS FOR D EARLYONTO HAVEAROLE INTHEPROTONATIONOF SUGGESTED 1"n AND 1"(n (2OBINSONAND!2#RO D F FTS PERSONA L COMMUNICATION  )N SUPPORT OF THIS VIEW A RECENT REPORT ,UPÓNKOVÈ ET AL  SHOWS A DRAMATIC F F MUTATINGTHISSITEONTHERATEOFOX F IDATION EFFECTO OF1!n /THERRESIDUESIDENTIlEDBYMUTAGENESISAS IMPORTANT FFOR 1" BINDING ARE $ 0HE AND D $ ,EU /HADD AND (IRSCHBERG   &OR FURT F HER DETAILSSEE#HAPTER 0ETROULEASAND#ROFTS 03)) ISAKNOWNTAR K GETFORANUM F BEROF DIFFEREN F T CLASSES OF HERBI R CIDES INCLUDING THE TRIAZINES AND UREAS(ERBICIDERESISTANCEISAFFORDEDBYMUTATION D ITHIN$ IN OFF ANNYONEOFF AMINO ACIDD RESIDUESW AREGIONEXTENDINGFROM$ 0 F HETO$ ,EU REVIEWED BY /ETTMEIER   !CCORDING TO THE

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS STRUCTURAL MODELS FFOR $ THESE MUTATIONS CLUSTER WITHIN A DElNED REGION OF $ %RICKSON ET AL F HE HERBI R CIDESIN03))  4HE BINDINGPOCKET FORT OVERLAPSWITH THATFOR1 F " SOTHATMANY BUTNOTALL HERBICIDE RESISTANT MUTANTS ALSO AFFECT THE BINDING D LATEITSREDOXPROPERTIES/HADD AND OF 1" ANDD MODU (IRSCHBERG   7* $PODMVEJOH
3FNBSLT 4HESTUDY OF$AN F D $MUTANTS HASPROVEDD TO BE F LAPPROACH TOPROBETHE LINK AREMARK R ABLYSUCCESSFU BETWEEN03))STRUCTUREANDFUNCTION.OTONLYHASIT PROVIDEDAMP D LEEVIDENCETOSUPPORTTHECLOSEANALO GIESBETWEEN03))ANDD THE"2#WITHRESPECTTOTHE ELECTRONACCEPTORSIDESOFBOTHCOMPLEXES BUTITHAS ALSOIDENTIlEDD SEVERALRESIDUES D IN$ WHI W CH COULD NOT HAVE BEENPREDICTED BYCOMPARISONTO"2#S THAT PLAYKEYROLESINPHOTOSYNTHETICWATER OXIDATION4HE IMPORTANCEOF$ !SP F F FORASSEM BLYOFT F HE-N CLUSTERLEDD TOTHESUGGESTIONTHATTHE-NCLUSTERWAS CLOSETOTHE#$LOOPOF$.IXONAND$INER   -UTAGENESIS EXPERIMENTS ALSO IDENTIlEDD RESIDUES D WITHINTHE# TERMINAL REGIONOF$T F HATWERE IMPOR TANTFORASSEMBLYOFTHE-NCLUSTER4HEEMERGING STRUCTURALMODELSOF03)) F HAVECONlRMEDD THATTHESE F INDEED INVOLVED INLIGATINGTHE-N REGIONSOF$ARE CLUSTER:OUNIETAL  +AMIYAAND3HEN  "IESIADKAETAL &ERREIRAETAL  .OWTHAT A DETAILEDD STRUCTURAL MODEL FOR03)) F ISAVAILABLE WITH SIDE CHAININFORMATION&ERREIRAETAL  THERE WILL BETREMENDOUS INTERESTINTHECONSTRUCTIONAND ANALYSISOFF NEWMUTANTSANDD POSSIBLYTHEREASSESS MENTOFPREVIOUSLYCONSTRUCTEDONES 4OTHISEND IT WOULD BEHIGHLY BENElCIAL TO DEVELOPATHERMOPHILIC CYANOBACTERIUM SUCH AS 4HERMOSYNECHOCOCCUS ELONGATUS AS A SUITABLE MODEL SYSTEM TO COMBINE MUTAGENESIS ANDD SPECTROSCOPIC EXPERIMENTS WITH STRUCTURALSTUDIES "DLOPXMFEHNFOUT 4HEAUTH T ORS GRATEFU F LLYY ACKNOW K LEDGETH T ElNANCIALSUP U PORTOFT F HE2OYAL3OCIETYANDD THE""32#TO0*.AND -3 ANDAGRANTFROMTHE.2)#'053$!PROGRAM TO"!$ GRANTNO n 

$IBQUFS

%
BOE
%
1SPUFJOT 3FGFSFODFT !LIZADEH 3 .IXON 0* 4ELFER! AND "ARBER *  )SOLATION ANDCHARACTERISATIONOFTHEPHOTOSYSTEMTWOREACTIONCENTRE COMPLEX FROMA F DOUBL U EMUTANTOF #HL # AMYDOMONASREIN D HARD R TII 0HOTOSYNTH 2ESn !LIZADEH3 -ORA H IS& "ARB R ER*ANDD .IXON0* )SOTOPICLAB A EL LING OFT F HEPOLYPEPTIDESUB U UNITSOFT F HE ISOLATEDD 0HOTOSYSTEM)) # AMYD M DOMONASREINHARD R TII SUG REACTION CENTRECOMPLEXOF #HL GESTS AN _` HETERODIMERIC STRUCTURE FOR CYTOCHROME B  *0HOTOCHEM0HOTOBIOLn !LT* -ORRIS* 7ESTHOFF0AND(ERRMANN2' .UCLEOTIDE SEQUENCEOFTHECLUSTEREDGENESFORTHEKDCHLOROPHYLL A APOPROTEIN ANDD THE @ KD LIKE PROTEIN OFF 0HOTOSYSTEM)) REACTION CENTER IN THE SPINACH PLASTIDD CHROMOSOME #URRENT 'ENETn !URED D - -OLINER% !LFONSOF 9RUELA) 4OON30 0 3EIBERT-AND F HE0HOTOSYSTEM)) 0ICOREL 2 0IGMENTSTOICHIOMETRY OFT REACTION CENTER FROM HIGHER PLANTS "IOCHIM "IOPHYS !CTA n "ABCOCK'4AND3AUER+ %LECTRONPARAMAGNETICRESONANCE SIGNAL))INSPINACHCHLOROPLASTS)+INETICANALYSISFORUNTREATED CHLOROPLASTS"IOCHIM "IOPHYS!CTAn H  "ARB R ER*A 2ETHINKING THESTRUCTUREOFF THE0HOTOSYSTEMTWO REACTIONCENTRE4RENDS"IOCHEM3CI n "ARB R ER * B 0HOTOSYNTHETIC REACTION CENTRES! COMMON LINK4RENDS"IOCHEM3CI n "ARBER* #HAPMAN$*AND4ELFER! #HARACTERISATIONOFA 03))REACTIONCENTREISOLATEDFROMTHECHLOROPLASTSOF 0ISUM SATIVUM&%"3,ETTn "ARRY"!AND"ABCOCK'4 4YROSINERADICALSAREINVOLVED INTHEPHOTOSYNTHETICOXYGEN EVOLVINGSYSTEM0ROC.ATL!CAD 3CI 53!n "IESIADKA * ,ULL " +ERN * )RRANG + $ ANDD :OUNI !  #RYSTAL STRUCTURE OF CYANOBACTERIAL 0HOTOSYSTEM )) AT  ¯ K HE-N CLUSTER0HYS#HEM#HEM RESOLUTION!CLOSERLOOKATT 0HYSn "ISHOP .)  )DENTIlCATION OF 0HOTOSYSTEM)) THYLAKOID PROTEINSTHROUGHMUTATIONALANALYSISWITH #HLAMYDOMONAS # AND 3 3CENEDESMUS PHENOTYPES)N)NOUE9 #ROFTS!2 'OVINDJEE -URATA. 2ENGER'ANDD 3ATOH + EDS 4HE/XYGENEVOLVING 3YSTEM PPn!CADEMIC0RESS ,ONDON K DEN*4AND3AUER+ D  "LANKENSHIP2% "ABCOCK'4 7AR A /BSERVATIONOFF ANEW%02TRANSIENT INCHLOROPLASTSTHATMAY REmECTTH T EELECTRONDONORTO0HOTOSYSTEM))ATROOMTEMPERATURE &%"3,ETTn "OERNER 2* AND "ARRY "!  )SOTOPIC LABELLING AND %02 SPECTROSCOPYSHOWTHATATYROSINERESIDUEISTHETERMINALELECTRON DONOR : INMANGANESE DEPLETED0HOTOSYSTEM))PREPARATIONS * "IOL #HEMn "OERNER2* .GUYEN!0 "ARRY "!ANDD $EBUS2* %VI DENCE FROM F DIRECTEDD MUTAGENESISTHATASPARTATEOFF THE$ POLYPEPTIDE INmUENCES THE ASSEMBLY ANDOR STABILITY OFF THE MANGANESECLUSTERINTHEPHOTOSYNTHETICWATER SPLITTING COM PLEX"IOCHEMISTRYn "OTTOMLEY7 3PENCER$AND7HITFELD02 0ROTEINSYNTHESIS INISOLATEDSPINACHCHLOROPLASTS#OMPARISONOFLIGHT DRIVENAND ! !40 DRIVENSYNTHESIS!RCH"IOCHEM"IOPHYSn F AND !RNZEN #*  2EDOX PROPERTIES "OWES * #ROFTS!2 ON THE REDUC D ING SIDE OFF 0HOTOSYSTEM)) IN CHLOROPLASTS WITH

 ALTERED HERBI R CIDE BINDINGPROPERTIES"IOCHIM"IOPHYS!CTA n "OUGES "OCQUET "  %LECTRON TRANSFER BETWEEN THE TWO PHOTOSYSTEMSINSPINACHCHLOROPLASTS "IOCHIM"IOPHYS!CTA  n "YLINA %* +IRMAIER # -C$OWELL , (OLTEN $ AND D 9OUVAN F INO ACIDD RESIDUEONT D HEOPTICAL $# )NmUENCEOFANAM PROPERTIESAND D ELECTRONTRANSFER F DYNAMICSOFAP F HOTOSYNTHETIC REACTIONCENTRECOMPLEX.ATUREn #AMM%,AND D 'REEN"2  2ELATIONSHIP BETWEENTHETWO CHLOROPHYLL A PROTEIN COMPLEXES AND THE PHOTOSYSTEM TWO REACTIONCENTER"IOCHIM"IOPHYS!CTAn #AMPBELL+! #ONTINUOUSWAVEANDPUSLSED%02STUDIESOF 0HOTOSYSTEM))AND. CONTAININGCOMPLEXES4HESIS #HEMISTRY F LIFORN F IA $AVIS $EPARTMENT 5NIVERSITY OF#A #AMPBELL+! 0ELOQUIN*- $INER"! 4ANG8 3 #HISHOLM$! "RITT2$  4HETAU NITROGENOFF $HISTIDINE ISTHE HY D GENBOND DONORTOTH DRO T ETYROSINERADICAL9$% OFF 0HOTOSYSTEM)) *!M#HEM3OCn #AMPBELL+! &ORCE$! .IXON0* $OLE& $INER"!AND"RITT 2$ $UAL MODE%02DETECTSTHEINITIALINTERMEDIATEIN PHOTOASSEMBLYOFTHE0HOTOSYSTEM))-NCLUSTER4HEINmU ENCEOFAMINOACIDRESIDUEOFTHE$POLYPEPTIDEON-N COORDINATION * !M #HEM 3OCn #AO * 6ERMAAS7&* AND D 'OVINDJEE  !RGININE RESIDUES D IN THE $ POLYPEPTIDE MAY A STABILIZE BICARB R ONATE BINDING IN 3 NECHOCY C STIS SP 0##  "IOCHIM 0HOTOSYSTEM)) OF 3Y "IOPHYS!CTAn #HANG( # *ANKOWIAK2 2EDDY.23 9OCUM#& 0ICOREL2 3EIBERT-AND3MALL'* /NTHEQUESTIONOFTHECHLORO PHYLL A CONTENTOFTHE0HOTOSYSTEM))REACTIONCENTER*0HYS #HEMn #HUA. (AND D 'ILLHAM.7  4HESITESOFSYNT F HESISOFT F HE PRINCIPAL THYL T AKOID D MEM MBRANEPOLYPEPT Y IDES IN #HL # LAM MYDDOMONAS R TII* #ELL "IOLn REINHARD #HU( ! .GUYEN!0AND D $EBUS2*A !MINOACIDD RESI D DUEST HATINmUENCETHE BINDINGOFMAN F GANESEORCALCIUMTO 0HOTOSYSTEM))4HELUMENALINTERHELICALDOMAINSOFTHE$ POLYPEPTIDE"IOCHEMISTRYn #HU( ! .GUYEN!0AND$EBUS2*B !MINOACIDRESI D DUESTHATINmUENCETHEBINDINGOFMANGANESEORCALCIUMTO R OXY TERMINAL DOMAIN OFF THE $ 0HOTOSYSTEM)) 4HE CARB POLYPEPTIDE"IOCHEMISTRY n #OLEMAN7*AND D 9OUVAN$#  3PECTROSCOPICANALYSISOF GENETICALLY MODIlED D PHOTOSYNTHETIC REACTION CENTERS !NNU 2EV"IOPHYS"IOPHYS#HEMn $ALLA#HIESA- $EÈK: 6ASS) "ARBER*AND.IXON0* 4HE LUMENALLOOPCONNECTINGTRANSMEMBRANEHELICES)AND))OFTHE $POLYPEPTIDEISIMPORTANTFORASSEMBLYOFTHEPHOTOSYSTEM TWOCOMPLEX0HOTOSYNTH2ESn $ANIELIUS26 3ATOH+ VAN+AN0*- 0LIJTER** .UIJS!-AND VAN'ORK R OM(* 4HEPRIMARYREACTIONOFF 0HOTOSYSTEM)) IN THE $ $ CYTOCHROME B  COMPLEX &%"3 ,ETT  n $EBUS2*  !MINOACIDD RESIDUEST D HATMODU D LATETHEPROP ERTIESOFTYROSINE9: ANDTHEMANGANESECLUSTERINTHEWATER OXIDIZINGCOMPLEXOF0HOTOSYSTEM)) "IOCHIM"IOPHYS!CTA n $EBUS2* "ARRY"! "ABCOCK'4AND-C)NTOSH,A 3ITE DIRECTEDMUTAGENES D ISIDENTIlESATYROSINERADICAL INVOLVED IN THEPHOTOSYNTHETICOXYGEN EVOLVINGSYSTEM0ROC.ATL!CAD

 3CI 53!n $EBUS2* "ARRY"! 3ITHOLE) "ABCOCK'4AND-C)NTOSH, B $IRECTED MUTAGENESIS INDICATES THAT THE DONOR TO 0 IN0HOTOSYSTEM))ISTYROSINE OFTHE$POLYPEPTIDE "IOCHEMISTRYn $EBUS2* #AMPBELL+! 'REGOR7 ,I: , "URNAP2,ANDD "RITT 2$  $OESHISTIDINEOFF THE$POLYPEPTIDELIGATETHE MANGANESECLUSTERIN0HOTOSYSTEM))!NELECTRONSPINECHO ENVELOPEMODU D LATIONSTUDY" Y IOCHEMISTRY n F IKI+ (UB U ER2ANDD -ICHEL( $EISENHOFER* %PP/ 3TRUCTUREOFTHEPROTEINSUBUNITSINTHEPHOTOSYNTHETICREACTION CENTREOF 2HODOPSEUDOMONASVIRIDIS AT¯ RESOLUTION.ATURE n $EKKER*0ANDVAN'RONDELLE2 0RIMARYCHARGESEPARATION IN 0HOTOSYSTEM))0HOTOSYNTH2ESn $ELEPELAIRE 0  #HARACTERISATION OFF ADDITIONAL THYLAKOID MEMBRANE POLYPEPTIDES SYNTHESIZED INSIDE THE CHLOROPLAST IN #HLAMYD M DOMONASREINHARD R TII0HOTOBIOCHEM0HOTOBIOPHYS n $ELEPELAIRE0 0ARTIALCHARACTERISATIONOFTHEBIOSYNTHESIS AND INTEGRATION OF THE 0HOTOSYSTEM)) REACTION CENTRE IN THE THYLAKOIDMEMBRANEOF#HLAMYDOMONASREINHARDTII%-"/ *n F D!7 D  3PIN LATTICERELAXATION $ELIGIANNAKIS9ANDD 2UTTHERFOR OFF THEPHEOPHYTIN 0HEOn RADICALOFF 0HOTOSYSTEM))"IOCHEM ISTRYn F DD!7  $ ANDD $ $ELIGIANNAKIS9 (ANLEY *ANDD 2UTHERFOR Y OF THESEMIQUINONERADICAL1!nOF0HOTOSYSTEM)) %3%%-STUDY *!M#HEM3OCn DE0AULA*# )NNES*"AND"RUDVIG'7 %LECTRONTRANSFER IN 0HOTOSYSTEM)) AT CRYOGENIC TEMPERATURES "IOCHEMISTRY  n DE6I 6 TRY # 7OLLMAN & ! ANDD $ELEPELAIRE 0  &UNCTION OFF THEPOLYPEPTIDESOFF THE0HOTOSYSTEM))REACTIONCENTERIN #HLAMYD M DOMONAS REINHARD R TII ,OCALIZATION OF F THE PRIMARY REACTANTS"IOCHIM"IOPHYS!CTAn DE6I 6 TRY # #ARLES#ANDD $INER"! 1UANTITATIONOFF PLAS TOQUINONE IN0HOTOSYSTEM))REACTIONCENTERPARTICLES&%"3 ,ETTn DE 6ITRY # $INER "! AND 0OPOT * ,  0HOTOSYSTEM)) PARTICLES FROM # #HLAMYDOMONAS REINHARDTII * "IOL #HEM n $INER"!  !MINOACIDD RESIDUES D INVOLVED INTHECOORDINA TIONAND D ASSEMBLY OFT F HEMANGANESECLUSTEROFF 0HOTOSYSTEM)) F HEREDOX ACTIVETYROSINES 0ROTON COUPLEDD ELECTRONTRANSPORTOFT AND ITSRELATIONSHIPTOWATEROXIDATION"IOCHIM"IOPHYS!CTA n $INER"!AND.IXON0* 4HERATEOFREDUCTIONOFOXIDIZED REDOX ACTIVETYROSINE : BYEXOGENOUS-N ISSLOWEDINA SITE DIRECTED MUTANT AT ASPARTATE  OF POLYPEPTIDE $ OF F HOTOSYNTHETICOXYGENEVOLUTION 0HOTOSYSTEM)) INACTIVEFORP "IOCHIM "IOPHYS!CTAn $INER"!AND D .IXON0* %VIDENCE FOR$ ( F ISASTHE PROTON ACCEPTOR IMPLICATED IN THE OXIDATION OF F REDOX ACTIVE F B 'ED 0HOTOSYNTHESIS-ECHA TYROSINE9: OF03)))N'ARA NISMSAND%FFECTS 6OL)) PPn+LUWER!CADEMIC 0UBLISHERS $ORDRECHT $INER"!AND0ETROULEAS6 1 THENON HEMEIRONOF THE 0HOTOSYSTEM)) IRON QUINONE COMPLEX ! SPECTROSCOPIC PROBEOFQU F INONEAND INHIBITORBINDINGTOTHEREACTIONCENTER "IOCHIM "IOPHYS!CTAn

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS $INER"!AND D 0ETROULEAS6  &ORMATIONBY./OFF NITROSYL ADDUCTSOFREDOXCOMPONENTSOFTHE0HOTOSYSTEM))REACTION CENTER))%VIDENCETHAT(#/n #/ BINDSTOTHEACCEPTOR SIDE NON HEMEIRON"IOCHIM"IOPHYS!CTAn $INER "! AND 2APPAPORT &  3TRUCTURE DYNAMICS AND ENERGETICSOFF THEPRIMARY PHOTOCHEMISTRY OFF 0HOTOSYSTEM))OF OXYGENICPHOTOSYNTHESIS!NNU2EV0LANT"IOLn $INER "! 2IES $& #OHEN ". AND D -ETZ *'  #//( F LYPEPTIDE$OFF THE0HOTOSYSTEM)) TERMINAL PROCESSING OFPO 3 D DESMUSO BLIQUUS ISNECESSARY FORT F HE REACTIONCENTEROF 3CENE ASSEMBLYOFTHEOXYGEN EVOLVINGCOMPLEX*"IOL#HEM n $INER "! .IXON 0* AND &ARCHAUS *7 A 3ITE DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC REACTION CENTERS #URR /PIN 3TRUCT"IOL n $INER"! 0ETROULEAS6AND D 7ENDOLOSKI ** B 4HE IRON Q INONEELECTRON ACCEPTORCOMPLEXOFF 0HOTOSYSTEM))0HYSIOL QU 0LANTn $INER"! 3CHLODDER% .IXON0* #OLEMAN7* 2APPAPORT& ,AVERGNE* 6ERMAAS7&*AND#HISHOLM$! 3ITE DI RECTEDMUTATIONSAT$ (ISAND$ (ISOF0HOTOSYS TEM)) IN 3YNECHOCYSTIS 0##  3ITES OF PRIMARY CHARGE SEPARATIONANDCATIONANDTRIPLETSTABILISATION"IOCHEMISTRY  n $ORLET 0 6ALENTIN -$ "ABCOCK K '4 -C#RACKEN *,  )NTERACTION OF 9:% WITH ITS ENVI N RONMENT IN ACETATE TREATED D REACTIONCENTERCORES*0HYS 0HOTOSYSTEM))MEMBRANESAND #HEM"n $ORLET 0 2UTHERFORD!7 AND 5N 3  /RIENTATION OF THE TYROSYL$ PHEOPHYTINANION ANDSEMIQUINONE1!s nRADICALSIN 0HOTOSYSTEM))DETERMINEDBYHIGH lELDELECTRONPARAMAGNETIC RESONANCE"IOCHEMISTRYn $ORLET0 8IONG, 3AYRE24AND D 5N3  (IGH lELDD %02 STUDYOFF THEPHEOPHYTINANIONRADICAL INWILD D TYPEAND D $ % # AMYD M DOMONAS REINHARD R TII MUTANTS OFF 0HOTOSYSTEM)) IN #HL *"IOL #HEMn $URRANT*2 (ASTINGS' *OSEPH $- "ARB R ER* 0ORTER'AND D +LUG $2 3UBPICOSECONDEQUILIBRATIONOFEXCITATIONENERGY INISOLATED0HOTOSYSTEM))REACTIONCENTERS0ROC.ATL!CAD3CI 53!n $URRANT*2 +LUG$2 +WA3,3 VAN'RONDELLE2 0ORTER'AND $EKKER *0  ! MULTIMERMODEL FFOR 0 THE PRIMARY ELECTRON DONOR OFF 0HOTOSYSTEM)) 0ROC .ATL !CAD D 3CI 53!  n %AGLESHAM!2*AND D %LLIS2*  0ROTEINSYNTHESIS INCHLORO PLASTS)),IGHT DRIVENSYNTHESISOFMEM F BRANEPROTEINSBY ISO LATEDPEACHLOROPLASTS"IOCHIM"IOPHYS!CTAn %ATON 2YE ** AND 'OVINDJEE  %LECTRON TRANSFER THROUGH THEQUINONEACCEPTORCOMPLEXOF0HOTOSYSTEM))AFTERONEOR TWOACTINICmASHESINBICARBONATE DEPLETEDSPINACHTHYLAKOID H MEMBRANES"IOCHIM "IOPHYS!CTAn %IJCKELHOFF#AN F D $EKKER*0  $ETERMINATIONOFT F HEPIG MENT STOICHIOMETRY OF F THE PHOTOCHEMICAL REACTION CENTER OF 0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn %RICKSON*- 2AHIRE- -ALNOE0 0 'IRARD "ASCOU* 0IERRE9 9 "ENNOUN0AND2OCHAIX* $ ,ACKOFTHE$PROTEININA # #HLAMYDOMONASREINHARDTII PSB$ MUTANTAFFECTS03))STABILITY AND$EXPRESSION%-"/*n %RICKSON *- 0lSTER + 2AHIRE - 4OGASAKI 2+ -ETS , AND H ICAL ANALYSIS OF 2OCHAIX * $  -OLECULAR AND BIOPHYS HERBICIDE R

RESISTANT MUTANTS OF #HL # AMYD M DOMONAS REINHARD R TII

$IBQUFS

%
BOE
%
1SPUFJOT STRUCTURE FUNCT F ION RELATIONSHIPS OF F THE 0HOTOSYSTEM)) $ POLYPEPTIDE0LANT#ELLn %RMAKOVA 'ERDES3AND6ERMAAS7 -OBILITYOFTHEPRI MARYELECTRON ACCEPTINGPLASTOQUINONE1! OF0HOTOSYSTEM)) IN A 3YNECHOCYSTIS 3 SP0##CARRYINGMUTATIONSINTHE$ PROTEIN"IOCHEMISTRYn D  4ARG R ETEDD RANDOM %RMAKOVA 'ERDES3 9U:AND6ERMAAS7 F IONALLY IMPORTANTRESIDUES D INTHE MUTAGENESISTOIDENTIFY FUNCT 3 NECHOCY CYSTIS SPSTRAIN0## $PROTEINOFF 0HOTOSYSTEM)) IN 3Y *"ACTERIOL n &ERREIRA+. )VERSON4- -AGHLAOUI+ "ARBER*AND)WATA3  !RCHITECTURE OF THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn 'ERMANO- 3HKUROPATOV!9 0ERMENTIER( DE7IJN2 (OFF!* 3HUVALOV6!ANDD VAN'ORRKOM(*  0IGMENTORGANISATION AND D THEIR INTERACTIONS IN REACTION CENTERS OFF 0HOTOSYSTEM)) F IONCENTERSWITHMODIlED /PTICALSPECTROSCOPYAT+OFREACT PHEOPHYTINCOMPOSITION"IOCHEMISTRY n 'ERMANO- 0ASCAL! 3HKUROPATOV!9 2OBERT" (OFF!*AND VAN 'ORKOM (*  0HEOPHYTIN PROTEIN INTERACTIONS IN 0HOTOSYSTEM))STUDIEDBYRESONANCE2AMANSPECTROSCOPYOF MODIlEDREACTIONCENTERS"IOCHEMISTRYn 'IORGI," .IXON0* -ERRY3!0 *OSEPH$- $URRANT*2 $E,AS R ER* 0ORTER'ANDD +LUG $2  #OMPARISON 2IVAS* "ARB OFF PRIMARY CHARGESEPARATION INTHE0HOTOSYSTEM))REACTION CENTRECOMPLEXISOLATED FROMW F ILDD TYPEANDD $ MUTANTS OFF THECYANOBACTERIUM 3Y 3 NECHOCY CYSTIS 0##*"IOL #HEM  n 'OUNARIS+ #HAPMAN$* "OOTH0 #RYSTALL" 'IORGI," +LUG $2 0ORTER'AND"ARBER* #OMPARISONOFTHE$$ CYTOCHROMEBREACTIONCENTRECOMPLEXOFPHOTOSYSTEMTWO ISOLATEDBYTWODIFFERENTMETHODS&%"3,ETTn 'REBANIER!% #OEN$- 2ICH!ANDD "OGORAD ,  -EM BRANEPROTEINSSYNTHESISED BUTNOTPROCESSED BY ISOLATEDD MAIZE CHLOROPLASTS*#ELL "IOL n (AY A S! -! 6ASSILIEV )2 'OLBECK K *( AND $EBUS 2*  2OLE OFF $ (IS IN THE PROTON COUPLEDD ELECTRON TRANSFER F REACTIONS IN 0HOTOSYSTEM)) ! CHEMICAL COMPLEMENTATION STUDY"IOCHEMISTRYn (AYS! -! 6ASSILIEV )2 'OLBECK *( AND $EBUS 2*  2OLEOF$ (ISINTHEPROTON COUPLEDOXIDATIONOFTYROSINE 9: INMANGANESE DEPLETEDD 0HOTOSYSTEM))"IOCHEMISTRY n (EARST*%AND D 3AUER+ 0ROTEINSEQUENCE HOMOLOGIESBE TWEEN PORTIONSOFT F HE,ANDD -SUUBUNITSOFREACT F IONCENTERSOF 2HODOPSEU D D DOMONASCAPSU L ANDD THE1" PROTEINOFC LATA F HLOROPLAST THYLAKOIDMEMBRANES!PROPOSEDRELATIONTOQUINONE BINDING SITES:.ATURFORSCHCn (IENERWADEL2AND"ERTHOMIEU# "ICARBONATE BINDINGTO THENON HAEMIRONOF0HOTOSYSTEM))INVESTIGATEDBY&OURIER F INFRARE F D DIFFERENCE F SPECTROSCOPY AND # LABELED TRANSFORM BICARB R ONATE"IOCHEMISTRY n (IENERWADEL 2 "OUSSAC! "RETON* $INER"!ANDD "ERTHOMIEU # &OURIERTRANSFORM F INFRARE F D DIFFERENCESPECTROSCOP F Y OF 0HOTOSYSTEM))TYROSINE$USINGSITE DIRECTEDD MUTAGENESISAND SPECIlCISOTOPELABELING"IOCHEMISTRYn (IRSCHBERG*AND-C)NTOSH, -OLECULARBASISOFHERBICIDE RESISTANCEIN !MARANTHUSHYBRIDUS3CIENCEn (OFMANN% 7RENCH0- 3HARPLES&0 (ILLER2' 7ELTE7AND $IEDERICHS +  3TRUCTURAL BASIS OF LIGHT HARVESTING BY CAROTENOIDS0ERIDININ CHLOROPHYLL PROTEIN FROM F !MPH M IDINIUM

 CARTERAE3CIENCEn (OGANSON#7AND"ABCOCK'4 %LECTRON TRANSFEREVENTS NEARTHEREACTIONCENTERIN/ EVOLVING0HOTOSYSTEM))PREPARA TIONS"IOCHEMISTRYn (OGANSON #7 'HANOTAKIS $& "ABCOCK '4 AND9OCUM #&  -N REDUCES9Z INMANGANESE DEPLETED D 0HOTOSYSTEM)) PREPARATIONS0HOTOSYNTH 2ESn (OLSCHUH+ "OTTOMLEY7AND D 7HITFE F LDD 02 3TRUCTUREOF F HE$AND D  KD D REACTION THESPINACH CHLOROPLAST GENES FORT CENTRE PROTEINS OFF 0HOTOSYSTEM)) AND FFOR T2.! 3ERR 5'!  .UCL!CIDS2ESn (UNDELT- (AYS! -! $EBUS2*AND*UNGE7 /XYGENIC 3 S SP 0HOTOSYSTEM))4HEMUTATION$ $.IN 3YNECHOCYSTI 0##RETARDS3 STATETRANSITIONSWITHOUTAFFECTINGELECTRON F OM 9:TO 0"IOCHEMISTRYn TRANSFER FR D )NOUE9 3PECIlC ) LABELING OFF $ HER )KEUCHI-AND BICIDE BINDING PROTEIN !N INDICATION THAT $ FUNCT F IONS ON BOTHTHE DONORANDACCEPTORS D IDESOFF 0HOTOSYSTEM))&%"3 ,ETTn )KEUCHI-AND)NOUE9A !NEW0HOTOSYSTEM))REACTION CENTERCOMPONENTK$APROTEIN ENCODEDBYCHLOROPLAST GENOME&%"3,ETTn )KEUCHI-AND)NOUE9B 0ARTIALCHARACTERIZATIONOFTHE IODINATION SITE IN $ PROTEIN OFF MANGANESE RETAINING AND D 0HOTOSYSTEM)) MEMBRANES 0LANT #ELL MANGANESE DEPLETED 0HYSIOLn D 0AKRAS K I("  4HECARB R OXYL )VLEVA." 3HESTAKOV36AND TERMINAL EXTENSIONOFF THEPRECURSOR$PROTEINOFF 0HOTOSYSTEM)) IS REQUIRED FOR OPTIMAL PHOTOSYNTHETIC PERFORMANCE OF THE CYANOBACTERIUM 3YNECHOCYSTIS 3 SP 0##  0LANT 0HYSIOL n +AMIYA.AND3HEN* 2 #RYSTALSTRUCTUREOFOXYGEN EVOLV ING 0HOTOSYSTEM)) FROM F 4 ERMOSYNECHOCOCCUS VULCANUS AT 4H  ¯RESOLUTION0ROC.ATL!CAD3CI53!n +ARABI A N'$ &ARLEY-AN Y D (ALLICK K 2"  #HLOROPLASTGENEFOR F -RPOLYPEPTIDEOFF 0HOTOSYSTEM))IN %UGL U ENAGRACI G LIS IS INTERRUPTED BY FOUR F INTRONSWITH TH CONSERVED BOUNDARY Y SEQUENCES .UCL!CIDS2ESn +EILTY!4 6AVILIN$6AND6ERMAAS7&* &UNCTIONALANALY + SISOFCOMBINATORIALMUTANTSWITHCHANGESINTHE# TERMINUSOF THE#$LOOPOFTHE$PROTEININ0HOTOSYSTEM))OF3YNECHOCYSTIS SP0##"IOCHEMISTRYn +IRMAIER # 'AUL $ $E"EY 2 (OLTEN $ AND D 3CHENCK K ##  #HARGE SEPARATION IN A REACTION CENTER INCORPORATING BACTERIOCHLOROPHYLL FORP F HOTOACTIVEBACTERIOPHEOPHYTIN3CI ENCE n +LESS( /REN 3HAMIR- -ALKIN3 -C)NTOSH,AND%DELMAN -  4HE $ % REGION OF THE $ PROTEIN IS INVOLVED IN MULTIPLEQUINONEANDHERBICIDEINTERACTIONSIN0HOTOSYSTEM)) "IOCHEMISTRYn +LIMOV 66 +LEVANIKK !6 3HUVALOV 6! AND D +RASNOVSK KY !!  2EDUCT D IONOFP F HEOPHYTIN INTHEPRIMARY LIGHTREACTION OFF 0HOTOSYSTEM))&%"3,ETTn + BAY +O A ASHI- -AEDA( 7ATANABE4 .AKANE(AND3ATO D H+  #HLOROPHYLL A AND ` CAROTENECONTENT INTHE $$CY TOCHROME B REACTIONCENTERCOMPLEXFROMSPINACH&%"3 ,ETTn + +OIVUNIEMI! !RO%-AND!NDERSSON" $EGRADATION OF $ AND $ PROTEINS OF 0HOTOSYSTEM)) IN HIGHER PLANTS ISREGULATED BYREVERSIBLEPHOSPHORYLATION"IOCHEMISTRY n

 +OULOUGLIOTIS $ )NNES *" AND "RUDVIG '7  ,OCA TION OF CHLOROPHYLL: IN 0HOTOSYSTEM)) "IOCHEMISTRY  n +OULOUGLIOTIS$ 4ANG8 3 $INER"!AND"RUDVIG'7 3PECTROSCOPICEVIDENCEFORTHESYMMETRICLOCATIONOFTYROSINES D : IN 0HOTOSYSTEM))"IOCHEMISTRY n $AND + E$*  4HE$ALTON1" PROTEINOFF 0HOTOSYSTEM)) +YL 0HOTOCHEM0HOTOBIOL n ,AKSHMI +6 %ATON33 %ATON'2ANDD "RUDVIG '7 /RI ENTATIONOFF THETETRANUCLEARMANGANESECLUSTERANDD TYROSINE: INTHE/ EVOLVINGCOMPLEXOF0HOTOSYSTEM))!N%02STUDY OFTHE 39:% STATEINORIENTEDACETATE INHIBITED0HOTOSYSTEM)) MEMBRANES"IOCHEMISTRYn ,ANCASTER #2$ AND -ICHEL (  4HE COUPLING OF LIGHT INDUCE D D ELECTRONTRANSFERAN F D PROTONUP U TAKEAS DERIVED FROM F CRYSTAL STRUCTURESOFREACT F IONCENTRESFROM F 2HODO DOPSEUDOMONAS D D ATTHEBINDING SITEOFF THESECONDARY QUINONE VIRIDIS MODIlED 1" 3TRUCTUREn ,ERS! (EIFETZ0" "O F OYNTON*% 'ILLHAM.7AND/SMON D D #"  4HE CARBOXYL TERMINAL EXTENSION OF THE $ PROTEIN OF0HOTOSYSTEM))ISNOTREQUIREDFOROPTIMALPHOTOSYNTHETIC PERFORMANCEUNDER#/ ANDLIGHT SATURATEDGROWTHCONDITIONS *"IOL#HEMn ,I : ,AND"URNAP2, D  -UTATIONSOFF ARGININEWITHIN R ELICAL A B LOOP OF THE PUTATIVE #A BINDING LUMENAL INTERH 0HOTOSYSTEM))$PROTEIN DISRUPT BINDING OFF THEMANGANESE STABILIZING PROTEIN ANDD CYTOCHROME C IN 3Y 3 NECHOCY CYSTIS SP 0##"IOCHEMISTRY n ,INCE-4AND6ERMAAS7 !SSOCIATIONOF(ISINTHE $PROTEINOF0HOTOSYSTEM))WITHACHLOROPHYLLTHATAFFECTS EXCITATION ENERGYTRANSFEREFlCIENCYTOTHEREACTIONCENTER%UR *"IOCHEMn R ƾ :* 3CHRÚDER70 0AKRAS K I (" )RRGANG+ $ (ERRMANN ,ORKOVI D /ELMàLLER 2  -OLECULAR CHARACTERIZATION OF 2' AND D COMPONENT OFF THE 0HOTOSYSTEM)) 0SB7 A NUCLEAR ENCODED REACTIONCENTERCOMPLEXINSPINACH0ROC.ATL!CADD 3CI53! n ,UBITZ7 7 )SAACSON2! /KAMURA-9 9 !BRESCH%# 0LATO-AND &EHER' %.$/2STUDIESOFTHEINTERMEDIATEELECTRON ACCEPTORRADICALANION )%nnIN0HOTOSYSTEM))REACTIONCENTERS "IOCHIM"IOPHYS!CTAn ,UPÓNKOVÈ, -ETZ*' $INER"! 6ASS)ANDD +OMENDA*  (ISTIDINE RESIDUE D  OF THE 0HOTOSYSTEM)) POLYPEPTIDE IS INVOLVED IN A LIGHT INDUCE D D CROSS LINKING OFF THE POLYPEPTIDE U UNITOFC F YTOCHROME B STUDYOFF ASITE DI WITHTHE _ SUB RECTED D MUTANTOF 3YNECHOCY CYSTIS 0##"IOCHIM"IOPHYS !CTAn -ANNA0 ,O"RUTTO2 %IJCKELHOFF# $EKKER*0AND6ERMAAS7  2OLEOF!RGOFTHE$PROTEININ0HOTOSYSTEM)) STRUCTUREANDFUNCTION%UR* "IOCHEMn -ARDER *" 'OLOUBI U NOFFF 0 AND %DELMAN -  -OLECULAR ARCHITECTUREOFT F HERAPIDLYMETABOLISEDD  KILODALTONPROTEIN OFF 0HOTOSYSTEM)))NDICATIONS FOR#//( TERM F INALPROCESSING OFF A CHLOROPLAST MEMBRANE POLYPEPTIDE * "IOL #HEM  n -ARDER*" #HAPMAN$* 4ELFER! .IXON0*AND"ARBER* )DENTIlCATIONOF PSB! ANDPSB$ GENEPRODUCTS $AND$ AS REACTIONCENTREPROTEINSOFPHOTOSYSTEMTWO0LANT-OL"IOL  n -ATOO!+ AND D %DELMAN -  )NTRAMEMBRANE TRANSLOCA TION AND D POSTTRANSLATIONAL PALMITOYL O ATION OF F THE CHLOROPLAST

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS  K$AHERBICIDE BINDING PROTEIN0ROC.ATL!CAD3CI53! n -ERRY3!0 .IXON0* "ARTER,-# 3CHILSTRA- 0ORTER' "ARBER * $URRANT*2AND+LUG$2 -ODULATIONOFQUANTUMYIELD OFPRIMARYRADICALFORMATIONIN0HOTOSYSTEM))BYSITE DIRECTED F ING RADICALCATIONSAND D ANIONS"IOCHEMISTRY MUTAGENESISAFFECT  n -ETZ*'AND D "ISHOP.) )DENTIlCATIONOFAC F HLOROPLAST MEMBRANEPOLYPEPTIDEASSOCIATED D WITHTHEOXIDISINGSIDEOF 0HOTOSYSTEM)) BYTHEUSEOFF SELECT LOW mUORESCENTMUTANTSOF 3CENEDESMUS"IOCHEM"IOPHYS2ES#OMMUNn -ETZ*'AND3EIBERT- 0RESENCEIN0HOTOSYSTEM))CORE COMPLEXESOFA KILODALTONPOLYPEPTIDEREQUIREDFORWATER PHOTOLYSIS0LANT0HYSIOLn -ETZ*' 7ONG*AND D "ISHOP.) #HANGESINELECTROPHORETIC TY OF ACHLOROPLASTMEMB M RANEPOLYPEPT Y IDEASSOCIATED D WITH T MOBILITY T E LOSSOFF THEOXIDISING SIDEOFF 0HOTOSYSTEM))INLOWmUORESCENT TH MUTANTSOF 3CENE 3 D DESMUS &%"3,ETTn -ETZ*' 0AKRAS K I(" 3EIBERT-AND D !RNTZEN#*  %VIDENCE FOR A DUAL FUNCTION OF THE HERBICIDE BINDING $ PROTEIN IN 0HOTOSYSTEM))&%"3,ETTn -ETZ*' .IXON0* 2ÚGNER- "RUDVIG'7AND$INER"! $IRECTEDALTERATIONOFTHE$POLYPEPTIDEOF0HOTOSYSTEM)) %VIDENCETHATTYROSINE  ISTHEREDOXCOMPONENT : CON NECTING THEOXYGEN EVOLVING COMPLEXTOTHEPRIMARY ELECTRON DONOR 0"IOCHEMISTRYn -ICHEL (AND D $EISENHOFER* F  8 RAY A DIFFRACT F IONSTUDIESON ACRYSTALLINE BACTERIAL PHOTOSYNTHETICREACTIONCENTER!PROG RESSREPORTANDCONCLUSIONSONTHESTRUCTUREOF0HOTOSYSTEM)) REACTIONCENTERS)N3TAEHELIN,!AND!RNTZEN#*EDS 0HOTO SYNTHESIS)))0HOTOSYNTHETIC-EMBRANESAND,IGHT(ARVESTING 3YSTEMS PPn3PRINGER 6ERLAG "ERLIN -ICHEL (AND D $EISENHOFER* F  2ELEVANCEOFF THEPHOTOSYN THETICREACTIONCENTERFROM F PURPLE BACTERIATOTHESTRUCTUREOF 0HOTOSYSTEM))"IOCHEMISTRY n -ICHEL ( (UNT$& 3HABANOWITZ*AND D "ENNETT*  4ANDEM MASSSPECTROMETRY REVEALSTHATTHREE0HOTOSYSTEM))PROTEINS OFSPINACHCHLOROPLASTSCONTAIN. ACETYL / PHOSPHOTHREONINE ATTHEIR.( TERMINI*"IOL#HEMn -OENNE ,OCCOZ0 2OBERT"AND,UTZ- 3TRUCTUREOFTHE PRIMARYREACTANTSIN0HOTOSYSTEM))2ESONANCE2AMANSTUDIES OFF $$PARTICLES)N"ALTSCHEFFS F KY- K ED #URRENT2ESEARCH IN 0HOTOSYNTHESIS 6OL) PPn+LUWER!CADEMIC0UB U LISHERS $ORDRECHT .ABEDRYK K % !NDRIANAMBININTSOA 3 "ERGER ' ,EONHARD D - -ÊNTELE7AND D "RETON*  #HARACTERISATIONOF BONDING INTERACTIONSOFTHEINTERMEDIARYELECTRONACCEPTORINTHEREAC TIONCENTEROF0HOTOSYSTEM))BY&4)2SPECTROSCOPY"IOCHIM "IOPHYS!CTAn .AKATANI (9 +E " $OLAN % AND!RNTZEN #*  )DENTITY OFF THE 0HOTOSYSTEM)) REACTION CENTRE POLYPEPTIDE "IOCHIM "IOPHYS!CTAn D H + )SOLATIONOFF A0HOTOSYSTEM))REACTION .ANB N A/AND3ATO CENTERCONSISTINGOFF $ AND$ PO D LYPEPTIDESAND D CYTOCHROME B 0ROC.ATL!CAD3C D I53! n .IXON0* !NINVESTIGATIONOFTHE PSB! B ANDPSB$ GENE PRODUCTSOFHIGHERPLANTS0H$4HESIS 5NIVERSITY,ONDON .IXON0*AND$INER"! 0ROTEINCOORDINATIONOFTHEPHO TOSYNTHETICOXYGEN EVOLVINGCOMPLEX AQUATERNARYELECTRON COUNTER)N0EDERSEN0#AND/NARE D L " EDS 0ROCEEDINGSOF F H)NTERNATIONAL#ONFERENCEO F F THE)%%%%NGINEER THE4WELFT

$IBQUFS

%
BOE
%
1SPUFJOT ING IN-EDICINEANDD "IOLOGY 3OCIETY 6O Y L PPn )%%% 0ISCATAWAY A .IXON0*AND$INER"! !SPARTATEOFTHE0HOTOSYSTEM)) REACTIONCENTERPOLYPEPTIDE$ISINVOLVEDINTHEASSEMBLYOF THE OXYGEN EVOLVING MANGANESE CLUSTER "IOCHEMISTRY  n .IXON0* $YER4! "ARB R ER*AND(UNTER#. D  )MMUNOLOGICAL EVIDENCEFORT F HEPRESENCEOFF THE$ANDD $ PROTEINS IN03)) CORESOF HIGHERPLANTS&%"3,ETTn .IXON0* 2ÚGNER-ANDD $INER"! %XPRESSIONOFF AHIGHER PLANT PSB! GENE IN 3YNECHOCYSTIS 3  YIELDS A FUNCTIONAL HYBRID 0HOTOSYSTEM)) REACTION CENTER COMPLEX 0LANT #ELL n .IXON0* 4ROST*4AND$INER"!A 2OLEOFTHECARBOXY F FUNCT F IONAL TERMINUSOFF POLYPEPTIDE$ INTHEASSEMBLY OFA WATER OXIDIZING MANGANESE CLUSTER IN 0HOTOSYSTEM)) OFF THE CYANOBACTERIUM 3Y 3 NECHOCY C STIS SP 0##  !SSEMBLY REQUIRES A FFREE CARRBOXYL GROUP AT # TERMINAL POSITION  "IOCHEMISTRYn .IXON 0* #HISHOLM $! AND $INER "! B )SOLATION AND FUNCTIONAL ANALYSIS OF RANDOM AND SITE DIRECTED MUTANTS OF 0HOTOSYSTEM)))N3HEWRY02AND'UTTERIDGE3EDS 0LANT 0ROTEIN%NGINEERING PPn#AMBRIDGE5NIVERSITY0RESS #AMBRIDGE D $INER "!  !NALYSIS OF F WATER OXIDATION .IXON 0* AND 3 NECHOCY CYSTIS SP MUTANTSCONSTRUCTED INTHECYANOBACTERIUM 3Y 0## "IOCHEM3OC4RANSn * +OMENDA* * "ARB R ER* * $EAKK : 6ASS)ANDD $INER"! .IXON0*  $ELETIONOFTHE0%34 LIKEREGIONOFPHOTOSYSTEMTWO MODIlES THE 1" BINDING POCKET BUT DOES NOT PREVENT RAPID TURNOVEROF$*"IOL#HEMn .OGUCHI4 )NOUE9AND3ATOH+ &4 )2STUDIESONTHE TRIPLET STATE OF 0 IN THE 0HOTOSYSTEM)) REACTION CENTER 4 IPLETEQUILIBRIUMWITHINACHLOROPHYLL DIMER"IOCHEMISTRY 4R  n .OGUCHI4 4OMO4ANDD )NOUE9 &OURIERTRANSFORM F INFRARE F D STUDYY OFT F THECATIONRADICALOF0 F INTH T E0HOTOSYSTEM))REACTION CENTER%VIDENCEFORCHARGEDELOCALIZATIONONTHECHLOROPHYLL DIMER"IOCHEMISTRYn /ETTMEIER7 (ERBICIDERESISTANCEANDSUPERSENSITIVITYIN 0HOTOSYSTEM))#ELL-OL,IFE3CIn /ETTMEIER7 3OLL ( * ANDD .EUMANN %  (ERBI R CIDE AND PLASTOQUINONE BINDING TO0HOTOSYSTEM)):.ATURFORSC F HC n /HAD D .AND (IRSCHBERG*  -UTATIONS INTHE$SUB U UNIT OFF 0HOTOSYSTEM)) DISTINGUISH BETWEENQUINONEAND HERBI R CIDE BINDINGSITES0LANT#ELLn /KAMURA-9 3ATOH+ )SAACSON2!AND&EHER' %VI DENCEOFTHEPRIMARYCHARGESEPARATIONINTHE$$COMPLEX OF0HOTOSYSTEM))FROMSPINACH%02OFTHETRIPLETSTATE)N "IGGINS*ED 0ROGRESS IN 0HOTOSYNTHESIS2ESEARCH 6OL  PP n-ARTINUS.IJHOFFF 0UUBLISHERS $ORDRECHT /-ALLEY 0* "ABCOCK'4AN K D 0RINCE2# 4HECATIONIC PLASTOQUINONERADICAL OFF THECHLOROPLASTWATERSPLITTINGCOMPLEX (YPERlNE SPLITTING FFROM A SINGLE METHYL GROUP DETERMINES THE %02 SPECTRAL SHAPE OF SIGNAL )) "IOCHIM "IOPHYS!CTA n 0ADDOCK-, -C0HERSON0( &EHER'AND/KAMURA-9 0ATHWAYOFPROTONTRANSFERINBACTERIALREACTIONCENTERS2E PLACEMENT OFF SERINE , BY ALANINE INHIBITS ELECTRON AND PROTONTRANSFERSASSOC F IATEDD WITH THEREDUCT D IONOF QU Q INONETO

 DIHY H DROQUINONE0ROC.ATL!CAD3CI53!n 0AKRASI (" AND 6ERMAAS 7&*  0ROTEIN ENGINEERING OF 0HOTOSYSTEM)))N"ARBER*ED 4HE0HOTOSYSTEMS3TRUCTURE &UNCTIONAND-OLECULAR"IOLOGY PPn%LSEVIER3CIENCE 0UBLISHERS !MSTERDAM 0ELOQUIN*- #AMPBELL+!AND D "RITT2$ -N PULSED %.$/2 DEMONSTRATES THAT THE 0HOTOSYSTEM)) @SPLIT %02 SIGNAL ARISES FFROM A MAGNETICALLY COUPLED D MANGANO TYROSYL COMPLEX*!M#HEM3OCn 0ELOQUIN*- 4ANG8 3 $INER"!AND"RITT2$ !NELEC TRON SPIN ECHO ENVELOPE MODULATION %3%%- STUDY OF THE 1! BINDINGPOCKETOF03))REACTIONCENTERSFROMSPINACHAND 3YNECHOCYSTIS "IOCHEMISTRYn 0ETROULEAS6 $ELIGIANNAKIS9AND$INER"! "INDINGOF CARB R OXYLATEANIONSATTHENON HEME&E)) OF03))))#OMPET F I TIONWITH BICARB R ONATEAND D EFFECTSONT F HE 1!1" ELECTRONTRANSFER RATE"IOCHIM"IOPHYS!CTAn 0lSTER+ 3TEINBACK+% 'AR K DNER'AND!RNTZEN#* D  0HO TOAFlNITY LAB A ELLING OFF A HERBI R CIDERECEPTORPROTEIN INCHLOROPLAST MEMBRANES0ROC.ATL!CAD3CI53!n 0RESTON#AND3EIBERT- 4HECARBOXYLMODIlER ETHYL  ; DIMETHYLAMINO PROPYL=CARBODIIMIDE%$# INHIBITSHALFOF THEHIGH AFlNITY-N BINDINGSITEIN0HOTOSYSTEM))MEMBRANE F FRAGMENTS" IOCHEMISTRYn 0UEYO** -OLINER% 3EIBERT-AND D 0ICOREL 2  0IGMENT CONTENTOF$ $ C F YTOCHROME BREACTIONCENTREPREPARA TIONSAFTERREMOVA F LOFF #0CONTAMINATION!N IMMUNOLOGICAL STUDY" Y IOCHEMISTRYn 1IAN - $AO , $EBUS 2* AND "URNAP 2,  )MPACT OF MUTATIONSWITHINTHEPUTATIVE#A BINDINGLUMENALINTERHELI CALAnBLOOPOFTHE0HOTOSYSTEM))$PROTEIN ONTHEKINETICS OF PHOTOACTIVATION AND (/ OXIDATION IN 3YNECHOCYSTIS 3 SP 0##"IOCHEMISTRYn 2ADMER2AND D #HENIAE'- -ECHANISMSOFF OXYGENEVO LUTION)N"ARB R ER*ED 0RIMARY 0ROCESSESOFF 0HOTOSYNTHESIS 6OL  PP n%LSEVIER !MSTERDAM 2ASMUSSEN /& "OOKJANS ' 3TUMMANN "- AND D (ENNINGSEN +7 ,OCALISATIONANDNUCLEOTIDESEQUENCEOFTHEGENE FORTHEMEMBRANEPOLYPEPTIDE$FROMPEACHLOROPLAST$.! 0LANT-OL"IOLn 2AO*+- (ARGROVE0!AND!RGOS0 7ILLTHESEVENHELIX BUNDLEBEACOMMONSTRUCTUREFOR F INTEGRAL MEMBRANEPROTEINS &%"3,ETTn 2HEE+ ( -ORRIS%0 "ARB R ER*AND D +àHLBRANDT7 4HREE DIMENSIONAL STRUCTURE OFF THE PLANT 0HOTOSYSTEM)) REACTION CENTREAT¯ RESOLUTION.ATUREn 2OCHAIX* $ /RGANISATION FUNCTIONANDEXPRESSIONOFTHE CHLOROPLAST$.!OF #HLAMYDOMONASREINHARDTII%XPERIENTIA n 2OCHAIX* $ $RON- 2AHIRE-AND-ALNOE0 3EQUENCE HOMOLOGY BETWEENTHE+$ALTONAND D THE$CHLOROPLAST MEMBRANEPOLYPEPTIDESOF #HL # AMYD M DOMONASREINHARD R TII 0LANT -OL"IOL  n 2OFFE F Y2! VAN7IJK 7 K +* 3AAYRE24AND3T D YRING3 3PECTRO SCOPICCHARACTERIZATIONOFF TYROSINE :IN HISTIDINEMUTANTS OFTHE$PROTEININ0HOTOSYSTEM))03)) IN #HLAMYDOMONAS # R REINHARDTI I*"IOL#HEMn 2 2UFmE36AND3AYRE24 &UNCTIONALANALYSISOF0HOTOSY S TEM)))N2OCHAIX* $ 'OLDSCHMIDT #LERMONT-AND-ER CHANT3 EDS !DVANCESIN 0HOTOSYNTHESIS 6OL 4HEMOLECULAR BIOLOGY OFC F HLOROPLASTSAND D MITOCHONDRIA IN #HLAMYD M DOMONAS

 PPn+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT 2UFmE36 $ONNELLY$ "LUNDELL4,AND.UGENT*(! ! THREE DIMENSIONALMODELOFTHE0HOTOSYSTEM))REACTIONCENTRE OF 0ISUM SATIVUM0HOTOSYNTH2ESn 2UFmE36 7ANG* *OHNSTON(' 'USTAFSON4, (UTCHISON23 2 F ANDD 3AAYRE 24  0HOTOSYSTEM)) -INAGAWA * #ROFTS! PERIPHERAL ACCESSORYCHLOROPHYLLMUTANTSIN #HL # AMYD M DOMONAS R TII "IOCHEMICAL CHARACTERIZATION ANDD SENSITIVITY TO REINHARD PHOTO INHIBITION0LANT0HYSIOL n 2 HERFOR 2UT F D!7 0ATERSON$2 -U D LLET*%  !LIGHT INDUCE D D SPIN POLARIZEDTRIPLETDETECTEDBY%02IN0HOTOSYSTEM))REAC TIONCENTERS"IOCHIM"IOPHYS!CTAn 2UTHERFORD!7 :IMMERMAN*,AND-ATHIS0 %02OF03)) INTERACTIONS HERBICIDEEFFECTSANDANEWSIGNAL)N3YBESMA# ED !DVANCESIN 0HOTOSYNTHESIS2ESEARCH 6OL) PPn -ARTINUS.IJHOFF$R7*UNKK 0UUBLISHERS 4HE(AGUE 3ARCINA- $INER"! "RETON*ANDD .IXON0*  &4)2STUDIES ONTHE0CATIONANDD TRIPLETSTATESIN74ANDD MUTANT03)) 3 NECHOCY CYSTIS )N'ARAB' ED 0HO REACTIONCENTRESOF 3Y TOSYNTHESIS-ECHANISMSAND%FFECTS 6OL)) PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT 3ATOH+ 0HOTOSYSTEM))PARTICLESLARGELYDEPLETEDINTHETWO INTRINSICPOLYPEPTIDESINTHEK$AREGIONFROM 3YNECHOCOCCUS SP)DENTIlCATIONOFASU F UBUNITWHI W CHCARRIESTHE0HOTOSYSTEM)) REACTIONCENTRE&%"3 ,ETTn 3ATOH + )SOLATIONANDPROPERT D IESOFT F HE0HOTOSYSTEM)) REACTIONCENTER)N4HE0HOTOSYNTHETIC2EACTION#ENTER 6OL  PPn!CADEMIC0RESS)NC 3AN$IEGO 53! 3ATOH + .AKATANI (9 9 3TEINBACK +% 7ATSON * AND!RNTZEN #* 0OLYPEPTIDECOMPOSITIONOFA0HOTOSYSTEM))CORE COMPLEX 0RESENCE OF A HERBICIDE BINDING PROTEIN "IOCHIM "IOPHYS!CTAn 3ATOH + &UJII U 9 !OSHIMA4ANDD 4ADO4  )MMUNOLOGICAL IDENTIlCATIONOFT F HEPOLYPEPTIDE BANDSINTHE3$3 POLYACRYL AMIDE GEL ELECTROPHORESIS OFF 0HOTOSYSTEM)) PREPARATIONS &%"3,ETTn 3AY A RE24 !NDERSSON"ANDD "OGORADD ,  4HETOPOLOGY OF AMEMBRANEPROTEIN4HEORIENTATIONOFTHEKD1B BINDING CHLOROPLASTTHYLAKOIDMEMBRANEPROTEIN#ELLn 3CHELVIS*0- VAN.OORT0) !ARTSMA4*ANDVAN'ORKOM(*  %NERGY TRANSFER CHARGE SEPARATION AND PIGMENT AR RANGEMENT INTHEREACTIONCENTEROFF 0HOTOSYSTEM))"IOCHIM "IOPHYS!CTAn 3CHRADER3AND D *OHANNINGMEIER5 4HECARB R OXY TERMINAL EXTENSIONOFT F HE$ PRECURSORPROTEINIS DISPENSABLEFORA F F C FUN TIONAL 0HOTOSYSTEM))COMPLEXIN #HL # AMYD M DOMONASREINHARD RDTII 0LANT-OL"IOLn 3EIBERT- 4AMURA. AND)NOUE9 ,ACKOFPHOTOACTIVA TIONCAPACITYIN3CENEDESMUSOBLIQUUS,& RESULTSFROMLOSS OFHALFTHEHIGH AFlNITYMANGANESE BINDINGSITE2ELATIONSHIP D $ PROTEIN "IOCHIM "IOPHYS!CTA H  TO THE UNPROCESSED n 3HARMA* 0ANICO- "ARB R ER*ANDD -ORRIS(2 0URIlCATION AND DETERMINATION OF INTACT MOLECULAR MASS BY ELECTROSPRAY A IONISATIONMASSSPECTROMETRY OFF THE0HOTOSYSTEM))REACTION CENTERSUBUNITS*"IOL#HEMn 3TEINBACK+% -C)NTOSH, "OGORAD,AND!RNTZEN#* )DENTIlCATIONOFTHETRIAZINERECEPTORPROTEINASACHLOROPLAST GENEPRODUCT0ROC.ATL!CAD3CI53!n 3TEWART$( #UA! #HISHOLM$! $INER"! "OCIAN$&AND "RUDVIG '7  )DENTIlCATIONOF HISTIDINE INTHE$

1FUFS
+
/JYPO
.BSZ
4BSDJOB
BOE
#SVDF
"
%JOFS POLYPEPT Y IDEOFF 0HOTOSYSTEM))ASTHEAXIAL LIGAND D TOCHLOROPHYLL :"IOCHEMISTRYn 3VENSSON" 6ASS) #EDERGREN%AND3TYRING3 3TRUCTURE OF DONOR SIDE COMPONENTS IN 0HOTOSYSTEM)) PREDICTED BY COMPUTERMODELLING%-"/*n 3VENSSON" %TCHEBEST# 4UFFER F Y0 VAN+AN0 3MITH TH*AND 3TYRING 3  !MODEL FORT F HE0HOTOSYSTEM))REACTIONCENTERCORE INCLUDINGTHESTRUCTUREOFF THEPRIMARY DONOR0"IOCHEMISTRY  n 4AKAHASHI9AND3ATO D H+ )DENTIlCATIONOFF THEPHOTOCHEMI CALLYIODINATEDAMINO ACIDRESIDUEONTHE$ PROTEININTHE 0HOTOSYSTEM)) CORE COMPLEX BY PEPTIDE MAPPING ANALYSIS "IOCHIM"IOPHYS!CTAn 4AKAHASHI9 4AKAHASHI-AND3ATOH+ )DENTIlCATIONOF THESITEOF IODIDEPHOTOOXIDATION INTHE0HOTOSYSTEM))REACTION CENTERCOMPLEX&%"3,ETTn 4AKAHASHI9 (ANSSON ½ -ATHIS0AND3ATOH+ 0RIMARY RADICAL PAIR IN THE 0HOTOSYSTEM)) REACTION CENTRE "IOCHIM "IOPHY H S!CTAn 4AKAHASHI- 3HIRAISHI4AND!SADA+ #//( TERMINAL RESIDUESOF$ANDK$A#0 AATSPINACH03))CORECOMPLEX &%"3,ETTn 4AKAHASHI9 .AKANE( +OJIMA(AND3ATOH+ #HROMATO GRAPHICPURIlCATIONAND DETERMINATIONOFF THECARB R OXY TERMINAL SEQUENCESOFF 0HOTOSYSTEM))REACTIONCENTERPROTEINS $AND $0LANT#ELL 0HYSIOL n 4AMURA. )KEUCHI -AND D )NOUE9 !SSIGNMENTOF HISTIDINE D IN$PROTEINASPOSSIBLELIGANDS FOR F FUNCT F IONAL MAN RESIDUES GANESEINPHOTOSYNTHETICWATER OXIDIZINGCOMPLEX"IOCHIM "IOPHYS!CTAn 4ANG8 3 &USHIMI+AND3ATOH+ $ $COMPLEXOF THE0HOTOSYSTEM))REACTIONCENTERFROMSPINACH)SOLATIONAND PARTIALCHARACTERISATION&%"3,ETTn D $INER 4ANG8 3 #HISHOLM$! $ISMUKES'# "RUDVIG '7AND "! 3PECTROSCOPICEVIDENCEFROMS F ITE DIRECTED D MUTANTSOF CYSTIS 0##IN FAVORO F F LOSE INTERACTIONBETWEEN FAC 3 NECHOCY 3Y HISTIDINEANDREDOX ACTIVETYROSINE BOTHOFPOLYPEP TIDE$OFTHE0HOTOSYSTEM))REACTIONCENTER"IOCHEMISTRY n 4ANG8 3 $INER"! ,ARSEN"3 'ILCHRIST*R-, ,ORRIGAN'! AND"RITT2$ )DENTIlCATIONOFHISTIDINEATTHECATALYTIC SITEOFF THEPHOTOSYNTHETICOXYGEN EVOLVINGCOMPLEX0ROC.ATL !CAD 3CI 53!n 4AOKA3AND D #ROFTS!2 F  4WO ELECTRONGATE INTRIAZINERE SISTANTAND D SUSCEPTIBLE !MARANTH T USHYBRIDU D S)N"ALTSCHEFFS F KY - ED #URRENT2ESEARCH IN0HOTOSYNTHESIS 6OL ) PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT 4AYLOR-! .IXON0* 4ODD#- "ARBER*AND"OWYER*2 #HARACTERISATIONOFTHE$PROTEININA0HOTOSYSTEM))MUTANT ,&  OF3CENEDESMUSOBLIQUUS BLOCKEDONTHEOXIDISINGSIDE F HE %VIDENCESUPPORTINGNON PROCESSINGOFF $ASTHECAUSEOFT LESION&%"3 ,ETTn D "RUDVIG '7  #YTOCHROME B  4HOMPSON ,+ AND MAY A FUNCT F IONTOPROTECT0HOTOSYSTEM)) FROMP F HOTOINHIBITION "IOCHEMISTRY n 4RACEWELL#! #UA! 3TEWART$( "OCIAN$&AND"RUDVIG'7  #HARACTERIZATIONOFCAROTENOIDANDCHLOROPHYLLPHOTO OXIDATIONIN0HOTOSYSTEM))"IOCHEMISTRYn 4REBST! 4HETOPOLOGYOFTHEPLASTOQUINONEANDHERBICIDE BINDINGPEPTIDESOFF 0HOTOSYSTEM)) INTHETHY H LAKOID D MEMBRANE :.ATURFORSCH C n

$IBQUFS

%
BOE
%
1SPUFJOT 4ROST*4 #HISHOLM$! *ORDAN$"ANDD $INER"! 4HE $ # TERMINAL PROCESSING PROTEASE OF 0HOTOSYSTEM)) FROM 3CENEDESMUSOBLIQUUS*"IOL#HEMn 5N3 "RUNEL, # "RILL4- :IMMERMANN* ,AND2UTHERFORD!7  !NGULARORIENTATIONOFTHESTABLETYROSYLRADICALWITHIN PHOTOSYSTEMTWO BY HIGH lELD '(ZE D LECTRONPARAMAGNETIC RESONANCE0ROC.ATL !CAD 3CI 53!n 5N3 4ANG8 3 $INER"! '(Z(IGH &IELDD %023TUDY OFF 4YROSINE $% ANDD 4YROSINE :% INMUTANTSOFF 0HOTOSYSTEM)) "IOCHEMISTRYn VAN"REDERODE-%ANDVAN'RONDELLE2 .EWANDUNEX PECTEDROUTESFORULTRAFASTELECTRONTRANSFERINPHOTOSYNTHETIC REACTIONCENTERS&%"3,ETTn VAN'ORKOM(*AND3CHELVIS*0- +OKSOXYGENCLOCK 7HATMAKESITTICK4HESTRUCTUREOFF 0AND CONSEQUENCES Q OF ITSOXIDIZING POWER0HOTOSYNTH2ESn VAN-IEGHAM&*% 3ATOH +ANDD 2UTHERFOR F D !7  !CHLORO PHYLLTILTED D  RELATIVETOTHEMEMBRANEINTHE0HOTOSYSTEM)) REACTIONCENTRE"IOCHIM"IOPHYS!CTAn VAN2ENSEN**3 4ONK4*-ANDDE"RUIJN3- )NVOLVEMENT OF BICARBONATE IN THE PROTONATION OF THE SECONDARY QUINONE ELECTRONACCEPTOROF0HOTOSYSTEM))VIATHENON HEMEIRONOFTHE Q QUINONE IRONACCE PTORCOMPLEX&%"3,ETTn D "RUCE$ 0ICOSECONDD TIME RESOLVED mUORES 6ASILEV3AND CENCESTUDIESONEXCITATIONENERGYTRANSFER F INAHISTIDINE F HE$PROTEINOFF 0HOTOSYSTEM)) IN 3Y 3 NECHOCY CYSTIS MUTANTOFT "IOCHEMISTRY n 6ELTHUYS "2  %LECTRON DEPENDENT COMPETITION BETWEEN PLASTOQUINONE AND INHIBITORS FOR BINDING TO 0HOTOSYSTEM)) &%"3,ETTn 6 6ELTHUYS"2AND!MESZ* #HARGEACCUMULATIONATTHE REDUCINGSIDEOFSYSTEMOFPHOTOSYNTHESIS"IOCHIM"IOPHYS !CTA  n 6 6ERMAAS7  -OLECULAR BIOLOGICAL APPROACHESTOANALYZE F ION!NNU2EV0LANT0HYS 0HOTOSYSTEM))STRUCTUREAND FUNCT -OL"IOL n 6 6ERMAAS7&* !RNTZEN#* 'U, 1AN D 9U# ! )NTERAC TION OF HERBICIDES AND AZIDOQUINONES AT THE 0HOTOSYSTEM)) BINDING SITE IN THE THYLAKOID MEMBRANE "IOCHIM "IOPHYS !CTAn 6ERMAAS7&* 7ILLIAMS*'+AND!RNTZEN#* 3ITE DIRECTED 6 D INTHE$PROTEIN INACTIVATE MUTATIONSOFF TWO HISTIDINERESIDUES AND DESTABILI A SE0HOTOSYSTEM))INTHECYANOBACTERIUM 3Y 3 NECHO CYST Y IS  :.ATURFORSCH C n 6 6ERMAAS7&* 2UTHERFORD!7AND(ANSSON ½  3ITE DI RECTED D MUTAGENESIS IN0HOTOSYSTEM))OFT F HECYCANOBACTERIUM 3YNECHOCYSTIS SP0##DONOR$ISATYROSINERESIDUEIN THE$PROTEIN0ROC.ATL!CAD3CI53!n 6 6ERMAAS7 #HARITÏ*AND3HEN' 'LU OFTHE$PROTEIN IN0HOTOSYSTEM))ISAPOTENTIALLIGANDTO-NINVOLVEDINPHOTO SYNTHETICOXYGENEVOLUTION"IOCHEMISTRYn

 D 3TYRING 3  -UTATION 6ERMAAS7 6ASS ) %GGERS " AND OFAPUTATIVELIGANDTOTHENON HAEMIRONIN0HOTOSYSTEM)) )MPLICATIONSFOR1! REACTIVITY ELECTRONTRANSFER ANDHERBICIDE BINDING"IOCHIM"IOPHYS!CTAn 7ANG* 'OSZTOLA$ 2UFmE36 (EMANN# 3EIBERT- 7ASIELEWSKI 7 -2 (ILLE2 'USTAFSON4,*O F HNSTON(' 'USTAFSON4,AN F D 3AY A RE24  &UNCTIONALASYMMETRY OFF 0HOTOSYSTEM))$ AND D $ PERIPHERAL CHLOROPHYLL MUTANTS OF #HL # AMYD M DOMONAS R TII 0ROC.ATL!CAD 3CI 53!n REINHARD 7 BBER!. 0ACKMAN, # 7E K HAPMAN$* "ARB R ER*AND'RA D AY *#  ! lFTH ENCODED ENCODED POLYPEPTIDE IS PRESENT IN THE0HOTOSYSTEM))REACTIONCENTRECOMPLEX&%"3,ETT n 7HITELEGGE*0 +OO$ $INER"! $OMIAN)AND%RICKSON* !SSEMBLY OF F THE 0HOTOSYSTEM)) OXYGEN EVOLVING COMPLEXIS INHIBITED IN PSB! B SITE DIRECTED D MUTANTS OF #HLAM YDOMONASREIN D HARD R TII!SPARTATEOFF THE$POLYPEPTIDE* "IOL #HEMn 7ILLIAMS *' AND D #HISHOLM $!  .UCLEOTIDE SEQUENCES OFBOTH PSB$ GENESFROMTHECYANOBACTERIUM 3YNECHOCYSTIS )N"IGGINS*ED 0ROGRESSIN0HOTOSYNTHESIS2ESEARCH PPn-ARTINUS.IJHOFF0UBLISHERS $ORDRECHT 7ILLIAMS *# 3TEINER ,! /GDEN 2# 3IMON -) AND &EHER '  0RIMARY STRUCTURE OF F THE - SUB U UNIT OFF THE REACTION CENTRE FR F OM 2HODO DOPSEUDOMONASS D SPHAEROIDES D  0ROC .ATL !CAD 3CI 53!n 7ILLIAMS *# 3TEINER ,! &EHER ' AND D 3IMON -)  0RI MARY STRUCTURE OFF THE , SUB U UNIT OF F THE REACTION CENTRE FROM F 2HODOPSEUDOMONAS SPHAEROIDES 0ROC .ATL !CAD 3CI 53! n 8IONG* 3UBRAMANIAM3AND'OVINDJEE !KNOWLEDGE BASEDTHREEDIMENSIONALMODELOFTHE0HOTOSYSTEM))REACTION CENTER OF #HL # AMYDOMONAS D REINHARD R TII 0HOTOSYNTH 2ES  n 9 9AMAMOTO9 )NA GAKI . 3ATOH &AND D 3ATOH+ 3OMEAS PECTSOFCAR F RBOXYL TERMINALPROCESSINGOFF PRECURSOR$PROTEIN OFF 0HOTOSYSTEM)))N030ROCEEDINGS 3  #3)2/ 0UBLISHING !USTRALIA 9OUVAN $# "YLINA %* !LBERTI - "EGUSCH ( AND (EARST *% 9  .UCLEOTIDEANDDEDUCEDPOLYPEPTIDESEQUENCESOFTHE PHOTOSYNTHETIC REACTION CENTER " ANTENNA AND mANKING POLYPEPTIDESFROM F 2CAP A SULATA T  #ELL n :OUNI! 7 7ITT ( 4 +ERN * &ROMME 0 +RAU” . 3AENGER 7 AND D /RTH0  #RYSTALSTRUCTUREOFF 0HOTOSYSTEM)) FROM F G AT ¯ RESOLUTION .ATURE  3 NECHOCOCCUS ELONGATUS 3Y n :URAWSKI' "OHNERT(* 7HITFELD02AND"OTTOMLEY7 .UCLEOTIDESEQUENCEOFTHEGENEFORTHE-R  THYLAKOID MEMBRANEPROTEINFROM 3PINACIAOLERACE 3 A AND.ICOTIANADE . B NEYI PREDICTSATOTALLYCONSERVEDPRIMARYTRANSLATIONPRODUCTOF -R  0ROC.ATL!CAD3CI53!n

$IBQUFS
 5IF &YUSJOTJD
1SPUFJOT
PG
1IPUPTZTUFN
** 5 Z
.
#SJDLFS

5FSS #JPDIFNJTUSZ SZ
BOE
.PMFDVMBS
#JPMPHZZ 4FDUJPO
%FQBSUNFOU
PG
#JPMPHJDBM
4DJFODFT -PVJTJBOB
4UBUF
6OJWFSTJUZ
#BUPO
3PVHF
-"

64"

3PCFSU
-
#VSOBQ %FQBSUNFOU
PG
.JDSPCJPMPHZ
BOE
.PMFDVMBS
(FOFUJDT
0LMBIPNB
4UBUF
6OJWFSTJU Z UZ 4UJMMXBUFS
0,

64"

4VNNBSZ   * *OUSPEVDUJPO  **

5IF

L%B
.BOHBOFTF4UBCJMJ[JOH 1SPUFJO
1TC0   " 4USVDUVSF   #
*OUFSBDUJPOT
PG
UIF
.BOHBOFTF4UBCJMJ[JOH
1SPUFJO
XJUIJO
1IPUPTZTUFN
**   $
3FTJEVFT
PG
UIF
.BOHBOFTF4UBCJMJ[JOH
1SPUFJO
3FRVJSFE
GPS
#JOEJOH  %
&WJEFODF
GPS
#JOEJOH
UP
$1 &
&WJEFODF
GPS
#JOEJOH
UP
0UIFS
$PNQPOFOUT  '
5IF
4UPJDIJPNFUSZ
PG
UIF
.BOHBOFTF4UBCJMJ[JOH
1SPUFJO  (
5IF
'VODUJPO
PG
UIF
.BOHBOFTF4UBCJMJ[JOH 1SPUFJO  ***

5IF

L%B
BOE

L%B
1SPUFJOT
1TC1
BOE
1TC2
JO
)JHIFS
1MBOUT  " 4USVDUVSFT
PG
UIF

L%B
BOE

L%B 1SPUFJOT #
'VODUJPOT
PG UIF

L%B
BOE

L%B 1SPUFJOT  $
1VUBUJWF
)PNPMPHVFT
PG
1TC1
BOE
1TC2 JO $ZBOPCBDUFSJB  *7

.VMUJQMF
&YQSFTTFE (FOFT
PG
UIF
&YUSJOTJD
1SPUFJOT
JO
)JHIFS
1MBOUT 7 $ZUPDISPNF D 1TC7 JO $ZBOPCBDUFSJB 7 " *EFOUJàDBUJPO
PG
$ZUPDISPNF D BT B $PNQPOFOU PG
1IPUPTZTUFN
** # 4USVDUVSF
PG
$ZUPDISPNF D  $
#JOEJOH
BOE 4UPJDIJPNFUSZ
PG $ZUPDISPNF D   % 'VODUJPO
PG
$ZUPDISPNF D   7*

5IF
1TC6
1SPUFJO
JO $ZBOPCBDUFSJB   7** $PODMVTJPOT  "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ )NTHISCHAPTERTHESTRUCTUREANDFUNCTIONOFTHEEXTRINSICPROTEINSOF0HOTOSYSTEM))03)) AREEXAMINED(IGHER PLANTSAND GREENALGAECONTAINTHEK$AMANGANESE STABILI A ZING PROTEINANDD THEK$AANDD THE K$AEXTRINSIC PROTEINSWHIL W ETHECYANOBACTERIACONTAINTHESAMEMANGANESE STABILIZINGPROTEIN CYTOCHROME C ANDD THE  K$AEXTRINSICPROTEIN4HESEPROTEINSSERVEASENHANCERSOF/ EVOLUTION OPTIMIZING03))ACTIVITYATPHYSI OLOGICAL CALCIUMAND D CHLORIDECONCENTRATIONS4HEY SHIELDD THEMANGANESECLUSTERFROMEXO F GENOUSREDUCTANTS D D REACTANTSINTHESURROUNDINGAQUEOUSPHASE!NUMBEROFMO F LECULAR BIOCHEMICAL ANDD STRUCTURALSTUDIES AND

!UTHORFORCORRESPON F DENCE EMAIL BTB T RIC LSUEDU 47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ

HAVE BEENUSED D TOPROBETHESTRUCTURESAND FUNCT F IONSOFT F HESEPROTEINSWITHINTHEPHOTOSYSTEM7EWILL DISCUSS THEPROPOSEDFUNCTIONALROLESFORTHESECOMPONENTS THEIRSTRUCTUREASDEDUCEDFROMBIOCHEMICALAND8 RAY D THE LOCATIONOFT F HEIRPROPOSED BINDING DOMAINSWITHINTHE03))COMPLEX CRYSTALLOGRAPHICSTUDIES AND *
*OUSPEVDUJPO )NHIGHERPLANTSANDCYANOBACTERIAATLEASTSIXINTRINSIC PROTEINSAPPEARTO BEREQUIRED FOR F / EVOLUTION -U RATAETAL "URNAP A AND D 3HERMAN "RICKER  4HESEARE#0 #0 THE$PROTEIN THE$ PROTEIN AND D THE _ AND ` SUB U UNITSOFC F YTOCHROME B SEE#HAPTERS AND D  FOR F DETAILSONTHESECOMPO NENTS )NSERTIONALINACTIVATIONORDELETIONOFTHEGENES F HESECOMPONENTSRESULTS INTHECOMPLETELOSSOF FORT / EVOLUTIONACTIVITY!DDITIONALLY ANUMBEROF LOW MOLECULARMASSCOMPONENTSAPPEARTOBEASSOCIATED WITH 0HOTOSYSTEM )) 03)) )KEUCHI ET AL  #HAPTER ALTHOUGH THE FUNCT F IONSOFT F HESEPROTEINS REMAINOBSCURE7HILE03))COMPLEXESCONTAINING ONLYTHESECOMPONENTSCANEVOLVE/ THEY DOSOAT VERY LOWRATESABOUTOFCONTRO F L AREEXTREMELY SUSCEPTIBLE TO PHOTOINACTIVATION AND REQUIRE HIGH NON PHYSIOLOGICAL LEVELS OFF CALCIUM AND D CHLORIDE -URATAETAL "RICKER   )NHIGHERPLANTS THREE EXTRINSICPROTEINS WITHAP PARENTMOLECULARMASSESOFF K$ATHEMANGANESE STABILIZINGPROTEINOR-30 K$AAND D K$AARE Q / EVOLUTIONATPHYSI REQUIREDFORMAXIMALRATESOF OLOGICAL INORGANIC COFACTOR F CONCENTRATIONS 4HESE PROTEINSAREENCODED BYTHE PSB/ PSB0 AND PSB1 GENES RESPECTIVELY4HEYWEREORIGINALLYIDENTIlED ININVERTED D 03))THYLAKOIDD VESICLES!KERLUND D ETAL D 03)) MEMBRANE FRAGMENTS F +UWABARA  AND AND -URATA   )N SOME ORGANISMS IT APPEARS THAT EACH OFF THESE GENES IS REPRESENTED BY A SMALL F ILY )N CYANOBACTERIA ONLY THE -30 MULTIGENE FAM ISPRESENTHOWEVER SEE#HAPTERANDTHEDISCUS SIONBELOWONPOSSIBLEPSB0AND D PSB1HOMOLOGUES INCYANOBACTERIA WITHTHE FUNCT F IONSOFF THEK$A !BBREVIATIONS#$nCIRCULARDICHROISM# TERMINALnCARB R OXY TERMINAL #YT n CYTOCHROME $430 n DITHIOBISSUCCINIMIDYL PROPIONATE  %$# n  ETHYL   DIMETHYLAMINOPROPYL CARBODIIMIDE %02 n ELECTRON PARAMAGNETIC RESONANCE %8 !&3nEXTENDED8 RAYABSORPTIONlNESTRUCTURE&4)2n&OURIER TRANSFORM INFRARED '-%n GLYCINE METHYLESTER K$An KILO $ALTON ,$!/n LAURYLDIMETHYLAMINE OXIDE -N  n CATA LYTICMANGANESECLUSTER-30nMANGANESE STABILIZINGPROTEIN .(3n . HYDROXYSUCCINIMIDE . TERMINAL n AMINO TERMINAL 03))n0HOTOSYSTEM))3N nSTATESOFT F HE/ EVOLVINGCYCLE N     OR4,nTHERMOLUMINESCENCE

ANDD  K$A PROTEINS APPEARING TO BE PROVIDED BY CYTOCHROME#YT C ENCODED BY PSB6 ANDD A K$A PROTEIN ENCODED BY PSB5 3HEN AND )NOUE A B 4HESECOMPONENTSAPPARENTLY INTERACTWITH INTRINSICMEMBRANEPROTEINSANDD EACHOTHERTOYIELD FULLY FUNCTIONAL / EVOLVING COMPLEXES /F THESE EXTRINSICPROTEINS THE-30APPEARSTOPLAY A ACENTRAL ROLE INTHESTABILIZATIONOFF THEMANGANESECLUSTERAND ISESSENTIALFOREFlCIENTANDSTABLE / EVOLUTION 4HE K$AANDD  K$APROTEINSANDD THEIRCYANOBACTERIAL COUNTERPARTS #YT C ANDTHEK$APROTEIN APPEAR TOBEREQUIREDFORO Q PTIMALRATESOF/ EVOLUTION AT PHYSIOLOGICALCALCIUMANDD CHLORIDECONCENTRATIONS )NTHISCHAPTERWEWILL SUMMARIZETHE INFORMAT F ION AVAILABLECONCERNINGTHESTRUCTURESANDFUNCTIONSOF THEEXTRINSICPROTEINSOF03)) F  **
5IF

L%B
.BOHBOFTF4UBCJMJ[JOH 1SPUFJO 1TC0

"
4USVDUVSF S ! SEQUENCE ALIGNMENT FOR THE MATURE MANGANESE STABILIZING PROTEIN -30 IS SHOWN IN &IG 4HE D ISRELATIVELYHIGHLYCONSERVED D AMINOACIDSEQUENCE WITH  OF THE RESIDUES BEING EITHER COMPLETELY CONSERVEDD ORCONSERVATIVELY REPLACEDD ACROSSTHE PROTEINS FFOR WHICH DERIVEDD AMINO ACID D SEQUENCE Q INFORMATION IS AVAILABLE )NTERESTINGLY ALL OF THE CYANOBACTERIAL SEQUENCES EXAMINEDD EXHIBIT A  OR D . TERMINAL TRUNCATION WITH  AMINO ACIDD RESIDUE RESPECTTOTHESPINACHSEQUENCEANDMOSTEXHIBITAN Q EIGHTAMINOACID INSERTIONBETWEENRESIDUES0RO D 'LUOFF THESPINACHSE H QQUENCE4HE PRESENCEOFF THESE CONSERVEDFEATURESINTHECYANOBACTERIALSEQUENCESIS F KNOWNCONSEQUENCE K HOWEVER SEEBELOW  OFUN F PINACHCONTAINSAMINO 4HEMATURE-30FROMS ACID RESIDUES AND POSSESSES A CALCULATED MASS OF  K$A /H /KAETAL  4HE-30 DOESNOT APPEARTOBEPOST TRANSLATIONALLY MODIlED OTHERTHAN THEREMOVALOFITSTRANSITSEQUENCES SINCE-!,$) MATRIX ASSISTED LASER DESORPTIONIONIZATION MASS SPECTROMETRY MEASUREMENTSYIELD D AMOLECULARMASS VERY SIMILAR TO THE PREDICTED VALUE :UBRZYCKI ET AL  4HISPROTEINMIGRATESANOMALOUSLYDUR

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 **



&IG  !LIGNMENTOFTHEAMINOACIDSEQUENCESOFPHYLETICALLYDIVERSE K$AMANGANESE STABILIZINGPROTEINS0SB/ 4HEALIGNMENTS & WERECALCULATEDUSING#LUSTAL7IMPLEMENTEDATTHE"IOLOGY7ORKBENCHAT3AN$IEGO3UPERCOMPUTERFACILITY4HE'ONNETSERIESWEIGHT MATRIXWASUSEDWITHTHEFOLLOWINGPARAMETERSETTINGSGAPOPENPENALTY GAPEXTENSIONPENALTY IDENTITYFORDELAY  RESIDUE SPECIlCGAPPENALTIES ONPENALIZEENDGAPS ONHYDROPHILICGAPPENALTIES ONGAPSEPARATIONDISTANCE HYDROPHILICRESIDUE '03.$1%+24HE lGUREWASRENDEREDD USING'ENE$OCPROGRAMS

ING 3$3 0!'%ANDGELlLTRATION YIELDINGAPPARENT MASSESOFF AND  K$A RESPECTIVELY ,YD , AKIS 3I MANTIRISETAL B !DDITIONALLY DYNAMIC LIGHT SCATTERINGEXPERIMENTS ANALYTICALULTRACENTRIFUGATION :UB U RZYCKI ET AL  AND SMALL ANGLE 8 RAY A

SCATTERING3VENSSONETAL  INDICATETHATTHE -30EXHIBITSANEXTENDEDD STRUCTUREINSOLUTIONWITH ANAXIALRATIOOFF   .OSUCCESSFUL8 RAYCRYSTALLOGRAPHICSTUDIESHAVE BEENREPORTED FOR F ISOLATED-30 A D LTHOUGH!NATIAND

 !DIR  HAVEREPORTED D OBSERVINGMICROCRYSTALS OFTHEPROTEINANDHAVEARGUEDTHATTHESECONSISTED OF DIMERS (OWEVER THESE CRYSTALS WERE TOO SMALL TO PERFORM F STRUCTURAL ANALYSIS 7I 7 THIN THE CRYSTAL STRUCTURES OF 03)) FROM 3 3YNECHOCOCCUS ELONGATUS AND 4H 4 ERMOSY S NECHOCOCCUS VULCANUS :OUNI ET AL D 3HEN "IESIADKAETAL  +AMIYAAND &ERREIRAETAL #HAPTERSn ANELECTRON DENSITY T HAS BEENIDENTIlED D WHICH H WAS HYPOT Y THESIZED D TO F HE-30!BOUT OFF THE-30HAS BEEN ARISEFROMT MODELEDASPOLYALANINE+AMIYAAND3HEN  F FORMIN GACYLINDRICALSTRUCTURE ¯ =  ¯ WHICH ISRICHIN ` SHEETSTRUCTURESEEBELOW  !VARIETYOFLOWRESOLUTIONBIOCHEMICALANDBIO PHYSICAL STUDIES HAVEBEENPERFORME F D ONTHE-30 4HESE INCLUDE FFAR 56 #$ MEASUREMENTS &4)2 STUDIES ANDSITE SPECIlCLABELINGANDMAPPINGEXPERI MENTS4HESE INVESTIGATIONS HAVE YIELDED INTERESTING D INLARGEMEASURE COMPLEMENTARYRESULTS&AR 56 AND SECONDARYSTRUCTUREDETERMINATIONS8UETAL  D THATTHEEXTRINSIC 3HUTOVAETAL  HAVEREVEALED -30CONTAINSA HIGHPROPORTIONOF ` SHEET^ ANDARELATIVELYLOWPROPORTIONOF _ HELICAL STRUCTURE ^ -OST&4)2STUDIES !HMED D ETAL ( :HANGETAL , 8:HANGETAL  ALSOINDICATEAHIGHPROPORTIONOF` SHEETn WITHVARYING AMOUNTSOFREPORTE F D _ HELICALSTRUCTURE n 3EVERALSTRUCTURAL MODELS BASED D ONTHESE LOWRESOLUTIONSTUDIESHAVEBEENPROPOSED"RICKER AND D &RANKEL  0AZOS ET AL  FFOR A MORE DETAILED DISCUSSION OF F THE SECONDARY AND D TERTIARY STRUCTUREOFTHE-30INSOLUTION SEE0OPELKOVAET AL A  (UTCHISON ET AL  HAVE REPORTED ISOTOPE EDITING EXPERIMENTS IN WHI W CH # LABELED 03))MEMBRANESWERERECONSTITUTEDWITH# LABELED -305PON BINDING OFF THE LABELED D -30TO03)) A D ADECREASE SUB U STANTIAL INCREASEIN ` SHEETCONTENTAND IN RANDOM ANDORTURN STRUCTURE WERE OBSERVED #
*OUFSBDUJPOT
PG
UIF
.BOHBOFTF4UBCJMJ[JOH 1 1SPUFJO
XJUIJO
1IPUPT TZTUFN
** 4HE INTERACTIONOFF THE-30WITHOTHER03)) PROTEINS ISQUITESTRONG-IYAOAND-URATA DETERMINED ASINGLE+D OFN-FOR-30BINDINGTO03))MEM BRANES,EUSCHNERAND D "RICKER OBSERVED D TWO -30SPER03))INHIGHERPLANTSSEEBELOW WHICH D +D OFANDN- RESPECTIVELY)NBOTH EXHIBITED OFF THESESTUDIES REBINDINGWASPERFORME F D INTHEPRES ENCEOFHIGHCHLORIDECONCENTRATIONSM- IN ORDERTOMAINTAINTHEMANGANESECLUSTERINAN INTACT

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ F IONALSTATE#ONSEQUENTLY THEACTUAL +D MAY AND FUNCT BE MUCH LOWER 3EVERAL SITE SPECIlC CHEMICAL LABELING STUDIES HAVEBEENPERFORME F D TO PROBETHEINTERACTIONSOFF THE -30WITH03))&RANKELAND"RICKER 3EIDLER A-IURAETAL &RANKELETAL  )N THESESTUDIES THE-30WASMODIlEDD WITH REAGENTS WHICHSPECIlCALLYLABELINDIVIDUALAMINOACIDRESI D DUES#OMPAR ISONOFT F HE DISTRIBUTIONOFF THEMODIlED D HENPERFORME F D AFTER F LABELINGOF RESIDUESPRESENTWAST THE-30EITHERINASSOCIATIONWITHTHEINTRINSICCOM PONENTSOF03))OR F INSOLUTION&RANKELANDD "RICKER  USEDD . HY H DROSUCC D INIMIDOBIOTINTOSELECTIVELY LABELLYSYLRESIDUESUNDERTHESETWOCONDITIONS&OUR DOMAINS 'LU ,Y , S , ,YS , ,YS ,Y , S AND , ,YS ,YS OFT F HE-30WERELABELED ONLY L WH W EN THEPROTEINWASFREEINSOLUTION4HISINDICATESTHAT THESEDOMAINSARESHIELDEDD WHENTHE-30IS INTERACT INGWITH 03))-IURAETAL UTILIZEDD ANUMBER OFAMINO GROUPMODIlCATIONREAGENTS INASIMILAR SERIESOFF EXPERIMENTS4HESERESULTS INLARG R EMEASURE WEREVERYCONGRUENTTOTHOSEOUTLINEDD ABOVE&RANKEL AND"RICKER   -IURAETAL ALSOMODIlEDD THEASPARTYLAND D HE-30WITHGLYCINEMETHY H L GLUTAMYLRESIDUESONT ESTER'-% INA  ETHYL   DIMETHYLAMINOPROPYL CARB R ODIIMIDE%$# CATALYZEDD REACTION4HISRESULTED INTHEMODIlCATIONOFMORET F HANEIGHTCARB R OXYLATE RESIDUES !FTER MODIlCATION THEY FOUND THAT AT A '-% LABELED-30TO03))REACT D IONCENTERRATIOOF  THE MODIlED PROTEIN COULD D STILL BIND D TO 03)) AND FULLY RECONSTITUTE / EVOLUTION ACTIVITY 4HEY R OXYLATE RESIDUES D ON THE -30 CONCLUDEDD THAT CARB DID D NOT PARTICIPATE IN THE BINDING OFF THE -30 TO 03)) /THER INVESTIGATORS HOWEVER HAVE COME TO OPPOSITECONCLUSIONS3EIDLERA EXAMINEDD THE ROLEOFF CARRBOXYLATESONTHE-30INTHEBINDINGOF THIS COMPONENT TO 03)) $IFFERENTIAL ACTIVATION OF R OXYLATES ON THE -30 ANDD ON THE INTRINSIC THE CARB PROTEINS OF F 03)) WAS PERFORME F D USING %$# AND SULFO .(3. HYDROXYSUCCINIMIDE 4HESESTUDIES INDICATEDD THATALLOFT F HECARB R OXYL GROUPS INVOLVED IN F IONWEREPRESENTONTHE-30&RANKEL CROSSLINK FORMAT ETAL DEMONSTRATEDTHAT-30WHICHHADBEEN LABELED INSOLUTIONWITH '-% %$#UNDERCONDITIONS VERYSIMILARTOTHOSEUTILIZED BY-IURAETAL EXHIBITEDVERYPOORBINDINGAND/ EVOLUTION RECON STITUTION ATLOW'-% LABELED-30TO03))REACT D ION CENTERRATIOS4HEMODIlEDD PROTEINEXHIBITED HIGHLY SIGMOIDAL BINDING AND / EVOLUTION REACTIVATION KINETICS4HE LOCATIONSOFT F HE DIFFERENT F IALLYMODIlED

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** CARB R OXYLATERESIDUESWEREMAPPE D D BYPROTEASEDIGES TIONANDMASSSPECTROMETRY4HESESTUDIESINDICATED THAT'-% %$#MODIlCATIONS WITHINTHE DOMAINS !SP !SPANDOR'LU 'LN # TERMINUS ARERESPONSIBLEFORTHEBINDINGKINETICSOBSERVEDFOR THECHEMICALLY MODIlED D -30 )TSHOULD lNALLY L BENOTED D THATCHEMICALMODIlCATION OF 4RP OF THE -30 WITH . BROMOSUCCINIMIDE LEADSTOA GREATLY DECREASED D AFlNITYOFF THEMODIlED PROTEINTO03))AND D ALOSSOFT F THEABILI A TYOFF THEMODIlED PROTEINTORECONSTITUTE/ EVOLUTION9UETAL   &AR 56#$MEASUREMENTS INDICATED D THATMODIlCATION D LED D TOA LOSSOFF SOLUTIONSTRUCTUREOFF THE OFF THISRESIDUE PROTEIN WITHTHECHANGESBEINGVERYSIMILARTOTHOSE OBSERVED D AFTERMUTA F GENICTRUNCATIONOFT F THE# TERMINUS ,YDAKIS 3IMANTIRISETAL ASEEBELOW  $
3FTJEVFT
PG
UIF
.BOHBOFTF4UBCJMJ[JOH 1 1SPUFJO
3FRVJSFE
G GPS
#JOEJO H %ATON 2Y 2 EANDD -URATA DEMONSTRATED D THATTHE D RESIDUESO D F THE-30  . TERMINAL AMINOACID WEREREQUIREDFORTHEBINDINGOFTHISPROTEINTO03)) #HYMOTRYPSINORR 6PROTEASEWASUSED TOREMOVETHE . TERMINAL TOAMINOACIDD RESIDUES RESPECT D IVELY FFROMTHE-304HESEPROTEOLYTICALLYMODIlEDPROTEINS D DEPLETED D 03))MEMBRANES COULDD NOT BINDTO-30 4HEYREPORTED D THATFAR 56#$ANA F LYSIS INDICATED D THAT NO MAJOR STRUCTURAL CHANGES OCCURRED IN THE -30 AFTERPROTEASETREATMENT4 F HESERESULTSARECONSISTENT WITHRESULTS INDICATINGTHATAN. TERMINAL DOMAINOF THE-30ISNOT.(3 BIOTINYLATEDWHENTHISPROTEIN ISASSOCIATED D WITH 03)) &RANKEL AND D "RICKER  AND D THATTH T E. TERMINAL DOMAINISCROSSLINKED D TO #0 WITH%$#/DOMAND"RICKER   4WOEXPERIMENTALSYSTEMSHAVE BEENUSED D TOINTRO D DUCES ITE DIRECTED D MUTATIONS INTOTHE$.!ENCODING THEEXTRINSIC-30INANATTEMPTTOIDENTIFYRESIDUES THATAREREQUIRED FORE F ITHERBINDING TO03))ORFUN F C TION&IRST MUTATIONS HAVE BEENINTRODUCE D D INTOTHE CLONED PSB/ GENE3EIDLERETAL "ETTSETAL D -30WAS A-OTOKI ETAL  4HEMODIlED THENEXPRESSED INAN %COLI EXPRESSIONSYSTEM THE PROTEIN PURIlEDANDREFOLDED ANDTHENEXTRINSICPRO TEIN DEPLETED D 03))WASRECONSTITUTED D WITH THTTHEPURIlED D IONSWERE INTRODUCE D D ALTERED PROTEIN3ECOND MUTAT INTOTHE PSB/ GENEOF 3YNECHOCYSTIS 3 "URNAP ETAL  4HEPHENOTYPICCONSEQUENCESOFT F HESE MUTATIONSWEREEVALUATED INVIVO ALTHOUGHSOME IN VITROSTUDIESHAVEBEENPERFORMED)NTHEFOLLOWING DISCUSSION THESPINACHAMINOACIDD NUMBERSWILL BE F BOTH SYSTEMS USED FOR

 3EIDLERETAL B IDENTIlEDD TWORESIDUES D INSPINACHWHICH WHENMODIlED APPEARTOLOWERTHE F LTERATIONOF!SPTO F AFlNITY OFF THE-30 FOR03))! D IELDSAMODIlEDPROTE D INTHATEXHIBITS A LYSYLRESIDUEY A FOLDLOWERAFlNITYFOR03))!LTERATIONOFTHIS RESIDUE D TO!SN HOWEVER RESULTS IN A MODIlED PROTEINWITH NORMAL BINDINGCHARACTERISTICS!LTERA TIONOF!SPTOEITHER!SNOR,YSYIELDSA PROTEIN WITH DECREASED BINDING AFlNITY! Y SIMILAR D D IN 3YNEC 3 HOCYSTIS BY"URNAP MUTATIONWAS INTRODUCE ETAL -ODIlCATIONOF!SPTO!LAIN D PROTEIN WITH EXTREMELY SPINACH YIELDS A MODIlED SIGMOIDAL BINDING CHARACTERISTICS # & 9OCUM PERSONALCOMMUNICATION WHICHCAN NEVERTHELESS ALMOSTFU F LLY RECONSTITUTE/ EVOLUTIONACTIVITY/T Y HER MODIlCATIONS NEAR THE # TERMINUS ALSO APPEAR TO PROFOUNDLYAFFECTBINDINGANDACTIVITY4HEREPLACE MENTOF6A F LWITH!LAYIELDSAMODIlEDPROTE D IN WHICHEXHIBITSADEFECT F INTHEABILITYTO BINDTO03)) D MEMBRANESANDRECONSTITUTE/ EVOLUTIONACTIVITYAT  # "ETTSETAL B !TROOMTEMPERATURETHE BINDINGCHARACTERISTICSOFT F HISALTEREDD PROTEINAPPEAR NORMAL)NCOMPETITIONREBINDINGEXPERIMENTSWITH THISALTEREDPROTE D INANDD WILD TYPEPROTE Y IN THEBINDING OFT F HEWILD TYPEPROTEIN ISHIGHLYPREFERRE F D "ETTSET AL  )NTERESTINGLY IFMODIlEDPROTEINISlRST BOUNDD TO03))MEMBRANESANDD THENWILD TYPEPRO TEIN ISADDED INANATTEMPTTO DISPLACETHEMODIlED COMPONENT ONLYHALFOFTHEALTEREDPROTEINISREMOVED SEEBELOW 4HEREQUIREMENTOFAN F INTACT# TERMINUS OFT F HE-30HAS BEENPROBED BY DELETIONMUTAGENESIS "ETTSETAL  2EMOVALOFTHE# TERMINALDIPEP TIDE DIDD NOTAFFECT F BINDING ORFUNCT F IONOFF THEPROTEIN WHILEREMOVAL OFT F HE# TERMINALTRIPEPTIDESERIOUSLY WEAKENEDBINDINGOFTHEPROTEINANDRESULTEDINPOOR F  EVOLUTIONACTIVITY$E Y LETIONOF RECONSTITUTIONOF/ THE# TERMINALTETRAPEPTIDERESULTED INAPROTEINTHAT COULDNEITHERBINDNORRECONSTITUTEACTIVITY7ITHIN A BETHEMOST THIS DOMAIN ITAPPEARSTHE,EUMAY CRITICALRESIDUE#ERTA D INLY REPLACEMENTOFF THISSINGLE RESIDUEWITHGLUTAMATERESULTSINADISTURBEDPHENO TYPE NEARLY AS SEVERE AS THE # TERMINAL TRIPEPTIDE DELETION ,YDAKIS 3IMANTIRISETAL A  !SNOTEDPREVIOUSLY THE. TERMINUSOFTHE-30 HAS BEEN IMPLICATED IN BINDING TO03)) %ATON 2Y 2 E ANDD -URATA /DOMANDD "RICKER  #ON TROVERSYEXISTSCONCERNINGTHEROLESOFRESIDUESNEAR THE. TERMINUS IN BINDINGOFF THE-30TO03)))N SPINACH 3EIDLERETALA REPORTEDD THATALTERATION OF!SPTOEITHER!SNOR,YSRESULTSINPROTEINS WHICHEXHIBITPHENOTYPESTHATARE INDISTINGUISHABLE F FROMT THATOFW F ILD TYPE3EIDLERANDD 2UTTHERFOR F D  D 

 4HEYALSOARGUED D THAT BECAUSETHEACIDICRESIDUEAT D POSITIONISNOTCONSERVED BEINGREPLACEDBYGLU TAMINEIN !NABAENA AND 3YNECHOCOCCUS "ORTHAKUR K AND D (ASELKORN -IURAETAL  THENIT ISNT INVOLVEDINBINDINGANDORFUNCTION(OWEVER INTHE 3YNECHOCY CYSTIS SYSTEM ALTERATIONOFF !SPTO, ,YSAND F !SPTO(IS !RG !SN OR3ER ALTERATIONOF ALLRESULTEDINSIGNIlCANTCHANGESINTHEPHENOTYPE OFF CELL LINES THAT CONTAIN THESE MUTATIONS 1IAN ET AL  !LL OFT F HESEMUTANTSEXHIBITED D An DECREASE IN / EVOLUTION RATES AND A DECREASE IN BINDINGSTRENGTH OFT F HE-30TO03))4HIS INDICATES THATALLORMOSTOFF THE03))CENTERSPRESENTCONTAIN THEMODIlED-30BUTTHATITISLESSTIGHTLYBOUND !FTERT F HE HYDROXYLAMINEEXTRACTIONOFMAN F GANESE THE !SP3ER AND D !SP(IS MUTANTS EXHIBITED D A PHOTOACTIVATIONRATEINTERMEDIATEBETWEENTHERATES F FORW ILD TYPEAND D THE 6PSB 6 /STRAIN 2ECENTLY SITE DIRECTEDMUTAGENES D IS HASBEENUSED TOINTRODUCESHORT. TERMINALDELETIONSINTOTHESPIN ACHPROTEIN 0OPELKOVAETAL A 4HE DELETIONOF THETHREE. TERMINAL AMINOACID D RESIDUESRESU D LTS INA PROTEINTHATISFULLYCOMPETENTINTHERECONSTITUTIONOF / EVOLUTIONACTIVITY4 Y HE DELETIONOFT F HE. TERMINAL TENAMINOACID D RESIDUES D HOWEVER YIELDSAPROTEIN THATEXHIBITSAMARKEDDECREASEINITSABILITYTORE CONSTITUTE /EVOLUTION!DDITIONALLY ON Y LYONECOPY D INCANBIND D TOTHEPHOTOSYSTEM OFF THISMODIlEDPROTE IN THESE STUDIES TWO COPIES OF WILD TYPE PROTEIN D TOBIND &URTHERRESULTS INDICATED D THAT AREOBSERVED . TERMINAL DELETIONS INVOLVING THE DOMAIN4HR 'LUESSENTIALLYCOMPLETELYABOLISHEDTHEBINDING OFF -30TO03)) 0OPELKOVAETAL B 4HUS TWO . TERMINAL BINDING DOMAINSWEREIDENTIlED INTHESE STUDIES$ELETIONOFTHElRST ,YS 'LU ABOLISHES THEBINDING OFF ONECOPY OFF THE-30TO03))WHIL W E THESECOND D 4HR 'LU AB A OLISHESTHE BINDINGOFT F HE SECONDCOPY&URTHERSTUDIESHAVEINDICATEDTHATTHE THREONYL N RESIDUES D LOCATED INTHESETWO DOMAINS 4HR AND D 4HR ARECRITICAL INDETERMININGTHESPECIlCITY OFTHESEINTERACTIONS0OPELKOVAETAL B )N TERESTINGLY C Y YANOBACTERIAL-30SEQUENCESTYPICALLY EXHIBITSEQUENCETRUNCATIONATTHEIR. TERMINI &IG ANDONLYPOSSESSONEOFTHESEIDENTIlED-30BINDING DOMAINS)T HASBEENSPECULATED D THATTHEABSENCEOF F HESEBINDING DOMAINS INCYANOBACTERIARESULTS ONEOFT INTHESEORGANISMSPOSSESSINGONLYONE-30PER03)) 0OPELKOVAETAL B  F THE CONSERVED D CYSTEINYL RESIDUES 4HE ROLES OF #YS AND #YS HAVE ALSO BEEN EXAMINED BY SITE DIRECTED D MUTAGENESIS%ARLY BIOCHEMICAL STUDIES

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ INDICATEDD THATTHESERESIDUES D F FORME D ADISULlDE BOND Q ANDTHATTHISINTERACTIONWASREQUIREDFORTHEINTERAC

TIONOFF THE-30WITH 03))4ANAKAAND7A D DA   ,ATERSTUDIESINSPINACH HOWEVER DEMONSTRATEDD THE DOUBLEMUTANT## !!#YS!LA #YS!LA F LLY RE EXHIBITEDD ESSENTIALLY NORMAL BINDING AND FU CONSTITUTED / EVOLVINGACTIVITY"ETTSETAL A  4HISSHOWSCONCLUSIVELYTHATTHEDISULlDEBRIDGEIS F BINDINGORACTIVITY Y NOTREQUIRED FOR %
&WJEFODF
GPS
#JOEJOH
UP
$1 #ONSIDERABLE BIOCHEMICAL EVIDENCE IS AVAILABLE DEMONSTRATINGTHEINTERACTIONOF#0WITHTHE-30 #0ISA LARGE AMINOACID INTRINSICMEMBRANE H LL A BINDING COMPONENT OFF 03)) )T IS A CHLOROPHY PROTEIN WHICH CONTAINS SIX TRANSMEMBRANE HELIXES ANDD A LARGE AMINOACID LUMENALLY EXPOSED LOOP TERMED LOOP% "RICKER "RICKERANDD &RANKEL #HAPTER %ATON 2YEAND0UTNAM %VANS  ,IMITEDPROTEO D LYSISSTUDIESPERFORME F D BOTH INTHE PRESENCEANDD ABSENCEOFT F HE-30INDICATETHE-30 SHIELDS#0FROMTRYPTICATTACK"RICKERAND&RAN KEL  (AY A ASHI ETAL  SHOWEDD THATBOTH THE/ EVOLVINGCAPACITYAND-30 D BINDINGCAPACITY OFENDOPROTEINASE ,YS # TREATED03))DECREASEDIN PARALLEL WITH CLEAVAGE AT ,Y , S ON #0 4HESE F #0 IN THE AUTHORS CONCLUDEDD THAT THE REGION OF VICINITY OF ,YS WAS NECESSARY FOR THE BINDING OFT F HE-30ANDD THEMAINTENANCEOF / EVOLUTIONBY EXTRINSICPROTEIN DEPLETEDD 03)) ! VARIETY OF PROTEIN CROSSLINKERS ARE CAPABLE OF CROSSLINKING #0 TO THE -30 IN SPINACH 03)) MEMBRANES 4HESE INCLUDE DITHIOBISSUCCINIMIDYL PROPIONATE $430 %NAMI ET AL  "RICKER ET AL   IMINOTHIOLANE % #AMM PERSONAL U ECARB R ODIIMIDE COMMUNICATION ANDD THEWATER SOLUBL %$# "RICKER ET AL  %NAMI ET AL   %$#ISPARTICULARLY INTERESTING BECAUSEITCROSSLINKS R OXYL GROUPSWHI W CHAREINVAN AMINOGROUPSTOCARB DER7AALSCONTACT(ACKETTAND3TRITTMATTER   0ROTEINSCROSSLINKED BY THISREAGENTAREASSUMEDD TO BEINTERACTINGVIAASALTBRIDGE/DOMANDD "RICKER  HAVEMAPPEDTHEDOMAINSONBOTHTHE-30 D HATARECROSSLINKEDD WITH %$#4HEDOMAIN AND#0T 'LU !SP OFF #0 IS CROSSLINKEDD TO THE . TERMINALDOMAIN'LU ,YS OFTHE-303EIDLER A HASARGUED D BASEDD ONMUTAGENESISSTUDIES D ICIPATE IN THATTHEMOST LIKELYRESIDUEON#0TOPART THEPRODUCTIONOFTHISCROSSLINKEDPRODUCTIS,YS )T HASALSO BEENSHOWNTHAT%$# GENERATESCROSSLINKS

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** BETWEENTHE LUMENALLYEXPOSED LOOP!OFF #0AND THE -30 /HTA ET AL   )N ADDITION TO THESE INTERMOLECULARCROSSLINKS INTRAMOLECULARCROSSLINKS HAVE BEENDOCUMENTED D WITHINTHE-30 "RICKERET AL /HTAETAL 3EIDLER A  )N ADDITION TO THESE CROSSLINKING STUDIES IT HAS BEENDEMONSTRATED D THATTHE-30SHIELDS LYSYL RESI DUESLOCATEDON#0FROMLABELINGWITHTHEAMINO GROUP MODIFYINGREAGENT.(3 BIOTIN &RANKEL AND "RICKER )SHIKAWAETAL  4REATMENTSTHAT REMOVETHE-30FROMSPINACH03))MEMBRANESALLOW THESPECIlC LABELINGOF#0W F ITHTHISREAGENT4WO DOMAINS ,YS ,YSAND D ,YS ,YS BOTH OFWHICHLIEINTHELARGEEXTRINSICLOOP%OF#0 N ATED D ON THIS PROTEIN ARE THE ONLY REGIONS BIOTINYL &RANKELAND D "RICKER  2ECENTLY )SHIKAWAETAL  DEMONSTRATEDTHAT.(3LABELINGOF#0IN 4RIS WASHED D 03))CORECOMPLEXESPREVENTSEFlCIENT RECONSTITUTIONOFF THE-30TO03))4HELABELED D RESI DUESWERENOTIDENTIlEDINTHISSTUDY HOWEVER THE LABELINGCONDITIONSWERESIMILARTOTHOSE DESCRIBED PREVIOUSLY &RANKEL AND D "RICKER   )F #0 IS INVOLVED IN THE BINDING OF THE -30 WHICHRESIDUESARE D INVOLVED7E HYPOTHESIZETHATA NUMBEROF POINTSOF INTERACTIONEXIST BETWEEN#0 AND POSSIBLY OTHER 03)) COMPONENTS SEE BELOW D THE-304HECOOPERATIONOFT F HESEINTERACTIONS AND YIELDSTHEEXTREMELYSTRONGOBSERVED BINDINGOFT F HE -30TO03))3ITE DIRECTEDMUTAGENESISEXPERIMENTS HAVE FOCUSE F D DONPRO D ING ALTERATIONSINTHELARGE DUC F LEITERETAL  EX EXTRINSICLOOP%OF#0' AMINEDANUMBEROFPHOTOAUTOTROPHICMUTANTSTRAINS CYSTIS THATCARRIED D SHORTDELETIONS INTHE OF 3YNECHOCY D 6ERMAAS (AAG EXTRINSIC LOOP%%ATON 2YEAND ETAL  4HEYFOUNDTHATTHEMUTANTS 6!LA !SP AND 6!RG 6AL EXHIBITED D AMARRKED DECREASEINTHEABILITYTO BIND D THE-303ITE DIRECTED MUTAGENESISRESULTS BY 0UTNAM %VANS AND "RICKER  DEMONSTRATED D THATTHERESIDUES!R D GAND !RGWERE IMPORTANT FORMA F INTAINING HIGH RATES OF/ EVOLUTION)NTERESTINGLY 03))PARTICLESISOLATED F FROMT THE22 ''MUTANTDIDD NOTCONTAINBOUND -30 #0UTNAM %VANSAND4-"R D ICKER UNPUBLI U SHED OBSERVATIONS  3UBSEQUENTLY 0UTNAM %VANS ET AL  SHOWEDD THATTHEONLYCONSERVED D CHARGED D RESI D DUES INTHEDOMAIN'LU !RGWH W OSEALTERATION INmUENCED / EVOLUTION ACTIVITY WERE!RG AND !RG4HEMOSTSERIOUSLY AFFECTE F D MUTANTEXAM INED D 22 %% EXHIBITED D A PHENOTYPE VERY SIMILARBUTNOTIDENTICAL SEEBELOW TOTHATEXHIBITED BY 6PSB/ 6P P STRAINS4HISINCLUDED D AREDUCT D ION IN

 THE/ EVOLUTIONRATE ANINCREASEINTHE 3 LIFET F IME AND A RETARDED 3A3=A3 TRANSITION 1IAN ET AL  HAVEDEMONSTRATEDD THATTHETHYLAKOIDSOFT F HIS MUTANT BINDD THE-30EXTREMELYPOORLY!DDITIONALLY THEY FOUND THAT DELETION OF THE PSB/ GENE IN THIS STRAIN YIELDING THE MUTANT 22 %% -30 HAD LITTLEADDITIONALEFFECTONT F HEPHENOTYPEOFT F HIS MUTANT4HISINDICATESTHATBOTHTHE22 %% MUTATIONANDD THEDELETIONOFT F HE PSB/ GENEAFFEC F T THE SAME PROCESSES4HE MOST DRAMATIC DIFFERENCE F 6 BETWEENTHE22 %%MUTANTANDTHE 6PSB/ STRAIN IS THE OBSERVEDD RATES OF DARK R INACTIVATION IN 7 TYPE 3YNEC 3 HOCYSTIS EXHIBITSA Tš THESEMUTANTS7ILD F R DARK INACTIVATION OF  HRS 4HE 6PSB/ FO 6PP STRAIN DARK R INACTIVATESVERYRAPIDLY EX Y HIBITINGATš OFA F BOUT HRSWHIL W E22 %%EXHIBITSANINTERMEDIATERATE OFHOURS4HETRIPLEMUTANT22 %% -30 HADD A DARK R INACTIVATIONRATEWHI W CHWASIDENTICAL TO THATOBSERVED INTHE 6PSB/ 6PP STRAIN4HISMAY INDICATE THATTHE MECHANISM OF DARK INACTIVATION IS DISTINCT F FROMT HOSEPROCESSESTHATAFFECTT F HE 3 LIFET F IMEAND 3A3=A3TRANSITION INTHESEMUTANTS 7E 7 BELIEVE THATTHESERESULTSINDICATETHATTHEARGINYLRESIDUES LOCATEDD ATPOSITIONSANDD INTHELARGEEXTRINSIC LOOP%OFF #0 FORMAMA F AJORBINDING DOMAINOFF THE -30TO03)))NTHEABSENCEOFTHESEINTERACTIONS THE-30 ISONLY WEAKLY BOUNDD TO03))AND ISONLY F IONAL PARTIALLYFUNCT &
&WJEFODF
GPS
#JOEJOH
UP
0UIFS
$PNQPOFOUT ! NUMBER OF OTHER 03)) COMPONENTS HAVE BEEN SUGGESTEDD TO BEINVOLVED INTHEBINDINGOFF THE-30 F HESEOTHER TO03)))TSHOULD BENOTEDD THATNONEOFT HYPOTHESIZEDINTERACTIONSHAVEBEENSUPPORTEDBYTHE W CH HASBEENPROVIDED FOR F SAMEWEIGHTOFF EVIDENCEWHI THE#0 -30 INTERACTION.EVERTHELESS ITREMAINS VERYPOSSIBLETHATOTHERCOMPONENTSAREINVOLVEDIN THE03)) -30INTERACTION H LL PROTEIN#0ISSTRUCTURALLYVERY 4HECHLOROPHY SIMILAR TO #0 "OTH PROTEINS APPEAR TO CONTAIN SIX TRANSMEMBRANE HELIXES ANDD TO POSSESS LARGE LUMENALLY EXPOSED DOMAINS BETWEEN THE lFT F H AND SIXTHHELIXES"RICKER #HAPTER %ATON 2YE ANDD 0UTNAM %VANS  )SOGAI ET AL  REPORTED U ONREMOVALOFT F HE-30 #0BECAMEMUCH THATUP MOREACCESSIBLETOTRYPSIN%NAMIETAL HAVE EXTENDEDD THESESTUDIES4HEY REPORTEDD THATREMOVAL OFT F HE-30 FO F LLOWED BYTRYPSINTREATMENTRESULTED IN CLEAVAGE OF #0 AT !RG 4HIS PATTERN OF R ABLY SIMILAR TO THAT OBSERVEDD TO CLEAVAGE IS REMARK

 OCCUR AT ,YS IN #0 (AYASHI ET AL   !DDITIONALLY THE _ SUBUNITOF#YT B WAS CLEAVED D THAT AT!RG2ECONSTITUTIONEXPERIMENTSINDICATED THE REBINDING OFF THE -30 TO TRYPSIN TREATED D 03)) MEMBRANESDECREASEDINPARALLELWITHTHEOBSERVED F D YT B0OSSIBLERESIDUESO D# D F CLEAVAGEOF#0AN #0THATMAY BEINVOLVED IN BINDINGTHE-30 HAVE NOTYETBEENIDENTIlED 4HE$AND$PROTE D INSOF03)) F HAVEALSO BEEN IMPLICATED IN BINDING OFF THE -30 3ITE DIRECTED MUTATIONS AT POSITION!RG IN THE $ PROTEIN AP PEARTODISRUPTBINDINGOFT F HE-30 AND D TOA LESSER EXTENT#YT C ,IAND"URNAP  4HEMUTANT !RG'LU ALTERED IN THE PUTATIVE CALCIUM BINDING D VERY SIMILAR DEFECTS F TO THOSE ! " LOOP EXHIBITED 6 / MUTANTS4HYLAKOIDSISOLATEDFROM OBSERVED IN 6PSB/ THIS MUTANT EXHIBITED AN  LOSS OF BOUND -30 T ATEITH T ER!RG R  ORRESIDUES D IN 4HESERESULTS INDICATETH ITSVICINITY CONTRIBUTESTOTHE FORMAT F IONOFA F BINDING DOMAINFORTHE-30ORTHATCONFORMATIONALCHANGES INTHE! "LOOP INDUCE D D BY THISMUTATION DISRUPTA DISTANTBINDINGSITE )N THE 03)) CRYSTAL STRUCTURE FROM 4HERMO S NECHOCOCCUS +AMIYAAND3 SY D HEN  THE-30 D D PERH R APS APPEARSTO INTERACTWITH #0AND#0AN OTHER INTRINSIC COMPONENTS OF THE PHOTOSYSTEM 4HESE lNDINGS ARE CONSISTENT WITH THE LARGE BODY OF BIOCHEMICAL DATA DESCRIBED D ABOVE4HERESIDUES D ON#0AND#0WHICHAREINVOLVEDINTHEINTER ACTION HOWEVER CANNOT BE IDENTIlED D AT THIS TIME D HE-30REQUIRED FO F R !DDITIONALLY THERESIDUESONT ITSINTERACTIONWITHTHEINTRINSICCOMPONENTSOFTHE PHOTOSYSTEMCANNOT BE DETERMINED FROMT F HECURRENT CRYSTAL STRUCTURE '
5IF 4UPJDIJPNFUSZ
PG
UIF
.BOHBOFTF4UB ' CJMJ[ J JOH
J 1SPUF 1 J JO 4HERE IS CONSIDERABLE CONTROVERSY CONCERNING THE STOICHIOMETRYOFF THE-30WITHIN03))%ARLYSTUD IESINDICATEDTHATTHEREWEREONETOTHREECOPIESOF THE -30 PER 03)) REACTION CENTER !NDERSSON ET AL -URATAETAL -ILLNERETAL   -URATAETAL AND%NAMIETAL REPORTED F BEROF ASTOICHIOMETRYOFONE-30PER03))!NUM LOWRESOLUTIONELECTRONMICROSCOPYSTUDIES "OEKEMA ETAL A(OLZENBURGETAL (ASLERETAL  AND D THECYANOBACTERIALCRYSTALSTRUCTURES:OUNI ETAL +AMIYAAND3 D HEN "IESIADKAET AL &ERREIRAETAL #HAPTERSn ARE INTERPRETED D AS INDICATING ONECOPYOFT F HE-30PER

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ 03)) REACTION CENTER 4HIS IS THE MOST COMMONLY ACCEPTEDVALUE .EVERTHELESS SIGNIlCANTBIOCHEMICALEVIDENCEIN DICATESTHATTHEREARETWOCOPIESOFF THIS PROTEINWITHIN 03)) ATLEASTINHIGHERPLANTS9AMAMOTOETAL  8UANDD "RICKER 3EIDLER ,EUSCHNERAND "RICKER "ETTSETAL FORAMORECOM F PLETE DISCUSSION SEE"RICKERAND&RANKEL  4HERECENT OBSERVATIONTHATTWOBINDING DETERMINANTS FOR03)) F EXISTONTHE-30ANDD THATONEOFF THESE ISLACKINGINTHE CYANOBACTERIALSEQUENCES0OPELKOVAETAL B W CH MAY A HELPEXPLAINTHEAPPARENTCONTRADICTIONWHI EXISTS BETWEENTHEHIGHERPLANT BIOCHEMICALEXPERI MENTSANDTHECYANOBACTERIALCRYSTALSTRUCTURES:OUNI D HEN "IESIADKAET ETAL +AMIYAAND3 AL &ERREIRAETAL #HAPTERSn )T HASBEENSUGGESTEDTHATINHIGHERPLANTS ONECOPYOF THE-30MAY A PLAY A AREGULATORY ROLEWHIL W ETHESECOND COPYMAY BESTRUCTURAL "ETTSETAL   (
5IF
'VODUJPO
PG
UIF
.BOHBOFTF4UBCJMJ[JOH 1SPUFJ 1S U JO 4HETERM@MANGANESE STABILI A ZING PROTEINWASAPPLIED TOTHEK$AEXTRINSICCOMPONENT BASEDD ONTHEOB SERVATIONTHATREMOVALOFTHISCOMPONENTFROM03)) LEADSTOADESTABILIZATIONOFT F HEMANGANESECLUSTER H IOLOGICAL CHLORIDECONCENTRATIONS5NDER ATLOW PHYS THESECONDITIONS TWOOFTHEFOURMANGANESEBECOME PARAMAGNETICALLY UNCOUPLED -AVANKALETAL  K DIA -IYAOAND ANDD AREEVENTUALLYLOSTTOTHEBULKME -URATA B/NOAND)NOUE  )NTHEPRES ENCEOF HIGHCHLORIDECONCENTRATIONSM- THE D OXYGEN CAN MANGANESE CLUSTER REMAINS INTACT AND BEEVOLVEDINTHEABSENCEOFTHE-30-IYAOAND -URATA B "RICKER  BUT AT SIGNIlCANTLY LOWERRATES n OFCONTRO F L  %ARLIERSTUDIESEXAMINEDTHEPOSSIBILITYTHATTHE -30WASINVOLVED INTHE LIGATIONOFE F ITHERMANGANESE ORCALCIUMATTHEACTIVESITEOFF 03))!BRAMOWICZ AND $ISMUKES  FOR INSTANCE REPORTED THAT EXTRACTEDD -30CONTAINED BOUNDD MANGANESE/THER STUDIES HOWEVER CLEARLYDEMONSTRATEDD THATTHE-30 COULD BE ESSENTIALLY COMPLETELY REMOVED WITH THE MANGANESECLUSTERREMAINING INTACTAND / EVOLUTION PROCEEDINGATASIGNIlCANT ALBEITLOWER RATE-IYAO AND-URATA B+UWABARAETAL "RICKER  !DDITIONALLY %8!&3EXPER Y IMENTSPERFORME F D ON03))MEMBRANESDEPLETEDD OFT F HE-30 INDICATED THATESSENTIALLYNOCHANGEOCCURREDINTHESTRUCTURE F HEMANGANESECLUSTERUPONREMOVAL OFT F HISCOM OFT

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** PONENT#OLEETAL  )TSHOULD BENOTED D THAT THESESTUDIESDONOTPRECLUDETHEPOSSIBILITYTHATTHE -30PROVIDESONEORAFEWTERM F INAL LIGANDSTOTHE MANGANESECLUSTER WITHTHESELIGANDSBEINGREPLACED BYWATERORHYDROXIDEUPONREMOVALOFTHEPROTEIN -UTANTSLACKING THE-30CANNOT GROWAT LOW CAL CIUM0HILBRICKETA K L  ORCHLORIDE%NGELSETAL  CONCENTRATIONS%ARLYSTUDIESSUGGESTEDTHATTHE -30WASINVOLVED INCALCIUM BINDING ATTHEACTIVE F #OLEMANAND'OV D INDJEE 7ALES SITEOF03)) ETAL 7EBBERAND'RAY  !NUMBEROF A MORERECENTSTUDIES HOWEVER INDICATETHATTHE-30 ISNOTDIRECTLY INVOLVED INCALCIUMBINDING SEE3E IDLER B FORANIN DEPTHDISCUSSION 3EIDLERAND 2 HERFOR 2UT F D  FOR F INSTANCE DEMONSTRATED D THAT INTHEABSENCEOFF THE-30 THEHIGH AFlNITY BINDING SITEFORCALCIUMASSOCIATEDWITHTHE/ EVOLVINGSITE PERSISTS4HESESTUDIES DONOT HOWEVER ELIMINATETHE POSSIBILITYTHATTHE-30MAYPROVIDEONEORAFEW F LIGANDSTOCALCIUM4HESELIGANDSCOULDHYPOTHETICALLY BEREPLACED BYWATERORHYDROXIDEUPONTHEREMOVAL OFF THE-30 &UNCTIONALLY WHATPARAMETERSOFTHE/ EVOLVING PROCESSAREAFFECTE F D BY THE BIOCHEMICAL ORGENETIC F HE-30-IYAOETAL  PERFORME F D REMOVAL OFT mASH/YIELDMEASUREMENTSON-30 DEPLETED03)) D THATTHE3 LIFET F IME IN MEMBRANES4HESE INDICATED D SAM MPLESWASINCREASED lVE FO F LD D AND K$A DEPLETED THATTHELIFETIMEOFTHE 3 STATE WAS ALSO INCREASED !DDITIONALLY T Y HE3A3 TRANSITIONWAS INHIBITED D AND THE3A3=A3 TRANSITIONWASRETARDED D TWO TOTTHREE F FOLD/NOAND)NOUE USINGTHERMOLUMINES

CENCEASAPROBE FOR3 STATETRANS F ITIONS OBSERVED D THAT -30DEPLETIONINHIBITED D THE 3A3=A3 TRANSITION 4HE 3 MULTILINESIGNALHASALSOBEENEXAMINEDIN -30DEPLETEDD SAMPLES -ILLERETAL 3TYRING ETAL  4HE INTENSITYOFF THE 3 MULTILINESIGNAL DECREASED-ILLERETAL FOUNDNOSIGNIlCANT DECREASEINTHESHAPEOFT F HEMULTILINESIGNALWHIL W E D A SIGNIlCANT SHAPE 3TYRING ET AL  REPORTED CHANGEOFTHISSIGNAL PARTICULARLYATLOWlELDVALUES )NTERPRETATION OF F THESE SPECTRA WAS COMPLICATED D HOWEVER BYTHERELATIVELYLOWSIGNAL INTENSITYOFT F HE MULTILINESIGNAL THEPRESENCEOFTHEGY SIGNAL ARISING FROM F LOWPOTENTIAL #YT B AND D THEPRESENCE OF HEXA AQUOMANGANESE 3IMILARRESULTSWEREOBTAINEDUP U ONEXAMINATION 6P P /MUTANTS"URNAPAND D 3HERMAN  OFF THE 6PSB DEMONSTRATED D THATTHI T SSTRAINCOULDD GROWPHOTOAUTOTRO PHICALLYINSTANDARD"' MEDIA ALTHOUGHITEVOLVED / ATASIGNIlCANTLY LOWERRATETHANWILDD TYPE4HESE

 STRAINS HOWEVER CANNOTGROWPHOTOAUTOTROPHICALLY ATLOWCALCIUM0HILBRICKETAL  ANDCHLORIDE CONCENTRATIONS %NGELSETAL  &LASH/ YIELD 6PP /MUTANTSHOWEDD AN IN MEASUREMENTSONTHE 6PSB CREASEDSTABILIZATIONOFTHE3 AND3 STATES ANDD THATTHE 3A3=A3TRANSITIONWASSLOWEDD AT LEAST lVE FO F LD "URNAPETAL  !PARALLEL RETARDATION INTHERATE OF 9Z REDUCTIONWASALSOOBSERVED2AZEGHIFARDET AL  4HERMOLUMINESCENCE 4, MEASUREMENTS ALSOINDICATEDD A HIGHERSTABILITYOFF THE 3 AND3 STATES "URNAPETAL 6ASSETAL   )T ISPOSSIBLETHATTHE-30MAY A PARTICIPATEMORE DIRECTLY INTHEWATER OXIDIZINGPROCESS (UTCHINSON ET AL   03)) MEMBRANES WERE RECONSTITUTED F LY # LAB A ELEDD -30 INAN ISOTOPEEDITING WITH UNIFORM EXPERIMENT&4)2WASTHENUSEDD TOMONITORCHANGES INTHEPROTONATIONSTATEOFTHE-30DURING3STATE TURNOVER$URING THE3A3 TRANSITION SPECTRAWERE OBTAINEDD WHICH WERECONSISTENTWITH THE DEPROTON ATIONOFTHE-304HEPROTONACCEPTORDIDNOTAP PEARTORESIDEONTHEPROTEIN4HESERESULTSSUGGEST THATTHE-30MAYSERVETOSTABILIZETHECHARGED 3 TRANSITION STATE )NTERESTINGLY T Y HEOXIDATIONSTATEOFT F HEMANGANESE F HESTRUCTUREOF BOUND-30 D CLUSTERAPPEARSTOMODIFYT (ONGETAL  4REATMENTOF03))MEMBRANES D ION WITH  +- HYDROXYLAMINELEADSTOTHEREDUCT F HEMANGANESECLUSTERWHI W CH HOWEVER REMAINS OFT INTACT ANDTHEEXPOSUREOFTRYPTICSITESONTHE-30 D D SAMPLES LEADSTOTHE 4RYPSINTREATMENTOFF REDUCE RAPID DIGESTIONOFT F HE-30 WHI W CH ISNORMALLYQUITE RESISTANTTOTRYPSINATTACKWHENASSOCIATEDWITHTHE 03))REACTIONCENTERINTHEPRESENCEOFF ANOXIDIZED MANGANESECLUSTER  ***
5IF

L%B
BOE

L%B
1SPUFJOT 1TC1 BOE
1TC2
JO
)JHIFS
1MBOUT "
4USVDUVSFT
PG
UIF

L%B
BOE

L%B 1 1SPUFJOT )NHIGHERPLANTS THEK$A ALSOTERMEDD  K$A AND K$AALSOTERMEDORK$A PROTEINSAPPEARTO BEINVOLVED INMODU D LATIONOFT F THECALCIUMANDD CHLORIDE REQU Q IREMENTS FOR F / EVOLUTION4HESE PROTEINS ARE 0 ANDPSB1 NUCLEARGENES WHICH ENCODEDBYTHE PSB0 ENCODEPROTEINSWITHPREDICTEDD MOLECULARMASSESOF  K$A ANDD  K$A IN SPINACH RESPECTIVELY *ANSENETAL  3IGNIlCANTLY FEWERBIOCHEMICAL F D ONTHESE ANDD MOLECULARSTUDIES HAVE BEENPERFORME



5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ

&IIG  !LIGNMENTOFTHEAMINOACIDSEQUENCESOFPHY & H LETICALLYDIVERSEEXTRINSICK$APROTEIN0SB0 !PPARENTHOMOLOGSFROMCYANO BACTERIAAREINCLUDEDBOTTOMSEQUENCES 4HEALIGNMENTSWEREPERFORMEDASDESCRIBEDINTHELEGENDOF&IG

COMPONENTSTHANONTHE-303EQUENCEALIGNMENTS FFORTHESEPROTEINSAREFOUNDIN&IGSAND!SISTHE CASEFORT F HE-30 THESTRUCTUREOFT F HESEPROTEINS HAS BEENPROBED BY#$AND D &4)24HE K$APROTEIN CONTAINSALOWPROPORTIONOF_ HELIX ANDHIGH PROPORTIONOF ` SHEET  AND D RANDOMTURN SECONDARY STRUCTURE :HANG ET AL   2ECENT PROGRESSINTHECRYSTALLIZATIONOFTHEK$APROTEIN D STRUCTURAL INFORMAT F ION IN MAY A PROVIDEMORE DETAILED THENEARFUTURE F )FU F KUETA K L   4HE  K$A COMPONENT IS ESTIMATED TO CONTAIN SIGNIlCANTLY HIGHERAMOUNTSOF _ HELICAL STRUCTURE n THANOBSERVED FORT F HE-30AND D  K$A PROTEINS AND LOWER AMOUNTS OF ` SHEET   :HANGETAL "ALSERAETAL  2ECENTLY T Y THE F HISPROTEIN HAS BEENDETERMINED CRYSTAL STRUCTUREOFT ATARESOLUTIONOF¯#ALDERONEETAL  4HE PROTEINISA FOUR F HELIX BUNDLEWHI W CH ISCONSISTENTWITH

THE HIGH AMOUNT OF _ HELICAL STRUCTURE PREVIOUSLY OBSERVED:HANGETAL "ALSERAETAL   F LY T Y HE. TERMINAL AMINOACID D RESIDUES D 5NFORTUNATE F THEPROTEINARENOTOBSERVED INTH T ECURRENTSTRUCTURE OFT )TIS UNCLEARIFTHIS DOMAIN IS UNORDERED ORIFITHAS BEENLOST DUETOPROTEO D LYSIS 4HE  K$A ANDD  K$A COMPONENTS CAN BE REMOVEDFROM03))BYTREATMENTWITH-.A#L!K ERLUNDD ETAL  WHIL W ETHE K$ACAN BEREMOVED TH M-.A#LAND D SPECIlCALLY BYTREATMENTWITH METHANOL9AMAMOTOAND+UBOTA  2EMOVALOF THESEPROTEINSAPPEARSTO INTRODUCEA D LTERATIONS INTHE L ISOFF 03)) STRUCTUREOFF THE03))COMPLEX)MAGEANALYS SUPERCOMPLEXESFROMHIGHERPLANTSWHICHRETAINTHE LIGHT HARVESTING ANTENNAEOFF THEPHOTOSYSTEM FROM F WHICH THESE COMPONENTS WERE REMOVED BY .A#L WASHING INDICATESTHAT#0MOVESNMTOWARDS THECENTRALCOREOFT F HE03))COMPLEX "OEKEMAET

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 **



& &IG !LIGNMENTOFF THEAMINOACIDD SEQUENCESOFP F HY H LETICALLY DIVERSEEXTRINSIC K$APROTEIN 0SB1 !PPARENT HOMOLOGSFROMCY F A NOBACTERIAAREINCLUDED BOTTOMSEQUENCES 4HEALIGNMENTSWEREPERFORME F D ASDESCRIBED INTHE LEGEND D OF &IG

AL  !LTERATIONS IN THE STRUCTUREFUNCTION OF THECOREPROTEINSOFF THE03))COMPLEXMAY A ALSOBE F D BYTHEREMOVAL OFT F HESEEXTRINSICPROTEINS AFFECTE !TRAZINE BINDINGATTHE 1" SITE WHICHISLOCATEDON THE$PROTEINAND D EXPOSED ATTHESTROMALSURFACE F R EDLYINCREASED INTHEABSENCEOF OFF THE03)) ISMARK THEK$AANDK$APROTEINS2ASHIDAND#ARPEN TIER  4HISMAY A INDICATETHATREMOVALOFT F HESE F HE LUMENAL FACEO F F HECOMPLEX FT COMPONENTSFROMT INDUCESTRANSMEMBRANE ALTERATIONSINTHE STRUCTURE OFF THE 1" BINDING SITE)TSHOULD BENOTED D HOWEVER THAT IT ISUNCLEARINTHISAND D THEPREVIOUSSTUDY IFF THE ALTERATIONS OBSERVED ARE DUETOTHE RELEASE OFTHE  K$AAND D  K$ACOMPONENTSPERSE ORDUETOT D HE CONCOMITANT DISRUPTIONOFF THEHIGH AFlNITYCALCIUM ANDCHLORIDE BINDINGSITESINDUCEDBYTHERELEASEOF THEPROTEINS SEE BELOW  4HE BINDINGSITESONTHE INTRINSICMEMBRANEPRO TEINSFORTHEK$AANDK$APROTEINSTO03))HAVE D HAVETHERESIDUESONT D HE NOT BEENIDENTIlED NOR K$AAND D K$ACOMPONENTSWHI W CHAREREQU Q IRED FOR F THESE PROTEIN PROTEIN ASSOCIATIONS WITH 03)) BEEN D THATTHREE DIMENSIONAL MAPPED)TSHOULD BENOTED CRYSTALSOF HIGHERPLANT03))UNIFORM F LY DIFFRACT8 F

RAYSPOORLY YIELDINGRESOLUTIONSOF¯!DIR   7HILETHECRYSTALSTRUCTUREOFF CYANOBACTERIAL03)) IS OBVIOUSLYQUITEIMPORTANT FOROURUN F DERSTANDINGOF THEPHOTOSYSTEM ITISOFLITTLEUTILITYINIDENTIFYINGTHE BINDING DOMAINSOFT F THE K$AAND D  K$APROTEINS IN

HIGHERPLANTS SINCETHEK$AANDK$APROTEINS HAVENORECOGNIZABLEHOMOLOGYWITHTHEIRAPPARENT CYANOBACTERIAL COUNTERPARTS #YT C ANDD THE K$A PROTEIN ,OWRESOLUTIONSTUDIESHAVEBEENPERFORMED BYY ELECTRONDIFFRACT F IONONTWO DIMENSIONALCRYSTALSOF HIGHERPLANT03))ORBYELECTRONMICROSCOPIC IMAGING OFDETERGENTSOLUBILIZED03))PARTICLESFOLLOWEDBY SIGNALAVERAGING "OEKEMAETAL B(OLZENB N URG R ETAL -ORRISETAL 3TOYLOVAETAL  "OEKEMAETAL "OEKEMAETAL .IELD ETAL #HAPTER (ANKAMERETAL !NUMBER F HESESTUDIESHAVEIDENTIlEDD AREASOF ELECTRONOR OFT IMAGEDENSITY WHICHHAVEBEENASSIGNEDTOTHE K$AANDD K$APROTEINS4HESEREGIONSAREEXPOSED F F THE03))COMPLEX4HE K$A ATTHE LUMENAL FACEO AND  K$A PROTEINS APPEAR TO BE POSITIONED OVER THE#0$SIDEOFF THEHETERODIMERWITH THE-30 BEINGPOSITIONEDD OVERTHE$#0SIDE  )N RECONSTITUTION EXPERIMENTS WITH 03)) MEM BRANESPREPARATIONS THE-30APPEAREDD TO BEREQUIRED FFOR THE BINDING OF F THE  K$A PROTEIN ANDD THE  K$APROTEINAPPEAREDTOBEREQUIREDFORTHEBINDING OFT F HE K$APROTEIN!NDERSSONETAL +A VELAKIANDD 'HANOTAKIS  (OWEVER DIFFERENT F IAL EXTRACTIONEXPERIMENTSUSINGEITHERABUTANOL WATER PHASESEPARATIONSYSTEM 9AMAMOTOETAL  OR (G "ERNIERANDD #ARPENTIER  INDICATEDD THAT THE-30COULDBEREMOVEDFROM03)) PREPARATIONS F THE  K$A WITHOUT THE CONCOMITANT EXTRACTION OF

 AND D  K$APROTEINS)TIS POSSIBLETHATTHE-30 IS REQUIREDFORBINDINGOFTHEK$ACOMPONENTBUT THAT UPON BINDING CONFORMAT F IONAL CHANGES OCCUR WITHINTHE INTRINSICCOMPONENTSOF03))W F WHICHAL LOWTHESELECTIVEREMOVALOFTHE-30ANDRETENTION OFF THEK$AAND D  K$APROTEINS)NANY N EVENT THE -30  K$AAND D K$APROTEINSMUST BEINCLOSE PROXIMITY CROSSLINKING EXPERIMENTS PERFORMED WITHHOMOBIFUNCTIONALCROSSLINKERSn ¯ SPAN INDICATEDTHATTHE-30ISWITHIN¯OFTHEK$A PROTEINANDTHATTHEK$APROTEINISWITHIN¯ OFF THE  K$A COMPONENT %NAMI ET AL   !DDITIONALLY IN CROSS RECONSTITUTION EXPERIMENTS SALT WASHED SPINACH 03)) MEMBRANES WHICH HAD BEENRECONSTITUTED D WITH EITHERCYANOBACTERIALORRED ALGAL -30COULD D ONLYPARTIALLYREBINDSP D INACH K$A PROTEINANDCOULDBINDNOSPINACHK$APROTEIN 4HIS INDICATESTHATTHEREARESTRUCTURAL DETERMINANTS PRESENTONTHESPINACH-30WHI W CHAREREQU Q IRED FO F R THEBINDINGOFTHEK$AANDK$APROTEINSAND WHICHAREABSENT INTHECYANOBACTERIAL AND D RED ALGAL PROTEINS %NAMI ETAL  #HEMICAL MODIlCATION EXPERIMENTS"RICKERAND&RANKEL  INDICATED THATCARB R OXYLATE GROUPSONTHE-30WEREREQUIRED F FORE FlCIENT BINDINGOFF THE K$APROTEINTO03)) 4HEMOSTLIKELYRESIDUESINVOLVEDINTHESEINTERAC TIONSWERETHERESIDUES' D LU 'LU 'LUANDOR 'LU WHI W CH ARENOTCONSERVED INCYANOBACTERIAL -30 0 4HERESIDUESPRESENTONT D HE K$AAND D  K$A PROTEINSTHATAREREQUIRED FORT F HEIRBINDINGTO03)) HAVENOTBEENRIGOROUSLYIDENTIlED,IMITEDPROTE OLYSISOFT F HE K$APROTEIN INDICATED D THATREMOVAL OFF THEEIGHT. TERMINALRESIDUES D !LA 0HE OFT F HIS COMPONENTSOMEWHATLOWEREDITSBINDINGAFlNITYTO 03)) TWICETHEAMOUNTOFPROTE F INWASREQUIRED D TO F ITSABILITYTOSUP SATURATE BINDING BUTDIDD NOTAFFECT PORT/ EVOLUTIONORCOFACTORSEQUESTRATION-IYAO ET AL   2EMOVAL OFF THE . TERMINAL THIRTEEN D 'LU ,EU FFROM THE  K$A PROTEIN RESIDUES HOWEVER ESSENTIALLYABOLISHEDBINDINGOFTHISCOM PONENTTOTHEPHOTOSYSTEM +UWABARAETAL   D LVED INTHECURRENTCRYSTAL 4HESERESIDUESARENOTRESO STRUCTUREOFTHISCOMPONENT#ALDERONEETAL   D HEINTRINSICPROTEINSREQUIRED FORT F HE 4HERESIDUESONT BINDINGOFF THEK$AAND D K$ACOMPONENTS HAVE NOT BEEN IDENTIlED4HE SAME IS TRUE FOR POSSIBLE RESIDUES D ON THE -30 REQUIRED FFOR  K$A PROTEIN BINDINGAND D POSSIBLERESIDUESONT D HE K$APROTEIN REQUIREDFORK$APROTEINBINDING

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ #
'VODUJP J OT PGG UIF 
L%B BOE  L%B 1 1SPUFJOT 2EMOVALOFF THEK$AANDD K$APROTEINSBY.A#L TREATMENTRESULTSINAMARKEDLOSSOF/ EVOLUTION ACTIVITY TOABOUTOFF CONTROL -UCHOFF THELOST ACTIVITYCAN BERESTORED BYRECONSTITUTIONOFF THEEX T TRACTEDPROTEINS TOPROTEIN DEPLETED03))!KERLUN D D BUTONLY IFT F HE ETAL -IYAOAND-URATA  F D INTH T E PRESENCEOFF CALCIUM RECONSTITUTIONIS PERFORME ANDCHLORIDE'HANOTAKISETAL B'HANOTAKIS ETAL  2ECONSTITUTIONOFF THE K$AANDD  K$ACOMPONENTS INTHEABSENCEOFT F HESEINORGANIC COFACTORSDOESNOTRESULTINTHERESTORATIONOF/ EVOLU TION)TSHOULD BENOTEDD THATHIGH NON PHYSIOLOGICAL CONCENTRATIONSOFF CALCIUMANDD CHLORIDECANSUPP U ORT HIGHRATESOF/ EVOLUTIONACTIVITYEVENINTHEABSENCE F HE K$AANDD  K$APROTEINS 'HANOTAKISETAL OFT A-IYAOANDD -URATA A.AKATANI  #HAPTER  VAN 'ORKOM AND 9OCUM  )NHIBITION OF / EVOLVING ACTIVITY BY EXTRACTIONOFF THE K$A ANDD K$APROTEINS ISACCELERATED INTHE LIGHT4HIS APPEARS TO BE DUE TO DIFFERENTIAL BINDING AFlNITIES F BOTH CALCIUM "OUSSAC ANDD 2UTHERFOR FOR F DD  !DELROTH ET AL  ANDD CHLORIDE 7I 7 NCENCJ C USZ ET AL 7INCENCJUSZ ET AL  WHICH ARE F F FOR 3 STATEDEPENDENT4HE BINDINGAFlNITY OF03)) CALCIUMISSIMILARINTHE PRESENCEANDD ABSENCEOFF THE K$AANDK$APROTEINS!DELROTHETAL   (OWEVER THERATEOF BINDINGOFF CALCIUMTO03))WAS ENHANCED INTHEABSENCEOFF THESETWOCOMPONENTS AS WASTHE RATE OF DISSOCIATION OFTHE COFAC F TOR FROM THE03))COMPLEX4HESERESULTSINDICATETHATTHE K$AANDD  K$APROTEINSMODU D LATETHEKON AND KOFF RATE CONSTANTS FOR CALCIUM BUT DO NOT SIGNIlCANTLY ALTERITS +D4HEK$AANDD  K$AALSOAPPEARTO F IONAL BARRIERFORC F HLORIDE 7I 7 NCENCJ C USZ ACTASA DIFFUS ETAL   $EPLETIONOFT F HE K$AANDD  K$APROTEINSMARK R EDLY INCREASES THE ACCESSIBILITY OF F THE MANGANESE CLUSTERTOEXOGENOUSREDUCTANTS'HANOTAKISETAL C  )N INTACT 03)) MEMBRANES HYDROQUINONE DOESNOTINHIBIT / EVOLUTIONWH W EREHY H DROXYLAMINE INHIBITS / EVOLUTIONACTIVITYWITHAN) OF ABOUT M-)N.A#L TREATEDD SAMPLES HOWEVER HYDROQUI NONEEXHIBITSAN) OFF ABOUT +-ANDD THE ) FOR F HYDROXYLAMINEISABOUT +-4HESE RESULTS DEMON STRATETHATTHE K$AANDD K$APROTEINSSHIELD D THE F BULKYEXOGENOUSRE K D DUCTANTS MANGANESECLUSTERFROM 7HILE SMALL REDUCTANTS CAN ACCESS THE MANGANESE CLUSTER IN THE PRESENCE OF F THE  K$A ANDD  K$A

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** PROTEINS THEIR ACCESS IS GREATLY FAC F ILITATED BY THE REMOVALOFTHESEEXTRINSICCOMPONENTS2EMOVALOF THESECOMPONENTSMAY A DIRECTLY INmUENCETH T ESTRUCTURE F THEMANGANESECLUSTER4,STUDIESAPP A EARTOINDICATE OFT THATTHE 31!n STATEISMORESTABLEINCALCIUM DEPLETED 03))PREPARATIONSLACKING THEK$APROTEIN/NO ETAL (OMANNAND D -ADABUSI  FORAN F ALTERNATIVEVIEWPOINT HOWEVER SEE*OHNSSONETAL  7HETHERTHISISDUETOMO D DIlCATIONSOFT F HE ENVIRONMENTOFF THEMANGANESECLUSTERORTOALTERATIONS INTHE1! SITE VIATRANSMEMBRANEEFFECTS ISUNCLEAR )TWAS NEVERTHELESS HYPOTHESIZED D THATTHEOBSERVED DIFFERENT F IALSTABILITYWASDUETOANA D LTERATIONOFF THE CONFORMATIONALSTATEOFTHEMANGANESECLUSTER4HE 3 STATEPARALLELPOLARIZATION@MULTILINE%02SIGNAL F FROMS PINACH03))PREPARATIONSCANALSO BEOBSERVED AFTERREMOVALOFTHESECOMPONENTS#AMPBELLETAL D PREVIOUSLY BEEN OBSERVED A 4HIS SIGNAL HAD ONLY IN CYANOBACTERIAL SAMPLES #AMPBELL ET AL B 4HEAUTHORSHYPOTHESIZEDTHATTHEREMOVAL D  K$APROTEINSMAY A DIRECTLYALTER OFF THE K$AAND THEMAGNETICPROPERTIESOFT F HEMANGANESECLUSTEROR ALTERNATIVELY REMOVALOFTHESECOMPONENTSMAYEFFECT THEINTERACTIONOFF CALCIUMANDORCHLORIDEWITHTHE MANGANESECLUSTERINTHE3 STATE4HESERESULTSSEEM TOINDICATEACLOSEINTERACTIONBETWEENTHEMANGANESE D THE K$AAND D  K$APROTEINS CLUSTERAND $
1VUBUJWF
)PNPMPHVFT
PG
1TC1
BOE
1TC2 $ JO
$ZBOPCBDUFSJB ",!340 SEARCHES OF CYANOBACTERIAL GENOMIC DA TABASES WITH SPINACH  K$A AND D  K$A PROTEIN F BEROFGENESW F WHICH ENCODE SEQUENCESIDENTIFYANUM PROTEINSTHATARESIMILARTOTHESEHIGHERPLANTCOM PONENTS&OREXAMPLE THE .OSTOC . GENE ALLAND THE 3YNEC 3 HOCYSTIS GENE SLLENCODE PROTEINSTHAT SHAREAnIDENTITY n SIMILARITY WITH THEK$APROTEIN WHIL W ETHE 3YNECHOCY CYSTIS SLL GENE ENCODES A PROTEIN WITH A  IDENTITY  SIMILARITY TO THE  K$A PROTEIN 7HAT IF ANY A IN THE FUNCT F ION ROLES THESE PUTATIVE PROTEINS PLAY ASSEMBLYORSTABILITYOFCYANO F BACTERIAL03))ISYET TOBEDETERMINED)NTERESTINGLY ARECENTPROTEOMIC STUDYOFC F YANOBACTERIAL03))+ASHINOETAL  IDENTIlED D PROTEINSWHI W CHWEREASSOCIATED D WITH A HIGHLYPURIlED(IS TAGGED03))PREPARATION"RICKER ETAL  !LLOFF THE KNOWNC K YANOBACTERIAL 03)) PROTEINS EXCEPT0SB. WERE IDENTIlED INTHISSTUDY #HAPTER #HANDLERETAL 4HE0SB1 HOMOLOGUE 3LL PROTEIN WAS ALSO FOUN F D TO BE ASSOCIATED WITHTHIS03))PREPARATION

 *7
.VMUJQMF
&YQSFTTFE (FOFT
PG
UIF
&YUSJO TJD
1SPUFJOT
JO
)JHIFS
1MBOUT )T SHOULD BE NOTEDD THAT TWO DIFFERENT F PSB/ GENES PP PSB/

 AN A D PSB/  AREEXPRESSEDIN !RABIDOP O SIS UN DERNORMAL GROWTHCONDITIONS YIELDING TWO DIFFEREN F T -30S-30 ANDD -30  RESPECTIVELY 4HESE HAVE BEEN IDENTIlED ONTWO DIMENSIONAL )%& 3$3 0!'% GELSOF LUMENALLY LOCALIZEDD PROTEINS +IESELBACHET U ERTETAL  ! HIGH mUORESCENCE AL 3CHUB MUTANTWASIDENTIlEDINWHICHASTOPCODONHADBEEN INTRODUCE D D ATAMINOACID D RESIDUEO D F THEMATURE PROTEIN 'LN A 3TOP INTHEPSB/  GENE WHI W CH LEDTOTHELOSSOFTHISCOMPONENT-URAKAMIETAL  4HEMUTANTPLANTSCOMPENSATED FORT F HIS DEFECT F BYACCUMULATINGAN INCREASEDD AMOUNTOFT F HE-30  PROTEIN4HEMUTANTEXHIBITEDALOWER&V & &M LOWER T 4WOHYPOT Y THESES RATESOF / EVOLUTIONANDD SLOWGROWTH D LAINTHISRESULT)FF ASINGLE-30ISASSOCIATED COULDEXP WITHEACH03))REACTIONCENTER THENTWO POPULATIONS OF03))WOU F LD D EXIST IN !RABIDOPSIS D THYLAKOIDS ONE W CH ISCAPABLEOF HIGH RATESOF CONTAINING-30  WHI ELECTRONTRANSPORTANDEXHIBITSAHIGH&V &M ANDASEC ONDD POPULATIONCONTAINING -30  WHI W CHEX H HIBITSTHE MUTANTCHARACTERISTICS)T ISUNCLEARWH W ATADVANTAGE THEPLANTWOULDACCRUEFROMSUCHANARRANGEMENT Y IFF TWOCOPIESOF-30WEREASSOC F IATED !LTERNATIVELY WITHTHE03))REACTIONCENTERSEEAB A OVE ONEEACH OF -30 AND-30  ONECOPYCOULDPLAYAPRIMARILY F FUNCT IONALROLE -30  ANDD THESECONDD COULDP D LAY A PRIMARILY A STRUCTURAL ROLE -30  AS PREVIOUSLY HYPOTHESIZED"ETTSETAL  )NTHEABSENCEOF A FUNCT F IONAL-30  03))REACTIONCENTERSCONTAIN INGTWOCOPIESOFF -30 WOULDD UNIFORM F LYEXHIBIT THE MUTANT PHENOTYPE &URTHER STUDY IS OBVIOUSLY F IATE REQUIREDD ON THIS INTERESTING MUTANT TO DIFFERENT BETWEENTHESEOROTHER HYPOT H HESES -ULTIPLE EXPRESSED GENES PSB1  PP AND PSB1  F HEK$APROTEIN HAVEALSOBEENOBSERVED IN FORT DOPSIS BYTWO DIMENSIONALELECTROPHORESISOF !RABIDO LUMENALEXTRACTS3CHUBERTETAL  ANDAPUTA TIVEGENEENCODINGASECONDK$APROTEINPSB0  PP HAS BEEN IDENTIlED BYGENOMICANALYSIS4HE0SB0  PROTEINDOESNOTAPP A EARTOBEEXPRESSEDUNDERNORMAL GROWTHCONDITIONSAND ITSPOSSIBLEEXPRESSIONUNDER STRESSCONDITIONSHASNOTBEENEXAMINED!DDITIONALLY L NUMEROUS EXPRESSED PROTEINS WHOSE GENES EXHIBIT HIGHH SIMILARITY TY TOTTHE K$AANDD K$APROTEINS HAVE BEENOBSERVED IN !RABIDO DOPSIS ANDD SPINACH LUMENAL EXTRACTS3CHUBERTETAL  0OSSIBLEFUNCTIONSFOR THESEPROTEINS IN03)) HAVENOT BEEN DETERMINED

 7 $ZUPDISPNF D 1TC7
JO $ZBOPCBDUFSJB 7

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ I SP0##CYTOCHROME+ANG OFF THE 3YNECHOCYSTIS ETAL  !NALIGNMENTOFTHEKNOWNCYANOBACTE RIALSEQUENCESISSHOWNIN&IG

"
*EFOUJàDBUJPO
PG
$ZUPDISPNF $ D BT B $PNQPOFOU
PG
1IPUPTZ TZTUFN
**

# 4USVDUVSF
PG
$Z $ UPDISPNF
D

4HE PRESENCE OF F A LOW POTENTIAL %M ^ n M6 C TYPECYTOCHROMEINCYANOBACTERIAHASBEENKNOWN D -EYERS  AND IS SINCETHES(OLTENAND VARIOUSLY DESIGNATED D AS#YT C ORC ACCORDINGTO ITSALPHAABSORBANCEBANDPEAKPOSITION#YT C HAS D IS AMOLECULARMASSOFF APPROXIMATELY AND AS TYPICAL OFC TYPECYTOC F HROMES BINDS ITS HEMEMOIETY VIATHIOETHERLINKAGESTOAPAIROFCYSTEINERESIDUES 4HEPROTEINWASORIGINALLY SUGGESTED D TOHAVEAROLE IN HY H DROGEN METABOLISM BASED D UPON ITS MID POINT POTENTIALANDTHEFACTTHATTHECYTOCHROMEAPPEARS TOACCUMULATEMOSTABUNDANTLY INALGALMATS WHI W CH AREOFTENC F HARACTERIZED BYSEMI ANAEROBICCONDITIONS ANDTHUS WHEREREDUCTIVEDISSIMILATORYMETABOLISM F LGAL SURVIVAL (OETAL  MAY A BEIMPORTANTFORA + +ROGMANN  4HEASSOCIATIONOFF #YT C WITH THE03))COMPLEXWAS HOWEVER NOTCLARIlEDUNTIL MORERECENTLY 3HENETAL 3HENAND D )NOUE A B )NTERESTINGLY #YT CWASALREADYEVIDENT IN THE lRST HIGHLY ACTIVE 03)) PREPARATION WHICH WASOBTAINED BY,$!/SOLUBILI U ZATIONOFF THYLAKOIDS F FROMT HETHERMOPHILICCYANOBACTERIUM 0HORMIDIUM LAMINOSUM 3TEWARTAND"ENDALL     4HECYTOCHROMEINTHISEARLY PREPARATIONWAS F FOUN D TO BEDIFlCULTTOREMOVE FROMT F HETHY H LAKOID MEMBRANES AND EXHIBITED APPROXIMATELY THE SAME F LD FO D ENRICHMENT DUR D INGTHEPURIlCATIONOFT F HE03)) PARTICLESASTHE#YT B 3TEWARTAND D "ENDALL   (OWEVER ITWASNOTUNTIL3HENCHARACTERIZEDAHIGHLY PURIlED D 03))PREPARATION FROM F 4 ERMOSY 4H S NECHOCOC CUSVULCANUS 3HENETAL  AND DELETED D THEGENE F OM 3YNECHOCYSTIS SP0##THATTHE PRESENCE FR OFF #YT C WASlNALLY DETERMINED D TOBEACOMPONENT OFF THE03))OFCYANO F BACTERIA 3HENETAL A B  D IN MATERIAL USEDD TO 4HIS CONCLUSION HAS BEEN VERIlED D RECENTLY OBTAINTHE03))CRYSTAL STRUCTURESOBTAINED FFROM CYANOBACTERIA :OUNI ET AL  +AMIYA AND3HEN "IESIADKAETAL &ERREIRAET AL  #HAPTERS n  .EVERTHELESS CURRENT EVIDENCE DOESNOTPRECLUDETHEPOSSIBILITYTHATTHE CYTOCHROMEISBIFUNCTIONALWITHASUBPOPULATIONOF THETOTALCELLULARPOPULATIONOFT F HEMOLECULEFUNCT F ION INGOUTSIDETHE LUMENAL DOMAINOF03)) F +ROGMANN  )NDEED THISPOSSIBILITYWASALREADYSUGGESTED D INGTHE INITIALMOLECULARGENETICCHARACTERIZATION DUR

4HESTRUCTURESOFF #YT C FROMTWOSPEC F IESOFCYANO F

BACTERIA 3YNECHOCYSTIS SP0##AND !RTHROSP S IRA MAXIMA HAVE BEENSOLVEDD TOATOMICRESOLUTIONSOF  AND  ¯ RESPECTIVELY &RAZAO ET AL  3AWAYA ET AL   #YT C EXHIBITS BIS HISTIDYL AXIALCOORDINATIONOFF THE HEME&EWHI W CHH MAAY ACCOUNT INPART FOR F ITSLOWMIDPOINTPOTENTIAL OF %M n M6(OLTENAND-EYERS .AVARROETAL  6RETTOSETAL  4HE IMIDAZOLERINGSOFF THEOP POSINGHISTIDINES LIEAPPROXIMATELY INTHESAMEPLANE W CH IS CONSISTENTWITHTHE %02 CHARACTERISTICSTHAT WHI SUGGESTTHATTHEPLANESOFF THE IMIDAZOLERINGSADOPTAN D LLEL ORIENTATION6RETTOSETAL   UNSTRAINED PARA !S DISCUSSED BELOW ADDITIONAL FACTORS BESIDES THE BIS HISTIDYLCOORDINATION LIKELY CONTRIBUTE HOWEVER TOTHE LOWMIDPO D INTPOTENTIALMEASURED IN#YT C #URIOUSLY THEHEMEGROUPINTHEPUBLISHEDSTRUCTURES ISNOTINARUFmEDD CONFORMAT F IONCHARACTERISTICOFF C TYPECYTOCHROMESANDPRE D DICTED BASEDUPONANA D LYSIS OF2AMANSPECTRA6RETTOSETAL  4HERUFmED CONFORMAT F IONISANTICIPATED DUETO D DISTORTIONCAUSED BYSTERICHINDRANCE D IMPOSED BYTHETHIOETHERLINKAGES WITHTHECYSTEINESWITHINTHEHIGHLYCONSERVED#YS F HARACTERISTICOF 8 8 #YS (IS HEME BINDINGMOTIFC C TYPECYTOCHROMES)T H ISNOTCLEARWH W ETH T ERTH T EAB A SENCE OFRUFmINGISAMODELINGOVERSIGHTORAGENUINEFEATURE F HE LOWPOTENTIAL CYTOCHROMES4HEPOLYPEPTIDE OFT CHAIN OF F THE CYTOCHROME EXHIBITS A FO F LD INVOLVING _ HELICESTHATLARGELYENVELOPSTHEHEMEANDIS N F FOUR TYP Y ICALOFF #LASS) C TYPEC Y YTOCHROMES4HISPOINTWAS ILLUSTRATED INTHECRYSTALLOGRAPHICSTUDYOF3AWAYA F ET AL  WHICH ALSO PRESENTS THE PARALOGOUS F HE !RTH T ROSPIRAMAXIMA CYTOCHROME C STRUCTUREOFT THATSERVESASANELECTRONDONORTO0HOTOSYSTEM) 4HE _ CARBONBACKBONESOFTHE#YT C AND#YT C F FROM !RTH T ROSPIRAMAXIMA ARESUPERIMPOSABLE WITH AROOTMEANSQUAREDEVIATIONOFONLY¯ DESPITE LOWOVERALLSEQUENCEIDENTITY )NTERESTINGLY THE D HOMOLOGY ISNOTDISTRIBUTED STRUCTURALANDSEQUENCE UNIFORM F LYACROSSTHETWOPARALOGS&IRST #YT C ISA CONSIDERABLYLONGERPOLYPEPTIDE HAVINGAMINO F YT C4OA LARGE ACIDSVERSUSTHEAMINOACIDSOF# D HECONSIDERABLYLONGER. TER MEASURETHISISDUETOT MINUS^AMINOACIDSLONGER OFTHEMATURE#YTC 4HISLONGER. TERMINUS ABSENT IN#YT C ADOPTSAN

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 **



&IG !LIGNMENTOFTHEAMINOACIDSEQUENCESOFCYTOCHROME C 0SB6 4HEALIGNMENTSWEREPERFORMEDASDESCRIBEDINTHELEGEND OF&IG

ANTIPARALLEL ` SHEETCONlGURATIONWITHANINTERVENING HAIRPINSTRUCTURECONNECTINGTHETWOSTRANDSOFT F HE SHEET&OLLOWINGTHE UNIQUE. TERMINUS HE#YTC AND #YT C HAVEVERYSIMILARFO F LDSUPTOAND INCLUD INGTHECONSERVED#YS X X #YS ( D ISHEME BINDING Q SEQUENCEMOTIFTHATFORMSTHETHIOETHERATTACHMENTS AND HISTIDYL lFT F H IRON LIGAND "EYOND D THIS REGION OF HIGHSEQUENCEAND D STRUCTURALSIMILARITY BETWEEN #YT C AND#YT C ANDTOWARDSTHE# TERMINUS THE Y RHAPS INDICATING SIMILARITY DECREASESDRAMATICALLY PER F HIFTTYPEMUTAT F IONEVENTUALLY LED D TOTHERE A FRAME S PLACEMENTOFTHEMETHIONINEBYHISTIDINEATTHESIXTH LIGAND D POSITIONOFF THEHEME/BVIOUSLY ANA Y LOGOUS ARGUMENTSCOULD BEMADEIFF ONEASSUMESTHAT LOW POTENTIALCYTOCHROMEEVOLUTIONARILYPREDATESTHEHIGH POTENTIALCYTOCHROME(OWEVER THISSCENARIOSEEMS LESS LIKELYGIVENTHE BROADERDISTRIBUTIONOFF THE HIGH POTENTIAL CYTOCHROMES! CLUE TO THE EVOLUTION OF THELOWPOTENTIAL CYTOCHROMEMAY A LIE INTHETANDEM ARRANGEMENTOF#Y F T C AND#Y D T C INTHEGENOMESOF 4 4HERMOSYNECHOCOCCUS AND !NABAENASP0## 4ANDEMCHROMOSOMAL ARRANGEMENTSOF HOMOLOGOUS GENESAREGENERALLYTHOUGHTTOBETHERESULTOFF GENE DUPLICATIONANDSUBSEQUENTDIVERGENCEEVENTS !SMENTIONED T D HE BIS HISTIDYL COORDINATIONOFF THE HEME IRONATLEASTPARTLYACCOUNTSFORT F THELOWPOTENTIAL OF#YT C 4HIS IS CONSISTENT WITHTHE OBSERVATION THATB TYPEC Y YTOCHROMESAND D SEVERAL TETRAHEMEC TYPE Y

CYTOCHROMES HAVERELATIVELYNEGATIVEMIDPO D INTPO TENTIALS^TOnM6 ANDD EXHIBIT BIS HISTIDYL COORDINATIONOFTHEHEME4HEIMINOMOIETYATTHE ¡ NITROGENOFF THEHISTIDYL ATTHESIXTH LIGANDD POSITION CONTRIBUTESTOTHENEGATIVITYOFTHEMIDPOINTBYOF F F FERINGMOREELECTRONDENSITYTOTHECOORDINATEDIRON IN COMPARISON TO A SIXTH POSITION SULFUR F LIGANDD OF METHIONINE ASISTYPICALLYFOUN F D INHIGHERPOTENTIAL C TYPECYTOCHROMESTYPICALLY%M nM6  /N THE OTHER HAND D OTHER FFACTORS CONTRIBUTE TO THE TUNINGOFF THEMIDPO D INTPOTENTIAL3OLVENTEXPOSUREOF THEHEME ESPECIALLYTOTHEPROPIONATES ISTHOUGHTTO HAVEAROLE INMODU D LATINGTHEMIDPO D INTPOTENTIAL WITH INCREASINGEXPOSURECORRELATEDD WITH DOWNSHIFT F INGTHE POTENTIAL! MORE NEGATIVE MIDPOINT POTENTIAL DUE TOINCREASEDD SOLVENTEXPOSUREOFF THEHEMEMOIETY IS DEDUCE D D BYANALYSISOFTREN F DSOFF MIDPO D INTPOTENTIAL VERSUS HEME EXPOSURE ACROSS DIFFERENT SPECIES OF CYTOCHROME THE EFFECT F OF MUTATIONS EXCLUDING OR INCLUDINGSOLVENTFROMT F HEHEME BINDINGPOCKET AND THEEFFECTOFPROTEINUNFOLDINGOF C TYPECYTOCHROMES #ONSISTENTWITH THESEANALYSES CALCULATIONOFSO F LVENT EXPOSUREOFTHEHEMESOF!RTHROSPIRAC F  AND C GIVES EXPOSUREVALUESOFVERSUSOFTHEIRSURFACE AREAS RESPECTIVELY 3AWAY A AETAL  )NTERESTINGLY Y F T C WASMEASURED W ENTHEREDOXPOTENTIAL OF#Y WH WHILETHEMOLECULEWASADSORBEDTOAGRAPHITEELEC TRODESURFACE AN F %M OFnM6WASO F BTAINEDD AND

 THEAUTHORSSUGGESTED D THATTHEAPPROXIMATELYM6 MOREPOSITIVEVALUECOMPAREDTO PREVIOUSSOLUTION D HEEXCLUSIONOFF SOLVENT MEASUREMENTSWAS DUETOT F FROMT HE INTERFACE F BETWEENADSORB R ED D CYTOCHROMEAND THEELECTRODESURFACE#OMPARATIVEINSPECTIONOFTHE D THEAXIAL  $STRUCTURESREVEALSTHATTHE HEMEAND LIGAND D AREACCESSIBLETOWATERINTHE C STRUCTURE BUTNOTTHE#YT C STRUCTURE4HISRAISESTHEPOSSIBIL ITYTHATTHEPROTONATIONSTATEOFT F HE HISTIDINE(IS D CONTROLTHEREDOXPOTENTIAL BUTTHISREMAINS COULD UNEXPLORED!DDITIONALLY THISHYPOTHETICALPROTON ATIONCOULD FAC F ILITATETHEDISSOCIATIONOF LIGAND D AND ALLOWREACTIONWITH ALTERNATE LIGANDSATTHISSIXTH&E COORDINATIONPOSITION(OWEVER DISPLACEMENTOFTHE HISTIDYL LIGAND BY CYANIDEISNOTOBSERVED 6RETTOSET AL  EVENUNDERAWIDERANGEOFF AMBIENTP(S 2,"URNAP UNPUBLISHED THEREFOREDISSOCIATION D LIKELY/T Y HERFACTORS F OFF THE HISTIDYL LIGANDAPPEARSUN INmUENCINGTHEMIDPO D INTPOTENTIAL INCLUDE HY H DROGEN BONDINGTOTHEHEMEANDITSLIGANDSASWELLASMORE GLOBAL CHARGE DENSITY DISTRIBUTIONS OFF THE HEME ENVIRONMENT&OREXAMPLE THE HEMEPROPIONATE$ GROUPSAREHYDROGENBONDEDTOTHEAMIDEBACKBONE IN #YT C OFFER F ING LITTLE OR NO ELECTRON DENSITY U ONREDUCT D IONOFF THEHEME WH W EREAS COMPENSATIONUP THEPROPIONATE$IN#YT C ISHYDROGENBONDEDTOA PAIROF LYSINES WHI W CHCOULD D STABILIZETHEADDITIONOF THEELECTRONTOTHE HEMEOCCURRINGUPONREDUCT D ION OFTHECYTOCHROME $
#JOEJOH
BOE 4UPJDIJPNFUSZ
PG
$ZUPDISPNF
D ! SINGLE #YT C MOLECULE IS MODELED D WITHIN THE CYANOBACTERIAL03))CRYSTALSTRUCTURES:OUNIETAL D 3HEN "IESIADKAETAL  +AMIYAAND &ERREIRAETAL #HAPTERSn 4HISASSIGN MENTWASMADEONTHEBASISOFDENSITYRESEMBLINGTHE Y YTOCHROMEFO F LDINGOF _ HELICES CHARACTERISTIC C TYPEC D AN APPARENT PORPHYR H IN4HE CYTOCHROME IS AROUND SITUATED LATERALLY TO THE REGION OF DENSITY ASSIGNED TO -30 RELATIVE TO THE AXIS PERPENDICULAR TO THE MEMBRANEPLANE4HE HEME&EOF#YT F C ISAPPROX  ¯ FROM THE CENTER OF ELECTRON DENSITY ASSIGNED TO THE -N CLUSTER "ASED D UPON THE POSITION OF F THE DElNED HELICES TH T E HEMEEDGEEXPOSED INTH T EISOLATED CYTOCHROMEAPPEARSTOBEORIENTEDTOWARDSTHE-N  CLUSTER (OWEVER THE CRYSTALLOGRAPHIC STRUCTURE IS PRESENTLYNOTATSUFlCIENTRESOLUTIONTODETERMINETHE COMPOSITIONOFTHEINTERVENINGREGION !LTHOUGH BIOCHEMICAL ANALYSISOFT F HEBINDINGAND

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ F C ISSPARSE SOMEINFORMAT F ION STOICHIOMETRYOF#YT IS AVAILABLE4HE BINDING OF ONE #YT C PER 03)) F ITRATIONCURVES 3HEN MONOMERWASESTIMATED FROMT ET AL  3HEN ANDD )NOUE A W WHICH IS IN AGREEMENTWITHESTIMATESOFCYTOCHROMECONTENTTAK ING INTOACCOUNT#YT B 4ANG ETAL 2EImER ETAL ,AKSHMI ETAL  ANDD THECRYSTAL STRUCTURE ANALYSIS MENTIONED ABOVE )N CONTRAST A MODEL CONTAINING TWO#YT C PER03))MONOMER WAS PROPOSED BASED UPON CONSIDERATIONS OFF THE ISOLATEDCYTOCHROMEANDITSDIMERICPACKINGINTHE CRYSTALSUSEDD TO DElNEITS HIGH RESOLUTIONSTRUCTURE WHENDISSOCIATED FROM03)) F 3AWAYAETAL   (OWEVER THETWO#YT C PER03))MONOMERMODEL SEEMSLESSLIKELY GIVENTHEABOVECONSIDERATIONSAND RECENTRESULTSINDICATINGTHATTHECYTOCHROMEEXISTSAS AMONOMERINSOLUTION,AKSHMIETAL  NOTA DIMERASSUPPOSED BY THEAUTHORSOFF THETWO#YT C PER 03)) MONOMER MODEL 3AWAYA ET AL   4HEBINDINGOF#YT C SHOWSADEPENDENCEUP U ON THEPRESENCEOFF -30 ALTHOUGH THE DEPENDENCEON -30BINDINGAPPEARSTO BESTRONGERINTHEREDD ALGA # #YANIDIUMCALDARIU M %NAMIETAL  COMPARED 4 ERMOSY S NECHOCOCCUS 3HEN TOTHECYANOBACTERIUM 4H ANDD )NOUE A $ESPITETHESEAPPARENTBINDING DIFFERENCES THE ABILITY OF #YT C TO PROMOTE THE F LLOWING REMOVALOF RECONSTITUTIONOFF / EVOLUTION FO THEEXTRINSIC03))PROTEINSWAS HOWEVER FOUN F D TO BESTRICTLYDEPENDENTUPONTHEPRESENCEOF-30IN 03)) FROM F BOTH OFF THESEORGANISMS3HENANDD )NOUE A%NAMIETAL  #HEMICALCROSSLINKING USING%$#FOLLOWEDBYGELELECTROPHORESISANDIM MUNOBLOTANALYSISLEDD TOTHE IDENTIlCATIONOFF THREE D HATCONTAINED#YTC MAJ A ORCROSSLINKEDD PRODUCTST AND0SB5#YTC 0SB5 AND-30#YT C 0SB5 ANDD $ (AN ET AL   /N THE BASIS OFF THESE H ICAL RESULTS ITWASCONCLUDEDD THAT#YT C ISINPHYS CONTACTWITHTHEOTHERTWOEXTRINSIC PROTEINS3INCE F YT C HAD BEENPREVIOUSLY ASSESSED THEBINDING OF# F -30 3HEN ANDD )NOUE TO BIND INDEPENDENTLY OF A THEAUTHORSALSOCONCLUDEDTHATTHE$PRO TEIN FORMSA F DIRECTBINDINGSITEFORT F HECYTOCHROME 7ITH THE IMPORTANTCAVEATTHATTHELUMENALLYEXPOSED PORTIONSOFTHEINTRINSIC03))POLYPEPTIDESREMAIN LARGELY UNRESOLVED T D HISCONCLUSIONISSUPPORTED BY THERECENT03))CRYSTALSTRUCTURESWHI W CHPLACES#YT C NEAR THE $ PROTEIN AND IN CONTACT WITH -30 ANDD 0SB5 :OUNI ET AL  +AMIYA ANDD 3HEN  "IESIADKAETAL  &ERREIRAETAL  #HAPTERSn  )NSPECTIONOFT F HEAVAILABLECRYSTAL STRUCTURESSUG

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** GESTS THAT AN ADDITIONAL CONTACT WITH THE LUMENAL PORTION OF #0 MAY EXIST )N THIS REGARD RECENT EXAMINATIONOF 3YNECHOCY CYSTIS 03))PARTICLES BEARING THEMUTATION!RG3ERIN LOOP%OF#0 F HAS DEM ONSTRATEDTHATTHEBINDINGOF#YT C BUTNOT-30 IS PERTURB R ED4HISOBSERVATIONWASTAK A ENASEVIDENCETH T AT #0FORMSPARTO F F THE#YT C BINDINGSITE"RICKER ETAL  )TISWORTHMENTIONINGTHATTHEFAILURE OFF THE%$#CROSSLINKINGTO IDENTIFY A#YT C #0 PRODUCTMAYRE D mECTTHEABSENCEOFCAR F RBOXYLATE PRI MARYAMINOGROUPSATTHEPROPOSEDBINDINGINTERFACE F HCONTACTSIFT F HEY ORTHESOLVENTINACCESSIBILITY OFSUC INDEED D EXIST!NANALOGOUSEXPERIMENTINWHI W CH ISO LATED 3Y 3 NECHOCY CYSTIS THY H LAKOIDSCONTAINING MUTATIONS INTHE LOOP%OFF #0CAUSED AWEAKENED BINDING F T C2 OFF -30 BUTLITTLELOSSINTHEBINDINGOF#Y ,"URNAP UNPUBLISHED 3UMMINGUP THESERESULTS D YT COLLECTIVELY SUPPORTTHEHYPOTHESISTHAT-30AND# C OCCUPY INDEPENDENT PERH R APSADJACENT BINDING SITES ON THE INTRINSIC PORTION OF THE 03)) COMPLEX AND D THATTHESTRUCTURAL INTERACTIONS BETWEENTHESETWO EXTRINSICPROTEINSARERELATIVELYWEAK 3TRUCTURALALIGNMENTOFTHEATOMICRESOLUTIONOF #YT C STRUCTURE WITHTHEMOREPOORLYRESOLVED D CY TOCHROMEMODELED D WITHINTHE03))CRYSTAL STRUCTURE SUGGESTSSEVERALINTERESTINGFEATURESOFTHE#YT C F HESPATIAL DISTRIBUTIONOFCONSERVE F D AND !NALYSISOFT D IN#YT C REVEALS NOTUNEXPECT MUTABLERESIDUES EDLY THAT RESIDUES PREDICTED TO BE AT THE INTERFACE BETWEEN#YT C AND ITSBINDING SITEONTHEINTRINSIC PORTIONOFF 03))ARETHEMOST HIGHLYCONSERVED4HE STRICTLYCONSERVED!RG 3YNECHOCYSTIS NUMBERING D TOWARDSTHEINTRINSICPORTIONOFT F HE APPEARSORIENTED 03))COMPLEXAND D THUSISACANDIDATEFOR F PARTICIPAT INGINANIONICBINDINGINTERACTION"OTHTHEAMINO D CARRBOXY TERMINI PROTRUDEANDAREPRE D DICTED D TO BE AND D TOWARDSTHE INTRINSICPORTIONOFF THECOMPLEX ORIENTED 4HE# TERMINALPOSITIONISOCCUPIEDBYPHENYLALANINE ORTHECONSERVATIVEREPLACEMENT TYROSINE % 'VODUJPO
PG
$ ' $ZUPDISPNF
D $ESPITE ITS LOW%M #YT C IS INTIMATELYASSOCIATED WITHTHEWATER OXIDATIONCOMPLEX WHICHOPERATESAT POTENTIALS GREATERTHANM64HEREISNOEVI DENCETHAT#YT C PARTICIPATES INTHEREDOXEVENTSOF T THEWATEROXIDATIONREACTION)NDEED CURRENTEVIDENCE WEIGHS HEAVILYAGAINSTSUCHAPARTICIPATIONINWATER OXIDATION ENZYMOLOGY .EITHER BIOCHEMICAL NOR GENETICREMOVALOF#YT C RESULTSINTHE ABOLITION D )NOUE B OFF WATEROXIDATIONACTIVITY 3HENAND

 %NAMIETAL 3HENETAL A B %NAMI ETAL  2EMOVALOF#YT C DOES AFFEC F TTHE KINETICS OF RKSTABILITY OFF THE 3 THE 3 STATETRANSITIONSANDD THEDARK AND 3 STATES BUT/ EVOLUTIONPERSISTSANDD EXHIBITS ITSCHARACTERISTIC PERIODFOUROSCILLATIONSUNDERmASH ILLUMINATION 3HENETAL  'ENETICREMOVALOF #YT C RESULTS INAPRONOUNCED DESTABILIZATIONOFT F HE ENTIRE03))COMPLEXb NORMALACCUMULATION 3HEN ETAL B3HENETAL  $ESTABILIZATIONOF THE03))COMPLEX ISAGENERAL PHENOTYPESHARED BYA VARIETYOFMUTATIONSINDIFFERENT03))PROTEINS6ER MAASETAL  ANDD THUSIT IS DIFlCULTTOASCRIBEA PARTICULARFUNCT F IONAL SIGNIlCANCETOTHIS FEATURE&UR F

THERMORE ITISIMPORTANTTONOTETHATWHENMAXIMAL WHOLECELL/ EVOLUTIONRATES INTHEDELETIONMUTANT ARENORMALIZEDD TOTHEREDUCE D D03))CONTENT D THENTHE MAXIMAL03))TURNOVERAPPEARSLARGELYUNAFFECTED 3HENETAL A 4HISlTSWITH THEOBSERVATION D INGTHE3 A 3 A 3 TRANSITION THAT / RELEASE DUR MEASUREDPOLAROGRAPHICALLYUNDERmASHILLUMINATION ISONLY SLIGHTLY RETARDED 3HENETAL  4HEREFORE F ITIS HIGHLYUNLIKELYTHAT#YT C IS DIRECTLY INVOLVED IN THEREDOXEVENTSOF3 STATECYCLINGDURINGTHEPROCESS OFWATEROX F IDATION/NTHEOTHERHAND D BINDING #YT C IS IMPORTANT FFOR THE STABILITY OFF THE -N  AND APPEARSTOMODULATEITSSTRUCTUREANDREACTIVITYINA MANNERNOTUNLIKETHE K$APROTEIN4HEPRESENCE F T C ISCORRELATEDD WITHTHEABILITYTO FORMT F HE OF#Y G%02SIGNALOFTHE-N  POISEDINTHE3 STATE IN 3YNECHOCY CYSTIIS 03))PARTICLES ,AK A SHM H IETAL   -ATERIAL DEPLETEDD OF#Y F T C LOSTTHEABILITYTO FORM F THESIGNALANDRECONSTITUTIONWITHPURIlED#YT C LEDD TOAPARTIALRESTORATIONOFT F HESIGNAL4HE G F HEEXPENSEOFT F HE SIGNAL HASBEENPROPOSEDD TOFORMATT MULTILINESIGNALANDTHEINTERCONVERSIONBETWEENTHE TWOSIGNALS ISTHOUGHTTORESULT FROMA F LTERATIONS INTHE EXCHANGECOUPLINGSWITHINTHE-N  DE0AULAETAL "OUSSACETAL  4HEYIELDOFTHEG SIGNAL DEPENDS INVERSELY UPONTHECHLORIDEBINDING /NOETAL ,INDBERGANDD !NDREASSON  ANDDIRECTLYUPONTHEBINDINGOFTHE  K$A ANDD  K$APROTEINSIN HIGHERPLANT03))PREPARATIONS DE 0AULAETAL  4HESIMILAREFFECTSO F F STABILIZA TIONOFTHEGSIGNALBYTHEBINDINGOF#YT C TO THECYANOBACTERIAL 03))ANDD THEK$AANDD K$A PROTEINS INPLANT03))LEDD TOTHECONCLUSIONTHATTHE K$APROTEINISFUNCTIONALLYHOMOLOGOUSTO#YTC ,AK A SHM H IETAL  7HETH T EROTH T ERFUNCT F IONAL PROP ERTIESOFT F HE K$APROTEINAREMIRRORED BY#YT C REMAINSLARGELYUNRESOLVED!SDISCUSSEDABOVE THE K$APROTEIN ISGENERALLY AGREEDD TOCONTROL ACCESS

 OFF CALCIUM TO ITS BINDING SITE NEAR THE -N  )T IS NOT CLEAR WHETHER #YT C EXERTS A SIMILAR CONTROL F LCIUMINCYANOBACTERIAL OVERTHEACCESSIBILITY OFCA AND D RED ALGAL 03)) .EVERTHELESS THERE ARE SOME INDICATIONS THAT THE FUNCTIONAL HOMOLOGIES EXTEND BEYOND D STABILIZATIONOFF THE GSIGNAL"IOCHEMI CAL DEPLETION RECONSTITUTIONSTUDIESWITH #YANI # DIUM DEMONSTRATEDTHATREMOVALOF#YT C AND0SB5MAKES / EVOLUTIONSTRONGLY DEPENDENTUPONCALCIUMAND CHLORIDE AS IS FOUN F D WITH HIGHERPLANT03)) DEPLETED OFTHEANDK$A PROTEINS(OWEVER ITISWORTH NOTING THAT THE LEVEL OFF TOTAL RECONSTITUTION IN THIS SYSTEM RARELY EXCEEDS  SO THAT MITIGATION BY THESEIONSOFDAMAGETO03))MAYCOMPLICATETHE ANALYSIS"ETTERLEVELS^ OFRESTORAT F IONOF INITIAL / EVOLUTIONACTIVITYAREOBTAINEDUSINGTHETHERMO PHILICCYANOBACTERIUM 4HERMOSYNECHOCCUSVULCANUS 3HENAND)NOUE  D B 5SINGTHISSYSTEM ITWAS D THATCALCIUMALONECANNOTSUB U STITUTEFO F R CONCLUDED #YT C WHICHDIFFERSFROMTHEABILITYOFCALCIUM TORESTOREACTIVITY LOSTUPONREMOVALOFF THEK$A D K$APROTEINSOFF PLANT03)) 3HENAND D )NOUE AND B #AREFULSTUDIESUSING #HELEX TREATEDBUFFERS TOEXAMINETHECALCIUMDEPENDENCEOFWATEROX F IDA TION INAMANNERALLOWINGKINETICDISCRIMINATIONOF HIGHAFlNITYCALCIUMBINDINGSITESREMAIN HOWEVER TOBEPERFORME F D WITH CYANOBACTERIALAND D RED ALGAL PREPARATIONS4HEREFORE F IT ISNOTYETPOSSIBLETOMAKE THOROUGHCOMPARISONSWITHEXPERIMENTSTESTINGTHE F F HEK$APROTEINONCALCIUMBINDING IN FT EFFECTSO HIGHERPLANTPREPARATIONS$ELETIONOFF #YT C IN 3YN 3 ECHOCYSTIS I INCREASESTHE DEMAND D FOR BOTHCALCIUM AND CHLORIDE INTHECULTUREMEDIUM FOR03)) F DEPENDENT GROWTH WHI W CH HASBEENTAKENASEVIDENCETHAT#YT C MODULATESEITHERTHEAFlNITYORACCESSIBILITYOF THE03))SITES FORT F HE INORGANICCOFACTORS F 3HENETAL F INTHE4,  4,MEASUREMENTSREVEALANUPSHIFT PEAKFORTHE 31!n AND31"nCHARGERECOMBINATIONS UPONREMOVAL OFF CALCIUMINHIGHERPLANT03)) INDICAT INGANALTERATION INTHEREDOXPROPERTIESOFT F HE -N  UPONCALCIUMDEPLETION/NOAND)NOUE /NO D )NOUE B/NOAND)NOUE A/NOAN D D AND )NOUE /NOETAL  4HISUPSHIFT F INTHE4, PEAKS HOWEVER ISONLYOBSERVEDINTHEPRESENCEOF THE K$APROTEIN /NOETAL  !NUPSHIFT F IN THE4, PEAK FORT F HE31!n AND31"nCHARGERECOMBINA TIONSISALSOOBSERVEDINTHE#YT C DELETION MUTANT 3HENETAL  4HESERESULTSARECONSISTENTWITH THEPROPOSITIONTHAT#YT C MODU D LATESTHE BINDING OFCALCIUMINCYANOBACTERIAL03))!GAINITISIM PORTANTTONOTETHATSIMILAROBSERVATIONSHAVE BEEN

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ F IT MADEWITH THE-30 DELETIONMUTANTANDD THEREFORE ISDIFlCULTTOEXCLUDEAMOREGENERALIZEDEFFECTDUE TOTHEABSENCEOFF EXTRINSICPROTEINSPERSE "ESIDES AFFECT F ING THE CALCIUM ANDD CHLORIDE RE QUIREMENTS #YTCAPPEARSTOMODIFYTHESTABILITY F HE -N 4HEABSENCEOF# F YT C DUETO D GENETIC OFT DELETION CAUSES A DRAMATIC IN VIVO DESTABILIZATION OFTHE/ EVOLUTIONACTIVITY INTHEDARKANDANIN CREASEDD SUSCEPTIBILITY TO PHOTOINHIBITION "OTH OF THESECHARACTERISTICSARESHARED BYDELETIONMUTANTS LACKING -30 "URNAP AND 3HERMAN  -AYES ETAL "RICKER  7HILESUSCEPTIBILITY TO PHOTOINHIBITION MAY BE A GENERAL PROPERTY SHARED BYMANYDONOR SIDEMUTANTS6ERMAASETAL -AY A ESETAL  THEINSTABILITY OF/ F  EVOLUTION ACTIVITY IN THE DARK R IS NOT BUT HAS BEEN ASSOCIATED WITHMUTANTSTHATLACK-30ORARESEVERELYIMPAIRED IN-30BINDING %NGELSETAL "URNAPETAL 'LEITERETAL "URNAPETAL   4HE / EVOLUTIONACTIVITYLOSTINTHEDARKISREADILYRESTORED BY RE ILLUMINATIONUNDERWEAK LIGHT4HEKINETICSOF F HOTO ACTIVITYRESTORATIONRESEMBLETHE KINETICSOFP ACTIVATION#HENIAEAND-ARTIN %NGELSETAL "URNAPETAL  SUGGESTINGTHATTHELOSS F IVITYISDUETOT D HE DISASSEMBLYOFF THE -N  AND OFACT THERESTORATIONOFACTIVITYISDUETOTHELIGHT DRIVEN F LS INTOTHEAPOPROTEIN3HENETAL ASSEMBLYOFMETA F ION INSTABILIZATIONOFF THEWATEROXIDA  !FUNCT TIONCOMPLEXISALSOINDICATEDBYEXPERIMENTSSHOW INGTH T ATBIOCHEMICALREMOVAL ANDD RECONSTITUTIONOFF #YT C IN 3YNEC 3 HOCOCCUS SP0##TH THYLAKOIDSRESULTS INALOWERTEMPERATURETHRESHOLDFORTHEINACTIVATION OF / EVOLUTIONBY HEATTREATMENT .ISHIYAMAETAL F INTAINING  #LEARLY #YT C IS IMPORTANT FORMA THESTRUCTURALINTEGRITYOFTHEWATEROXIDATIONCOMPLEX D LATETHEACTIVITYOF AND INDOINGSO ITAPPEARSTOMODU F HE-N 7HATISNOTCLEARIS THECATALYTICACTIVITYOFT W WHETHERORNOTITMAYHAVEADDITIONALFUNCTIONSTHA T WOULD INVOLVETHE HEMEMOIETY OFF THEPROTEIN)T IS POSSIBLETH T AT#YTC ISMERELYANEVO L LUTIONARYVESTIGE OFAPREVIOUSLYOPERATINGDONOR SIDEELECTRONTRANSFER A THATNO LONGEROPERATES INTHECONTEMPORARY PATHWAY WATEROXIDIZINGPHOTOSYSTEM(OWEVER ITISDIFlCULT TORATIONALIZETHERETENTIONOFTHEHEMEMOIETYTHOUGH THEEONSAND INSOMANY N TAXONOMICGROUPS 7*
5IF
1TC6
1SPUFJO
JO
$ZBOPCBDUFSJB !SWITH#YTC THE 0SB5PROTEINWASALREADYEVIDENT IN EARLY 03)) CORE PARTICLE PREPARATIONS OBTAINED

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 **



&IG !LIGNMENTOFT &I F HEAMINOACIDSEQUENCESO D F THEK$APROTEIN0SB5 4HEALIGNMENTSWEREPERFORME F D AS DESCRIBED INTHE LEGENDOF&IG

FFROM THERMOPHILIC CYANOBACTERIA (OWEVER UNLIKE THECYTOCHROME ASPECIlCFUNCTIONINTHERETENTIONOF WATEROXIDATIONACTIVITYWASREALIZED FOR0S F B5DUR D ING THESEEARLYINVESTIGATIONS3TEWARTAND D "ENDALL  3TEWARTETAL A B 3PECIlCALLY ITWASNOTICED THAT/ EVOLVINGACTIVITYWAS DIMINISHEDUPONT D HE LOSS OFF 0SB5 SO CALLED D  K$A OR  K$A PROTEIN DEPENDINGUPONTHESPECIES ANDTHATTHERETENTION D 0SB5DEPENDEDUPONT D HEINCLUSION OFF ACTIVITY AND OF HIGH CONCENTRATIONS OF F GLYCEROL IN THE ISOLATION BUFFERS3TEWARTETAL A B  #OMPARED D TO THE OTHER 03)) EXTRINSIC PROTEINS INFORMAT F IONONTHE BINDINGAND FUNCT F IONOF0S F B5 IS SPARSE4HEMOSTRECENT03))CRYSTALSTRUCTUREMODELS +AMIYAAND D 3HEN  AND D EARLIERMODELSBASED UPONELECTRONMICROSCOPICEXAMINATIONOFF POLYPEP TIDE DEPLETEDANDRECONSTITUTED03))PARTICLESHAVE PLACED D 0SB5INASTRUCTURALLY BRIDGINGPOSITION BE TWEEN-30AND D #YT C +UHLETAL .IELD D ETAL  !CONTACTWITHBOTH-30ANDCYTOCHROME C ISALSO INDICATED BYY THE%$#CROSSLINKING EXPERIMENTS D ABOVE (ANETAL  AND D THUSSUPPORT MENTIONED THECRYSTALSTRUCTUREASSIGNMENT4HISASSESSMENTIS ALSOCONSISTENTWITH TH THEOBSERVATIONFROM F BIOCHEMICAL DEPLETION RECONSTITUTION EXPERIMENTS SHOWING THAT THEBINDINGOF0SB5COMPLETELYDEPENDSUPONTHE PRESENCEOF BOTH -30AND# D YT C 3HENAND D )NOUE A B%NAMI ETAL   !SWITH THEOTHER EXTRINSICPROTEINS 0SB5INmUENCESTHEDEMANDFOR 03))CALCIUMAND D CHLORIDE)N #Y # ANIDIUMCA D LDARIU D M

ATHERMOPHILIC ACIDOPHILICREDALGA THE0SB5PRO TEIN FFUNCTIONS TO MINIMIZE THE CALCIUM AND CHLORIDE REQUIREMENTSOFF THEWATEROXIDATIONREACTION!SWITH #YT C BIOCHEMICAL DEPLETION RECONSTITUTIONSTUD IES WITH 3YNECHOCOCCUS SP0## DEMONSTRATED THAT0SB5 ISREQUIRED FORMAX F IMAL STABILIZATIONOFT F HE WATER OXIDATION COMPLEX AGAINST HEAT INACTIVATION .ISHIYAMAETAL   0SB5FROM #YANIDIUM HAS BEEN HETEROLOGOUSLY EXPRESSED IN % COLI AND F LYPEPT L IDE DEPLETED SHOWNTORECONSTITUTEACTIVITYOFPO 03))CORECOMPLEXESTOASIMILAREXTENTASTHENATIVE PROTEIN/HTAETAL  !NALIGNMENTOFF THEKNOWN K CYANOBACTERIAL SEQUENCESISSHOWNIN&IG 7**
$PODMVTJPOT $ESPITECONSIDERABL A EPROGRESSINOURUNDERSTANDINGOF F IONAL ANDD STRUCTURAL ATTRIBUTESOFT F HEEXTRINSIC THE FUNCT PROTEINSOF03)) ITISCLEARTHATSIGNIlCANTQUESTIONS REMAINUNANSWERED4HEMECHANISMS BYWHI W CHTHESE PROTEINSFAC F ILITATE/ EVOLUTIONATPHYS H IOLOGICALCOFAC F TORCONCENTRATIONSREMAINTOBEDETERMINED 7HILE F HE BASICSTRUCTURALORGANIZATION ANUNDERSTANDING OFT OFT F HECYANOBACTERIAL EXTRINSICSUB U UNITSAPPEARSTOBE NEARATHAND:OUNIETAL +AMIYAAND3HEN "IESIADKAETAL &ERREIRAETAL  #HAPTERS n THE ORGANIZATION OFF THE HIGHER PLANTEXTRINSICSUBUNITSREMAINSLARGELYSPECULATIVE Y THE ONGOING DEBATE CONCERNING THE !DDITIONALLY

 STOICHIOMETRYOFT F THE-30INCYANOBACTERIAL VS HIGHER PLANT03))REMAINSUNRESOLVED!RATIONALEFORTHE PRESENCEOFF THELOWPOTENTIAL #YT C INTHECYANOBAC TERIAL PHOTOSYSTEMISALSOLACKING4HEPOSSIBLEROLES AND D FUNCTIONS OFTHE PSB0 0 ANDPSB1 HOMOLOGUESIN THECYANOBACTERIAAREONLY BEGINNINGTO BEEXPLORED &INALLY THEROLEIFF ANY OFT F HEOBSERVED HETEROGENEITY OFTHEEXTRINSICSUBUNITSOFHIGHERPLANT03))HASYET TOBEDETERMINED4HEREISUNDOUB U TEDLYMUCHWORK R THATNEEDSTOBEDONE "DLOPXMFEHNFOUT 4HISWORK RKWASSUPPORTED BY GRANTSFROMT F HE.ATIONAL 3CIENCE&OUNDATIONTO4-"AND2,"ANDBYAGRANT FFROM THE $EPARTMENT OF %NERGY TO4-" 3PECIAL THANK N STO-S,AURIE+&RANK N EL FOR F HERCRITICAL READING OFF THISCHAPTERAND HERMANY HELPFU F LSUGGESTIONS 3FGFSFODFT !BRAMOWICZ$!AND$ISMUKES'# -ANGANESEPROTEINS ISOLATEDFROMSPINACHTHYLAKOIDMEMBRANESANDTHEIRROLEIN / EVOLUTION))!BINUCLEARMANGANESE CONTAININGK$A PROTEIN A PROBABLE COMPONENT OF THE WATER DEHYDROGENASE SYSTEM"IOCHIM "IOPHYS!CTAn H  !DELROTH TH 0 ,INDBERG R +AND!N D DREASSON, % D  3TUDIESOF# F A BINDING INSPINACH 0HOTOSYSTEM))USING #A"IOCHEMISTRY n !DIR.  #RYSTALLIZATIONOFF THEOXYGEN EVOLVINGREACTION CENTREOF0HOTOSYSTEM))INNINEDIFFERENTDETERGENTMIXTURES !CTA#RYSTALn !HMED! 4AJMIR 2IAHI(!AND#ARPENTIER2 !QUAN TITATIVESECONDARYSTRUCTUREANALYSISOFTHEK$AEXTRINSIC PROTEINOFF 0HOTOSYSTEM))BY&4)2SPECTROSCOPY&%"3,ETT  n !KERLUND D ( % *ANSSON#AND!N D DERSSON"  2ECONSTITUTION OFF PHOTOSYNTH T ETICWATERSPLITTING ININSIDE OUTTHYL T AKOIDD VESICLES AND IDENTIlCATION OFF A PARTICIPATING POLYPEPTIDE "IOCHIM "IOPHYS!CTAn !NATI 2 AND !DIR .  #RYSTALLIZATION OF DIMERS OF THE MANGANESE STABILIZINGPROTEINOF0HOTOSYSTEM))0HOTOSYNTH 2ES  n !NDERSSON" ,ARSSON# *ANSSON# ,J , UNGBERG5ANDD !KERLUND ( %  )MMUNOLOGICAL STUDIES ON THE ORGANIZATION OF PROTEINS INPHOTOSYNTHETICOXYGENEVOLUTION"IOCHIM"IOPHYS !CTAn "ALSERA- !RELLANO*" 'UTIERREZ*2 (EREDIA0 0 2EVUELTA*, AND $E ,AS 2IVAS *  3TRUCTURAL ANALYSIS OF THE PSB1 PROTEINOF0HOTOSYSTEM))BY&OURIERTRANSFORMINFRAREDAND CIRCULARDICHROICSPECTROSCOPYANDBYBIOINFORMATICMETHODS "IOCHEMISTRYn D IER2 4HEACTIONOFF MERCURYON "ERNIER-AND#ARPENT THE BINDINGOFF THEEXTRINSICPOLYPEPTIDESASSOCIATEDD WITH THE

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ WATEROXIDIZING COMPLEXOFF 0HOTOSYSTEM))&%"3,ETT n "ETTS3 2OSS*2 (ALL+5 0ICHERSKY%AND9OCUM#&A &UNCTIONALRECONSTITUTIONOF0HOTOSYSTEM))WITHRECOMBINANT MANGANESE STABILIZING PROTEINS CONTAINING MUTATIONS THAT REMOVE THE DISULlDE BRIDGE "IOCHIM "IOPHYS !CTA  n "ETTS3$ 2OSS*2 0ICHERSKYY %AND D9OCUM#& B #OLD SENSI TIVEASSEMBLY M OFAMUTANTMAN F GANESE STABILI A ZINGPROTEINCAUSED BY AVAL TOALAREPLACEMENT"IOCHEMISTRY n "ETTS3$ 2OSS*2 0ICHERSKY%AND9OCUM#& -UTATION 6 6AL!LAWEAKENSBINDINGOFTHE K$AMANGANESESTABILIZ

INGPROTEINOF0HOTOSYSTEM))TOONEOFTWOSITES"IOCHEMISTRY  n "ETTS3$ ,YD , AKIS 3IMANTIRIS. 2OSS*2AND9OCUM#& D  4HECARB R OXYL TERMINALTRIPEPTIDEOFF THEMANGANESE STABILIZING PROTEINISREQUIRED FORQUANT F ITATIVEASSEMBLY M INTO0HOTOSYSTEM)) AND FOR F HIGHRATESOFF OXYGENEVOLUTIONACTIVITY" Y IOCHEMISTRY  n "IESIADKE* ,OLL" +ERN* )RRGANG+ $AND:OUNI! #RYSTAL STRUCTURE OF CYANOBACTERIAL 0HOTOSYSTEM )) AT  ¯ RESOLUTION!CLOSERLOOKATTHE-N CLUSTER0HYS#HEM#HEM 0HYSn "OEKEMA%* (ANKAMER" "ALDD $ +RUIP * .IELD* "OONSTRA!& D D 2OGNER-A 3UPRAMOLECULARSTRUCTUREOFT F HE "ARB R ER*AND F GREENPLANTSAND D CYANOBACTERIA 0HOTOSYSTEM))COMPLEX FROM 0ROC.ATL !CAD 3CI 53!n "OEKEMA%* (ANKAMER" "ALD$ +RUIP* .IELD* "OONSTRA!& "ARBER*AND2OGNER-B 3UPRAMOLECULARSTRUCTUREOFTHE 0HOTOSYSTEM))COMPLEXFROMGREENPLANTSANDCYANOBACTERIA 0ROC.ATL!CAD3CI53!n "OEKEMA%* .IELD* (ANKAMER"AND"ARBER* ,OCALIZA TIONOFF THE K$ASUB U UNITOFT F HEOXYGEN EVOLVINGCOMPLEX OFF 0HOTOSYSTEM )) BY ELECTRON MICROSCOPY Y %UR * "IOCHEM  n "OEKEMA %* * VAN "REEMAN *&, VAN 2OON ( AND D $EKKER *0  #ONFORMAT F IONALCHANGES IN0HOTOSYSTEM))SUPERCOM PLEXESUPONREMOVALOFEXTRINSICSUBUNITS"IOCHEMISTRY n "ORTHAKUR$AND(ASELKORN2 .UCLEOTIDESEQUENCEOF THEGENEENCODINGTHEK$AWATEROXIDIZINGPOLYPEPTIDEIN !NABAENA SPSTRAIN0##AND ITSEXPRESSIONIN % COLI 0LANT-OL "IOL  n "OUSSAC!AND D 2UTHERFOR F D!7 D  .ATUREOFF THE INHIBITION OFF THEOXYGEN EVOLVINGENZYMEOFF 0HOTOSYSTEM))INDUCE D D BY .A#L WASHING AND D REVERSED BY THE ADDITION OFF #A OR 3R &%"3,ETTn "OUSSAC! +UHL( 5N3 2OGNER-AND2UTHERFORD!7 %FFECTOFNEAR INFRAREDLIGHTONTHE3 STATEOFTHEMANGANESE COMPLEX OF 0HOTOSYSTEM )) FROM 3 3YNECHOCOCCUS ELONGATUS "IOCHEMISTRYn "RICKER4- 4HESTRUCTUREAND FUNCT F IONOF#0A AN F D D#0A  IN0HOTOSYSTEM))0HOTOSYNTH2ESn "RICKER4- /XYGENEVOLUTION INTHEAB A SENCEOFF THEK$A MANGANESE STABILIZING PROTEIN"IOCHEMISTRY n  "RICKER4-AND&RANKEL,+ 5SEOFAMONOCLONALANTI BODYINSTRUCTURALINVESTIGATIONSOFTHEK$APOLYPEPTIDEOF 0HOTOSYSTEM))!RCH"IOCHEM"IOPHYSn "RICKER4-AND&RANKEL,+ 4HESTRUCTUREANDFUNCTION OFF THEK$AEXTRINSICPROTEINOFF 0HOTOSYSTEM))!CRITICAL REVIEW0HOTOSYNTH 2ESn

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** "RICKER4-AND D &RANKEL,+ 4HESTRUCTUREAND FUNCT F ION OF #0 AND #0 IN 0HOTOSYSTEM )) 0HOTOSYNTH 2ES  n "RICKER4-AND&RANKEL,+ #ARBOXYLATEGROUPSONTHE MANGANESE STABILIZINGPROTEINAREREQUIREDFOREFlCIENTBIND ING OFF THE  K$A PROTEIN TO 0HOTOSYSTEM )) "IOCHEMISTRY  n "RICKER4- /DOM72ANDD 1UEIROLO#" #LOSEASSOCIATION OFF THEK$AEXTRINSIC PROTEINWITH THEAPOPROTEINOFF #0A  IN0HOTOSYSTEM))&%"3,ETTn "RICKER4- -ORVANT* -ASRI. 3UTTON(AND&RANKEL,+ )SOLATIONOFAHIGHLYACTIVE0HOTOSYSTEM))PREPARATIONFROM 3YNECHOCYSTIS USINGAHISTIDINE TAGGEDMUTANTOF#0 "IOCHIM"IOPHYS!CTAn "RICKER4- 9OUNG! &RANKEL ,+ANDD 0UTNAM %VANS#  )NTRODUCTION OFF THE !RGTO 3ERMUTATIONINTHE LARGEEXTRIN SIC LOOP%OFT F HE#0 PROTEINOF 3Y 3 NECHOCY CYSTIS SP0## LEADSTOTHELOSSOFF CYTOCHROME C BINDING TO0HOTOSYSTEM)) "IOCHIM"IOPHYS!CTAn "URNAP 2, AND 3HERMAN ,!  $ELETION MUTAGENESIS IN 3YNECHOCYSTIS SP0##INDICATESTHATTHE-N STABILIZING PROTEINOF0HOTOSYSTEM))ISNOTESSENTIALFOROXYGENEVOLUTION "IOCHEMISTRYn "URNAP2, 3HEN* 2 *URSINIC0! )NOUE9ANDD 3HERMAN,!  /XYGENYIELDD AND THERMOLUMINESCENCECHARACTERISTICS OFF ACYANOBACTERIUM LACKING THEMANGANESE STABILIZINGPROTEIN OFF 0HOTOSYSTEM))"IOCHEMISTRY n "URNAP2, 1IAN- 3HEN* 2 )NOUE9AND3HERMAN,! 2OLEOFDISULlDELINKAGEANDPUTATIVEINTERMOLECULARBINDING RESIDUESINTHESTABILITYANDBINDINGOFTHEEXTRINSICMANGA NESE STABILIZINGPROTEINSTOTHE0HOTOSYSTEM))REACTIONCENTER "IOCHEMISTRYn + ALDI 3ANDD 0IERCE# 0HOTOACTIVA "URNAP2, 1IAN- !L +H TIONAND3 STATEC D YCLING KINETICS IN 0HOTOSYSTEM))MUTANTS IN CYSTIS SP0##)N-ATHIS0 ED 0HOTOSYNTHESIS 3 NECHOCY 3Y &ROM,IGHTTO"IOSPHERE 6OL )) PPn+LUWER!CADEMIC 0UBLI U SHERS $ORDRECHT "URNAP2, 1IAN-AND0IERCE# 4HEMANGANESE STABILIZ INGPROTEINOF0HOTOSYSTEM))MODIlESTHEINVIVODEACTIVATION ANDPHOTOACTIVATIONKINETICSOFTHE (/OXIDATIONCOMPLEXIN 3YNECHOCYSTIS SP0##"IOCHEMISTRYn #ALDERONE6 4RABUCCO- 6UJI U CIC! "ATTISTUTTA2 'IACOMETTI R ATO 2 ANDD :ANOTTI '  #RYSTAL '- !NDREUCCI & "ARB STRUCTUREOFT F HE0SB1PROTEINOFF 0HOTOSYSTEM)) FROM F HIGHER PLANTS%-"/2EPORTSn #AMPBELL+! 'REGOR7 0HAM$0 0ELOQUIN*- $EBUS2*AND "RITT2$A 4HEANDK$APROTEINSOF0HOTOSYSTEM)) MODULATETHEMAGNETICPROPERTIESOFTHE3 STATEMANGANESE CLUSTER"IOCHEMISTRYn #AMPBELL+! 0ELOQUIN*- 0HAM$0 $EBUS2*AND"RITT2$ Q B 0ARALLEL POLARIZATION %02 DETECTION OFF AN 3n STATE @MULTILINE%02SIGNAL IN 0HOTOSYSTEM))PARTICLESFROM F 3YN ECHOCY CYSTIS SP0##*!M#HEM3OCn D -ARTIN)&  %FFECTSO F F HYDROXYLAMINEON #HENIAE'-AND F INGTHEDECAY A OFF / EVOLUTION 0HOTOSYSTEM)))&ACTORSAFFECT 0LANT0HYSIOLn #OLE* "OSKA-"LOUGH.6AND3AUER+ 2EVERSIBLEAND IRREVERSIBLEEFFECTSOFALKALINEP(ON0HOTOSYSTEM))ELECTRON TRANSFERREACTIONS"IOCHIM"IOPHYS!CTAn #OLEMAN7*AND D 'OVINDJEE  !MODEL FORT F HEMECHANISM OFF CHLORIDEACTIVATIONOFOX F YGENEVOLUTIONIN 0HOTOSYSTEM))

 0HOTOSYNTH2ESn DE0AULA*# ,I0- -ILLER!& 7U"7AND"RUDVIG'7 %FFECTOFTHE AND KILODALTONPOLYPEPTIDES CALCIUM AND CHLORIDEONELECTRONTRANSFERIN0HOTOSYSTEM))"IOCHEMISTRY  n %ATON 2Y 2 E**AND D -URATA.  %VIDENCETHATTHEAMINO TER MINUSOFF THEK$AEXTRINSICPROTEIN ISREQUIRED FOR F BINDING TOTHE0HOTOSYSTEM))COMPLEX"IOCHIM"IOPHYS!CTA n %ATON 2Y 2 E**AND D 6ERMAAS7&* /LIGONUCLEOTIDE DIRECTED MUTAGENESISOF PSB" THEGENEENCODING#0 EMPLOYINGA DELETIONSTRAINOFTHECYANOBACTERIUM 3YNECHOCYSTIS SP0## 0LANT-OL"IOLn %NAMI) 3ATOH+AND+ATOH3 #ROSSLINKINGBETWEENTHE D THE K$ACHLOROPHYLL CARRYING K$AEXTRINSICPROTEINAND PROTEINOFT F HE03))REACTIONCENTERCORECOMPLEX&%"3,ETT  n %NAMI) -OCHIZUKI9 9 4AKAHASHI3 +AKUNO4 K 4 (ORIO4 4 3ATOH + AND D +ATOH 3 %VIDENCE FROMCROSS F LINKING FORT F HENEAR ESTNEIGHBORRELATIONSHIPSAMONGTHETHREEEXTRINSICPROTEINS OFSPINACH0HOTOSYSTEM))COMPLEXESTHATAREASSOCIATEDWITH OXYGENEVOLUTION0LANT#ELL0HYSIOLn %NAMI) +ANEKO- +ITAMURA. +OIKE( 3ONOIKE+ )NOUE9 AND D +ATOH3 4OTAL IMMOBILIZATIONOFT F HEEXTRINSIC K$APROTEIN INSPINACH 0HOTOSYSTEM))MEMB M RANEPREPARATIONS D STABILIZATIONOFOX F YGENEVOLUTION 0ROTEINSTOICHIOMETRYAND "IOCHIM"IOPHYS!CTAn %NAMI) -URAY A AMA( /HTA( +AMO- .AKAZATO+AND3HEN *2 )SOLATIONANDCHARACTERIZATIONOFA0HOTOSYSTEM)) COMPLEXFROMTHEREDALGA #YANIDIUMCALDARIUM #  !SSOCIATION OFCYTOCHROME C ANDAK$APROTEINWITHTHECOMPLEX "IOCHIM"IOPHYS!CTAn %NAMI ) 4OHRI ! +AMO - /HTA ( AND D 3HEN * 2  )DENTIlCATIONOF DOMAINSONTHEK$ACHLOROPHYLL CARRYING PROTEIN#0 THATARESHIELDED FROMTR F YPTICATTACK BY BINDING OFF THEEXTRINSIC K$APROTEINWITH 0HOTOSYSTEM))COMPLEX "IOCHIM"IOPHYS!CTAn %NAMI ) +IKUCHI 3 &UKUDA 4 /HTA ( AND 3HEN *2  "INDING AND FUNCTIONAL PROPERTIES OF FOUR EXTRINSIC PROTEINS OF0HOTOSYSTEM))FROMAREDALGA #YANIDIUMCALDARIUM AS STUDIED BY RELEASE RECONSTITUTION EXPERIMENTS "IOCHEMISTRY  n %NAMI) 9OSHIHARA3 4OHRI ! /KUMURA! / K HTA(AND D 3HEN*2  #ROSS RECONSTITUTIONOFF VARIOUSEXTRINSICPROTEINSAND F YANOBACTERIA RED D ALGAEAND 0HOTOSYSTEM))COMPLEXESFROMC HIGHERPLANTS0LANT#ELL0HYSIOLn %NGELS$( ,OTT! 3CHMID'(AND0ISTORIUS%+ )NACTIVA TIONOFTHEWATEROXIDIZINGENZYMEINMANGANESESTABILIZING PROTEIN FREEMUTANTCELLSOFTHECYANOBACTERIUM 3YNECHOCOCCU 3 S 0##AND 3YNECHOCYSTIS 0##DURINGDARKINCUBA TIONAND D CONDITIONS LEADINGTOPHOTOINACTIVATION0HOTOSYNTH 2ES  n &ERREIRA+. )VERSON4- -AGHLAOVI+ "ARB R ER*AND)WATA3 D  !RCHITECTURE OF F THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn &RANKEL,+AND"RICKER4- -APPINGOF.(3 BIOTINYL ATIONSITESANDTHEEPITOPEOFTHEMONOCLONALANTIBODY&!# ONTHEAPOPROTEINOF#0A )N"ALTSCHEFFSKY-ED #URRENT 2ESEARCHIN0HOTOSYNTHESIS 6OL) PPn +LUWER!CA DEMIC0UBLI U SHERS $ORDRECHT &RANKEL,+AND D "RICKER4-  )NTERACTIONOF#0A W F ITH THE

 MANGANESE STABILI A ZINGPROTEINOFF PHOTOSYSTEM)))DENTIlCATION OFDOMAINSON#0A WHICHARESHIELDEDFROM. HYDROXYSUC CINIMIDEBIOTINYLATIONBYTHEMANGANESE STABILIZINGPROTEIN "IOCHEMISTRYn &RANKEL,AND"RICKER4- )NTERACTIONOFTHE K$AEX TRINSICPROTEINWITH TH 0HOTOSYSTEM)))DENTIlCATIONOF DOMAINSON F BIOTINYL N ATION THE K$APROTEINTHATARESHIELDED FROM.(3n BY 0HOTOSYSTEM))"IOCHEMISTRY n R OXYLATEGROUPS &RANKEL,+ #RUZ*!ANDD "RICKER4- #ARB ONTH T EMANGANESE STABILI A ZINGPROTEINAREREQUIRED FOR F ITSEFlCIENT BINDINGTO0HOTOSYSTEM))"IOCHEMISTRYn &RAZAO # %NGUITA &* #OELHO 2 3HELDRICK '- .AVARRO *! (ERVAS- $ELA2OSA-!AND#ORRONDO-! #RYSTAL STRUCTUREOFLOW POTENTIALCYTOCHROME C FROM 3YNECHOCYS TIS SP0##AT¯RESOLUTION*"IOL)NORG #HEM n K D 9OCUM#& A #ALCIUM 'HANOTAKIS$& "ABCOCK'4AN F OXYGEN EVOLUTION IN POLYPEPTIDE RECONSTITUTES HIGH RATES OF DEPLETED 0HOTOSYSTEM )) PREPARATIONS &%"3 ,ETT  n 'HANOTAKIS$& 4OPPER*. "ABCOCK'4AND9OCUM#&B 7 7ATER SOLUBLE K$AAND K$APOLYPEPTIDESRESTOREOXY

GENEVOLUTIONACTIVITYBYCREATINGAHIGH AFlNITYBIDING SITE F #A ONTHEOXIDIZINGSIDEOFF 0HOTOSYSTEM))&%"3,ETT FOR  n 'HANOTAKIS $& 4OPPER *. AND9OCUM D #& C 3TRUCTURAL ORGANIZATION OFF THE OXIDIZING SIDE OFF 0HOTOSYSTEM )) %X OGENOUS REDUCTANTS D REDDUCE AND DESTROY O THE -N COMPLEX IN 0HOTOSYSTEM)) MEMBRANES DEPLETED OF THE  AND  K$A POLYPEPTIDES"IOCHIM"IOPHYS!CTAn 'HANOTAKIS$& "ABCOCK'4AND9OCUM#& /NTHEROLEOF WATER SOLUBLEPOLYPEPTIDES K$A CALCIUMANDCHLORIDE INPHOTOSYNTHETICOXYGENEVOLUTION&%"3,ETTn &RAZAO # %NGUITA &* #OELHO 2 3HELDRICKK '- .AVARRO *! D DO-! #RYSTAL (ERVAS- $E LA2OSA-!AND#ORRON STRUCTUREOF LOW POTENTIAL CYTOCHROME C FROM F 3 NECHOCY 3Y CYSTIS SP 0##  AT  ¯ RESOLUTION * "IOL )NORG #HEM  n 'LEITER(- (AAG% 3HEN* 2 %ATON 2YE** )NOUE9AND6ERMAAS 7&* &UNCTIONALCHARACTERIZATIONOFMUTANTSTRAINSOFTHE CYANOBACTERIUM3YNECHOCYSTIS 0##LACKINGSHORTDOMAINS F HECHLOROPHY H LL PROTEIN WITHINTHELARGE LUMEN EXPOSED LOOPOFT #0 IN 0HOTOSYSTEM))"IOCHEMISTRYn 'LEITER(- (AAG % 3HEN* 2 %ATON 2Y 2 E** 3EELIGER!' )NOUE 9 6ERMAAS7&*AND 2ENGER'  )NVOLVEMENTOFF THE#0 9 PROTEIN IN STABILIZATION AND D PHOTOACTIVATION OFF A FUNCT F IONAL WATER OXIDIZINGCOMPLEXINTHECYANOBACTERIUM 3YNECHOCYSTIS SP0##"IOCHEMISTRYn (AAG % %ATON 2YE ** 2ENGER ' AND 6ERMAAS 7&*  &UNCTIONALLYIMPORTANTDOMAINSOFTHELARGEHYDROPHILICLOOP OFF #0ASPROBED BYOLIGONUCLEOTIDE DIRECTEDMUTAGENES D ISIN CYSTIS SP0##"IOCHEMISTRY n 3 NECHOCY 3Y D ITTMATTER0  #OVALENTCROSSLINKING OF (ACKETT#3AND3TR THE ACTIVE SITES OF F VESICLE BOUNDD CYTOCHROME B ANDD .!$( CYTOCHROMEB REDUCTASE*" D IOL #HEMn (AN+ # 3HEN* 2 )KEUCHI-AND)NOUE9 #HEMICAL CROSSLINKING STUDIES OF EXTRINSIC PROTEINS IN CYANOBACTERIAL 0HOTOSYSTEM))&%"3,ETTn (ASLER, 'HANOTAKIS$ &EDTKE" 3PYRIDAKI! -ILLER- -ULLER 3! %NGLE!ANDD 4SOTIS' 3TRUCTURAL ANALYSISOFF 0HOTO SYSTEM))#OMPARATIVESTUDIESOFC F YANOBACTERIAL AND HIGHER

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ PLANT0HOTOSYSTEM))COMPLEXES*3TRUCT"IOLn (AYASHI( &UJIMURA9 -OHANTY03AND-URATA. 4HE ROLEOF#0INTHEEVOLUTIONOFOXYGENANDTHEBINDINGOF THE EXTRINSIC  K$A PROTEIN TO THE CORE COMPLEX OF 0HOTO SYSTEM )) AS DETERMINED BY LIMITED PROTEOLYSIS 0HOTOSYNTH 2ES  n (O++ 5LRICH %, +ROGMANN$7AND D 'OMEZ ,O OJERO#  )SOLATIONOFF PHOTOSYNTHETICCATALYSTS FROMC F YANOBACTERIA"IO CHIM"IOPHYS!CTAn (OLTEN27AND D -EYERS* #YTOCHROMESOFA F BLUE GREEN ALGAE%XTRACTIONOFA C TYPEWITHASTRONGLYNEGATIVEREDOX POTENTIAL3CIENCEn (OLZENBURG ! &LINT 4$ 3HEPARD &( AND &ORD 2#  0HOTOSYSTEM))-APPINGTHELOCATIONSOFTHEOXYGENEVOLU TION ENHANCING SUB U UNITS BY ELECTRONMICROSCOPYY ICRON n (OMANN0(AND D -ADABUSI ,-  -ODIlCATIONOFF THETHER MOLUMINESCENCEPROPERTIESOF# F A DEPLETED D 0HOTOSYSTEM)) MEMBRANESBYTHE K$AEXTRINSICPOLYPEPTIDEAND BY OLIGO CARBOXYLICACIDS0HOTOSYNTH2ES  (ONG3+ 0AWLIKOWSKI3! 6ANDER-EULEN+!AND9OCUM#&  4HEOXIDATIONSTATEOFTHE0HOTOSYSTEM))MANGANESE CLUSTERINmUENCESTHESTRUCTUREOFTHEMANGANESE STABILIZING PROTEIN"IOCHIM "IOPHYS!CTAn H  (UTCHISON2 "ETTS3$ 9OCUM#&AND D "ARRY "!  #ONFO F R MATIONAL CHANGESINTH T EEXTRINSICMANGANESE STABILI A ZINGPROTEIN UPONBINDINGTOTHE0HOTOSYSTEM))REACTIONCENTER!N ISOTOPE EDITING AND D &4)2STUDY" Y IOCHEMISTRY n (UTCHINSON23 3TEENHUIS** 9OCUM#& 2AZEGHIFARD-2AND "ARRY ""  $EPROTONATION OF THE  K$A EXTRINSIC MANGANESE STABILIZING SUBUNIT ACCOMPANIES PHOTOOXIDATION OF THE MANGANESE IN 0HOTOSYSTEM )) * "IOL #HEM  n )FU F KU+ .A K KATSU4 +ATO(AND D 3ATO&  #RYSTALLIZATIONAND PRELIMINARY CRYSTALLOGRAPHICSTUDIESONTHEEXTRINSICK$A PROTEIN IN THE OXYGEN EVOLVING COMPLEX OFF 0HOTOSYSTEM)) !CTA#RYSTAL $n )KEUCHI- +OIKE(AND)NOUE9 . TERMINALSEQUENCING OF 0HOTOSYSTEM )) LOW MOLECULAR MASS PROTEINS  AND  K$ACOMPONENTSOFTHE/ EVOLVINGCORECOMPLEXFROMHIGHER PLANTS&%"3,ETTn U O - 4HEG 3- AND D 4AMURA. )SHIKAWA9 9AMAMOTO9 /TSUB  #HEMICAL MODIlCATION OF F AMINE GROUPS ON 03)) PROTEINS RETARDSPHOTOASSEMBLYOFT F HEPHOTOSYNTHETICWATER OXIDIZINGCOMPLEX"IOCHEMISTRY n )SOGAI9 9AMAMOTO9 AND D .ISHIMURA -  !SSOCIATION OF THE  K$A POLYPEPTIDE WITH THE  K$A COMPONENT IN 0HOTOSYSTEM))PARTICLES&%"3,ETTn *ANSEN 4 2OTHER # 3TEPPUHN * 2EINKE ( "EYREUTHER + *ANSSON# !NDERSSON"AND(ERRMANN2' .UCLEOTIDE SEQUENCEOFC$.!C F LONESENCODINGTHECOMPLETEK$AAND D WITH THEOXYGEN EVOLVING K$APRECURSORPROTEINSASSOCIATED COMPLEXFROMS F PINACH&%"3,ETTn *OHNSSON'. "OUSSAC!AND D 2UTHERFOR F D !7  4HEORIGIN OF  n  # THERMOLUMINESCENCE BANDS IN 0HOTOSYSTEM )) "IOCHIM"IOPHYS!CTAn +AMIYA.AND3HEN* 2 #RYSTALSTRUCTUREOFOXYGEN EVOLV ING0HOTOSYSTEM))FROM 4HERMOSYNECHOCOCCUSVULCANU 4 S AT ¯NGSTROMRESOLUTION0ROC.ATL3CI!CAD53!n +ANG # #HITINIS 02 3MITH 3 AND D +ROGMANN $7  D SEQUENCEANALYSISOFT F HE GENEENCODING THELOW #LONINGAND

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** POTENTIAL CYTOCHROME C OF 3YNECHOCY CYSTIS 0##  &%"3 ,ETTERSn +ASHINO9 ,AUBER7- #ARROLL*! 7ANG1 7HITMARSH* 3ATOH +AND0AKRASI(" 0ROTEOMICANALYSISOFAHIGHLYACTIVE 0HOTOSYSTEM))PREPARATIONFROMTHECYANOBACTERIUM 3YNECHO CYSTIS Y SP0##REVEALSTHEPRESENCEOFNOVE F LPOLYPEPTIDES "IOCHEMISTRYn D 'HANOTAKIS$&  %FFECTO F F THEMANGANESE +AVELAKI+AND COMPLEXONTH T E BINDINGOFT F THEEXTRINSICPROTEINS K$A  K$A AND D  K$A OFP F HOTOSYSTEM0HOTOSYNTH 2ESn +IESELBACH4 "YSTEDT - (YNDS 0 2OBINSON # AND 3CHRODER 70 !PEROXIDASEHOMOLOGUEANDNOVELPLASTOCYANIN LOCATEDBYPROTEOMICSTOTHE !RABIDOPSIS CHLOROPLASTTHYLAKOID LUMEN&%"3 ,ETTn F YANO +ROGMANN$7  4HELOWPOTENTIAL CYTOCHROME C OFC BACTERIAAND D ALGAE"IOCHIM "IOPHYS!CTAn +UHL( 2OGNER- 6AN"REEMEN*&ANDD "OEKEMA%* ,OCALIZATION OFF CYANOBACTERIAL 0HOTOSYSTEM )) DONOR SIDE SUB U UNITS BYELECTRONMICROSCOPY ANDD THESUPRAMOLECULAROR GANIZATIONOF0HOTOSYSTEM))INTHETHYLAKOIDMEMBRANE%UR *"IOCHEMn +UWABARA4AND-URATA. #HARACTERIZATIONOF KILO DALTON  KILODALTONAND KILODALTON PROTEINSINTHE PHO TOSYNTHETICOXYGEN EVOLVINGSYSTEMOFSP F INACH)N)NOUE9 F INDJEE -URATA. 2ENGER'ANDD 3ATOH + EDS #ROFTS!2 'OV 4HE/XYGEN %VOLVING 3YSTEMOFF 0HOTOSYNTHESIS PPn !CADEMIC0RESS 4OKYO +UWABARA 4 -IYAO - -URATA 4 ANDD -URATA .  4HE FUNCTIONOFTHEK$APROTEININTHEOXYGENEVOLUTIONSYSTEM STUDIEDBYRECONSTITUTIONEXPERIMENTS"IOCHIM"IOPHYS!CTA n +UWABARA4 -URATA4 -IYAO-AND-URATA. 0ARTIAL DEGRADATIONOFT F HEK$AOFT F HEPHOTOSYNTHETICOXYGEN EVOLV ING COMPLEX!STUDY OFF ABINDING SITE"IOCHIM "IOPHYS!CTA  n ,AKSHMI +6 2EImER-* #HISHOLM$! 7ANG*9 $INER"!AND "RUDVIG '7  #ORRELATIONOFF THECYTOCHROME C CONTENT OFCYANOBACTERIAL0HOTOSYSTEM))WITHTHE%02PROPERTIESOFTHE OXYGEN EVOLVINGCOMPLEX0HOTOSYNTH2ES n ,EUSCHNER#AND"RICKER4- )NTERACTIONOFTHEK$A EXTRINSICPROTEINWITH0HOTOSYSTEM))2EBINDINGOFTHEK$A EXTRINSICPROTEINTO0HOTOSYSTEM))MEMBRANESWHI W CHCONTAIN F FOUR TWO ORZEROMAN GANESEPER0HOTOSYSTEM))REACTIONCENTER "IOCHEMISTRYn D "URNAP2,  -UTATIONSOFF ARGININEWITHINTHE ,I :,AND PUTATIVECALCIUM BINDINGINTERHELICALA BLOOPOFTHE0HOTOSYS TEM))$PROTEINDISRUPTBINDINGOFTHEMANGANESE STABILIZING PROTEIN AND CYTOCHROME C IN 3YNECHOCYSTIS SP 0## "IOCHEMISTRYn ,INDBERG + AND!NDREASSON , %  ! ONE SITE TWO STATE F HE BINDINGOFAN F IONS IN 0HOTOSYSTEM))"IOCHEM MODEL FORT ISTRYn D A ,EUCINE , AKIS 3IMANTIRIS. "ETTS3$AND9OCUM#& ,YD D FOR F FO F LDINGAND FUNCT F IONOFT F HEMAN ISACRITICALRESIDUE GANESE STABILIZING PROTEIN OFF 0HOTOSYSTEM )) "IOCHEMISTRY n ,YDAKIS 3IMANTIRIS. (UTCHISON23 "ETTS3$ "ARRY"!AND 9 9OCUM#&B -ANGANESESTABILIZINGPROTEINOF0HOTO

SYSTEM )) IS A THERMOSTABLE NATIVELY UNFOLDED POLYPEPTIDE "IOCHEMISTRYn -AY A ES 32 #OOK K +- 3ELF F 3* :HANG : ANDD "ARRBER * 

 $ELETION OFF THE GENE ENCODING THE 0HOTOSYSTEM ))  K$A PROTEINFROM 3YNECHOCYSTIS 3 SP0##DOESNOTINACTIVATE WATER SPLITTINGBUTINCREASESVULNERABILITYTOPHOTOINHIBITION "IOCHIM"IOPHYS!CTAn -AVANKAL ' -C#AIN $# AND "RICKER 4-  %FFECTS OF CHLORIDEONPARAMAGNETICCOUPLINGOFMAN F GANESE INCALCIUM CHLORIDE WASHED 0HOTOSYSTEM )) PREPARATIONS &%"3 ,ETT    -ILLER!& $E0AULA*#AND D "RUDVIG '7  &ORMATIONOF D STRUCTUREOF THEMANGANESECOMPLEXIN0HOTO THE 3 STATEAND SYSTEM))LACKINGTHEEXTRINSICK$APOLYPEPTIDE0HOTOSYNTH 2ESn -ILLNER0! 'OGEL'AND"ARBER* )NVESTIGATIONOFTHE SPATIALRELATIONSHIPSBETWEENPHOTOSYSTEMPOLYPEPTIDESBY REVERSIBLE CROSSLINKING AND DIAGONAL ELECTROPHORESIS 0HOTO SYNTH 2ESn -IURA+ 3HIMAZU4 -OTOKI! +ANI 3 (IRANO-AND D +ATOH 3  .UCLEOTIDESEQUENCEOFT F HE-N STABILIZING PROTEINGENE OFF THETHERMOPHILICCYANOBACTERIUM 3Y 3 NECHOCOCCUSELONGATU G S "IOCHIM"IOPHYS!CTAn -IURA4 4 3HEN* 2 4AKAHASHI3 +AMO- .AKAMURA% /HTA( +AMEI! )NOUE9 $OMAE. 4AKIO+ .AKAZATO+ )NOUE9AND %NAMI) )DENTIlCATIONOFDOMAINSONTHEEXTRINSIC K$APROTEINPOSSIBLY INVOLVED INELECTROSTATICINTERACTIONWITH 0HOTOSYSTEM))COMPLEX BY MEANSOFF CHEMICAL MODIlCATION *"IOL #HEMn -IYAO - AND D -URATA .  0ARTIAL DISINTEGRATION AND D RE CONSTITUTIONOFT F HEPHOTOSYNTHETICOXYGENEVOLUTIONCOMPLEX "IOCHIM"IOPHYS!CTAn -IYAO-AND-URATA.A #ALCIUM IONSCANBESUBSTITUTED FORTHEK$APOLYPEPTIDEINPHOTOSYNTHETICOXYGENEVOLUTION &%"3,ETTn -IYAO-AND D -URATA. B 2OLEOFF THEK$APOLYPEPTIDE INPRESERVING -N INPHOTOSYNTHETICOXYGEN EVOLUTION&%"3 ,ETT n -IYAO-AND D -URATA.  4HEMODEOF BINDINGOFT F HREE EXTRINSICPROTEINSOF F K$A  K$AAND D  K$A INTHE0HO TOSYSTEM))COMPLEXOFSPINACH"IOCHIM"IOPHYS!CTA n -IYAO- -URATA- ,AVOREL* -AISON 0ETRI" "OUSSAC!AND %TIENNE! , %FFECTSOFTHEK$APROTEINONTHE3 STATETRANSITIONSINPHOTOSYNTHETICOXYGENEVOLUTION"IOCHIM "IOPHYS!CTAn -IYAO- &UJI U MURA9AND D -URATA.  0ARTIAL DEGRADATION OFF THEEXTRINSICK$APROTEINOFT F HE0HOTOSYSTEM))COMPLEX OFF SPINACH"IOCHIM"IOPHYS!CTAn -ORRIS%0 (ANKAMER" :HELEVA$ &RISO'AND"ARBER* 4HE THREE DIMENSIONAL STRUCTURE OF A 0HOTOSYSTEM )) CORE COMPLEX DETERMINED BY ELECTRON CRYSTALLOGRAPHY 3TRUCTURE  n -OTOKI ! 5SUI - 3HIMAZU4 (IRANO-AND D +ATOH 3  ! DOMAINOFT F HE-N STABILIZING PROTEIN FROM F 3 NECHOCOCCUS 3Y ELONGATUS G INVOLVED IN FUNCT F IONAL BINDINGTO0HOTOSYSTEM))* "IOL #HEM  -URAKAMI 2 )FU FUKU+ 4AKAB A AY A ASHI! 3HIKANAI4 %NDO4AND3ATO D & #HARACTERIZATIONOFAN !RABIDOPSISTHALIANA MUTANT WITHIMPAIREDPSB/ ONEOFTWOGENESENCODINGEXTRINSIC K$A PROTEINSIN0HOTOSYSTEM))&%"3,ETTn -URATA. -IYAO- /MATA4 -ATSUNAMI(AND+UWABARA4  3TOICHIOMETRY OF F COMPONENTS IN THE PHOTOSYNTHETIC OXYGENEVOLUTIONSYSTEMOFF 0HOTOSYSTEM))PARTICLESPREPARED

 WITH4RITON8 FROMSP F INACH CHLOROPLAST"IOCHIM"IOPHYS !CTAn .AVARRO*! (ERVAS- $ELA#ERDA"AND$ELA2OSA-! 0URIlCATIONANDPHYSICOCHEMICALPROPERTIESOFTHELOWPOTENTIAL CYTOCHROME# FROMTHECYANOBACTERIUM 3YNECHOCYSTIS SP 0##!RCH "IOCHEM"IOPHYSn .AKATANI (9  0HOTOSYNTHETICOXYGENEVOLUTION DOESNOT REQUIRE THE PARTICIPATION OFF POLYPEPTIDES OFF  AND  K$A "IOCHEM"IOPHYS2ES#OMMn 6 -ORRIS%0 0 'OWEN" VAN(EEL -ANDD "ARRBER .IELDD * /RLOVA%6 * $MAPOFTHEPLANT0HOTOSYSTEM))SUPERCOMPLEXOB TAINEDBYCRYOELECTRONMICROSCOPYANDSINGLEPARTICLEANALYSIS .AT3TRUCT"IOLn .IELD* +RUSE/ 2UP U RECHT* DA&ONSECA0 "UCHEL#AND"ARBER # AMYD M DOMONAS *  4HREE DIMENSIONAL STRUCTURE OF #HL R AND 3Y RDTII 3 NECHOCOCCUSELONGATUS G 0HOTOSYSTEM))COM R REINHA PLEXESALLOWSFORCOMPAR F ISONOFT F THEIROXYGEN EVOLVINGCOMPLEX ORGANIZATION*"IOL #HEMn .ISHIYAMA9 (AY A ASHI ( 7ATANABE 4 ANDD -URATA .  0HOTOSYNTHETICOXYGENEVOLUTIONISSTABILIZEDBYCYTOCHROME C AGAINSTHEATINACTIVATIONIN 3YNECHOCOCCUS SP0## 0LANT0HYSIOLn .ISHIYAMA9 ,OS$! (AYASHI(AND-URATA. 4HERMAL PROTECTION OFF THE OXYGEN EVOLVING MACHINERY BY 0SB5 AN EXTRINSICPROTEINOFF 0HOTOSYSTEM)) IN 3YNECHOCOCCUS SPECIES 0##0LANT0HYSIOLn .ISHIYAMA9 ,OS$!ANDD -URATA. 0SB5 APROTEINAS SOCIATED D WITH 0HOTOSYSTEM)) ISREQUIRED FORT F HEACQUISITIONOF CELLULARTHERMOTOLERANCEIN 3YNECHOCOCCUS SPECIES0## 0LANT0HYSIOLn /DOM72AND"RICKER4- )NTERACTIONOF#0A WITHTHE MANGANESE STABILIZINGPROTEINOF0HOTOSYSTEM)))DENTIlCATION OF DOMAINS CROSSLINKED BY  ETHY H L  ; DIMETHY H LAMINO PROPYL=CARB R ODIIMIDE"IOCHEMISTRY n /H /KA( 4ANAKA3 7ADA+ +UWABARA4ANDD -URATA.  #OMPLETEAMINOACID D SEQUENCEOF  K$APROTEIN ISOLATED FROM F SPINACH0HOTOSYSTEM))PARTICLES&%"3,ETTn /HTA( 9OSHIDA. 3ANO- (IRANO- .AKAZATO+AND%NAMI)  %VIDENCEFORELECTROSTATICINTERACTIONOFTHELOOP!ON #0  WITH THE EXTRINSIC  K$A PROTEIN )N -ATHIS 0 ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL)) PPn U SHERS $ORDRECHT +LUWER!CADEMIC0UBLI /HTA( /KUMURA! / K K YAMA3 !KIYAMA! )WAI- 9OSHI KU HARA3 3HEN*2 +AMO-ANDD %NAMI)  #LONING EXPRES SIONOFT F THE PSB5GENE AND FUNCT F IONALSTUDIESOFF THERECOMBI M NANT  K$APROTEINOFF 0HOTOSYSTEM)) FROMARE F D ALGA #Y # ANIDIUM CALDARIUM"IOCHEM"IOPHYS2ES#OMMn /NO4AND)NOUE9 #A DEPENDANTRESTORATIONOF/ EVOLV INGACTIVITYIN#A#L WASHED03))PARTICLESDEPLETEDOF  ANDK$APOLYPEPTIDES&%"3,ETTn /NO4AND D )NOUE9  3 STATETURNOVERINTHE/ EVOLVING SYSTEMOFF #A#L WASHEDD 0HOTOSYSTEM))PARTICLES DEPLETEDD OF THREEPERIPHERAL PROTEINSASMEASURED BYTHERMOLUMINESCENCE F K$A PROTEIN INHIBITS3 TO 3 TRANSITION"IOCHIM 2EMOVALOF "IOPHYS!CTAn /NO4!AND)NOUE9 $ISCRETEEXTRACTIONOFTHECALCIUM ATOM FUNCTIONAL FOR OXYGEN EVOLUTION IN HIGHER PLANT 0HO TOSYSTEM )) BY A SIMPLE LOW P( TREATMENT &%"3 ,ETT  n /NO4! AND D )NOUE9 A 2EMOVAL OFF CALCIUM BY P(  TREATMENT INHIBITS3 TO 3 TRANSITIONINPHOTOSYNTHETICOXYGEN

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ EVOLUTIONSYSTEM"IOCHIM"IOPHYS!CTAn /NO4!AND)NOUE9B 2OLESOFCALCIUMIONINOXYGEN EVOLUTIONINHIGHERPLANT0HOTOSYSTEM))%FFECTSOFREPLACEMENT OFCALCIUMIONSITEBYOTHERCATIONS!RCH"IOCHEM"IOPHYS  n /NO4!AND D )NOUE9  !BNORMALREDOXREACTIONS INTHEPHO TOSYNTHETIC/ EVOLVING CENTERSIN.A#L%$4! WASHED03)) D R STABLE%02MULTILINESIGNALAND D ANUNKNOWNPOS K ITIVE ! DARK CHARGEACCUMULATOR"IOCHIM"IOPHYS!CTAn /NO4 :IMMERMAN*, )NOUE9AND D 2UTHERFOR F D !7  %02 EVIDENCEFORAMODIlED3 STATETRANSITIONINCHLORIDE DEPLETED 0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn /NO4 )ZAWA 3 AND )NOUE9  3TRUCTURAL AND FUNCTIONAL MODULATIONOFTHEMANGANESECLUSTERIN#A DEPLETED0HOTOSYS TEM)) BY BINDING OFF THEK$AEXTRINSICPROTEIN"IOCHEMISTRY  n 0AZOS& (EREDIA0 6ALENCIA!AND DELAS2IVAS* 4HREAD ING STRUCTURAL MODEL OFF THE MANGANESE STABILIZING PROTEIN 0SB/REVEALSPRESENCEOFTWOPOSS F IBLE BETA SANDWICH DOMAINS 0ROTEINSn 0HILBRICK*" $INER"!AND:ILINSKAS"! #ONSTRUCTION AND CHARACTERIZATION OF CYANOBACTERIAL MUTANTS LACKING THE MANGANESE STABILIZINGPROTEINOF0HOTOSYSTEM))*"IOL#HEM  n 0OPELKOVA( )M-- $!URIA* "ETTS3$ ,YD , AKIS 3IMANTIRIS D A . TERMINUSOFF THE0HOTOSYSTEM)) .AND9OCUM#& F F FSEQUENCEE LONGATION MANGANESE STABILIZING PROTEIN%FFECTSO AND D TRUNCATION"IOCHEMISTRYn 0OPELKOVA( )M--AND9OCUM#&B . TERMINALTRUNCA TIONSOFMANGANESESTABILIZINGPROTEINIDENTIFYTWOAMINOACID SEQUENCES REQUIRED FOR BINDING OF THE EUKARYOTIC PROTEIN TO 0HOTOSYSTEM))ANDREVEALTHEABSENCEOFONEBINDING RELATED SEQUENCE INCYANOBACTERIA"IOCHEMISTRYn 0OPELKOVA ( 7Y 7 MAN ! AND D 9OCUM #& A !MINO ACID SEQUENCES AND D SOLUTION STRUCTURES OFF MANGANESE STABILIZING PROTEINTHATAFFECTSRECONST F ITUTIONOFF 0HOTOSYSTEM))ACTIVITY Y 0HOTOSYNTH2ESn 0OPELKOVA( )M--AND9OCUM#&B "INDINGOFMAN GANESE STABILIZING PROTEIN TO 0HOTOSYSTEM )) )DENTIlCATION OFESSENTIAL. TERMINALTHREONINERESIDUESANDDOMAINSTHAT PREVENTNONSPECIlCBINDING"IOCHEMISTRYn 0UTNAM %VANS # AND D "RICKER 4-  3ITE DIRECTED D MUTA GENESIS OFF THE #0A  PROTEIN OFF 0HOTOSYSTEM ))!LTERATION OFF THE BASIC RESIDUE D PAIR  2 TO  ' LEADS TO A DEFEC F T ASSOCIATED D WITHTHEOXYGEN EVOLVINGCOMPLEX"IOCHEMISTRY  n 0UTNAM %VANS # 7U * "URNAP 2 7HITMARSH * AND "RICKER 4- 3ITE DIRECTEDMUTAGENESISOFTHE#0PROTEINOF 0HOTOSYSTEM))!LTERATIONOFCONSERVEDCHARGEDRESIDUESIN THE DOMAIN % 2"IOCHEMISTRYn 1IAN- !L +H + ALDI3 0UTNAM %VANS# "RICKER4-AND D "URNAP 2,  0HOTOASSEMBLY M Y OFT F THE0HOTOSYSTEM))-N  CLUSTER IN SITE DIRECTED D MUTANTS IMPAIRED INTHEBINDINGOFF THEMANGANESE STABILIZING PROTEIN"IOCHEMISTRYn 2ASHIDD !AND #ARPENTIER2 4HEAND D  K$AEXTRINSIC POLYPEPTIDESANDTHEASSOCIATED#A AND#LnMODIFYATRAZINE INTERACTIONWITHTHE0HOTOSYSTEM))CORECOMPLEX0HOTOSYNTH 2ESn 2AZEGHIFARD-2 7YDRZYNSKI4 0ACE2*AND"URNAP2, 9Z REDUCT D ION KINETICSINTHEABSENCEOFT F HEMANGANESE STABILIZING PROTEINOFF 0HOTOSYSTEM))"IOCHEMISTRYn

$IBQUFS

5IF
&YUSJOTJD
1SPUFJOT
PG
14 ** 2EImER-* #HISHOLM$! 7ANG* $INER"!ANDD "RUDVIG'7  %NGINEERINGANDRAPIDPURIlCATIONOFHISTIDINE TAGGED 3 0##)N'ARAB'ED 0HOTOSYSTEM))FROM 3YNECHOCYSTIS 0HOTOSYNTHESIS-ECHANISMSAND%FFECTS 6OL)) PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT 3AWAY A A-2 +ROGMANN$7 3ERAG ! (O 9EATES4/ANDD +ERFE F LD F YTOCHROME C ANDD CYTOCHROME C #! 3TRUCTURESOFC F FROMT HECYANOBACTERIUM !RTH T ROSP S IRAMAXIMA"IOCHEMISTRY n 3CHUB U ERT- 0ETERSSON5 ! (ASS"* * &UNK#AN K D HRODER70 D3C  0ROTEOMEMAPOFTHECHLOROPLASTLUMENOF !RABIDOPSIS THALIANA*"IOL#HEMn 3EIDLER! )NTRODUCTIONOFAHISTIDINETAILATTHE. TERMINUS OFASECRETORYPROTEINEXPRESSEDIN % COLI0ROTEIN%NGINEERING  n 3EIDLER ! A )NTERMOLECULAR AND INTRAMOLECULAR INTERAC TIONSOFT F HEK$APROTEINOFF 0HOTOSYSTEM))%UR*"IOCHEM  n 3EIDLER! B 4HEEXTRINSICPOLYPEPTIDESOFF 0HOTOSYSTEM)) "IOCHIM"IOPHYS!CTAn 3EIDLER!AND2UTHERFORD!7 4HEROLEOFTHEEXTRINSIC K$APROTEININ#A BINDINGIN0HOTOSYSTEM))"IOCHEMISTRY  n 3EIDLER! 2OLL +ANDD -ICHEL (  #HARACTERIZATIONOFF THE K$APROTEINOFF THEOXYGEN EVOLVINGCOMPLEXOF HIGHERPLANTS BY SITE DIRECTEDD MUTAGENESIS)N-URATA.ED 2ESEARCH IN 0HOTOSYNTHESIS 6OL )) PPn+LUWER!CADEMIC0RESS $ORDRECHT 3EIDLER! 2UTHERFORD!7AND-ICHEL(A 4HEEXTRINSIC K$APROTEINOF0HOTOSYSTEM)))MPROVEDEXPRESSIONPLAS MIDSANDPROGRESSINMUTATIONALANALYSIS)N-ATHIS0ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL)) PPn +LUWER!CADEMIC0RESS $ORDRECHT 3EIDLER! 2UTHERFOR F DD!7AND -ICHEL ( B /NTHEROLEOFT F HE F HEEXTRINSICK$APROTEINOFF 0HOTOSYSTEM)) .TERMINUSOFT 0LANT-OL"IOL   3HEN ' %ATON 2Y 2 E ** AND6ERMAAS D 7&*  -UTATION OF HISTIDINE RESIDUES IN #0  LEADS TO A DESTABILIZATION OF THE 0HOTOSYSTEM )) COMPLEX AND TO IMPAIRMENT OF LIGHT ENERGY TRANSFER"IOCHEMISTRYn 3HEN* 2AND)NOUE9A #ELLULARLOCALIZATIONOFCYTOCHROME C )TSSPECIlCASSOCIATIONWITHCYANOBACTERIAL 0HOTOSYSTEM )) * "IOL #HEMn 3HEN* 2AND D )NOUE9B "INDINGAND FUNCT F IONAL PROPERTIES OFF TWONEWEXTRINSICCOMPONENTS CYTOCHROME C ANDD A K$APROTEININCYANOBACTERIAL0HOTOSYSTEM))"IOCHEMISTRY n 3HEN* 2 )KEUCHI-AND)NOUE9 3TOICHIOMETRICASSOCIA TIONOFEXTRINSICCYTOCHROME C ANDTHEK$APROTEINWITH A HIGHLY PURIlED OXYGEN EVOLVING 03)) CORE COMPLEX FROM 3YNECHOCOCCUSVULCANUS &%"3 ,ETTn 3HEN * 2 "URNAP 2, ANDD )NOUE9 A !N INDEPENDENT ROLEOFF CYTOCHROME C INCYANOBACTERIAL 0HOTOSYSTEM))AS U E DELETION MUTAGENESIS OF F THE PSB/ AND REVEALED BY DOUBL PSB6 GENES IN 3YNECHOCY CYSTIS SP0##"IOCHEMISTRY  n 3HEN* 2 6ERMAAS7&*AND)NOUE9B 4HEROLEOFCYTO CHROME C AS STUDIED THROUGH REVERSE GENETICS AND MUTANT CHARACTERIZATION IN 3YNECHOCYSTIS SP0##*"IOL#HEM  n 3HEN* 2 1IAN- )NOUE9AND"URNAP2, D  &UNCTIONAL CHAR A

 ACTERIZATIONOF 3YNECHOCY CYSTIS SP0## 6PSB 6P P 5 AND 6PSB 6 6 MUTANTSREVEALSIMPORTANTROLESOFCYTOCHROME C INCYANOBAC TERIALOXYGENEVOLUTION"IOCHEMISTRYn 3HUTOVA4 )RRGANG + $ 3HUBIN6 +LIMOV66 AND 2ENGER '  !NALYSISOFP( INDUCEDSTRUCTURALCHANGESOFTHEISOLATED EXTRINSIC KILODALTONPROTEINOFF 0HOTOSYSTEM))"IOCHEMISTRY  n 3TEWART!AND D "ENDALL$ 0REPARATIONOFACT F IVE FRACT F IONS ENRICHED IN0HOTOSYSTEM)AND D 0HOTOSYSTEM)) FROMT F HETHER MOPHILICBLUE GREENALGA 0HORMIDIUMLAMINOSUM"IOCHEM 3OC4RANSn 3TEWART!AND"ENDALL$ 0ROPERTIESOFOXYGEN EVOLVING PHOTOSYSTEM ))PARTICLESFROM 0HORMIDIUM LAMINOSUM ATHER MOPHILLICBLUE GREENALGA"IOCHEM*n D "ENDALL $  0HOTOSYNTHETICELECTRONTRANSPORT 3TEWART!AND INACELL FREEPREPARAT F ION FROMT F HETHERMOPHILIC BLUE GREENALGA 0HORMIDIUM LAMINOSUM"IOCHEM * n D "ENDALL$ 0ROPERTIESOFOX F YGEN EVOLVING 3TEWART!AND 0HOTOSYSTEM ))PARTICLES FROM F 0HORMIDIUM LAMINOSUM ATHER MOPHILICBLUE GREENALGA"IOCHEM*n 3TEWART!# ,JUNGBERG5 !UKERLUND( AND!NDERSON"A 3TUDIES ON THE POLYPEPTIDE COMPOSITION OF THE CYANOBACTE RIAL OXYGEN EVOLVING COMPLEX "IOCHIM "IOPHYS!CTA  n 3TEWART!# 3ICZKOWSKI-AND D ,JUNGBERG5B 'LYCEROL STABILIZESOXYGENEVOLUTIONAND D MAINTAINSBINDING OFF A K$A POLYPEPTIDE IN0HOTOSYSTEM))PARTICLES FROMT F HECYANOBACTE RIUM 0HORMIDIUM LAMINOSUM &%"3,ETTn 3TOYLOVA33 &LINT4$ &ORD2#AND(OLZENBURG! 0ROJEC TIONSTRUCTUREOF0HOTOSYSTEM))INVIVODETERMINEDBYCRYO ELECTRONCRYSTALLOGRAPHY-ICRONn 3TYRING3 -IYAO-AND2UTHERFORD!7 &ORMATIONAND mASH DEPENDENTOSCILLATIONOFT F HE 3 STATEMULTILINE%02SIGNAL INANOXYGEN EVOLVING 0HOTOSYSTEM))PREPARATIONLACKING THE T EOXYGEN EVOLVING SYSTEM"IOCHIM T REEEXTRINSICPROTEINSINTH TH "IOPHYS!CTAn 3VENSSON " 4IEDE $- AND D "ARRY "!  3MALL ANGLE 8 RAYSCATTERINGSTUDIESOFTHEMANGANESE STABILIZINGPROTEININ 0HOTOSYSTEM))*0HYS#HEM"n 4ANAKA 3 AND7ADA +  4HE STATUS OF CYSTEINE RESIDUES INTHEK$APROTEINOFSPINACH0HOTOSYSTEM))COMPLEXES 0HOTOSYNTH2ESn 4ANG83 #HISHOLM$! $ISMUKES'# "RUDVIG '7AND D $INER F ITEDIRECTED D MUTANTSOF "! 3PECTROSCOPICEVIDENCE FROMS CYSTIS 0##IN FAVORO F F LOSE INTERACTIONBETWEEN FAC 3 NECHOCY 3Y HISTIDINEAND D REDOXACTIVETYROSINE BOTH OFPO F LYPEP TIDE$OFTHE0HOTOSYSTEM))REACTIONCENTER"IOCHEMISTRY n 6ASS) #OOL+- :SUZSANRA$ -AYES32AND"ARBER* 4HERMOLUMINESCENCE AND mASH OXYGEN CHARACTERIZATION OF 3 HOCYSTIS SP0##LACK THE)# DELETIONMUTANTOF 3YNEC INGTHE0HOTOSYSTEM))KDAPROTEIN"IOCHIM "IOPHYS!CTA  n 6ERMAAS7&* )KEUCHI -AND D )NOUE9  0ROTEINCOMPOSITION OFF THE0HOTOSYSTEM))CORECOMPLEXIN GENETICALLY ENGINEERED 3 0##  MUTANTS OF THE CYANOBACTERIUM 3YNECHOCYSTIS 0HOTOSYNTH2ESn 6ERMAAS 7&* #HARITE * AND 3HEN '  'LU  OF THE $ 0ROTEIN IN 0HOTOSYSTEM )) IS A POTENTIAL LIGAND TO -N INVOLVED IN PHOTOSYNTHETIC OXYGEN EVOLUTION "IOCHEMISTRY  n

 6RETTOS*3 2EImER-* * +IEVIT/ ,AKSHMI +6 6 DE0AULA*#AND "RUDVIG '7  &ACTORS THAT DETERMINE THE UNUSUALLY LOWREDUCTIONPOTENTIALOFCYTOCHROME C INCYANOBACTERIAL 0HOTOSYSTEM))*"IOL)NORG#HEMn 7 7ALES2 .EWMAN"* 0APPIN$AND'RAY*# 4HEEXTRINSIC  K$APROTEINOFF THEOXYGEN EVOLVINGCOMPLEXOFF 0HOTOSYS TEM))ISAPUTATIVECALCIUM BINDINGPROTEINAND ISENCODED BY AMULTI GENEFAM F ILY INPEA0LANT-OL "IOLn 7 BBER!.AND 7E D 'RAAY*# $ETECTIONOFCA F LCIUM BINDING BY 0HOTOSYSTEM))POLYPEPTIDES IMMOBILIZEDD ONTONITROCELLULOSE MEMBRANES&%"3,ETTn 7 7INCENCJUSZ( 9OCUM#&ANDVAN'ORKOM(* 3 STATE DEPENDENCEOFCHLORIDEBINDINGAFlNITIESANDEXCHANGEDYNAM ICSINTHEINTACTANDPOLYPEPTIDEDEPLETED/ EVOLVINGCOMPLEX OFF 0HOTOSYSTEM))"IOCHEMISTRY n 7INCENCJ C USZ( 9OCUM#&ANDVAN'OR D RKOM(*  !CTIVATING ANIONSTHATREPLACECHLORIDEINTHEOXYGEN EVOLVING COMPLEXOF 0HOTOSYSTEM))SLOWTHE KINETICSOFF THETERMINALSTEPINWATER OXIDATIONAND DESTABILIZETHE 3 ANDD 3 STATES"IOCHEMISTRY n 8U1AND"RICKER4- 3TRUCTURALORGANIZATIONOFPROTEINS ONTHEOXIDIZINGSIDEOF0HOTOSYSTEM))4WOMOLECULESOFTHE K$AMANGANESE STABILIZINGPROTEINPERREACTIONCENTER* "IOL #HEMn 8U1 .ELSON*ANDD "RICKER4- 3ECONDARYSTRUCTUREOFF THE F IRCULAR  K$A EXTRINSICPROTEINOFF 0HOTOSYSTEM))!FAR 56C DICHROISMSTUDY" Y IOCHIM"IOPHYS!CTAn 9AMAMOTO9 AND +UUBOTA &  3PECIlC RELEASE OFF THE EX 9 TRINSIC K$APROTEINFROMSPINACH0HOTOSYSTEM))PARTICLES BYTHETREATMENTWITH.A#LANDMETHANOLANDITSAPPLICATION FORLARGESCALEPURIlCATIONOFTHETHREEEXTRINSICPROTEINSOF 0HOTOSYSTEM )) WITHOUT CHROMATOGRAPHY "IOCHIM "IOPHYS !CTA  n 9 9AMAMOTO9 .A KAY A AMA3 #OHN#,ANDD +ROGMANN$7 

5FSSZ
.
#SJDLFS
BOE
3PCFSU
-
#VSOBQ (IGHLY Y EFlCIENTPURIlCATIONOFT F THE  AND D  K$APROTEINS INSPINACH0HOTOSYSTEM))BYBUTANOLWATERPHASEPARTITIONING ANDHIGH PERFORMANCELIQUIDCHROMATOGRAPHY!RCH"IOCHEM "IOPHYSn 9 9U9 ,I2 8U#( 2UAN+# 3HEN9+AND'OVINDJEE . BROMOSUCCINIMIDE MODIlCATION OFF TRYPTOPHAN  AT THE C TERMINUS OF F THE MANGANESE STABILIZING PROTEIN OFF PLANT 0HOTOSYSTEM)) INmUENCES ITSSTRUCTUREAND FUNCT F ION0HYSIOL 0LANTn :HANG( &ISCHER'AND D 7YDRZYNSKI4  !N&4)2STUDY OFCALCIUMINTERACTIONSIN0HOTOSYSTEM)))N-ATHIS0ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL)) PPn +LUWER!CADEMIC0RESS $ORDRECHT :HANG ( )SHIKAWA9 9AMAMOTO9 AND #ARPENTIER 2  3ECONDARYSTRUCTUREAND D THERMALSTABILITY OFT F HEEXTRINSIC K$APROTEINOFF 0HOTOSYSTEM))STUDIED BY &OURIERTRANSFORM F INFRARE F D DSPECTROSCOP Y Y&%"3,ETTn D #ARPENTIER 2  :HANG ( 9AMAMOTO9 )SHIKAWA9 AND #HARACTERIZATIONOFTHESECONDARY STRUCTUREANDTHERMOSTABILITY OFTHEEXTRINSICKILODALTONPROTEINOFSPINACH0HOTOSYSTEM)) BY&OURIERTRANSFORMINFRAREDSPECTROSCOPY*-OL3TRUCT n :HANG, 8 ,IANG( ' 7ANG* ,I72AND9U4: &LUORES CENCEAND D &OURIER TRANSFORM F INFRARE F D SPECTROSCOPICSTUDIESON THEROLEOF DISULlDEBOND INTHECALCIUMBINDING INTHEK$A PROTEINOFF 0HOTOSYSTEM))0HOTOSYNTH 2ESn 7 TT ( 4 +ERN * &ROMME 0 +RAUSS . 3AENGER7 :OUNI! 7I AND D /RTH 0  #RYSTAL STRUCTUREOFF 0HOTOSYSTEM))FROM F 3YNECHOCOCCUS ELONGATUS AT ¯ RESOLUTION .ATURE  n :UBRZYCKI ): &RANKEL ,+ 2USSO 03 AND "RICKER4-  (YDRODYNAMICSTUDIESONTHEMANGANESE STABILIZINGPROTEIN OFF 0HOTOSYSTEM))"IOCHEMISTRYn

$IBQUFS
 5IF -PX
.PMFDVMBS
8FJHIU
1SPUFJOT
PG
1IPUPTZTUFN
** -FFBOO
&
5IPSOUPO
+PIOOB
-
3PPTF
BOE
)JNBESJ
#
1BLSBTJ

%FQBSUNFOU
PG
#JPMPHZ
8BTIJO Z HUPO
6OJWFSTJUZ UZ
#PY
 
4BJOU
-PVJT
.0

64"

.BTBIJLP *LFVDIJ %FQBSUNFOU
PG
-JGF
4DJFODFT
#JPMPHZ


6OJWFSTJUZ
PG
5PLZP ,PNBCB

.FHVSP
5PLZ LZP

+BQBO

4VNNBSZ   *
*OUSPEVDUJPO  **
.FNCSBOF 4QBOOJOH 4VCVOJUT   "
1TC&' ‡ $ZUPDISPNF C   #
1TC)  $
1TC*   %
1TC+  &
1TC,   ' 1TC-   '
(
1TC.   )
1TC/  *
1TC4   +
1TC5D   ,
1TC5/   - 1TC8   .
1TC9   /
1TC:  0
1TC;   ***

&YUSJOTJD 4VCVOJUT  "
1TC3  #
1TC   $
1TC   *7
$PODMVTJPO  "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 0HOTOSYSTEM))03)) HASACOMPLEXARRANGEMENTOFF MEMBRANESPANNING ANDD SOLUBL U ESUB U UNITSRESPONSIBLEFOR F ITSUNIQUEROLEINOXYGENICPHOTOSYNTHESIS!DVANCES INSEVERALAREASOF BIOLOGY HAVECONTRIBUTEDD SIGNIlCANTLY TOOURUNDERSTANDINGOFINDIVIDUALSUBUNITSANDTHE03))COMPLEXASAWHOLE)NRECENTYEARS THEGENOME SEQUENCESOFSEVERA F L PLANTSAND D MAN NYCYANOBACTERIAHAVE BEENCOMPLETEDD ALLOWING FORCOMPAR F ISONSOFT F HEIR F ION ALONGWITH PREVIOUSBIOCHEMICALEVIDENCE HASSTRENGTHENEDD THECONCLUSIONTHATTHE GENES4HIS INFORMAT

!UTHORFORCORRESPON F DENCE EMAILPAKRAS K I WUSTLEDU D

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

03))PROTEINCOMPONENTSAREHIGHLYCONSERVED D FROMT F HEPRIMITIVECYANOBACTERIUM 'LOEOBACTERVIOLACEUS 0##TOTHEVASCULARPLANT !RABIDOP O SISTHALIANA!DVANCESIN PURIlCATIONOF03))SAMPLESANDPROTEIN IDENTIlCATIONHAVECONlRMED D THEPRESENCEOFF SEVERALNOVEL PROTEINSANDD PROVIDENEWTOOLS FORT F HEANALYSISOF 03))INVARIOUSMUTANTSAND D UNDERDIFFERENTGROWT F H CONDITIONS2ECENTSTRUCTURAL STUDIESHAVEALSOCONTRIB UTEDTREMENDOUSLYTOOURUNDERSTANDINGOFTHEORGANIZATIONOF03))SUBUNITSANDCOFACTORS4HECOMBINATION OF GENOMIC PROTEOMIC AND D STRUCTURALSTUDIESWILL CONTINUETOBEASOURCEOFF NEW INSIGHTS INTOTHEASSEMBLY Y F IONOF03))4 F HISCHAPTERHIGHLIGHTSTHECONTRIBUTIONSOFSUC F H STUDIESTOOURUNDERSTANDING REGULATIONAND FUNCT OFTHENUMEROUSLOWMOLECULARWEIGHT03))COMPONENTS *
*OUSPEVDUJPO 0HOTOSYSTEM )) 03 )) IS A LARGE PROTEIN COMPLEX CONSISTINGOFATLEASTSUBUNITSTHATCONTRIBUTETO D / EVOLUTION03))IS OPTIMALCHARGESEPARATIONAND MADEUPOFF MANYSUUBUNITSTHATAREHIGHLYCONSERVED INALLTHEORGANISMSFROMWHICHITHASBEENISOLATED 4HECOMPLEXITYOFF 03)) ISCONSERVED BETWEENTHE PRIMITIVESINGLE MEMBRANESYSTEMOF 'LOEOBACTER VIOLACEUS 0##  AND THE INTRICATELY STRUCTURED THYLAKOIDD MEMBRANES OFF CHLOROPLASTS $ESPITE ITS BULK 03))ISSTILLASSEMBLED FREQUENT F LY INPHOTOSYN THETICORGANISMSBECAUSEITHASAVERYHIGHTURNOVER RATE 03)) IS THE TARGET OFF EXTENSIVE PHOTODAMAGE D SU UBUNITS REQUIRING RAPIDD REMOVAL OF ITS DAMAGED AND REASSEMBLY OF NEW ONES 5NDERSTANDING 03)) N ASAWH W OLEREQUIRESEXAMINING THECOMPLEXONMANY DIFFERENT F LEVELS FROMU F LTRASTRUCTURETOTHE FUNCT F IONOF INDIVIDUALCOMPONENTSINANUMBEROFPHOTOSYNTHETIC ORGANISMS 2ECENTLY GREATADVANCES HAVEBEENMADETOWARDS DETERMINING THE STRUCTURAL COMPONENTS OF 03 )) )NNOVATIVETECHNOLOGIES INPROTEINPURIlCATIONAND CRYSTALLIZATION RECENT DEVELOPMENTS IN GENOME Q ANNOTATION AND THE IMPLEMENTATION OF SEQUENCE HIGHTHROUGHPUTPROTEOMEANALYSISHAVESHED D NEW LIGHTONTHE BIGPICTUREOFF 03)) BOTH FOR F ITSFORM F AND D ITS FU F NCTIONS :OUNI AND COWORKERS ACHIEVED  ¯ RESOLUTION CRYSTAL STRUCTURE OF 03)) FROM 4HERMOSYNECHOCOCCUS ELONGATUS "0  WHICH PROVIDESANARRANGEMENTFORMANYOFTHEMEMBRANE !BBREVIATIONS#YTnCYTOCHROME$#"1n  DICHLORO P

P BEN ZOQUINONE(IS TAGnHISTIDINETAG(0ANDD ,0n HIGH AND LOW REDOXPOTENTIAL RESPECTIVELY,(#))nLIGHTHARVESTINGCOMPLEX ASSOCIATEDWITH03))-!,$)-3nMATRIXASSISTEDLASERDESORP TIONIONIZATIONMASSSPECTROMETRY/2&nOPENREADINGFRAME 0nPRIMARYELECTRONDONORIN03))01nPLASTOQUINONE03) AND D 03))n0HOTOSYSTEM)AND)) RESPECT D IVELY 1! nTHEPRIMARY Q INONEACCEPTOROF03))ONT QU F HE$SUB U UNIT1" nTHESECONDARY Q INONEACCEPTOROFF 03))ONTHE$SUB QU U UNIT3ECnSECRETION F 'ENOMIC2ESEACH 4')2n4HE)NSTITUTE FOR

SPANNING HELIXES :OUNI ET AL  #HAPTER  7ITT  03)) FFROM ANOTHER CYANOBACTERIUM 4HERMOSY S NECHOCOCCUSVULCANUS WASCRYSTALLIZEDD TO  ¯ RESOLUTION WITHASIMILARSUBUNITARRANGEMENT ASWASSEENINTHE:OUNI STUDY +AMIYAAND3 D HEN  #HAPTERE  3HEN ANDD +AMIYA ! SECOND STRUCTUREWASDETERMINEDFOR03))FROM 4ELONGATUS AT  ¯ RESOLUTION WITH DIFFERENT ASSIGNMENTS FOR F THE SMALL MEMBRANE SPANNING SUB U UNITS SOME OF &ERREIRAETAL #HAPTER "ARBERAND)WATA  #ONTINUEDD EFFORTS F INPURIlCATIONANDD CRYSTALLIZATION F ILLSHED FURT F HERLIGHTONTHEVARIABILITY IN OF03))W THEARRANGEMENTOFSUBUNITSBETWEENORGANISMSAND F GROWTH CONDITIONS"IESIADKAET ALSOUNDERDIFFERENT AL  0ROGRESS IN THE lELD OF GENOMICS HAS ALLOWED BROADD COMPARISONS OFF THE 03)) SUB U UNITS FOUN F D IN CYANOBACTERIAANDD PLANTS BASEDD ONPREDICTEDD AMINO ACIDSEQUENCESIMILARITIES4ABLESHOWSTHELISTOF Q 03)) GENES WHI W CHWEREOBTAINED FROMT F HECOMPLETE F DINGFRAMESO F F 3YNEC 3 HOCYSTIS SP0## SETSOFOPENREA +ANEKOETAL  !NABAENA SP0## +ANEKO ET AL  4 ELONGATU G S .AKAMURA ET AL  0ROCHLOROCOCCUSMARINUS -%$ 2OCAP ET AL  0ROCHLOROCOCCUS MARINUS -)4 2OCAPETAL  'VIOLACEUS .AKAMURAETAL K B AND 3YNECHOCOCCUS SP7( 0ALENIKET AL  OREXTRACTEDFROMNUCLEOTIDESEQUENCES OFF THE COMPLETE OR INCOMPLETE GENOME DATA OF .OSTOC PUNCTIIFORME !4 . ! ##  4RIC 4 HODESMIUM D O SIS THALIANA )NCLUSION ERYTHRAEUM AND !RABIDOP OF !RABIDOPSI D S IN THIS COMPARISON MOSTLY AMONG CYANOBACTERIAALLOWSCOMPARISONOFSEVERA F L ADDITIONAL 03))SUBUNITS BECAUSEPLANTSHAVEACQUIREDGENES THATAPPEARNOTTO BEPRESENTINCYANOBACTERIA/NTHE OTHERHAND SOMECYANO D BACTERIA HAVE03))GENESTHAT ARENOTPRESENTINOTHERCYANOBACTERIA&OREXAMPLE ' VIOLACEUS LACKS PSB9 PSB: : PSB  ANDTHESECOND COPYOF PSB$ INSPITEOF ITSRELATIVELY LARGEGENOME SIZE-B 0HYLOGENETICANALYSISOF3R2.! INDICATESTH T AT' VIOLACEUS ISONEOFT F THEMOSTANCIENTLY

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** BRANCHED D SPECIES AMONG CYANOBACTERIA )F F SO THE GENECOMPOSITIONOF' VIOLACEUS MAYSUGGESTTHAT THE ANCESTRAL 03)) CONSISTED D OFF A SIMPLER SUB U UNIT COMPOSITION !LTERNATIVELY COMPONENTS WHICH AREMISSINGIN ' VIOLACEUS MAYBERELATEDTOTHE DEVELOPMENTOFT F THETHYL T AK A OIDD MEM MBRANESYSTEM SINCE ITISTHEONLYORGANISMTHATLACKSTHY H LAKOIDS!NOTHER ANOMALOUSORGANISMIS 0 MARINUS -%$ WHICH LACKS PSB5AND 5 PSB6 6 SINCE0 MARINUS -)4  A CLOSERELATIVE RETAINSBOTH OFF THEM 0 MARINUS -%$ ISKNOWNTOBEAHIGH LIGHTADAPTEDECOTYPEWITH MULTIPLE HLIP I AND D PHOTOLYASE GENES(ESSETAL   D WITH THEABSENCEOF 3UCHADAPTATIONMAYBECOUPLED TWOEXTRINSICPROTEINSIN03)) D S IN4ABLEREVEALSTHE 4HE INCLUSIONOF !RABIDOPSI CHANGESTH T ATHAVETAK A ENPLACE IN03))GENESDUR D INGTH T E EVOLUTIONFROMCYANOBACTERIATOCHLOROPLASTS-ANY OFF THE03))COMPONENTSAREENCODED BYSINGLECOPY GENESINTHECHLOROPLAST4HEREAREALSO03))GENES IN THE PLANT NUCLEAR GENOME THAT ENCODE SUBUNITS F D INCYANOBACTERIA 0SB7 0SB2 0SB3 AND NOT FOUN 0SB4. ANDPRESUMA D ABLLYNOTNECESSARYFORA F F LIV FREE I ING PHOTOSYNTHETICORGANISM&URTHERMORE THEREAREPLANT 03))PROTEINS 0SB0AND 0SB1 WH W OSEFUNCT F IONALROLE HAS BEENEXAMINED D THOROUGHLY WH W EREASTHEFUNCT F ION OFHOMOLOGSINCYANOBACTERIAREMAINSANOPENQUES TION4ABLE ISANEXAMPLEOFF THETYPEOF INFORMAT F ION THATCAN BEGATHERED FROMGENOM F ICANALYSISOF03)) F

CONTAININGORGANISMS3UCHINFORMATIONISUSEFULFOR ESTABLISHINGTHEDIFFERENTCOMPONENTSUSE F D IN03)) ACTIVITYINVARIOUSMODEL ORGANISMS 3TUDIESONTHEBIOCHEMICALCOMPOSITIONOF03)) AREPROVIDINGCORRELATIONS BETWEENTHESTRUCTUREAND F IOUSORGANISMS#OMPREHENSIVE GENETICDATAFROMVAR ANALYSISOFAHIGHLYACTIVE HISTIDINE(IS TAGPURI lED03)) D F FROM 3YNECHOCY CYSTIS USINGTHELATEST PROTEINIDENTIlCATIONTECHNOLOGIESREVEALED D ANUM MBER OF03 ))SUBUNITSTHATHADNEVERBEENSEENINSUCHA PURIlED D PROTEINCOMPLEX +ASHINOETAL A ! SIMILARSTUDYIN 4VULCANUS +ASHINOETAL B U OF03))INTHETWO REVEALEDTHATTHEPROTEINMAKEUP Y IMILAR WITH THONLYY AFEWSU F UBTLEVARIA ORG R ANISMSISVERYS TIONS SUCHASADIFFERENTAPPARENTMO F LECULARWEIGHT OFTHE CYTOCHROME#YT B LARGESUBUNIT)RRGANG ETAL  EXAMINED03))CORECOMPONENTS D F FROM BOTH 4ELONGATUS AND SPINACH TOSHOWTHATTHEREARE EXPECTEDDIFFERENCESINSOMEOFTHESUBUNITCOMPO NENTS BUTTHEBEHAVIOROFF THECOMPLEXES IN FORM F ING MONOMERSAND DIMERS ISESSENTIALLYTHESAME )NTHISCHAPTER WEWILLDISCUSSTHELOWMOLECULAR WEIGHTPROTEINSUB U UNITSOF03))W F ITHRESPECTTOTHE

 NEW DISCOVERIES THAT CONTRIBUTE TO A BETTER OVERALL UNDERSTANDINGOFTHEFUNCTIONOFTHEENTIREPROTEIN COMPLEX -OST OFF THE PROTEINS DISCUSSEDD ARE LESS THAN K$A BUTAFEWARE F BETWEENANDD  K$A 4HISGROUPINCLUDESTHEHEME BINDINGHETERODIMER #YT B WH W OSE FUNCT F IONHAS BEENASUBJ U ECTOF INTENSE DEBATE FORA F LMOSTTHIRTYYEARS #HAPTER &ALLERAND 2 2UTHERFORD 4HEMAJORITYOFTHEPROTEINSCOVEREDIN THISCHAPTERCONSISTSOFS F INGLE HELIX MEMBRANE SPAN NINGPROTEINSTHATAREUSUALLYORGANIZEDD PERIPHERALLY TOTHELARGEINTEGRALPROTEINS-ANYOFTHESEPROTEINS ARENOTESSENTIAL FOR03))ACT F IVITY Y BUTTHEY APPEARTO BEIMPORTANT INREGULATIONORBIOGENESIS03))ALSO HASSMALLSOLUBLE EXTRINSICPROTEINS ASSOCIATEDWITH EITHERTHESTROMALCYTOPLASMICORLUMENAL SURFACEO F F 03))4HESMALLEXTRINSICPROTEINS DISCUSSED HERE DO NOTHAVEAKNOWNROLEINOXYGENEVOLUTIONACTIVITYAS DOTHE LARGEREXTRINSICPROTEINSDESCRIBED IN#HAPTER "RICKERANDD "URNAP -OSTOFT F THESMALL03))SUB U UNITS F FOUNDINCYANOBACTERIAAREALSOPRESENTINPLANTSAN D ENCODED BY GENESINTHECHLOROPLASTGENOME ** .FNCSBOF
4QBOOJOH 4VCVOJUT "
1TC&' ‡ $ $ZUPDISPNF
C 4HIS ENIGMATIC MEMBER OF F THE 03)) COMPLEX IS A HEME LINKED HETERODIMER OF THE SINGLE MEMBRANE SPANNINGPROTEINS 0SB%ANDD 0SB&#YT B WAS lRST BIOCHEMICALLYCHARACTERIZED BY'AREWAL AND7ASSER D

MANB WHODEVELOPEDANEFlCIENT4RITON BASED PURIlCATIONMETHOD5SING THISPURIlCATIONMETHOD 'AREWAL AND7ASSERMAN A D #YT B WASISO LATEDANDSEQUENCED(ERRMANNETAL 7IDGERET AL  4HEPROTEINCONTAINSONE _ SUB U UNIT 0SB% ANDD ONE ` SUB U UNIT 0SB& & EACHOFW F WHICH CONTRIBUTES ONEHISTIDINERESIDUETOCOORDINATEASINGLEHEMETO GIVEITSUNIQUETWO CHAINSTRUCTURE0SB% ISK$A ANDD 0SB& ISK$AANDD THEPROTEINSAREANDD  AMINOACIDS RESPECTIVELY Y )NMOST03)) CONTAINING BACTERIA THEPROTEINSARE ENCODED BYTH T E PSB%&,* ,* OPERON4HEGENE PSB& WAS & ACTUALLYFOUNDBECAUSEITSRIBOSOME BINDINGREGION OVERLAPSTHESTOPCODON FO F R PSB% % (ERRMANNETAL  'ENETICANALYSISOF #YT B IN 3YNEC 3 HOCYSTIS  0AKRASI ET AL    AND #HLAMYD M DOMONAS REINHARD R TII -ORAIS ET AL  F IVITY BUT  INDICATEDD THATITISREQUIRED FOR03))ACT MAYNOTBEINVOLVEDINPRIMARYELECTRONTRANSPORT! RECENTSTUDY OFTO F BACCOCHLOROPLASTMUTANTS INDICATED

'05  '05

600 605 

5 665 5  615 5 660  610 615 5 610

615 5 610 600

600

600  %& %& 615 5

46(  (*!&%
%*(
'(&*!%

46(
.*& (&$

#(
)++%!* 46(
.*& (&$
 )$##
)++%!* 46(
)$##
$$(%)'%%!% '(&*!% 46(
)$##
$$(%)'%%!%
'(&*!% 46(
)$## $$(%)'%%!%
'(&*!% 46(
)$##
$$(%)'%%!%
'(&*!% 46(
)$## $$(%)'%%!%
'(&*!% 46( )$##
$$(%)'%%!% '(&*!%

46(
$%%))*!#!/!%
'(&*!%

46(  "
-*(!%)!
'(&*!%

46(

"
-*(!%)!
'(&*!%

46(

"
-*(!%)!
'(&*!% 46( #! *
(,)*!% &$'#-
(+#*&( 46( ) )$##
$$(%)'%%!% '(&*!%

600  600 %& 610 610

600 605  600 

46(!

"
-*(!%)!
'(&*!%

46("
.*& (&$
 

46(#
)$##
$$(%)'%%!%
'(&*!% # # 46($
)$##
$$(%)'%%!%
'(&*!% $ 46(%
)$##
$$(%)'%%!%
'(&*!% % % 46(&
)$##
$$(%)'%%!%
'(&*!%

46(   "
-*(!%)!
'(&*!%

46(
"
-*(!%)! '(&*!%

46( 2
-*(!%)! '(&*!%

%&

'00 '05 

605  600

46(

 #&(&' .##!%!%
'(&*!% 46(

 #&(&' .##!%!%
'(&*!%

.) .)

'00 '00

%& .) .) .)

.)

.)

%&

%& %& .)

.)

.)

.)

.) .) .) .) .) .) .) .)

.)

'00

%& '65 5  '65 '65 

'00

'05

%&

%& %& '60

'00

'00

'00

'65 5  '65 5  '60 '65

'65 5  '60 '65 5 

'60

 %)

'00  '05 5 

'05  '05 '05 5 

605 600

605

46( 

(*!&%
%*(
'(&*!%

.) .)

 482)7/,351+     

2'('+2' )'  

 &*&).)*$  %

:2+).3):67/66 )'  

4 4ABLE  ,ISTOFF 0HOTOSYSTEM))GENES INREPRESENTATIVE GENOMES

.)

.)

%& .) .) .)

 %)

.)

%&

%& %& .)

.)

.)

.)

.) .) .) .) .) .) .) .)

.)

.) .)

.)

5/).3*+61/81 +5: 5:7.5'+81

705 5  

7700 

 



%&   

 

%&

700 700 %& 7  765 7  760 765

%&

%&

%& %& 

%&

 





        



 



1'5/286 

7 700 

%&

%& %& 7765

7 

700

705 

7  700

765 7  765 7  760 7   765 7  765 760 7  765 7  760

705  7 5 705

705 705 

705 7  705 705


+032-'786 

 



       

  

  

%&       

 

  

%&

%& %&  

%&

  

 

       

 

    

   

1'5/286   

)# 

%&

%& -65  %& %&

-00

-00

0   -00

%&

%& %& -65

%&

-00

-05 

-65 5

-65 -65 -60  -65 -65

5 -65 -60

-05

-05 -05

-00 -05

-05  -05 5 -05 

9/30')+86  

%# 

%#



%& %# #  %#  # %#

%#

%# 

%&

%& %& %# 

%#

%# 

%#

%# %# #  %# %# #  %# %# %# #  %# 

%# #

%#

%#  %# #

%#  # %#  %#  %#

:2+).3)3))86 )'  

#&(&'#)* #&(&'#)* #&(&'#)* #&(&'#)* #&(&'#)* #&(&'#)* #&(&'#)* #&(&'#)*

*



*  * 

*  *  *   #&(&'#)*

%&

%&

*  * 

*  *   #&(&'#)*

*  *   *

*

 *   *  *  *  *  *  *

 *  *  

* 

 *  

       

 #&(&'#)*

 #&(&'#)*  #&(&'#)*

 #&(&'#)*

5'(/*346/6 7 '0/'2 7. / '

 -FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** THAT 0SB%AND 0SB&AREEACH REQU Q IRED FORT F HESTABLE INSERTIONOFTHEOTHER ANDWITHOUTTHESE PROTEINS THE $SUB U UNITISNOTSTABL A E3WIATEKETA K L  &URTH T ER SUPPORTINGTHECONNECTIONBETWEEN $STABILITYAND #YT B IS EVIDENCE FROM ,UPINKOVA ET AL  INDICATING THAT 3YNECHOCY CYSTIS  $ (IS IS REQU Q IRED FORA F DDUCT D F FORMAT IONWITH THE. TERMINAL SERINEOF#YT B UNDERDAMAGINGCONDITIONS (ISTORICALLY ONEO Y F HEDILEMMASINUNDERSTAND FT INGTHESTRUCTUREOF#YT F B WASTHEORIENTATIONOF THESUBUNITSRELATIVETOEACHOTHERANDTHETHYLAKOID D THAT THE #YT HAS AN MEMBRANE )T WAS POSTULATED _` TOPOLOGYWITHTHE ` SUBUNIT# TERMINUSINTHE STROMA ANDTH D E ASSOCIATED D HEME CLOSERTO THE LUMEN -C.AMARAETAL   7I 7 TH THEPUBLI U CATIONOFT F HE 03))CRYSTAL STRUCTUREFROM F 4ELONGATUS ITSEEMSTHAT THEDEBATEISlNALLYRESOLVEDINFAVOROFAPARALLEL HETERODIMERWITHTHE. TERMINIOF BOTH SUB U UNITSIN THESTROMA :OUNIETAL   !NOTHERCONTROVERSYTHATISSTILLUNRESOLVEDREGARDS THE NUMBER OF F CYTOCHROME HETERODIMERS PER 03)) A ORDIFlCULTIESINVOLVESPURIFY F ING CENTER/NEOFF THEMAJ THE03))CENTERWITHOUTDISTURBINGTHECYTOCHROMES BOUND D TO THE SYSTEM )N TWO RECENT STUDIES USING PURIlED D 03)) WITH A POLY HISTIDINE TAGGED #0 THERE ARE DIFFE F RENTRATIOS OFF #YT B TO03))CENTERS ,AKSHMI AND D COLLEAGUES ,AKSHMI ETAL  FOUN F D D 03))PREPARATION  #YT B HEMESINA HIS TAGGED W WHILE+ASHINOETALA FOUNDHEMESPER03 ))4HECURRENTCRYSTAL STRUCTURE INDICATESTHATTHERE IS ONE #YT B PER 03)) &ERREIRA ET AL  SO W WHETHERONEISREMOVEDMOREEASILYORTHEEXTINCTION COEFlCIENTOFF ASINGLE HEMEISVARIABLEREMAINSAN Q ION OPEN QUEST Q !NATTRIBUTEOF#YT B THATMAKESITUNIQUEASWELL AS DIFlCULT TO UNDERSTAND IS ITS CHANGING MIDPO D INT 4 4ABLE ,EGEND @9ESMEANSTHEPRESENCEOFASPECIlEDGENE WHICHISCONlRMEDBY",!34SEARCHBUTISNOTYETANNOTATEDAS AN/2&.UCLEOTIDEANDDEDUCEDSEQUENCEDATAWEREDOWNLOADED FROMTHEWEB.AKAMURAETAL  #OMPLETENAMESFORTHE ORGANISMSINCLUDED INTHETABLEARE 3YNEC 3 HOCYSTIS SP0##  . C PUNCTIF IFORME !4 ! ##  !NABAENA SP 0##  .OSTO 4 HODESMIUM 4RIC D ERRYTH T RAEUM 4H 4 ERMOSY S NECHOCOCCUS ELONGATU G S "0  0ROCHLOROCOCCUSMARINUS -%$ 0ROCHLOROCOCCUSMARI NUS -)4 'LOEOBACTERVIOLACEUS 0## 3YNECHOCOCCUS SP7(AND!RABIDOPSISTHALIANA !RABIDOPSIS GENESWERE IDENTIlEDBYSEARCHINGTHE !RABIDOPSIS GENOMEWITH 3YNECHO CYSTIS 03))GENES WHEREAPPLICABLE4HENOMENCLATUREFOR THENUCLEARGENESREFERSTOLOCUSNUMBERSASPUBLISHEDINTHE4HE F 'ENOMIC2ESEARCH !RABIDO DOPSIS DATAB A ASE  .O )NSTITUTE FOR MENCLATURE FORC F YANOBACTERIAL GENES ISASTHE/2&SAREANNOTATED IN#YANOBASE .AKAMURAETAL A 4HETABLEWASUPDATED $EC  SOANNOTATIONSARECURRENTASOFT F HAT DATE

 POTENTIAL4HETWOPREDOMINANTFORMSARET F HE HIGH POTENTIAL(0 ANDLOWPOTENTIAL,0 WITHTHEIRREDOX MIDPO D INTATnM6ANDnM6 D RESPECTIVELY Y !N INTERESTINGCORRELATIONBETWEEN #YT B ANDD THE / EVOLUTION ACTIVITY OF 03)) CAN BE DRAWN FROM THE CONTRIBUTION OFF ELECTRONS FFROM THE MANGANESE CLUSTERIN FORC F INGASHIFT F FROM,0TO(0CYTOC F HROME -IZUSAWAETAL  )NTHISSTUDY 03))COMPLEXES F WHICH ABOLISHED / EVOLUTION WERE DEPLETEDD OF-N W ACTIVITYANDD CAUSED THE(0CYTOCHROMETOCONVERT TO ,0 -IZUSAWA ET AL  SHOWED THAT DURING PHOTOACTIVATIONCONDITIONS ELECTRONDONATIONFROM F THE -N CLUSTER WAS SUFlCIENT TO CONVERT ,0 CYTO CHROME TO (0 &URTHERMORE THIS STUDY LINKED THE INTERCONVERSION BETWEEN CYTOCHROME REDOX FORMS F TO 1!nOXIDATION !NEARLYHYPOTHESISFORTHEROLEOF#YT B IN 03 )) WASTHAT ITCYCLEDD EXTRAELECTRONSAROUNDD THEREACTION CENTERTORELEASEEXCESSENERGY!STUDY INSPINACH CHLOROPLASTS REVEALED THAT OXIDATION OF #YT B IN THEPRESENCEOFF WATEROXIDATION INHIBITORSCOULD BE H IOLOGICALLYRELEVANTUNDERCONDITIONS CONSIDEREDD PHYS W WHENTHEWATER SPLITTINGMACHINERYCANNOTKEEPUP WITH THEOXIDATIONCAPACITYOFF THECHLOROPHYLL (EBER ET AL   )T HAS BEEN PROPOSEDD THAT #YT B IS PHOTOOXIDIZEDBY0THROUGH#HL:AmUORESCENCE T EWATEROXIDATIONCOMPLEXFROM F QUENCHER TOPROTECTTH DAMAGEWH W ENEXCESSENERGY ISABSORB R ED 4HOMPSON AND "RUDVIG   4HE CYTOCHROME CAN BE RE REDUCE D D BY 1" UNDERSOMECONDITIONS SUPPORTING THE IDEAFOR F ITSINVOLVEMENTINANELECTRONPATH ALTERNATIVE TOTHEWATEROXIDATIONPATH"USERETAL  %VEN F AY FROMT F HE THOUGHTHE B HEME ISRELATIVELY FARAWA 1" SITE FOR F DIRECT ELECTRON TRANSFER F :OUNI ET AL +AMIYAAND3HEN &ERREIRAETAL  #HAPTERSn RECENTEVIDENCE BY,UPINKOVAAND COLLEAGUES,UPINKOVAETAL  SUGGESTSTHATTHE LINK BETWEENTHE. TERMINAL SERINE OF #YT B AND (ISON$COULDD ALLOWFORANE F LECTRONTUNNEL FROM F 1" TOTHE#YT B HEME !NOTHERMODELFORTHEFUNCTIONOF#YTB ALLOWING F FORP HOTOPROTECTIONANDENCOMPASS D ING THECHANGING MIDPOINTPOTENTIAL HASBEENCALLEDD AMOLECULARSWITCH MECHANISM%VIDENCESUPPORTINGASINGLEELECTRON REDOXPROTECTIONMECHANISMIN03)).EDBAL ETAL R ERANDD $E,AS2IVAS  0OULSONETAL  "ARB  SUGGESTSTHATUNDERINCREASINGLIGHTINTENSITY THE,0FORMO F F #YT B ACCUMULATESASANALTERNATE PATH FFOR ELECTRONS FROM F 0 THROUGH PHEOPHYT H IN !S EVIDENCED IN 0OULSON ET AL  THE SLOW PHOTOCHEMISTRY OF #YT B INVIVO IS JUSTIlED BYTHE



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

SLOWBUILDUPO D F REDUC D INGEQUIVALENTSONTHEACCEPTOR SIDEDURINGILLUMINATION)NTHISMODEL THEACCEPTOR D THE(0 SIDEOFF 03)) ISOXIDIZED BY,0#YT B AND FFORM RED DUCES THE DONOR SIDE ! MAJ A OR DIFFERENCE F BETWEENTHISMODELANDTHE#HL:QUENCHINGMODEL ISTHATITIS BESTAPPLIED D UNDERLIMITEDP D HOTO STRESSAS D TOCONSTANT ILLUMINATION OPPOSED "OTHMODELSINCORPORATE #YTB INTO03))ACTIVITY F LTERNATEELECTRONPATHWAY AY)TAPPEARS ASPARTOFANA Q A EVIDENTTHAT#YT B ISNOTREQUIREDFORWATEROXID TION EVENTHOUGHITISIMPORTANTINTHEFUNCTIONOF 03))UNDERPHYSIOLOGICAL CONDITIONS!RECENTSTUDY BY-ORAISETAL USED D MUTATIONS INTHEHEME BINDINGSITEOF# F YT B IN #REINHARDTI # I TO SHOWTHAT REDOX ACTIVITY OFF THE CYTOCHROME IS NOT NECESSARY F / EVOLUTIONFROM03 ))4HEAUTHORSMENTIONIN FOR THEDISCUSSIONTHATTHERECOULDBEAVERYLOWLEVEL W CHWOULDD ALLOWFORT F HE OF HEME BINDING INVIVOWHI SMALL NUMBER OFF ASSEMBLED D 03 )) CENTERS IN THEIR MUTANT-ORAISETAL  )TISUNCLEARIFTHEREIS NORMAL 03))ACTIVITY INTHECOMPLETEABSENCEOFF A #YTB HEME-UCHPROGRESSHASBEENMADETOWARDS SPECULATINGTHEROLEOF#YT B IN03)) WHICHALLOWS F GRAND DEBATES &URTHER STUDIES ARE REQUIRED FOR D TO DETERMINEHOWTHEPRESENCEOF#YT F B AND ITSREDOX CONTRIBUTIONSINmUENCETHEACTIVITYOF03))FORMORE DETAILSSEE#HAPTER !HRLINGETAL  #
1TC) 4HE0SB(SUB U UNITHASAMOLECULARWEIGHTINTHERANGE OF nK$A)TWASIDENTIlEDIN03))PREPARATIONS F FROM HIGHERPLANTS SPINACHAN H D WHEAT TH T E GREENALGA # #REIN HARD RDTII AND D THECYANOBACTERIUM 4VU 4 LCANUS BY . TERMINALSEQUENCING(IRDETAL -ICHELAND "ENNETT $EDNERETA D L +OIKEETAL   4HE PSB( GENE WAS IDENTIlED BASED D ON SEQUENCE HOMOLOGYIN 3YNECHOCYSTIS !BDEL -AWGOOD D $ILLEY  Y AND D MORERECENTLY0SB(WAS IDENTI AND lED D ASA PROTEININ 3YNEC 3 HOCYSTIS  THY H LAKOIDSBY -!,$)-3ANALYSIS!BDEL -AWGOODAND$ILLEY  3ZABO ET AL  +ASHINO ET AL A  5SINGAMUTANTWITH A(IS TAGATTHE. TERMINUSOF THE0SB(PROTEIN "UCHELETAL INVESTIGATED THE LOCATION OFF THE 0SB( PROTEIN IN #REINHARD R TII 03)) BYGOLD LABELINGTHEPROTEINUSINGA.I .4! LINKER4HEIRRESULTSINDICATEAPOSITIONCLOSETOTHE TWO TRANSMEMBRANE HELICES OF #YT B !LTHOUGH THEYWEREUNABLETO IDENTIFYCROSS F LINKED D PRODUCTS D BETWEEN0SB(AND#YT B 0SB(DIDCROSS LINKTO 0SB8 WHI W CH HASBEENSHOWNTOBECLOSETO #YT B

-ORERECENTLY 0SB(WASASSIGNEDTOAHELIXADJACENT TO 0SB8INTHE03))STRUCTURE&ERREIRAETAL   ( #YT B F 4HE PSB(GENEISCO TRANSCRIBEDWITHTHE COMPLEXGENES PET#AND # PET! T IN HIGHERPLANTS BUT IS 3 S -AYESETAL MONOCISTRONIC IN 3YNECHOCYSTI  )N #REINHARD R TII THE PSB" PSB4 AND PSB( GENESARELOCATED INTHESAMEGENECLUSTER HOWEVER DELETIONMUTAGENESISSHOWEDTHAT PSB"4AND PSB( D LYSINCEDISRUPTIONATEITHER ARETRANSCRIBEDSEPARATE LOCUS HADD NOEFFECTONT F THEOTH T ER3UMMERETAL   ( CLUSTERISALSOFOUNDINSOMECYA ! SIMILAR PSB"4( NOBACTERIASUCHAS ' VIOLACEUS SUGGESTINGTHATTHE CLUSTERMAYREmECTANANCIENTGENEARRANGEMENT )NSPINACHAND #REINHARDTII # 0SB(UNDERGOESLIGHT DEPENDENT PHOSPHORYLATION AT A THREONINE RESIDUE D POSITION  OR  IN THE MATURE PEPTIDE DEPENDING ONTHESPECIES ONTHESTROMALSIDEOFTHETHYLAKOID W CH ISUSUALLY FO F LLOWED BYAREGIONOF MEMBRANE WHI BASICAMINOACIDS$EDNERETAL -ICHELAND "ENNETT  2ECENTMASSSPECTROSCOPICANALYSES D PROTEINS HAVE IDENTIlED OF HIGHER PLANT THYLAKOID TWOPHOSPHORYLATIONSITESON 0SB()N !RABIDO DOPSIS 0SB( IS PHOSPHORYLATED AT 4HR AND 4HR 6ENER ETAL  'OMEZETAL ALSO FOUN F D DIRECT F D HOSPHORYLATIONSITEON0SB( DP EVIDENCE FORASECON FROMSPINACHANDPEA)NTHEIR-3ANALYSIS TWO$A D B(WEREIDENTIlED CORRESPON D DING TOA ADDUCTSON0S DOUBL U YPHOSPHORYLATEDD 0SB(PEPTIDE!DDITIONALLY THEYOBSERVEDTWO$AADDUCTSASSOCIATEDWITHTHE DOUBLY U PHOSPHORYLATEDD 0SB( PEPTIDE W WHICH THEY H HYPOT HESIZE ISTHERESULTOFOX F IDATIVEMODIlCATION 3UNDBYETAL REPORTEDTHATSODIUMBICAR BONATEHASANEGATIVEEFFECTONT F HEPHOSPHORYLATION F B( INSPINACHANDD THATDEPLETIONOFF ENDOGENOUS OF0S BICARBONATE STIMULATED PHOSPHORYLATION OF 0SB( 4O ACCOUNT FFOR THE OBSERVEDD COMPETITION BETWEEN BICARB R ONATEANDP D HOSPHORYLATION THEYSUGGESTTHAT BASICAMINOACIDSMAYPROVIDEABINDINGSITEFORTHE BICARB R ONATEANION #YANOBACTERIAL0SB(DOESNOT HAVETHECONSERVED THREONINERESIDUETHATSERVESASTHEPHOSPHORYLATION SITE IN PHOTOSYNTHETIC EUKARYOTES (OWEVER 0SB( F FROM 3 3YNEC HOCYSTISISPHOSPHORYLATED INVITRO 3 3YNECHOCYSTIS  0SB( CONTAINS A THREONINE RESIDUE ATPOSITION BUTTHEREHASBEENNO DIRECTEVIDENCEFO F R ITSPHOSPHORYLATION0HOSPHORYLATIONWASLIGHT INDE PENDENT BUTWASINHIBITEDBYOXIDIZINGCONDITIONS 7HEN03))MEMBRANESFROMP F LANTSWERESUBJ U ECTED TOHIGH LIGHTSTRESS 0SB(WASDEGRADED 2ACEAND 'OUNARIS   $ELETION OF PSB( ( IN 3Y 3 NECHOCY CYSTIS  RESULTS

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** IN A MUTANT CAPABLE OFF PHOTOAUTOTROPHIC GROWTH AT A REDUCED RATE / EVOLUTION mUORESCENCE AND THERMOLUMINESCENCE MEASUREMENTS INDICATE THAT ELECTRON mOW FROM F 1! TO 1" ISIMPAIRED -AYESETAL Q  )TWASCONCLUDEDTHAT0SB(ISNOTREQUIREDFOR CYANOBACTERIAL03))ASSEMBLY AND FUNCT F ION INVIVO BUTTHEPOLYPEPTIDEPROBABLYOPTIMIZESELECTRON mOW BYINTERACTINGWITHTHE1" SITE ONTHE $ PROTEIN )N F HER ANALYSIS OFF THE PSB( FURT ( DELETION MUTANT +O MENDAAND D COLLEAGUES+OMENDAETAL  ALSO FOUND WEAKENED ATTACHMENT OF #0TOTHE$ $ HETERODIMERSASWELL ASWEAKENED BINDING OF BICAR BONATETOTHEACCEPTORSIDEOF03)) 1UITEADIFFERENTPHENOTYPEWASOBSERVEDINTHE # REINHARD # R TII MUTANT LACKING 0SB( /g#ONNOR ET AL 3UMMERETAL  !LTHOUGH03))CORE PROTEINSARETRANSLATEDANDINSERTEDINTOTHETHYLAKOID MEMBRANEASINWILD D TYPECELLS 03))CENTERS DONOT ACCUMULATE4HE03)) DElCIENCYISALSOOBSERVED IN DARKGROWNCELLSSUGGESTINGTHEEFFECTISNOTDUETO PHOTOINHIBITION4HEREFORE 0S F B(PLAY A SAN IMPORTANT ROLE IN03))ASSEMBLYAND D STABILITYIN #REIN # HARD R TII )NTERESTINGLY /#ONNORETAL ALSOFOUNDTHAT THESITE DIRECTED D MUTANT4HR!LAOF 0SB( IN #REIN HARD R TII WHI W CH ELIMINATESTHEIDENTIlED D PHOSPHORYLA TIONSITE HASAPHENOTYPEIDENTICALTOWILDTYPE! SLIGHT BUTCONSISTENTDECREASE IS/ EVOLUTIONACTIVITY WASOBSERVED IN4HR!LACOMPARED D TOWILD D TYPE4HIS MAYRESULTFROMASLIGHTSTRUCTURALCHANGEIN0SB( F IVITY! Y LSO IT UPONPHOSPHORYLATIONTHATEFFECTSACT IS LIKELYTHATANOTHERPHOSPHORYLATIONSITEEXISTS IN THE #REINHARDTII # PROTEINSINCETWOPHOSPHORYLATION SITESWERE IDENTIlED INHIGHERPLANT0SB( )NSUMMARY THE0SB(PROTEINFUNCT F IONS DIFFERENT F LY IN03))OFCYANOBACTERIAANDTHATOFPHOTOSYNTHETIC EUKARYOTES AS EVIDENCED BY THE DIFFERENT F MUTANT PHENOTYPES7HILETHEREISSOME DESTABILIZATIONOF CYANOBACTERIAL 03)) IN THE ABSENCE OF 0SB( THE PROTEIN ISABSOLUTELY REQUIRED FOR03))ASSEM F BLY AND STABILITY IN #REIN # HARD R TII4HEOBSERVED IMPAIRMENT OF 1! TO 1" ELECTRON mOW INTHE 3YNECHOCYSTIS  PSB(MUTANTMAYINFACTBEDUETOSLIGHTSTRUCTURAL ( CHANGESIN03))0HOSPHORYLATIONOFF 0SB(ISPROB ABLYAREGULATORYMECHANISMFORELECTRONmOWWITHIN 03))OR03))TURNOVERUPONPHOTOINHIBITION%ITHER SITUATION IS LIKELY TO DIFFER F BETWEEN CYANOBACTERIA ANDHIGHERPLANTS SODIFFERENCESINPHOSPHORYLATION AREEXPECTED $
1TC* 4HE PSB)) GENEINTHECHLOROPLASTSOFHIGHERPLANTS

 ANDD THEGENOMEOFF CYANOBACTERIAENCODESA K$A PROTEIN nRESIDUESINLENGTHDEPENDINGONTHE SPECIES)TWAS IDENTIlEDD ASAN INTRINSIC03))REAC TION CENTER COMPONENT IN SPINACH ANDD . TERMINAL SEQUENCEDATAREVEALEDITTOBEENCODEDBYACHLORO PLAST GENE)KEUCHI ANDD )NOUE  0SB)WAS LATER F FOUN D IN03))REACTIONCENTERPREPARATIONS FROM F 4 4 VULCANUS )KEUCHIETAL A #ROSSLINKING STUD IESSHOWEDD THATTHE. TERMINAL DOMAINOF0S F B)FROM F SPINACH IS INCLOSECONTACTWITH THE$PROTEINAND THE _ SUBUNIT OF#YT B 4OMOETAL  )NCON TRAST 03))STRUCTURALANALYSISHASPLACEDD 0SB)NEAR THEDIMERINTERFACE F WHI W CH ISCLOSERTO $AND #0 THAN $ ANDD #YT B &ERREIRAETAL   ! #REINHARD R TII PSB)) DELETIONMUTANTISCAPABLE OFP F HOTOAUTOTROPHICGROWTHUNDERLOWLIGHTCONDI TIONS BUTNOTHIGHLIGHTCONDITIONS4HEAMOUNTOF D D / EVOLUTIONACTIVITY IS ASSEMBLED03))CENTERSAN nTHATOFF WILDD TYPEAS DETERMINED BYVARIABLE mUORESCENCE YIELD AND STEADY STATE / EVOLUTION ACTIVITIES5NLIKEOTHER03)) DElCIENTMUTANTS THE HIGH LIGHT SENSITIVITY OFF THE PSB) ) DELETION MUTANT PERSISTED WHEN THE MUTANT WAS GROWN ON MEDIUM CONTAINING ACETATE +UNSTNERETAL  4HUS IN # #REIN HARD R TII 0SB) ISREQUIRED FOR F BOTHASSEMBLYAND FUNCTIONOF03 )) !DIFFERENTP F HENOTYPEWASOBSERVED FORT F HE 3YN ECHOCYSTIS  PSB)) DELETIONMUTANT4HECYANOBAC TERIALMUTANTHADALOSSOFONLYn03))ACTIVITY D TYPE /THER 03)) PROTEINS COMPAREDD TO THE WILD ACCUMULATEDD TOWILD D TYPE LEVELSWITH NOSIGNIlCANT DESTABILIZATIONOF03))CENTERS ANDTHE 3YNECHOCYSTIS I  MUTANTISONLY SLIGHTLYMORELIGHTSENSITIVETHAN THEWILDD TYPE)KEUCHI ETAL B 4HECONCLUSION 3  MUTANTINDICATES THAT FROMTHE 3YNECHOCYSTIS 0SB) IS NOT ABSOLUTELY REQUIRED FFOR 03)) ASSEMBLY AND FUNCT F ION INCYANOBACTERIA BUTOPTIMIZES03)) ACTIVITY 03)) CENTERS HAVE NOT BEEN ISOLATED FOR F FURT F HER BIOCHEMICAL ANALYSIS FFROM EITHER OFF THE MUTANTS DESCRIBEDABOVE"ECAUSEOFTHEIRMORESTABLE03 )) 4 LONGA G CENTERS THETHERMOPHILICCYANOBACTERIUM 4E TUS REPRESENTSA BETTERSYSTEMFOR F ISOLATINGMUTANT 03))CENTERSFORMOREDETAILEDBIOCHEMICALANALY SES+ATOHANDD )KEUCHI A CREATEDD SUCH A PSB) DELETIONMUTANTAND FOUN F D THE ISOLATEDD 03))CENTERS TOBEEXCLUSIVELYMONOMERIC4HEMUTANTALSOHAD DECREASED LIGHT HARVESTINGEFlCIENCY4 Y HESERESULTS DEMONSTRATETHAT0SB)PLAYSAMAJ A ORSTRUCTURAL ROLE IN03))DIMERASSEMBLYANDTHEFUNCTIONALASSEMBLY F HEANTENNACOMPLEX BUTISNOTREQUIRED FOR03)) F OFT F FUNCT IONINVITRO



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

%
1TC+

& 1TC, &


4HE 0SB* SUB U UNIT IS ENCODED BY THE PSB* * GENE A MEMBEROFF THE PSB%&,** OPERONTHATISCONSERVEDIN MOSTOXYGENICPHOTOSYNTHETICORGANISMS4HE PSB* B GENEWASDETERMINED D TOBETHESECONDOPENREA D DING F FRAMEC LOSELY DOWNSTREAMOFF THEGENESENCODINGTHE SUBUNITS OF #YT B ANDWASGIVENITSNAMEBECAUSE OFF APRESUMED ASSOCIATIONWITH 03)) #USHMANETAL  4HEPROTEINENCODED BY PSB*IN * 3YNEC 3 HOCYSTIS WASDETERMINEDTOBELOCALIZEDINTHETHYLAKOID D ET MEMBRANEAND HAVEAROLEIN03))ACTIVITY ,IND AL  0SB* ISPREDICTED D TO BE K$AAND IS AMINOACIDSLONGWITHAMINOACID LONG.# TERMI NAL EXTENSIONSmANKINGASINGLEMEMBRANESPANNING HELIX%VENTHOUGH0SB*WASPREDICTED D TOBEPARTOF 03))FORMANYYEARS IT WASlRSTSEENINANISOLATED 03))PREPARATION INARECENTSTUDY BY +ASHINOETAL A -ORERECENTLY IT HASBEENIDENTIlED D TO BE ACOMPONENTOFTHECRYSTALLIZED03))COMPLEX&ER REIRAETAL   -UTATIONS IN PSB** HAVEBEENMADEINBOTH3YNEC 3 HO CYSTIS ,INDETAL  ANDTOBACCO(AGERET D W CHSHOWED D THAT0SB* AL 2EGELETAL  WHI ISREQUIRED FORP F HOTOAUTOTROPHICGROWTH INTOBACCO BUTNOTIN 3YNECHOCYSTIS !MAJORDIFFERENCEIN 03)) ACTIVITY BETWEEN CYANOBACTERIAL AND D TOBACCO 6 * MUTANTSISTHATTHEELECTRONmOWFROM 1! TO 6PSB* 1" ISSLOWEDBYONLYINTHE 3YNECHOCYSTIS 3  MUTANTAND BYALMOSTTWOORDERSOFMA F GNITUDEINTHE TOBACCOMUTANT 2EGEL ETAL  3INGLEmASH/ EVOLUTIONEXP X ERIMENTSINBOTH3YNECHOCYSTIS  AN A D TOBACCO INDICATED D THAT0SB*ISREQUIRED FORT F THESTABILI A TY T F HE-NCLUSTERINTHE / EVOLUTION OFF THE 3N STATESOFT 3 APPARATUS2EGELETAL  )NTHE 3YNECHOCYSTIS  PSB** MUTANT THEDECAYOFTHE3 STATEVIA BACK F HEACCEPTORSIDE ISTWENTYTIMES ELECTRONmOWFROMT F FASTERTHANINWILD TYPECELLS2EGEL ETAL  )N TOBACCO YOUNG 6PSB* 6 * LEAVESHAVESLIGHTLYIMPAIRED 03)) DRIVENPHOTOCHEMISTRYWHIL W EMATURELEAVESARE SEVERELYDAMAGED(AGERETAL  SUGGESTING THATLIGHT INDUCE D D PHOTODAMAGE ISACCUMULATED INTH T E ABSENCEOFF 0SB*4HEDAMAGEACCUMULATED INMATURE 6 * TOBACCOLEAVESINCLUDESTHELOSSOFOXYGENEN 6PSB* HANCER/% PROTEINS BUTTHISAPPEARSTO BEA GENERAL D NOT DIRECTLYRESULTINGFROM F RESULTOFF THEDAMAGEAND THE ABSENCE OF 0SB*SINCETHOSEPROTEINSAREPROPERLY ASSEMBLED IN YOUNGERLEAVES,IKEMANY N OFT F HEOTHER U UNITSOF03)) F IT ISUNCLEARWH W ATTHEEXACT SMALLSUB ROLEOF0SB*ISINTHECOMPLEX

4HE PSB+GENEENCODESAnK$APOLYPEPTIDE + PREDICTEDD TOCONTAINASINGLETRANSMEMBRANEREGION WITHITS. TERMINALDOMAININTHETHYLAKOIDLUMEN )NSPINACH THETRANSLATIONPRODUCT D HASRESIDUES D BUT THE MATURE PROTEIN IS ONLY  AMINO ACIDS IN LENGTH 0SB+ WAS lRST IDENTIlED AS A CHLOROPLAST ENCODEDD SPINACH 03)) COMPONENT -URATA ET AL   4HE CYANOBACTERIAL 0SB+ SUB U UNIT WAS lRST 4 VULCANUS 03))PREPARATIONS+OIKE IDENTIlEDD IN 4 + IDENTIlCATIONANDCLONING ETAL  ,ATER PSB+ IN 3YNEC 3 HOCYSTIS REVEALEDD AGENECODING FORA F RESIDUEMATURE PROTEINWITHASHORTAMINOACID PRESEQUENCETHAT ISCLEAVEDD UPON INSERTION INTOTHE H LAKOIDD MEMBRANE)KEUCHIETAL  $E6I 6 TRY THY ETAL REPORTEDDETECTABLEAMOUNTSOF0SB+ IN # # REINHARD R TII 03)) CORE COMPLEXES 0SB+ WAS LATERFOUN F D INTHEDIMERFORMO F F #0 DEPLETED03)) D FROMSPINACHINDICATINGATIGHTERASSOCIATIONWITHTHE 03))CORECOMPLEXTHANORIGINALLYREPORTED 2H 2 EE ETAL  )NCONTRAST ITWASREPORTEDD THAT0SB+ PRESENTIN03))CORECOMPLEXESFROM #REINHARDTII # ISSPECIlCALLY BOUNDTO#0 D 3UGIMOTOANDD 4AKA HASHI  )NTERESTINGLY THE0SB+HELIX ISCLOSELY ASSOCIATED WITH #0 IN THE CYANOBACTERIAL 03)) CRYSTAL STRUCTURE &ERREIRAETAL   "ECAUSE 0SB+ REMAINS BOUND TO 03 )) AFTER F REMOVAL OF ,(#)) BUT IS NOT PRESENT IN THE 03)) REACTION CENTER CORE IT WAS CONCLUDEDD THAT 0SB+ WAS NOT ESSENTIAL FFOR 03)) ACTIVITY -URATA ET AL  (OWEVER 0SB+INACTIVATIONIN #REINHARDTII # CHLOROPLASTS RESULTS IN MUTANTS THAT ARE UNABLE TO ACCUMULATE NORMAL AMOUNTS OFF 03 )) ANDD CANNOT GROW PHOTOAUTOTROPHICALLY 'ROWTH RATES RETURNED TO WILD TYPE LEVELS ON ACETATE CONTAINING MEDIUM 0ULSE LABELING EXPERIMENTS CONlRMEDD A 03)) DE STABILIZATIONINTHE MUTANTRATHERTHAN A REDUCTION IN POLYPEPTIDESYNTHESIS4HISPHENOTYPESUGGESTSTHAT THE 0SB+PROTEINISNECESSARY FOR03))STA F BILIZATION INVIVO4AKAHASHIETAL   + DELETION MUTANTS IN THE CYANOBACTERIUM PSB+ 3 3YNEC HOCYSTIS GROWPHOTOAUTOTROPHICALLYATA TWO FOLDREDUCEDRATEANDHAVESLIGHTLYREDUCED03)) TY COMPARED TOTTHEWILD D TYPE4HESERESULTS DEM ACTIVITY ONSTRATETHAT0SB+ISNOTESSENTIAL FOR03))ACT F IVITY INPROKARYOTICCELLS 0SB+MAYHAVEOTHERFUNCTIONS INCYANOBACTERIABECAUSEADDITIONOF GLUCOSETOTHE MEDIUM DOESNOTFU F LLYRESTOREWILD TYPEGROWTH RATES ASOBSERVEDINOTHER03))DElCIENTMUTANTS)KEUCHI ETAL  

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** ! DETAILED BIOCHEMICALANALYSISOFT F HE03))CEN TERSINTHESEMUTANTSWASNOTPOSSIBLE BUTRECENTLY A DELETION MUTANT IN THE THERMOPHILIC BACTERIUM 4 LONGATUS AFFOR 4E F DED SUCH A STUDY +ATOH AND )KEUCHI A 4HEMUTANTPHENOTYPEWASSIMILAR TOTHATOBSERVED FORT F HE 3Y 3 NECHOCY CYSTIS  MUTANT #ELLS WERE CAPABLE OFF PHOTOAUTOTROPHIC GROWTH AT SLIGHTLY BUTREPRODUCIBLYSLOWERRATESUNDERVARIOUS TEMPERATURECONDITIONS4HE/ EVOLUTIONACTIVITY OF THE 0SB+ DEPLETED D 03)) PARTICLES WAS COMPARABLE TOTHEWILDTYPEATLOWCONCENTRATIONSOF$#"1!T HIGHER$#"1CONCENTRATIONS 0SB+ DEPLETED D 03)) PARTICLESHAD D ALOWER/ EVOLUTIONACTIVITY+ATOHAND )KEUCHI A !SIMILARACCEPTORDEPENDENCYWAS ALSOOBSERVED FOR0S F B8INTHESAMEORGANISM +ATOH AND D )KEUCHI B  ' 1TC!NOTHER MEMBER OFTHE PSB%&,* ,* OPERONISTHEGENE ENCODINGTHESMALL 03))SUB U UNIT 0SB,)TISPREDICTED TOBEAMINOACIDS LONGWITH ASINGLEMEMBRANE SPANNINGHELIXANDA AMINOACIDSOLUBLE. TER MINAL EXTENSION4HE 0SB, HELIXIS LOCATED D NEARTHE DIMERINTERFACEW F ITH TH 0SB4AND D 0SB- INCYANOBACTERIA 3 S  6PSB, 6P P &ERREIRAETAL  ! 3YNECHOCYSTI MUTANT LACKSAFUNCT F IONAL 03))COMPLEX!NBUDURA D I D 0AKRAS K I  +ITAMURAETAL /ZAWAET AND AL  WHILETHE TOBACCO MUTANTRETAINS RESIDUAL 03))ACTIVITY 3WIATEKETA K L  4OBACCO 6PSB, 6P P CANNOTGROWPHOTOAUTOTROPHICALLYAND IS HYPERSENS H I TIVETOLIGHT3WIATEKETAL  !DDITIONALLY THE 0SB*SUB U UNITISLOSTINTHETOBACCO 6PSB, 6P P MUTANT BUT 0SB*DOESNOTSEEMTOBEREQU Q IRED FORT F HEPRESENCE OF0SB,3WIATEKETAL   )NVITRORECONSTITUTIONOF HYDROP D HOBICCOMPONENTS INAPARTIALLY DISINTEGRATED D 03))COMPLEXHASONLY BEENSUCCESSFULFOR0SB,PROTEIN.AGATSUKAETAL D 03)) PARTIAL COMPLEX   7HEN 01 DEPLETED CONSISTING OF #0$$0SB)0SB7 WAS RECON STITUTED WITH 01 AND 0SB, FUNCTIONAL 1! ACTIVITY WASRESTORED3UCH RECONSTITUTIONISSPECIlCALLY DE PENDENT ON THE ADDITION OFF 0SB, +ITAMURA ET AL   &URTHER ANALYSIS USING RECOMBINANT 0SB, SHOWED D THATTHE# TERMINALPARTOF0S F B,ISCRUCIAL F FORRECOVER INGTHE1! ELECTRONTRANSFERACT F IVITY INTHE RECONSTITUTEDCOMPLEXES/ZAWAETAL  4HESE F LECTRON STUDIESSUGGESTTHAT0SB, ISNECESSARY FORE TRANSFER F FROM F 1!

 (
1TC. 4HE 0SB- SUB U UNIT WAS ORIGINALLY IDENTIlED BY . TERMINAL SEQUENCING AS A  K$A PROTEIN IN 03)) PREPARATIONSFROM 4 4 VULCANUS ISOLATEDINTHE PRES ENCEOFF 4RITON8 ANDD UREA)TWAS FOUN F D TO BE HOMOLOGOUSTO/2& FFROM TOBACCOAND LIVERWORT CHLOROPLAST$.!ANDWASPREDICTEDTOHAVEASINGLE TRANSMEMBRANESEGMENT)KEUCHIETAL B 4HE 0SB- PROTEINWASALSOIDENTIlED IN03)) PARTICLES # I DE6ITRYETAL  !NALYSIS FROM #REINHARDTI CYSTIS  03))BY +ASHINOETALA OF 3YNECHOCY CONlRMEDD THE PRESENCEOFT F HE0SB- PROTEINFORT F HE lRSTTIMEINTHATORGANISM4HE0SB-HELIXWASALSO 4 LONGATU G S 03))CRYSTAL STRUCTURE &ER SEEN INTHE 4E REIRAETAL  2ECENTLY PSB-DISRUPTIONMUTANT WAS CREATEDD IN 4 4 ELONGATUS BUTNOCLEARPHENOTYPEWAS DETECTED #!OY O AMA -)WAI -)KEUCHI PERSONAL COMMUNICATION !LTHOUGH ITISASSOCIATEDD WITH03)) FROMALLMODELORGANISMSSTUDIED NOFUNCTIONSHAVE BEENHYPOTHESIZED FOR0S F B- )
1TC/ "ASEDD ONAPARTIAL . TERMINAL SEQUENCEO Q F A K$A 4 POLYPEPTIDE IN ISOLATED 03)) PREPARATION FROM 4 VULCANUS )KEUCHI ETALB NAMED ITTHE0SB. PROTEIN4HE PSB.GENEISPRESENTINCYANOBACTERIA . ASWELLASTHECHLOROPLASTGENOMESOFANUMBEROF PLANTSANDD ALGAE:OUNIETAL HAVESTATEDD THAT THE CRYSTALLIZED D 03)) COMPLEX FROM F 4 ELONGATUS 4 HAS THE 0SB. PROTEIN ALTHOUGH THE PRIMARY DATA U SHED SUPPORTING THIS STATEMENT HAS NOT BEEN PUBLI YET3UB U SEQUENTLY A HELIXNEAR0SB+WASTENTATIVELY NAMED0SB.INTHEMOSTRECENT03))CRYSTALSTRUC TURE &ERREIRAETAL  )NCONTRAST +ASHINOETAL B HAVESHOWNTHATTHEORIGINALASSIGNMENTOF THE0SB. PROTEININ 4VULCANUS WASINCORRECT AND THISPROTEIN ISACTUALLY 0SB4C SEE BELOW 4HEREFORE F F B.IN03))REMAINSAMBIGUOUS THEPRESENCEOF0S *
1TC4 4HEINTEGRAL03))SUBUNIT 0SB3 ISAK$A PROTEIN F FOUN D INPLANTSANDD NOTINCYANOBACTERIA0SB3WAS F D ASACOPRECIPITANTINANANTIBODYPU D LL ORIGINALLY FOUN DOWNEXPERIMENTWITH 0SB/ANDD 0SB0 FROM SPINACH THYLAKOIDS ,J , UNGBERG ET AL A  )T HAS AMINO D SEQUENCE HOMOLOGYTO CHLOROPHY H LL AB BINDING ACID PROTEINSTHATMAKEUPTHELIGHTHARVESTINGCOMPLEX ,(# 7EDEL ETAL  -OST,(#PROTEINS HAVE



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

THREEMEMB M RANESPANNINGDOMAINS BUT 0SB3 HASFOUR F SUCHSPANS%VENTHOUGH 0SB3ISSUGGESTEDTOBIND K L  ITWASRECENTLY SHOWN CHLOROPHYLL &UNKETA TOHAVEAVERYWEAK K AFlNITY FORP F IGMENTS$OMINICI ETAL  !N !RABIDOP O SIS MUTANTDElCIENTIN03 )) D AND NONPHOTOCHEMICALQUENCHING NPQ WASCLONED D TOTHE PSB3LOCUS,IETAL  &URTHER 3 MAPPED ANALYSISOFTHISMUTANTREVEALEDTHAT0SB3DElCIENT PLANTS HAD D NORMAL QUANTUMYIELD D OFE F LECTRONTRANSPORT AND D NORMAL / EVOLUTION BUTDECREASEDNON PHOTO CHEMICALQUENCHINGWHENEXPOSEDTOHIGHLIGHT,I ETAL  /THERSTUDIESONTHETRANSCRIPTLEVELSOF PSB3INDICATETHATITMAYPLAYAROLEINTHEGREENING 3 PROCESSOFHYPOCOTYLSANDISPROBABLYREGULATEDBY THEPHYTOCHROMESYSTEM!DAMSKAETAL   0SB3 DOESNOTAPPEARTO HAVEADIRECTROLE INTHEACTIVITY OF03)) ANDWILLBEDISCUSSEDFURTHERFORITSROLEIN PIGMENTASSOCIATIONIN#HAPTER0OGSONETAL  +
1TC5 5D )NTHEEARLYS TWO03))PROTEINSWEREALMOST SIMULTANEOUSLYNAMED0SB4!MONGTHEM THElRST ONEISENCODED BY THE YCF C /2& INTHECHLOROPLAST # HARD R TII AND IS HENCE GENOMESOFF PLANTSAND #REIN REFE F RRED D TO ASTHE 0SB4C4HISGENEISUSUALLYLOCATED DOWNSTREAMOFT F HE PSB" GENE FORT F HE #0PROTEIN 0SB4C ISA^K$A INTRINSICMEMBRANEPROTEIN WITH A SINGLE PREDICTED MEMBRANE SPAN4HIS PROTEIN IS ALSOPRESENTINCYANOBACTERIAL 03))+ASHINOETAL A  4HE PSB4 4C SMR GENE HAS BEEN INACTIVATED IN 3YNECHOCY CYSTIS  ( +ATOH AND D - )KEUCHI PERSONAL COMMUNICATION BUT THE MUTANT HAS NOT BEEN FUNCTIONALLY CHARACTERIZED PSB4 4C WAS ALSO INACTIVATED IN 4 4 ELONGATU G S CAUSING A DECREASEINTHE DIMERFORMO F F F03)) BUTLITTLEDEFECTWAS F DETECTED IN THE / EVOLVINGACTIVITYOFTHECOMPLEXORINPHOTOAU TOTROPHIC GROWTHRATE H )WAI ETAL  )NTERESTINGLY Y THEMOSTRECENT03 ))STRUCTURAL MODELSHOWS 0SB4C AT THE DIMER INTERFACE CONSISTENT WITH A PROPOSED 4C ROLE IN DIMERIZATION &ERREIRAETAL  ! PSB4 INACTIVATIONMUTANTSTRAINOF #REIN # HARD R TII HAS BEEN STUDIEDINCONSIDERABLEDETAIL-ONODETAL  /HNISHI AND D 4AKAHASHI  4HIS MUTANT GROWS WELLUNDERMODERATELIGHTINTENSITIES BUTDIESUNDER HIGHLIGHT-OREOVER THERATESOFPHOTO INACTIVATION ASWELLASPHOTO DEGRADATIONOFF THE03))COMPLEX INTHISMUTANTARESIMILARTOTHOSEINTHEWILD TYPE STRAIN)NCONTRAST THERECOVERYOFPHOTO DAMAGED 03)) ISSIGNIlCANTLY SLOWERINTHISMUTANT2ADIOAC

TIVEPULSE CHASEANALYSISSHOWEDTHATTHEABSENCEOF 0SB4C DOESNOTAFFECTTHERATEOFSYNTHESISOFMAJOR 03)) PROTEINS SUCH AS $ $ #0 AND #0 SUGGESTINGTHAT0SB4C IS INVOLVED INAPOST TRANSLA TIONALSTEPDURINGTHEREPAIROF03))"IOCHEMICAL F IONATIONOF #REIN FRACT # HARD R TII THYLAKOID D MEMBRANES HASSHOWNTHAT0SB4C COFRACT F IONATESWITHTHE $$ HETERODIMERCOMPLEXTHATISDEPLETEDOF#0AND #0"ASEDD ONTHESEDATA ITHAS BEENSUGGESTED THAT I 0SB4C ISCLOSELYASSOCIATEDWITH$ ANDII 0SB4C MAY A ACTASASCAFFOLDFORTHEREPLACEMENTOF PHOTO INACTIVATEDD $ ,
1TC5 5/ 4HESECONDD 0SB4PROTEINISA^K$A K HY H DROP D HILICPRO TEIN)KEUCHIETAL C+APAZOGLOUETAL   F HISPROTEIN IS LOCALIZED INTHENUCLEAR 4HE GENEFORT GENOMEOFP F LANTS AND HASRECENTLYBEENNAMEDD THE 0SB4 4. GENE4HEPRECURSORFORMOFTHISPROTEINHASA RELATIVELY LONG BIPARTITETRANSITPEPTIDETHAT LOCALIZES H LAKOID LUMEN(ENCE THEMATUREPROTEIN INTHETHY 0SB4. ISALUMENALEXTRINSICPROTEININHIGHERPLANT 03))(OMOLOGSOFT F HISPROTEIN HAVENOT BEEN FOUN F D INCYANOBACTERIA4ABL A E 4HEFUNCT F IONOFF THEPROTEIN IN03))ISCURRENTLYUNKNOWN - 1TC8 0SB7 IS A NUCLEAR ENCODEDD nK$A PROTEIN AS SOCIATEDD WITH 03))PARTICLESINPHOTOSYNTHETICEU KARYOTES)TWASlRSTIDENTIlEDINSPINACHANDWAS PREDICTEDD TO BEAN INTEGRAL MEMBRANEPROTEINWITH ASINGLETRANSMEMBRANEDOMAIN)TSTOPOLOGY INTHE MEMBRANEISTHEOPPOSITEOFMOST03))MEMBRANE PROTEINSASIT HASALUMENAL. TERMINUS )RRGANGET AL  2ECENTLY PSB7 HAS BEENCHARACTERIZED IN THEGREENALGAE#REINHARDTII # ASWELL4HEGENECODES F FORAPROTE INOFF RESIDUESCONTA D INING ABIPARTITE TRANSITPEPTIDEOFF RESIDUESAN D D ARESIDUEMATURE D PROTEIN)WAIETAL  4HE PSB7 7 GENEISABSENT F FROMT HE 3Y 3 NECHOCY CYSTIS GENOMEANDD ALL OTHER 3 HOCYSTIS PROKARYOTESEXAMINED .OTE)NTHE3YNEC  DATABASE PSB IS MISTAKENLY ANNOTATED AS 7 7 PSB73EEBELOW 3TUDIES BY,ORK R OVICETAL ON SPINACH0SB7 REVEALEDAGENETHATCODESFORARESIDUEMATURE PROTEINWITH ANRESIDUE D BIPARTITEPRESEQUENCE4HE F LD BYLIGHT BUT M2.!LEVELSWEREUPREGULATEDD TEN FO THETRANSCRIPTANDTHEPROTEINWEREALSODETECTABLEIN ETIOLATEDD SEEDLINGS&URTHERCHARACTERIZATIONINSPIN

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** ACH FOUN F D THATOFF THE 0SB7PROTEIN LOCALIZESTO THEGRANAREGIONOFTHETHYLAKOIDS0SB7ISLOCATED D ON ITSPRESENCE CLOSETOTHE $ $ HETERODIMERBASED IN REACTION CENTER PREPARATIONS 3HI AND D 3CHRODER   5SING THE BLUE NATIVE GEL ELECTROPHORESIS SYSTEMTOSEPARATE03))MONOMERSAND DIMERSFROM F PEATHY H LAKOIDS 4HIDHOLMETAL FOUN F D 0SB7 EXCLUSIVELYASSOCIATEDWITHDIMERIC03))4HEYALSO D THAT0SB7 ISDIRECTLY ASSEMBLED INTO03)) REPORTED DIMERS IN A PROCESS DEPENDENT ON ITS NEGATIVELY CHARGED . TERMINUS 4HESE EXPERIMENTAL DATA ARE CONTRADICTORYTOTHEPREVIOUSEVIDENCETHAT0SB7IS D WITHTHE $ $ HETERODIMER&UR CLOSELYASSOCIATED THEREVIDENCEISNEEDEDTOEXPLAINHOW0SB7COULD BEASSOCIATED D WITH THE03))DIMERINTERFACEAN F D BE TIGHTLY BOUND D TO$ $ATTHESAMETIME !N !RABIDOP O SIS ANTISENSE MUTANT OFF 0SB7 WITH 7 TRANSCRIPT WAS GENER A   DECREASE IN PSB7 ATED BY3HI ETAL 4HEMUTANTCONTAINED D NO DIMERIC 03)) SUP U ERCOMPLEX BUT RETAINED NORMAL F PROPERTIES / EVOLUTION ACTIVITY ELECTRON TRANSFER WAS DECREASED BY DUETOT D HE DISAPPEARANCEOF U TOOFTHE$AND $ PROTEINS4HEAMOUNTS UP OFF OTHER03))PROTEINSWEREALSOAFFECTE F D TODIFFER F ING DEGREES)NAMOREDETAILED INVESTIGATIONOFT F HE0SB7 ANTISENSEMUTANT4HIDHOLMETAL  QUANTUM YIELDD MEASUREMENTS AFFTER PERIODS OF LIGHT STRESS D THEANTISENSE PLANTSTOBEMUCH H MORESENSITIVE SHOWED TOPHOTOINHIBITIONTHANWILDTYPEPLANTS4HUS LACK OFF 0SB7RESULTSINLESSSTABLE03)) DIMERS"ASED ON THIS PHENOTYPE THE CONCLUSION IS THAT 0SB7 IS REQUIREDFOR03))DIMERFORMATIONAND03))STABILITY INHIGHERPLANTS 0SB7ALSOUNDERGOES LIGHT INDUCE D D PROTEOLYSIS IN A PROCESSSIMILARTOTHATDESCRIBEDFORTHE$ PROTEIN !LTHOUGH 0SB7 CONTAINS A NUMBER OFF SERINES AND THREONINES IT IS NOT PHOSPHORYLATED D UNDER CONDI TIONSTHATLEADTOTHEPHOSPHORYLATIONOFOTHER03)) PROTEINS $ESPITE ITS LACK K OF PHOSPHORYLATION IT IS PROTECTED FROM F DEGRADATIONUNDERCONDITIONSWH W EN OTHER03))PROTEINSAREPHOSPHORYLATED4HESERESULTS SUPPORTTH T ECONCLUSIONTH T ATTH T E0SB7PROTEINBECOMES L ISWH W EN03)) IS DESTABILI A ZEDD AS SUSCEPTIBLETOPROTEOLYS A RESULT OF $PHOTODAMAGE(AGMANETAL  

 CHEMICALANDGENETICANALYSIS)NPLANTS ITCROSS LINKS TO #YT BHOWEVER ITISNOTPRESENTINISOLATED03)) REACTIONCENTERS3HIETAL  )NCYANOBACTERIAL 03))STRUCTURES 0SB8 HASBEENASSIGNEDD TOTHEHELIX THATLIESCLOSESTTOTHE#YT B HELICES&ERREIRAETAL +AMIYAANDD 3HEN  4HE0SB8M2.! LEVELSWEREALSO FOUN F D TO BETIGHTLYLIGHTREGULATED SUCHTHATNOM2.!ISDETECTABLEINTHEDARK3HIET AL   4HE DELETIONOF PSB8 B8INTHECYANOBACTERIUM 3YN 3 ECHOCYSTIS DIDNOTAFFECTGROWTHRATE ELECTRON TRANSPORT OR WATER OXIDATION BUT THE NUMBER OF F FUNCT IONALLYACT L IVE03))CENTERSWASONLYO L F ILD FW T TYPE0SB8M2.!SWEREOBSERVEDUNDERVARIOUSLIGH T CONDITIONSSUGGESTINGTHATTHETRANSCRIPTSARENOTAS TIGHTLY LIGHT REGULATED INCYANOBACTERIACOMPAREDD TO HIGHERPLANTS&UNK  !0SB8DELETIONMUTANT INTHETHERMOPHILICCYANOBACTERIUM 4E 4 LONGATU G S ALSO HADD A PHOTOAUTOTROPHIC PHENOTYPE BUT SHOWEDD A GROWTHDEFECTUNDERLOW#/ CONDITIONS 4HE MUTANT HADD ALOWER/ EVOLUTIONACTIVITY INTHEPRESENCEOF HIGH CONCENTRATIONSOFF ARTIlCIALQUINONEACCEPTORS COMPAREDTOWILDTYPE SUGGESTINGTHE0SB8PROTEIN IS INVOLVED INQUINONETURNOVERATTHE 1" SITE +ATOH ANDD )KEUCHI B  ,IKETHE 0SB7SUBUNIT THE0SB8 PROTEINISINSERTED INTOTHEMEMBRANEBY AMECHANISMINDEPENDENTOF THEPREVIOUSLYCHARACTERIZEDD TRANSLOCATIONMACHINER IESDESPITETHEPRESENCEOFABIPARTITETRANSITPEPTIDE +IMETAL  4ISSIERANDD COLLEAGUES 4ISSIERET AL  REPORTEDD THATTHE# TERMINAL ACIDICRESIDUES D ARE ESSENTIAL FOR THE MATURATION OF 0SB8! SINGLE 'LUTO6AL SUB U STITUTIONATPOSITIONPREVENTSPROPER INSERTIONINTOTHEMEMBRANE0ROCESSING ISPARTIALLY RESTOREDBYTHEHYDROPHILICRESIDUE!SN IMPLYINGTHAT THIS DOMAIN ISIMPORTANTFORC F LEAVAGE3UB U STITUTION OFT F HE# TERMINAL 'LURESIDUESW D ITH 6A 6 L CAUSESTHE CLEAVAGESITETOBEBURIEDINTHEMEMBRANE BUTINSER TIONISNOTAFFECTE F D4HESERESULTSSUGGESTTHATTHERE ARESTRINGENTREQUIREMENTSONTHEPEPTIDESEQUENCEOF 0SB8FORINSERTIONINTOTHEMEMBRANEANDCLEAVAGE BY THE THYLAKOIDPROCESS D ING PROTEASE EVEN THOUGH THEMECHANISMOF INSERTIONISNOT DElNED / 1TC:

.
1TC9 0SB8 ANUCLEAR ENCODED  D K$AMEMBRANEPROTEIN ASSOCIATED D WITH03)) WASlRSTIDENTIlED INSPINACH )KEUCHIETAL C 4HE0SB8PROTEINHASBEEN CHARACTERIZED IN PLANTS AND D CYANOBACTERIA BY BIO

4HESMALL03))SUBUNIT 0SB9 WASlRSTIDENTIlEDAS F INACH ANDD TOBACCO03))PREPARA ACOMPONENTOFSP TIONS BY'AUETAL  )TISAnK$A SINGLE MEMBRANE SPANNING PROTEIN WITH A n AMINO U E DOMAIN )N SPINACH AND ACIDD # TERMINAL SOLUBL



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

!RABIDO DOPSIS PSB9 9 ENCODES A n K$A PRECURSOR PROTEINWITHABIPARTITESIGNALPEPTIDETARGETINGITTO THECHLOROPLASTTHYL T AKOIDD MEM MBRANE 'AUETAL  -ANT AND D 2OBINSON  4HE PRECURSOR PROTEIN CONTAINS FOUR HYDROPHOBIC REGIONS TWO OF WHICH RESEMBLETHESIGNAL PEPTIDESPRECEDINGTHE 0SB7AND 0SB8MATUREPROTEINSEQUENCET Q HAT ISUSED FOR3EC F

INDEPENDENTINSERTIONINTOTHETHYLAKOIDMEMBRANE 4HOMPSONETAL  )NITIAL ACTIVITY STUDIESOF 0SB9CONCLUDED D THATITWASA MANGANESEREQUIRING , ARGININE METABOLIZING ENZYME SUSPECTED TO BE IMPORTANTTOTHEFUNCT F IONOFT F HEMANGANESECLUSTER INTHE/ EVOLUTIONCENTEROF03))'AUETAL   CYSTIS  MUTANT IN PSB9 9 WAS ABLE TO ! 3Y 3 NECHOCY GROW NORMALLY UNDER PHOTOAUTOTROPHIC CONDITIONS D NORMAL RATESOFF 03 )) / EVOLUTION INDICATING AND HAD THATITISNOTESSENTIALFORPHOTOSYNTHESISANDISNOT AN IMPORTANTMANGANESE LIGAND INTHE/ EVOLVING F D TO BE CENTER-EETAMETAL  0SB9WASFOUN PARTOFAPURIlED03))COMPLEXFROM 3YNECHOCYSTI 3 S  +ASHINOETAL A BUTWASNOTIDENTIlED IN THEMOSTRECENT03))CRYSTAL STRUCTURE&ERREIRAETAL  4HEFUNCTIONALROLEOF0SB9IN03))REMAINS TOBEDETERMINED 0
1TC; 0SB: IS A RELATIVELY NEW ADDITION TO THE GROUP OF PROTEINSKNOWNTOBEPARTOF03))%NCODING0SB:IS N CHLOROPLASTGENOMES A GENETHATWASLOCATED INMANY D WASNAMED YCF C FORHY HY H AND YP Y POTHETICAL CHLOROPLASTOPEN P F FR READING FRAME4HEGENEUSUALLYENCODESAPROTEIN OFF ABOUTAMINOACIDSWITHAPREDICTED D MOLECULAR F K$A)TISPREDICTED D TO HAVETWOMEMB M RANE MASSOF SPANNING DOMAINS BUT IS MOST LIKELY CLEAVED BY A LUMENALPEPTIDASEBETWEENTHEMEMBRANESPANNING REGIONS 4HEBRIEFHISTORYOFTHISOPENREADINGFRAMEWAS SUMMED D UPINARECENTREVIEW %CKARDT  WHI W CH DISCUSSED D THEDIFlCULTYINDETERMININGTHEFUNCT F IONOF THEPROTEINBECAUSEOFTHELACKOFHOMOPLASMICMU TANTSINCHLOROPLASTS/NEOFF THElRSTTOBACCO 6PSB: 6P P MUTANTS DIDD NOTEXHIBITAPHENOTYPEINNORMALGROWTH CONDITIONS BUTTHEAUTHORSFOUNDTHATTHEPROTEINWAS D WITHTHELIGHT HARVESTINGCOMPLEXOFF 03)) ASSOCIATED 2UFF ETAL  4WOMOREPAPERSPUBLI U SHED D ALMOST CONCURRENTLYEXAMINEDTOBACCO 6PSB: 6 : MUTANTSWITH DIFFERENT F INTERPRETATIONSOFF THEFUNCT F IONOF0S F B:4HE MUTANTSTUDIED BY"AENA 'ANZALESETAL STILL CONTAINEDATLEASTOFTHEWILD TYPEGENE WHICH D D ASUFlCIENTAMOUNTOFT F HEWILD COULD HAVEPRODUCE

TYPEPROTEINTO INmUENCETHEPHENOTYPEOFT F HEMUTANT 4HISSTUDYFOUNDTHATTHERATEOF03)) / EVOLUTION 6 : MUTANTANDTHERATEOF WASUNCHANGED INTHE 6PSB: ELECTRON mOWFROM03))TO03)WASEN F HANCED AND T SUGGESTEDTHAT0SB:ISINVOLVEDINREGULATINGELECTRON TRANSPORTDEPENDINGONTHEPREVAILINGCONDITIONS4HE 6PP : PAPER PUBLISHED ALMOST SIMULTANE SECOND 6PSB: OUSLY AS THE ABOVE STUDY EXAMINED TRANSPLASTOMIC K KOUTS IN BOTH TOBACCO AND # REINHARD KNOC R TII AND CONCLUDEDD THAT 0SB: IS NECESSARY FFOR THE STABILITY OFTHE03)) ,(#))SUPERCOMPLEX3WIATEKETAL  "ECAUSETWOORGANISMSWEREEXAMINEDD AND F D INEACH COMPLEMENTARYEXPERIMENTSWEREPERFORME THE3WIATEKSTUDYCONCLUSIVELYSUPPORTSAROLEFOR 0SB:THATWASSUGGESTED INTHEOTHERMANUSCRIPTS R BY3WIATEKETA K L INDICATESTHATAS 4HEWORK ACONNECTIONBETWEEN03))AND,(#)) 0SB:HELPS W EN 03)) MEDIATE NONPHOTOCHEMICAL QUENCHING WH IS EXPOSEDD TO LIGHT EXCEEDING THE WATER OXIDATION CAPACITYOF03)) EITHERINHIGHERTHANNORMALLIGHT INTENSITIESORINLOWTEMPERATURES)NTHECYANOBACTE RIAL03))STRUCTURE THETWO0SB: HELICESARE LOCATED ONTHE PERIMETEROFTHEDIMERNEAR#0&ERREIRAET AL  4HUS ANANALOGOUSLOCATIONINPLANT03)) WOULD BE CONSISTENT WITH 0SB: COORDINATING LIGHT HARVESTINGPROTEINASSOCIATION-OREEVIDENCEISRE QUIREDD TO DETERMINETHEMODEOFCOOR F DINATIONOF0S F B: BETWEEN03))AND,(#)) D INPLANTS4HEMECHANISM OF0SB:ACTIVITYISYETTOBEDISCOVERED ***
&YUSJOTJD
4VCVOJUT "
1TC3 0SB2 IS A  K$A EXTRINSIC PROTEIN FOUN F D ONLY IN PLANTSAND IDENTIlEDD ASASOLUBL U E PROTEINRELEASED FROM F SPINACH03))PREPARATIONS,JUNGBERGETAL B  !NTISENSEPOTATOPLANTSWEREGENERATEDD CONTAINING nOFF THENORMAL 0SB2PROTEIN LEVELSTODETERMINE IFTHEPROTEINWERENECESSARYFOR03))ACTIVITY BUT THEREWASNO DECREASEINEITHER03))ACCUMULATIONOR GROWTHRATE 3TOCKHAUSETAL  3O FAR ANA F LYSIS OFTHISPROTEINHASNOTINDICATEDAFUNCTIONALROLEIN WATEROXIDATION #
1TC 4HE 0SB SUB U UNIT WAS ORIGINALLY IDENTIlED FROM F THE. TERMINALSEQUENCEOFA^K$APOLYPEPTIDE COMPONENT OFF A PURIlEDD 03)) PREPARATION FROM F

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** 3 3YNEC HOCYSTIS  )KEUCHI ETAL A 4HISPRO TEINWASORIGINALLYNAMED0SB:!SDISCUSSEDABOVE F D SMALLER03))PROTEINISNOW A DISTINCTLY DIFFERENTAN D 0SB: 3WIATEK K ETAL  !CCORDINGTOA CALLED RECENTNOMENCLATURE THEK$APROTEINOF03))HAS BEENNAMED D 0SB+ASHINOETAL A  4HE0SBPOLYPEPTIDEIN 3YNECHOCYSTIS  HAS ONEPREDICTEDMEMBRANE SPANNINGDOMAINFOLLOWED BY ACLEAVAGESITE FORS F IGNALPEPTIDASE))4HUS THE 0SBPROTEINISEXPECTED D TO BEALUMEN LOCALIZED EXTRINSICPROTEINOF03)) 4HE!RABIDOPSISHOMOLOGS !TGAND D !TG OFF 0SBWERE FOUN F D IN APROTEOMICSSTUDYOFF THETHY H LAKOID LUMEN 0ELTIERET AL  !LTHOUGHITISASSOCIATEDWITH03))FROM A E THISPROTEINWASNOT MANY N MODEL ORGANISMS 4ABL IDENTIlED INTHECYANOBACTERIAL 03))CRYSTAL STRUCTURE &ERREIRAETAL  &URTHERMORE NOMUTANTHAS BEENDESCRIBED FORT F HISSUB U UNIT AND D NO FUNCT F IONHAS BEENHYPOT H HESIZED

 *7 $PODMVTJPO #LEARLY T Y HEREISSTILLMUCH TOBE LEARNEDD ABOUTTHE ROLE OFF EACH SUB U UNIT AND HOW IT CONTRIBUTES TO THE ACTIVITY OF THE 03)) COMPLEX4HERE ARE  SMALL PROTEINS DISCUSSED INTHISCHAPTER ANDD THEREAREPOS SIBLY MORE POLYPEPTIDES IN VARIOUS ORGANISMS THAT AREIMPORTANTTO03))FUNCTION03))ISANEXTREMELY W CH EVOLVED BEFORET F HEINTRICATE COMPLEXENZYME WHI MEMBRANE SYSTEMS FOUN F D IN MOST PHOTOSYNTHETIC ORGANISMSWEREDEVELOPED SUGGESTINGTHATTHETASKOF LIGHT INDUCE D D WATEROXIDATIONWASNOTASMALL HURDLE TOJUMP&UNCTIONALSTUDIES BIOCHEMICALANALYSES AND STRUCTURAL ARRANGEMENTS FROM MULTIPLE PHOTO SYNTHETICORGANISMSARENECESSARY FORUN F DERSTANDING HOWALLOFT F HE INDIVIDUA D LUNITSOFF 03))WORRK TOGETHER INUNIQUEINTRICACY "DLOPXMFEHNFOUT

$
1TC 4HE 0SB SUBUNIT WAS ORIGINALLY IDENTIlED FROM THE. TERMINALSEQUENCEOFA^ F K$APOLYPEPTIDE D PREPARATION FROM F 3 3YN COMPONENTOFF APURIlED03)) ECHOCYSTIS )KEUCHIETAL A 4HISPROTEIN WASORIGINALLY NAMED D 0SB! HOMOLOGUEOFF THIS PROTEIN IS THE DEDUCE D D PRODUCT D OF YCF CF AN OPEN READINGFRAMEINTHECHLOROPLASTGENOMEOF 0ORPHYR 0 RA &INALLY T Y HEPROTEINWASALSONAMED 0SB7!CCORD INGTOARECENTLYPUBLI U SHED D NOMENCLATURE 0SB HAS BEEN RENAMED 0SB AND A DIFFERENT AND SMALLER 03))PROTEINISCALLED D 0SB7SEEABOVE +ASHINO ETAL A  4HE0SBPROTEINISPREDICTEDTOHAVENOHYDROPHO BICMEMBRANE SPANNING DOMAINAND D TO BE LOCALIZED INTHESTROMACYTOPLASM)FF THISPREDICTIONISCORRECT 0SBISTHEONLYKNOWNEXTRINSICSTROMALCYTOPLAS MICPROTEININ03))4HEREWASNOUNASSIGNED DENSITY INTHERECENTLYPUBLI U SHED03))CRYSTA D LSTRUCTURETHAT COULD BE ATTRIBUTED D TO 0SB ONTHE STROMAL SURFACE F OFF 03))&ERREIRAETAL   ! KNOC K KOUTMUTATIONOFT F THE PSB GENE IN3YNECHO CYSTIS  RESULTS IN REDUCED GROWTH RATE UNDER PHOTOSYNTH T ETICCONDITIONSWH W ENCALCIUMAND D CHLORIDE ISNOTADDED D TOTHEGROWTH MEDIUM(+ATOH AND D - )KEUCHI PERSONALCOMMUNICATION &UTUREDETAILED F HIS INTERESTINGMUTANTSTRAINMAY A UNRAVEL STUDIESOFT THEFUNCT F IONALROLEOFF THE0SB PROTEININ03))

4HE PREPARATION OFF THIS CHAPTER WAS SUPPORTED BY FFUNDING FROM THE .ATIONAL 3CIENCE &OUNDATION $EPARTMENT OF F %NERGY AND D 5NITED D 3TATES $EPART MENTOF!GR F ICULTURE,%4AND*,2WERESUPPORTE D D BYGRADUATESTUDENTFELLOWSHIPSFROMTHE.ATIONAL 3CIENCE&OUNDATION 3FGFSFODFT !BDEL -AWGOOD!,AN ! D D $ILLEY2! Y  #LONINGAND D NUCLEOTIDE SEQUENCEOFTHEPSB(GENEFROMCYANOBACTERIUM 3YNECHOCYSTIS 3 0LANT-OL"IOLn !DAMSKA) &UNK# 2ENGER'AND!NDERSSON" $EVELOP MENTALREGULATIONOFTHE0SB3GENEEXPRESSIONINSPINACHSEED LINGS4HEROLEOFF PHYTOC H HROME0LANT-OL "IOLn D I02AND D 0AKRAS K I ("  -UTATIONALANALYSISOFT F HE !NBUDURA 0SB,PROTEINOFF 0HOTOSYSTEM)) INTHECYANOBACTERIUM 3YNECHO CYSTIS SP0##:.ATURFORSC CY F H Cn "AENA 'ONZALEZ% 'RAY A *# 4YY 4 STJ T ARVI % !RO%-AND D -AENPAA 0  !BNORMAL REGULATION OF PHOTOSYNTHETIC ELECTRON F INACTIVATIONMUTANT*"IOL#HEM TRANSPORTINACHLOROPLASTYCF n "ARBER*AND$E,AS2IVAS* !FUNCTIONALMODELFORTHE F HROME BINTHEPROTECTIONAGAINSTDONORAND ROLEOFCYTOC ACCEPTOR SIDE PHOTOINHIBITION 0ROC .ATL !CAD D 3CI 53!  n "IESIADKA* ,OLL " +ERN* )RRGANG + $AND D :OUNI!  #RYSTAL STRUCTURE OF CYANOBACTERIAL 0HOTOSYSTEM )) AT  ¯ RESOLUTION!CLOSERLOOKATTHE-N CLUSTER0HYS#HEM#HEM 0HYSn "UCHEL# -ORRIS% /RLOVA%AND"ARBER* ,OCALISATION OFTHE0SB(SUBUNITIN0HOTOSYSTEM))!NEWAPPROACHUSING



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

LAB A ELLING OFF (IS TAGSWITHA.I .4! GOLD D CLUSTERANDD SINGLE PARTICLEANALYSIS*-OL"IOLn "USER#! $INER"!AND"RUDVIG'7 0HOTOOXIDATION OFCYTOCHROME BINOXYGEN EVOLVING0HOTOSYSTEM))"IO CHEMISTRYn #USHMAN*# #HRISTOPHER$! ,ITTLE-# (ALLICKK 2"AND 0RICE & ORF RF AND ORF RF #! /RGANIZATIONOFF THE PSB% PSB& GENE LOCI ONTHE %UGL U ENAGRACI G LIS CHLOROPLASTGENOME#URR 'ENETn DE6ITRY # $INER"!AND0OPO*, D  0HOTOSYSTEM))PARTICLES FROM #HLAMYDOMONAS REINHARDTII 0URIlCATION MOLECULAR WEIGHT SMALLSUBUNITCOMPOSITION ANDPROTEINPHOSPHORYLA TION*"IOL#HEMn $EDNER. -EYER(% !SHTON#AND7ILDNER'& . 4ERMI NALSEQUENCE ANALYSISOFF THE+DAPROTEIN IN #HL # AMYD M DOMONAS R ˆLOCALIZATIONOFT RDII F HE PHOSPHOTHREONINE&%"3,ETT R REINHA  n $/% *OINT 'ENOME )NSTITUTE  *') 0ROGRAMS -ICROBIAL 'ENOMICSHTTPWWWJGI  DOEGOV*')?MICROBIALHTMLINDEX HTML!UGUST  $OMINICI 0 0 #AFFARRI 3 !RMENANTE & & #EOLDO3 #RIMI - AND "ASSI2 "IOCHEMICALPROPERTIESOFTHE0SB33UBUNITOF 0HOTOSYSTEM))EITHERPURIlEDFROMCHLOROPLASTORRECOMBINANT * "IOL #HEMn F B: INTHECORECOMPLEXOFF 0HO %CKARDT.! !ROLEFOR0S TOSYSTEM))0LANT#ELLn R ER*ANDD )WATA3 &ERREIRA+. )VERSON4- -AGHLAOUI + "ARB  !RCHITECTURE OF F THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn &UNK# &UNCTIONALANALYSISOFTHE0SB8PROTEINBYDELE TIONOFTHECORRESPONDINGGENEIN 3YNECHOCYSTIS 3 SP0## 0LANT-OL "IOL  n &UNK K # 3CHRODER70 .APIWOTZKI ! 4J 4 US3% 2ENGER'AND !NDERSSON "  4HE 03 )) 3 PROTEIN OF HIGHER PLANTS ! NEW TYPE OFF PIGMENT BINDING PROTEIN "IOCHEMISTRY  n 'AREWAL (3AND7ASSERMAN D !2A 4RITON8  -UREAAS ANEXTRACTIONMEDIUMFORMEMBRANEPROTEINS)0URIlCATIONOF CHLOROPLASTCYTOCHROME B"IOCHEMISTRYn 'AREWAL(3AND7ASSERMAN!2B 4RITON8  -UREA ASANEXTRACTIONMEDIUMFORMEMBRANEPROTEINS))-OLECULAR PROPERTIESOFPURECYTOC F HROME B! LIPOPROTEINCONTAINING SMALL POLYPEPTIDE CHAINS ANDD A LIMITED LIPID D COMPOSITION "IOCHEMISTRYn 'AU!% 4HOLE((ANDD 0ISTORIUS%+ )SOLATIONANDD PARTIAL CHARACTERIZATIONOFF AMANGANESEREQUIRING , !RGININEMETABO LIZINGENZYMEBEINGPRESENTIN0HOTOSYSTEM))COMPLEXESOF SPINACHANDTOBACCO:.ATURFORSCHCn 'AU!% 4HOLE(( 3OKOLENKO! !LTSCHMIED, (ERMANN2' AND 0ISTORIUS %+  0SB9 A NOVEL MANGANESE BINDING LOW MOLECULAR MASSPROTEINASSOCIATEDD WITH 0HOTOSYSTEM)) -OL 'EN'ENETn 'OMEZ3- .ISHIO*. &AULL +&ANDD 7HITELEGGE*0 4HE CHLOROPLAST GRANAPROTEOMEDElNED BY INTACTMASSMEASURE MENTS FROM F LIQUIDD CHROMATOGRAPHY MASS SPECTROMETRY Y -OL #ELL0ROTEOMICSn (AGER- (ERMANN- "IEHLER+ +RIEGER ,ISZKAY!AND"OCK2  ,ACKOFTHESMALLPLASTID ENCODED0SB*POLYPEPTIDERE SULTSINADEFECTIVEWATER SPLITTINGAPPARATUSOF0HOTOSYSTEM)) D D 0HOTOSYSTEM)LEVELS AND HYPERSENS H ITIVITYTO LIGHT* REDUCE "IOL #HEMn

(AGMAN! 3HI,8 2INTAMAKI % !NDERSSON"AND D 3CHRODER70  4HENUCLEAR ENCODED0SB7PROTEINSUBUNITOF0HOTO SYSTEM))UNDERGOESLIGHT INDUCEDPROTEOLYSIS"IOCHEMISTRY n (EBER5 +IRK-2AND"OARDMAN.+ 0HOTOREACTIONSOF CYTOCHROME B AND D CYCLICELECTRON mOWIN 0HOTOSYSTEM)) OF INTACTCHLOROPLASTS"IOCHIM "IOPHYS!CTAn (ERRMANN2' !LT* 3CHILLER" 7IDG 7 ER72AND D #RAMER7!  .UCLEOTIDESEQUENCEOFT F HE GENEFORAPOC F YTOCHROME B ONTHESPINACHPLASTIDD CHROMOSOME)MPLICATIONSFORT F HE STRUCTUREOFTHEMEMBRANEPROTEIN&%"3,ETTn (ESS72 2OCAP' 4ING#3 ,ARIMER& 3TILWAGEN3 ,AMERDIN* AND #HISHOLM 37  4HE PHOTOSYNTHETIC APPARATUS OF 0ROCHLO R R ROCOCCUS  )NSIGHTS THROUGH COMPARATIVE GENOMICS 0HOTOSYNTH2ESn (IRD3- $ D YER4!AND D 'RAAY *# 4HE GENE FORT F HEK$A PHOSPHOPROTEIN OFF 0HOTOSYSTEM )) IS LOCATED IN CHLOROPLAST $.! . &%"3,ETTn )KEUCHI-AND D )NOUE9  !NEW0HOTOSYSTEM))REACTION CENTERCOMPONENTK$APROTEIN ENCODEDBYCHLOROPLAST GENOME&%"3,ETTn )KEUCHI- +OIKE(AND)NOUE9A )DENTIlCATIONOFPSB) F ) AND PSB, GENEPRODUCTSINCYANOBACTERIAL0HOTOSYSTEM))REACTION CENTERPREPARATION&%"3,ETTn )KEUCHI- +OIKE(AND D )NOUE9 B . TERMINALSEQUENC ING OF LOW MOLECULAR MASS COMPONENTS IN CYANOBACTERIAL D TO 0HOTOSYSTEM))CORECOMPLEX4WOCOMPONENTSCORRESPOND D OPENREADING FRAMESO F F LANTCHLOROPLAST$.! FP UNIDENTIlED &%"3,ETTn )KEUCHI- 4AKIO+AND)NOUE9C . TERMINALSEQUENCING OF0HOTOSYSTEM))LOW MOLECULAR MASSPROTEINSANDK$A COMPONENTS OF THE / EVOLVING CORE COMPLEX FROM HIGHER PLANTS&%"3,ETTn )KEUCHI - %GGERS " 3HEN ': 7EBBER! 9U ** (IRANO! D  #LONING OF F THE PSB+ GENE )NOUE9 AND6ERMAAS7 F FROM 3YNECHOCY CYSTIS SP 0##  AND D CHARACTERIZATION OF 0HOTOSYSTEM))INMUTANTS LACKING03)) +*"IOL#HEM n )KEUCHI- )NOUE9AND6ERMAAS7A #HARACTERIZATIONOF 0HOTOSYSTEM))SUBUNITSFROMTHECYANOBACTERIUM3YNECHOCYSTIS SP0##)N-ATHIS0ED 0HOTOSYNTHESIS&ROM,IGHT TO "IOSPHERE PP n +LUWER !CADEMIC 0UBLISHERS $ORDRECHT )KEUCHI- 3HUKLA6+ 0AKRAS K I ("AND D )NOUE9B $IRECTED INACTIVATIONOFT F HE PSB)GENEDOESNOTAFFECT0HOTOSYSTEM)) ) INTHECYANOBACTERIUM 3Y 3 NECHOCY CYSTIS SP0##-OL 'EN 'ENETn )RRGANG+$ 3HI,8 &UNK#AND3CHRODER70 !NUCLEAR ENCODEDSUBUNITOFTHE0HOTOSYSTEM))REACTIONCENTER*"IOL #HEMn )RRGANG +$ 'EORGALIS9 &RANKE 0 "EHLKE * 3AENGER7 AND :OUNI !  3TRUCTURAL COMPARISON OF F OXYGEN EVOLVING 0HOTOSYSTEM))CORECOMPLEXESFROM F 3 NECHOCOCCUSELONGATUS 3Y G AND 3PINACIA OLERACEA )N 03 0ROCEEDINGS TH )NTERNATIONAL #ONGRESS ON 0HOTOSYNTHESIS 3  #3)2/ 0UBLISHING -ELBOURNE#$ 2/- )WAI - +ATOH ( AND )KEUCHI -  &UNCTIONAL ANALYSIS OF 03 )) 4 PROTEIN OF 0HOTOSYSTEM )) COMPLEX FROM THE 4 FORMERLY THERMOPHILICCYANOBACTERIUM 4HERMOSYNECHOCOCCUS 3YNECHOCOCCUS ELONGATUS "0 )N030ROCEEDINGSTH )NTERNATIONAL#ONGRESSON0HOTOSYNTHESIS 3 #3)2/

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** 0UBLI U SHING -ELBOURNE#$ 2/- +AMIYA . AND 3HEN * 2  #RYSTAL STRUCTURE OF OXYGEN EVOLVING0HOTOSYSTEM))FROM 4HERMOSYNECHOCOCCUSVULCANUS AT¯ RESOLUTION0ROC.ATL!CAD3CI53!n +ANEKO4 3ATO3 +OTANI( 4ANAKA! !SAMIZU% .AKAMURA 9 IYAJI 9 A MA. (IROSAWA- 3UGIURA- 3ASAMOTO3 +IMURA 4 4 (OSOUC HI4 -ATSUNO! -URAKI ! .AKAZAKI. .ARUO+ K 3 3HIMPO 3 4AKEUCHI # 7ADA 4 7ATANABE ! /KUMURA 9 9AMA DA- 9ASUDA-ANDD 4ABATA3  3EQUENCEANALYSIS OFF THE GENOMEOFT F HEUNICELLULARCYANOBACTERIUM 3YNECHOCY CYSTIS SPSTRAIN0##))3EQUENCEDETERMINATIONOFTHEENTIRE GENOMEANDASSIGNMENTOFPOTENTIALPROTEIN CODINGREGIONS $.!2ESn +ANEKO4 .AKAMURA9 7OLK#0 +URITZ4 3ASAMOTO3 7ATANABE ! )RIGUCHI - )SHIKAWA! +AWASHIMA+ +IMURA4 +ISHIDA 9 9 +O HARA- -ATSUMOTO- -ATSUNO! -URAKI! .AKAZAKI . 3HIMPO3 3UGIMOTO- 4AKAZAWA- 9AMADA- 9ASUDA D 4ABATA 3  #OMPLETE GENOMIC SEQUENCE OF F THE - AND lLAMENTOUS NITROGEN lXING CYANOBACTERIUM !NABAENA SP STRAIN0##$.!2ESnn +APAZOGLOU! 3AGLIOCCO&AND$URE, RD 03)) 4 A NEW NUCLEAR ENCODED LUMENAL PROTEIN FROM 0HOTOSYSTEM )) 4 4ARGETINGANDPROCESSINGINISOLATEDCHLOROPLASTS*"IOL#HEM  n +ASHINO9 ,AUB U ER7- #ARROLL*! 7ANG 1 7HITMARSH * 3ATOH D 0AKRAS K I (" A 0ROTEOMIC ANALYSIS OFF A HIGHLY + AND ACTIVE 0HOTOSYSTEM )) PREPARATION FFROM THE CYANOBACTERIUM CYSTIS SP 0##  REVEALS THE PRESENCE OF F NOVEL 3 NECHOCY 3Y POLYPEPTIDES"IOCHEMISTRYn +ASHINO9 +OIKE( 9OSHIO- %GASHIRA( )KEUCHI- 0AKRASI (" AND 3ATOH + B ,OW MOLECULAR MASS POLYPEPTIDE COMPONENTS OF A 0HOTOSYSTEM )) PREPARATION FROM THE U 4H 4 ERMOSY S NECHOCOCCUSVULCANUS T ERMOPHILICCYANOBACTERIUM TH 0LANT#ELL 0HYSIOLn +ATOH ( AND )KEUCHI - A #HARACTERIZATION OFF 03 F )) ) OR 03 )) + PROTEIN DEPLETED 0HOTOSYSTEM )) FROM S NECHOCOCCUSELONGATUS G STRAIN"0 03)) )PROTEIN 4 ERMOSY 4H PLAYS AN ESSENTIAL ROLE IN DIMERIZATION OF 0HOTOSYSTEM )) )N 03 0ROCEEDINGS TH )NTERNATIONAL #ONGRESS ON 0HOTOSYNTHESIS 3 #3)2/0UBLISHING -ELBOURNE#$ 2/- +ATOH(AND)KEUCHI-B 4ARGETEDDISRUPTIONOFPSB8 F P 8AND BIOCHEMICALCHARACTERIZATIONOFF 0HOTOSYSTEM))COMPLEX INTHE 3 NECHOCOCCUSELONGATU G S 0LANT THERMOPHILICCYANOBACTERIUM 3Y #ELL0HYSIOLn D +IM 3* 2OBINSON $ ANDD 2OBINSON #  !N !RABIDOPSIS THALIANA C$.!ENCODING03)) 8 AK$ACOMPONENTOF 0HOTOSYSTEM))!BIPARTITEPRESEQUENCEMEDIATES3EC!DELTA P( INDEPENDENT TARGETING INTO THYLAKOIDS &%"3 ,ETT  n +ITAMURA + /ZAWA 3 3HIINA 4 ANDD 4OYOSHIMA9  , PROTEIN ENCODED BY PSB, RESTORESNORMAL FUNCT F IONING OFT F HE PRIMARY QUINONEACCEPTOR 1! IN ISOLATEDD $$#0#YTB T )0HOTOSYSTEM))REACTIONCENTERCORECOMPLEX&%"3,ETT  n +OIKE ( -AMADA + )KEUCHI - AND )NOUE 9  ,OW MOLECULAR MASS PROTEINS IN CYANOBACTERIAL 0HOTOSYSTEM )) IDENTIlCATIONOFPSB( ( ANDPSB+ + GENEPRODUCTSBY. TERMINAL SEQUENCING&%"3,ETTn K  !BSENCEOFF THE +OMENDA* ,UPINKOVA,ANDD +OPECKY* PSB(GENEPRODUCTDESTABILIZES0HOTOSYSTEM))COMPLEXAND (

 BICARB R ONATEBINDINGON ITSACCEPTORSIDEIN 3YNECHOCY CYSTIS 0## %UR*"IOCHEMn +UNSTNER 0 0 'UARDIOLA ! 4AKAHASHI 9 AND 2OCHAIX *$  ! MUTANT STRAIN OF #HLAMYDOMONAS REINHARDTII LACKING THE CHLOROPLAST 0HOTOSYSTEM )) PSB) GENE GROWS PHOTOAUTOTROPHICALLY*" Y IOL#HEMn ,AKSHMI +6 2EImER-* #HISHOLM$! 7ANG*9 $INER"!AND "RUDV D IG'7 #ORRELATIONOFT F THECYTOCHROME H C CONTENT OFF CYANOBACTERIAL0HOTOSYSTEM))WITH THE%02PROPERTIESOFT F HE OXYGEN EVOLVINGCOMPLEX0HOTOSYNTH 2ESn ,I 80 "JORKMAN / 3HIH # 'ROSSMAN !2 2OSENQVIST - *ANSSON3AND.IYOGI++ !PIGMENT BINDINGPROTEIN ESSENTIAL FOR REGULATION OF PHOTOSYNTHETIC LIGHT HARVESTING .ATURE  n ,IND ,+ 3HUKLA 6+ .YHUS +* AND 0AKRASI ("  'ENETICAND IMMUNOLOGICALANALYSESOFF THECYANOBACTERIUM CYSTIS SP0##SHOWTHATTHEPROTEINENCODED BY 3 NECHOCY 3Y THE PSB** GENEREGULATESTHENUMBEROF0HOTOSYSTEM))CENTERS INTHYLAKOIDD MEMBRANES*"IOL#HEMn ,JUNGBERG5 !KERLUND(% ,ARSSON5+AND!NDERSSON"A , )DENTIlCATIONOFPOLYPEPTIDESASSOCIATEDWITHTHEAND K$A PROTEINS OF PHOTOSYNTHETIC OXYGEN EVOLUTION "IOCHIM "IOPHYS!CTAn ,JUNGBERG5 !KERLUND D (%AND !NDERSSON" B 4HERELEASE OFF A K$APOLYPEPTIDE FROMEVERTE F D 0HOTOSYSTEM))THYLAKOID MEMBRANESBY ALKALINE4RIS&%"3,ETTn ,ORK R OVIC:* 3CHRODER70 0AKRAS K I (" )RRGANG +$ (ERRMANN 2' AND /ELMàLLER 2  -OLECULAR CHARACTERIZATION OF 0SB7 A NUCLEAR ENCODED COMPONENT OF THE 0HOTOSYSTEM )) REACTIONCENTERCOMPLEXINSPINACH0ROC.ATL!CAD3CI53! n ,UPINKOVA , -ETZ * $INER " 6ASS ) AND +OMENDA *  D F HE0HOTOSYSTEM))$POLYPEPTIDE FT (ISTIDINERESIDUEO IS INVOLVED INALIGHT INDUCE D D CROSS LINKINGOFF THEPOLYPEPTIDE U UNITOFC F YTOCHROME B 3TUDYOFAS F ITE WITHTHEALPHASUB DIRECTED D MUTANTOF 3YNECHOCY CYSTIS 0##"IOCHIM"IOPHYS !CTA -ANT!AND2OBINSON# !N !RABIDOPSIS C$.!ENCODESAN A APPARENTPOLYPROTEINOFTWONON IDENTICALTHYLAKOIDMEMBRANE PROTEINSTHATAREASSOCIATEDWITH0HOTOSYSTEM))ANDHOMOLOGOUS TOALGAL YCF OPENREADINGFRAMES&%"3,ETTn -AYES32 $UBB U S*- 6ASS) (IDEG% .AGY,AND D "ARRBER* ( LOCUSOF 3Y 3 NECHOCY CYSTIS &URTHER CHARACTERIZATION OFF THE PSB( SP 0##  ˆ INACTIVATION OF PSB( ( IMPAIRS 1A TO 1" ELECTRON TRANSPORT IN PHOTOSYSTEM  "IOCHEMISTRY  n -C.AMARA60 3UTTERWALA&3 0AKRASI("AND7HITMARSH* 3TRUCTURALMODELOFCYTOCHROME BIN0HOTOSYSTEM))BASED ONAMUTANTWITHGENETICALLYFUSEDSUBUNITS0ROC.ATL!CAD 3CI 53!n -EETAM- +EREN. /HAD D )AND 0AKRAS K I ("  4HE0SB9 PROTEIN IS NOT ESSENTIAL FFOR OXYGENIC PHOTOSYNTHESIS IN THE CYANOBACTERIUM 3YNECHOCY CYSTIS SP 0##  0LANT 0HYSIOL  n -ICHEL (0AND"ENNETT* D  )DENTIlCATIONOFF THEPHOSPHORYLA TIONSITEOFANK$APROTEINFROM0HOTOSYSTEM))OFSPINACH &%"3,ETTn -IZUSAWA. -IYAO-AND9AMASHITA4 2ESTORATIONOFTHE HIGH POTENTIALFORMOFCYTOCHROME B BYELECTRONTRANSPORT D 0HOTOSYSTEM REACTIONSTHROUGH 0HOTOSYSTEM))INTRIS TREATED ))"IOCHIM "IOPHYS!CTAn



-FFBOO
&
5IPSOUPO +PIOOB
-
3PPTF )JNBESJ
#
1BLSBTJ
BOE
.BTBIJLP
*LFVDIJ

-ONOD D # 4AKAHASHI9 9 'OLDSCHMIDT # D LERMONT-ANDD 2OCHAIX*$  4HECHLOROPLAST YCF OPENREADINGFRAMEENCODESA0HO TOSYSTEM))POLYPEPTIDEWHICHMAINTAINSPHOTOSYNTHETICACTIVITY UNDERADVERSEGROWTHCONDITIONS%MBO*n -ORAIS & "ARBER * AND .IXON 0*  4HE CHLOROPLAST EN CODED D ALPHA SUB U UNIT OF F CYTOCHROME B  IS REQU Q IRED FO F R ASSEMBLY OFT F HEPHOTOSYSTEMTWOCOMPLEXIN BOTHTHELIGHT AND D THE DARK R IN #HL # AMYD M DOMONAS REINHARD R TII * "IOL #HEM  n -ORAIS& +UHN+ 3TEWART$( "ARB R ER* "RUDVIG '7ANDD .IXON 0* 0HOTOSYNTHETICWATEROXIDATIONINCYTOCHROME B MUTANTSCONTAININGADISRUPTEDHEME BINDINGPOCKET*"IOL #HEMn -URATA . -IYAO - (AYASHIDA . (IDAKA4 AND 3UGIURA  )DENTIlCATIONOFF ANEW GENEINTHECHLOROPLASTGENOME ENCODINGA LOW MOLECULAR MASSPOLYPEPTIDEOFF 0HOTOSYSTEM ))COMPLEX&%"3,ETTn .AGATSUKA4 &UKU K HARA3 !KABORI+ANDD 4O OYOSHIMA9  $ISINTEGRATIONANDD RECONSTITUTIONOFF 0HOTOSYSTEM))REACTION CENTERCORECOMPLEX))0OSSIBLEINVOLVEMENTOFLOW MOLECU LAR MASSPROTEINSINTHEFUNCTIONINGOF1! INTHE03))REACTION CENTER"IOCHIM"IOPHYS!CTAn .AKAMURA9 +ANEKO4 3ATO3 )KEUCHI- +ATOH( 3ASAMOTO3 7 7ATANA BE! )RIGUCHI - +AWASHIMA+ +IMURA4 +ISHIDA9 +IYOKAWA# +OHARA- -ATSUMOTO- -ATSUNO! .AKAZAKI . 3HINPO 3 3UGIMOTO - 4AKEUCHI # 9AMADA - AND D 3 4  #OMPLETE GENOME STRUCTURE OF F THE THERMOPHILIC CYANOBACTERIUM 4HERMOSY S NECHOCOCCUSELONGATUS G "0 $.! 2ESn .AKAMURA9 (IROSAWA- +ANEKO4AND4ABATA3A #YA NOBASEHTTPWWWKAZUSAORJPCYANOBASE$EC  .AKAMURA 9 +ANEKO 4 3ATO 3 -IMURO - -IYASHITA ( 4SUCHIYA4 3ASAMOTO3 7ATANAB A E! +AWASHIMA+ +ISHIDA9 +IYOKAWA# +OHARA- -ATSUMOTO- -ATSUNO! .AKAZAKI . 3HIMPO3 4AKEUCHI # 9AMADA-ANDD 4ABATA3 B #OMPLETE GENOME STRUCTURE OF 'LOEOBACTER VIOLACEUS 0##  ACYANOBACTERIUMTHATLACKSTHYLAKOIDS$.!2ES n .EDBAL, 3AMSON'AND7HITMARSH* 2EDOXSTATEOFA ONE ELECTRONCOMPONENTCONTROLSTHERATEOFPHOTOINHIBITIONOF 0HOTOSYSTEM))0ROC.ATL!CAD3CI53!n /g#ONNOR(% 2UFmE36 #AIN!* $EAKK : 6ASS) .UGENT*(AND 0URTON3 4HE K$APHOSPHOPROTEINOFF 0HOTOSYSTEM)) 'ENERATIONANDD CHARACTERISATIONOF #HL # AMYD M DOMONAS MUTANTS LACKING 03 )) ( AND A SITE DIRECTED MUTANT LACKING THE PHOSPHORYLATIONSITE"IOCHIM"IOPHYS!CTAn /HNISHI.AND4AKAHASHI9 0SB4POLYPEPTIDEISREQUIRED FOREFlCIENTREPAIROFPHOTODAMAGED0HOTOSYSTEM))REACTION CENTER*"IOL#HEMn /ZAWA3 +OBAYASHI4 3UGIYAMA2 (OSHIDA( 3HIINA4AND GENE 4OYOSHIMA 9  2OLE OF 03 )) , PROTEIN PSB, PP PRODUCT D ONTHEELECTRONTRANSFER F IN 0HOTOSYSTEM))COMPLEX D IONOFW F ILD TYPEANDD MUTANTVERSIONSOF03)) , F /VER PRODUCT PROTEINAND D RECONSTITUTIONINTOTHE03))CORECOMPLEX0LANT -OL"IOL n 0AKRASI (" 7ILLIAMS *' AND !RNTZEN #*  4ARGETED % ANDPSB&GENESBLOCKSPHOTOSYNTHETIC & MUTAGENESISOFTHE PSB% ELECTRONTRANSPORT%VIDENCEFORAFUNCTIONALROLEOFCYTOCHROME BIN0HOTOSYSTEM))%-"/*n K I(" .YHUS+*ANDD 'RANOK (  4ARGETED DELETION 0AKRAS F HE BETASUB U UNITOFC F YTOCHROMEBPROTEIN MUTAGENESISOFT

DESTABILIZESTHEREACTIONCENTEROF0HOTOSYSTEM)):.ATURFORSCH Cn 0AKRASI(" $E#IECHI0AND7HITMARSH* 3ITEDIRECTED MUTAGENESIS OF THE HEME AXIAL LIGANDS OF CYTOCHROME B AFFECTSTHESTABILITYOFTHE0HOTOSYSTEM))COMPLEX%-"/*  n 0ALENIK" "RA K HAMSHA" ,ARIMER&7 ,AND- (AUSER, # D HAIN 0 ,AMERDIN * 2EGALA7 !LLEN %% -C#ARREN * 0AULSEN ) F KY & 7EBB%!AND D 7ATERRBURY *  $UFRESNE! 0ARTENS 4HEGENOMEOFF AMOTILEMARINE 3Y 3 NECHOCOCCUS.ATURE n 0ELTIER *" %MANUELSSON / +ALUME $% 9TTERBERG * &RISO ' 2UDELLA! ,IBERLES$! 3ODERBERG, 2OEPSTORFF0 VON(EIJNE 'ANDVAN7IJK+* #ENTRALFUNCTIONSOFTHELUMENAL AND D PERIPHERAL THYLAKOIDPROTEOMEO D F !RABIDO DOPSIS DETERMINED BY EXPERIMENTATIONAND GENOME WIDEPREDICTION0LANT#ELL  n 0OULSON - 3AMSON ' AND 7HITMARSH *  %VIDENCE THAT CYTOCHROME B PROTECTS 0HOTOSYSTEM )) AGAINST PHOTOINHIBITION"IOCHEMISTRYn 2ACE(,AND'OUNARIS+ )DENTIlCATIONOFTHE PSB(GENE ( PRODUCT AS A  K$A PHOSPHOPROTEIN IN THE CYANOBACTERIUM 3YNECHOCYSTIS &%"3 ,ETTn 2EGEL2% )VLEVA." :ER( -EURER* 3HESTAKOV36 (ERRMANN K I("AND/ D HAD D )  $EREGULATIONOFF ELECTRON 2' 0AKRAS mOWWITHIN0HOTOSYSTEM))INTHEABSENCEOFF THE0SB*PROTEIN *"IOL #HEMn 2HEE+( -ORRIS%0 "ARB R ER*AND D +àHLBRANDT7 4HREE DIMENSIONAL STRUCTURE OF THE PLANT 0HOTOSYSTEM )) REACTION CENTREAT¯ RESOLUTION.ATUREn 2OCAP' ,ARIMER&7 ,AMERDIN* -ALFATTI3 #HAIN0 !HLGREN .! !RELLANO! #OLEMAN- (AUSER, (ESS72 *OHNSON :) ,AND D - ,INDELL $ 0OST!& 2EGALA7 3HAH - 3HAW 3, 3TEGLICH# 3ULLIVAN-" 4ING#3 4OLONEN! 7EBB %! :INSER%2AND# D HISHOLM37 'ENOMEDIVERG R ENCE INTWO 0ROCHLOROCOCCUS ECOTY TYPESREmECTSOCEANICNICHEDIFFERENT F IATION .ATURE n 2 2UF3 "IEHLER+AND"OCK2 !SMALLCHLOROPLAST ENCODED PROTEIN AS A NOVEL ARCHITECTURAL COMPONENT OF THE LIGHT HARVESTINGANTENNA*#ELL"IOLn 3HI,8AND3CHRODER70 #OMPOSITIONALANDTOPOLOGICAL STUDIESOFT F HE0SB7PROTEIN INSPINACHTHY H LAKOIDD MEMBRANE 0HOTOSYNTH2ESEARCH n D 3CHRODER70 3HI ,8 +IM3* -ARCHANT! 2OBINSON#AND F HOTOSYSTEM))AND #HARACTERISATIONOFF THE0SB8PROTEIN FROM0 LIGHTREGULATIONOF ITS GENEEXPRESSIONIN HIGHERPLANTS0LANT -OL"IOLn 3HI,8 ,ORKOVIC:* /ELMàLLER2AND3CHRODER70 4HE LOWMOLECULARMASS0SB7PROTEINISINVOLVEDINTHESTABILIZA TION OF THE DIMERIC 0HOTOSYSTEM )) COMPLEX IN !RABIDOP O SIS THALIANA* "IOL #HEMn F - 2ENGER ' 7ESTHOFFF 0 7YDRZYNSKI 4 3TOCKHAUS * (OFER AND D 7ILLMITZER, !NTI SENSE2.!EFlCIENTLY INHIBITS F FORMAT ION OF F THE  KD D POLYPEPTIDE OFF 0HOTOSYSTEM )) IN D PROTEIN TRANSGENICPOTATOPLANTSANALYSISOFF THEROLEOFF THE KD %-"/*n 3UGIMOTO ) AND 4AKAHASHI 9  ,OCALIZATION OF A SMALL CHLOROPLAST ENCODED POLYPEPTIDE 0SB+ IN 0HOTOSYSTEM )) CORECOMPLEXES)N030ROCEEDINGSTH)NTERNATIONAL #ONGRESS ON 0HOTOSYNTHESIS 3  #3)2/ 0UBLISHING -ELBOURNE #$ 2/-

$IBQUFS

5IF 4NBMM 4VCVOJUT
PG
1IPUPTZTUFN
** 3UMMER %* 3CHMIDD 6( "RUNS "5 ANDD 3CHMIDT '7  2EQUIREMENTFORTHE(PHOSPHOPROTEININ0HOTOSYSTEM))OF # #HLAMYDOMONASREINHARDTII 0LANT0HYSIOLn 3UNDBY # ,ARSSON 5+ AND (ENRYSSON 4  %FFECTS OF BICARBONATE ON THYLAKOID PROTEIN PHOSPHORYLATION "IOCHIM "IOPHYS!CTAn 3WIATEK- +URAS2 3O K KOLENKO! (IGGS$ /LIVE* #INQUE' -ULLER" %ICHACKER,! 3TERN$" "ASSI 2 (ERRMANN2' AND D 7OLLMAN&! 4HECHLOROPLAST GENE YCF C ENCODESA U UNIT 0SB: THATPARTICIPATESIN 0HOTOSYSTEM)) 03)) CORESUB 03))SUPRAMOLECULARARCHITECTURE0LANT#ELLn 3WIATEK- 2EGEL2% -EURER* 7ANNER' 0AKRASI(" /HAD) AND (ERRMANN 2'  %FFECTS OF SELECTIVE INACTIVATION OF INDIVIDUAL GENES FOR LOW MOLECULAR MASS SUBUNITS OF THE F ION4HE 0HOTOSYSTEM))ASSEMBLY BYCHLOROPLASTTRANSFORMAT PSB%&,** OPERONIN.ICOTIANATA . BACUM-OL 'EN'ENET n 3ZABO) 3ERAGLIA2 2IGONI& 4RALDI0AND' D IACOMETTI '-  $ETERMINATIONOFF 0HOTOSYSTEM))SUB U UNITSBYMATRIX ASSISTED LASERDESORPTIONIONIZATIONMASSSPECTROMETRY*"IOL#HEM n 4AKAHASHI 9 -ATSUMOTO ( 'OLDSCHMIDT #LERMONT - AND 4 # S 2OCHAIX*$ $IRECTEDDISRUPTIONOFTHE #HLAMYDOMONA CHLOROPLAST PSB+ + GENEDESTABILIZESTHE0HOTOSYSTEM))REACTION CENTERCOMPLEX0LANT-OL "IOL n 4HE )NSTITUTE FFOR 'ENOMIC 2ESEARCH  4)'2 $ATABASE !RABIDO DOPSISTH T ALIANAHTTPWWWTIGRORGTDBEKLATHATH SHTML!UG  4HIDHOLM% 3HI,8 ,INDSTROM6 6 &UNK#AND3CHRODER70  4HE 0SB7 PROTEIN )TS LOCATION AND INVOLVEMENT IN PHOTOINHIBITION)N030ROCEEDINGSTH)NTERNATIONAL #ONGRESS ON 0HOTOSYNTHESIS 3  #3)2/ 0UBLISHING -ELBOURNE #$ 2/- 4HIDHOLM% ,INDSTROM6 4ISSIER# 2OBINSON# 3CHRODER70

 AND D &UNK# K  .OVEL APPROACHREVEALSLOCALISATIONAND ASSEMBLY PATHWAY OF THE 0SB3 AND 0SB7 PROTEINS INTO THE 0HOTOSYSTEM))DIMER&%"3,ETTn 4HOMPSON ,+ AND "RUDVIG '7  #YTOCHROME B  MAYFUNCTIONTOPROTECT0HOTOSYSTEM))FROMPHOTOINHIBITION "IOCHEMISTRY n D -ANT! $UALSIGNALPEP 4HOMPSON3* 2OBINSON#AND TIDESMEDIATETHESIGNAL RECOGNITIONPARTICLE3EC INDEPENDENT INSERTIONOFAT F HYLAKOIDD MEMBRANEPOLYPROTEIN 0SB9*"IOL #HEMn 4ISSIER# 7OOLHEAD#!AND2OBINSON# 5NIQUESTRUC TURAL DETERMINANTS IN THE SIGNAL PEPTIDES OF @SPONTANEOUSLY INSERTINGTHYLAKOIDMEMBRANEPROTEINS%UR*"IOCHEM n 4OMO4 %NAMI)AND D 3ATOH +  /RIENTATIONAND D NEAREST NEIGHBORANALYSISOF PSB)GENEPRODUCTINTHE0HOTOSYSTEM)) ) REACTIONCENTERCOMPLEXUSING BIFUNCT F IONALCROSS LINKERS&%"3 ,ETTn 6ENER!6 (ARMS! 3USSMAN-2AND6 D 6IERSTRA2$  -ASS SPECTROMETRICRESOLUTIONOFREVERSIBLEPROTEINPHOSPHORYLATION INPHOTOSYNTHETICMEMBRANESOF !RABIDOPSISTHALIANA*"IOL #HEMn 7 7EDEL. +LEIN2 ,JUNGBERG5 !NDERSSON"AND(ERRMANN2'  4HESINGLE COPYGENEPSB3CODES FORAP F H LOGENETICALLY HY INTRIGUING  K$APOLYPEPTIDEOFF 0HOTOSYSTEM))&%"3,ETT  n 7IDGER 72 #RAMER 7! (ERMODSON - AND D (ERRMANN 2'  %VIDENCEFORA F HETERO OLIGOMERICSTRUCTUREOFT F HECHLO ROPLASTCYTOCHROME B &%"3,ETTn :OUNI! 7ITT (4 +ERN * &ROMME 0 +RAUSS . 3AENGER 7 AND/RTH0 #RYSTALSTRUCTUREOF0HOTOSYSTEM))FROM 3YNECHOCOCCUS ELONGATUS AT  ! RESOLUTION .ATURE  n

$IBQUFS
 1SJNBSZ
&MFDUSPO
5SBOTGFS (FSOPU
3FOHFS

.BY7PMNFS-BCPSBUPSJVN
GÛS
#JPQIZTJLBMJTDIF
$IFNJF
*OTUJUVU
GÛS
$IFNJF Z 
+VOJ

%
#FSMJO
(FSNBOZ 5FDIOJTDIF
6OJWFSTJUÅU
#FSMJO
4USB“F
EFT


"MGSFE 3
)PM[XBSUI .BY1MBODL*OTUJUVU
GÛS
#JPBOPSH S BOJTDIF
$IFNJF
4UJGUTUSB“F


 %
.ÛMIFJN
BO
EFS
3VIS
1PTUGBDI (FSNBOZ 4VNNBSZ   * *OUSPEVDUJPO  ** $PGBDUPST
PG 4UBCMF $IBSHF 4FQBSBUJPO JO 1IPUPTZTUFN **  *** 1IPUPQIZTJDBM
1SPQFSUJFT
PG
1JHNFOU
1SPUFJO $PNQMFYFT   " $IMPSPQIZMM
BOE
1IFPQIZUJO  # 1JHNFOU1JHNFOU
*OUFSBDUJPOT   &YDJUFE 4UBUF
&OFSHZ 5SBOTGFS 5   B
8FBL
$PVQMJOH
'ÕSTUFS5ZQF 5 &OFSHZ 5SBOTGFS   C 4USPOH $PVQMJOH
&YDJUPO $PODFQU   
&MFDUSPO
5SBOTGFS  $
1JHNFOU1SPUFJO
*OUFSBDUJPOT  *7

/BUVSF
BOE
1SPQFSUJFT
PG
1
BOE
1IFP  "
"SSBOHFNFOU
PG
UIF
$PGBDUPST
XJUIJO
UIF %%
)FUFSPEJNFS  # 4USVDUVSBM
$PNQBSJTPOT
#FUXFFO
1
1
BOE
UIF
#BDUFSJBM
4QFDJBM
1BJS   $
5IF
&MFDUSPOJD
4UBUF 1 BOE
UIF
-PXFTU
5SJQMFU
4UBUF 1   %
5IF
$BUJPO
3BEJDBM 1 r   
&MFDUSPO
&KFDUJPO GSPN 1 BOE
%JTUSJCVUJPO
PG
UIF
)PMF
XJUIJO 1 r   
1PTTJCMF
'BDUPST
GPS
UIF
)JHI 0YJEJ[JOH
1PXFS
PG 1 r   & 5IF
"OJPO
3BEJDBM 1IFPmr   7

,JOFUJDT
BOE
&OFSHFUJDT
PG $IBSHF 4FQBSBUJPO  7 "
.FDIBOJTN
BOE
,JOFUJDT
PG 'PSNBUJPO
PG UIF
3BEJDBM
*PO
1BJS 1 r 1IFPmr   #
.FDIBOJTN
BOE
,JOFUJDT
PG 'PSNBUJPO
PG UIF
3BEJDBM
*PO
1BJS 1 r 2"mr   $
&OFSHFUJDT
PG 1 r 2"mr 'PSNBUJPO
BOE 1SPUFJO 3FMBYBUJPO 1SPDFTTFT   
&OFSHJFT
PG
UIF
*OJUJBM 1
BOE
A'JOBM
1 r 2"mr 4UBUFT   &GGFDUT
PG
UIF
1SPUFJO
&OWJSPONFOU    &OFSHFUJDT
BOE 3FMBYBUJPO 1SPDFTTFT %VSJOH UIF -JGFUJNF
PG 1    &OFSHFUJDT
BOE 3FMBYBUJPO 1SPDFTTFT %VSJOH UIF
-JGFUJNF
PG 1 r 1IFPmr   
&OFSHFUJDT
BOE
5VSOPWFS
PG 1 r 2"mr   7*

'PSXBSE
#BDL
BOE 4JEF
3FBDUJPOT
PG
3BEJDBM
*PO
1BJS 1 r 2"mrm   " 1 r 2"mr 3FDPNCJOBUJPO
BOE 1 'PSNBUJPO   # 1 r 1IFPmr 3FDPNCJOBUJPO   $ 3FEVDUJPO
PG 1 r   7** $PODMVEJOH
3FNBSLT
BOE
'VUVSF 1FSTQFDUJWFT  "DLOPXMFEHNFOUT  3FGFSFODFT  

!UTHORFORCORRESPON F DENCE EMAILRENGER PC WSCHEMTU BERLINDE

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI

 4VNNBSZ

4HISCHAPTERREVIEWSOURCURRENTSTATEOF KNOW K LEDGEONTHEPRIMARYELECTRONTRANSFER F IN0HOTOSYSTEM)) 03))  0ROPERTIESOFF CHLOROPHY H LL #HL A INSOLUTIONAND D THE BASIC FEATURESO F F PIGMENT PIGMENTANDP D IGMENT PROTEIN INTERACTIONSASWELLASTHEPRINCIPLESUNDERLYINGEXCITATIONENERGYTRANSFERANDELECTRONTRANSFERAREBRIEmY OUTLINED5SINGTHISDESCRIPTIONASASTARTING POINT AND BASEDD ONRECENT INFORMAT F IONAVAILABLEFORT F HESPATIAL ARRANGEMENTOFF THECOFACTORS T F HEGENERAL FEATURESO F F LIGHT INDUCE D D CHARGESEPARATIONIN03))AREPRESENTED 3PECIALATTENTIONISGIVENTOTHEUNIQUEPROPERTIESOFTHEPHOTOACTIVEPIGMENT0 WHICHCONSISTSOFASPECIAL F HE FORM F #HL A  0HEO X WITH X OR4HEPOSSIBLEELECTRONICSTRUCTURES MULTIMERICPIGMENTCOMPLEXOFT D 0 s ASWELLASTHEUNDERLYINGFEATUREST F HATESTABLISH THEEXTRAORDINARILYHIGHOXIDIZING OF 0 0AND POWEROF0 s AREDISCUSSED%VIDENCEISPRESENTEDTHATINTHElRSTELECTRONTRANSFEREVENTA@MONOMERICTYPE #HL A WITHINTHEMULTICHROMOPHORIC0TRANSFERSANE F LECTRON FROM F ITSEXCITEDD SINGLETSTATETOANASSOCIATED PHEOPHYT H IN 0HEO A MOLECULEWHI W CHACTSASTHEPRIMARYELECTRONACCEPTOR4HISEVENT ISFO F LLOWED BYRAPIDSP D IN REDISTRIBUTION LEADINGTOPREDOMINANTLOCALIZATIONOFTHEELECTRONHOLEONA#HL A IN0DESIGNATEDAS0$ WHICH ISPARTOFF A@DIMERICSTRUCTURALMOTIFTERME F D 0$0$ AND ISINCLOSEPROXIMITYTOTHEREDOX ACTIVETYROSINE 9:4HEPROCESSLEADINGTOTHEFORMAT F IONOFT F HERADICAL IONPAIR0 s0HEOn s COMPRISESACASCADEOFRA F DICAL PAIRSTATESOFDECREASINGENERGYTHROUGHASEQUENCEOFRELAXATIONREACTIONSWITHTHEPROTEINENVIRONMENT4HE ROLEOFF THEPROTEINENVIRONMENT INTHEPRIMARY CHARGESEPARATIONPROCESSISEMPHASIZED * *OUSPEVDUJPO 4HE ESSENTIAL PROCESSES OF PHOTOSYNTHETIC WATER CLEAVAGE TAKE PLACE IN A MULTIMERIC PIGMENT PRO TEINCOMPLEXREFERRE F D TOAS0HOTOSYSTEM)) 03)) W CH ACTS AS A WATERPLASTOQUINONEOXIDOREDUCTASE WHI Q U UNITSAND 4HISCOMPLEXCONTAINSMORETHANSUB IS ANISOTROPICALLY INCORPORATED INTO THE THY H LAKOID MEMBRANE4HEOVERALLPROCESSCOMPRISESTHREETYPES OFF REACTIONSEQUENCES FORREV F IEWS SEE'2ENGER    35& $FF $F $FFF  v 5&

KN

m  35& $FF $ $FF FF 

v m 3 $FF $FF  m 35& $FF $FF v 



  (/ A /(,UMEN





v 

 (3TROMA 01 A 01(



WHERE02# SYMBOLIZESTHEPHOTOACTIVEPIGMENTINTHE !BBREVIATIONS " #HL n BACTERIO CHLOROPHYLL " 0HEOn BACTERIO PHEOPHYTIN"2#nBACTERIALREACTIONCENTER$$ #YT B nMINIMUM03))COMPLEXCONTAININGCOFACTORSOFPRIMARY CHARGESEPARATIONOF03))%%4nEXCITEDSTATEENERGYTRANSFER %4nELECTRONTRANSFER$n&RANCK #ONDONWEIGHTEDDENSITY OFF STATES.()nNONHEMEIRON,(nLIGHTHARVESTING0nSPECIAL PAIR0nPRIMARYELECTRONDONORIN03))0nPRIMARY ELEC TRON DONORIN03)03nPHOTOSYSTEM1! nPRIMARY PLASTOQUINONE ACCEPTOR 1" nSECONDARYPLASTOQUINONEACCEPTOR2#nREACTION CENTER74nWILDD TYPE

REACTIONCENTER2# THAT LEADSBY ELECTRONEJ E ECTION FFROMITS LOWESTEXCITEDD SINGLETSTATETO FORMAT F IONOFT F THE ns PRIMARYCATION ANIONRADICALPAIR0s 2# !CC WHICHIS STABILIZED BY RAPID D ELECTRONTRANSFER F FROMT F HEREDUCE D D PRIMARY ACCEPTOR!CC n s TO THE SECONDARY ACCEPTOR !CCTHESYMBOLS  AND ARE USEDD FORTHE REDOX EQUIVALENTSTHAT GIVERISETOOXIDATIVEWATERCLEAVAGE SEQUENCE  ANDD PLASTOQUINOL FORMAT F IONSEQUENCE  RESPECTIVELY (,UMEN ANDD (3TROMA REPRESENT THE PROTONRELEASE INTOTHE LUMENANDUPTA D KE FROMT F HE F HETHY H LAKOID D MEMBRANE!LL STROMA RESPECTIVELY OFT COFACTORSTHATCATALYZEREACTIONSEQUENCES n ARE INCORPORATED INTOASUPRAMOLECULARPROTEINCOMPLEX F HE$ANDD $POLYPEPTIDES #HAPTER CONSISTINGOFT  .IXONETAL !CCORDINGTORECENTSTRUCTURALDATA #HAPTERS n BINDING OF SOME COFACTORS F F FOR SEQUENCE INVOLVESAT LEASTONEADDITIONAL POLY PEPTIDE4HISCHAPTERDESCRIBESTHENATURE FUNCTIONAL PROPERTIESAND GEOMETRICALARRANGEMENTOFF THECOFA F C TORSTHATARE INVOLVED INREACTIONSEQUENCE  $ETAILS F FORREACTIONSEQUENCE AREDESCRIBEDIN#HAPTERS nANDD AND REACTIONSEQUENCE IS DESCRIBED IN#HAPTER ** $PGBDUPST
PG 4UBCMF $IBSHF 4FQBSBUJPO JO 1IPUPTZTUFN
** F F FORSTA BLECHARGESEPARA 4HENATUREOFF THECOFACTORS TIONIN03)) 02# !CC ANDD !CC WEREIDENTIlEDBY D D ABSORPTIONDIFFERENCESPE F C MEASURING LIGHTINDUCE

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



TRATHATCHARACTERIZETHETURNOVEROFEAC F H INDIVIDUA D L COMPONENT +E   &IGURE  SUMMARIZES THE D TO IDENTIFYTHECHEMICALSPECIES ORIGINAL DATAUSED THATCONSTITUTE02# !CC AND D !CC&IGURE!SHOWS THEDIFFERENCESPECTRUMOFmASHINDUCEDABSORPTION CHANGESMEASURED INDIGITONIN TREATEDSP D INACH THY LAKOIDS4HESEDATAREmECTTHESPECTRAL CHARACTERISTICS OF THE TURNOVER OF THE PHOTOACTIVE PIGMENT 02# OF 03)) $ÚRING ET AL   "ASED D ON THIS FEATURE F 02# WASIDENTIlEDASASPECIALCHLOROPHYLL#HL A NOW GENERALLY REFERRED TO AS 0 !T NEARLY THE SAMETIME THEACCEPTORCOMPONENT!CC COULD BE D TO A SPECIALLY BOUND D PLASTOQUINONE 1! ASSIGNED ASILLUSTRATEDBYTHE DIFFERENCESPECTRUMFOR1!ns1! IN &IG # 3TIEHL  SEE ALSO 3TIEHL AND7 D 7ITT H IN A 0HEO WASFOUNDTOACT  ,ATER PHEOPHYT ASTHEELECTRONACCEPTOR!CC FORTHEPRIMARYCHARGE F SEPARATION IN03)) +LIMOVETAL  4HE DIFFER ENCESPECTRUMFOR0 F HEOn0HEOISDEPICTEDIN&IG" "ASEDONTHISKNOWLEDGE THEOVERALLREACTIONPATTERN OFF STABLECHARGESEPARATIONACCORDINGTOSEQUENCE CAN BEMORESPECIlCALLYFORMU F LATED BYTHE FO F LLOWING Q SEQUENCE 33KHR4$ N3& N±

K




3 3KHR4$

3‡3KHR±‡4$

NVWDE N±

3‡3KHR4$±‡



WHERETHERATECONSTANTSK0# AND KSTAB A DESCRIBEPRIMARY CHARGESEPARATIONANDITSSTABILIZATION RESPECTIVELY ANDKKn ANDKKn ARETHE RATE CONSTANTS OFTHE CORRE SPONDING BACKK REACTIONS !LTHOUGHTHEBASICPRINCIPLESOFTHEPHOTOSYNTHETIC 2#SAREVERY Y SIMILARANDHIGHLY LY CONSERVEDTHROUGHOUT U EVOLUTION THEPHOTOACTIVEPIGMENT0OFF 03)) IS Q 4HECATIONRADICAL0s FO F RMED AS A RESULTOF UNIQUE THEPRIMARY CHARGESEPARATIONISONEOFTHESTRONGEST D THEENERGETICPREREQUISITE FOR F OXIDANTSINBIOLOGYAND THE OXIDATION OF WATER TO MOLECULAR OXYGEN7ITH ANESTIMATEDREDUCTIONPOTENTIALOFTO6 *URSINICAND'OV D INDJEE +LIMOVETAL  2APPAPORTETAL  0s EXCEEDS THE OXIDIZ INGPOWEROFTHECATIONRADICALSINALLOTHERNATURAL PHOTOSYNTHETICREACTIONCENTERS BYATLEAST64HE UNDERLYING MOLECULAR MECHANISM FOR THE UNUSUAL REDOX PROPERTIES OF 0 IS STILL NOT SATISFACTORILY D AND REMAINS A CHALLENGE FFOR CURRENT AND CLARIlED F FUTURERESEARCHACTIVITIES3ECTION)6$  4HIS PARTICULAR FEATURE AS WELL AS THE ENERGETIC BEHAVIORAND KINETICSOFT F HEREACTIONPATTERN DEPENDS

&IG IG$IFFERENCEA F ABSORPTIONSPECTRAOF 0s 0 ! 0HEO ns0HEO " AND1!n s1! # 4HESPECTRAWEREREDRAWNFROMDATAPUBLISHED IN$ÚRINGETAL +LIMOVETAL AND3TIEHL RESPECTIVELY4OPWAVELENGTHSCALEREFERSTOPANEL!AND" THE BOTTOMSCALETOPANEL#

ONTHESPECIlCSTRUCTURAL ARRANGEMENTOFT F HECOM PONENTSWITHINTHE PROTEINMATRIX WHICHTUNESTHE F HECOFACTORS F ENERGETICSANDD REACTIONCOORDINATESOFT !DVANCEMENTINOURUNDERSTANDINGOFT F HEFUNCT F IONAL AND STRUCTURAL ROLE OF THE PROTEIN MATRIX IN LIGHT INDUCE D D CHARGESEPARATIONWASONLYACHIEVEDD AFTE F R THE DEVELOPMENT OF ISOLATION PROCEDURES D FFOR 2#S PREPARATIONSWITHAMINIMUMCONTENTOFPROTEINSAND COFACTORSW F WHILERETAINING FU F LL FUNCT F IONAL ACTIVITY4 Y HE lRSTMILESTONE INTHIS LINEOFF RESEARCHCAMEALMOST YEARSAGOWITHTHEISOLATIONOFREACTIONCENTERS F FROMANOX YGENICPURPLE BACTERIA 2EEDD AND #LAY A TON

   ! SIMILAR ACHIEVEMENT FFOR 03)) WAS ONLY REACHEDABOUTYEARSLATERWHENAMINIMALPREPA RATION HAD BEEN DEVELOPED D THAT COULD D PERFFORM THE PRIMARYRADICALPAIRFORMAT F ION0s0HEOns.ANBA AND3ATOH   D 2#S ! COMPARISON WITH THE WELL CHARACTERIZED F FROMPURP LEBACTERIA "2#S LED D TOTHECONCLUSION THATTHECOFACTORSOFREACTIONSEQUENCE IN03)) ARE INCORPORATED INTOTH T E HETERODIMERICPROTEINMATRIX D $POLYPEPTIDES -ICHEL AND D $EISEN OFF THE$AND HOFER  4HOROUGHPIGMENTANALYSESREVEALED THAT $$#YT B PREPARATIONS CONTAIN ABOUT  #HL A PER0HEO A +OBAYASHIETAL %ICKEL HOFFETAL :HELEVAETAL +URRECKETAL F A  HOWEVER NON STOICHIOMETRICRATIOS FROMn #HLSPER0HEOAREOFTENO F BTAINED +ONERMANNAND (OLZWARTH  )NADDITIONTOTHECHLORINPIGMENTS THESEPREPARATIONSALSOCONTAINTWOSPECTRALLY DISTINCT ` CAROTENE MOLECULES +OBAYASHI ET AL   )N ORDERTOUNRAVELTHEPROPERTIESOFSUCHAMULTIPIGMENT ARRANGEMENT ITISNECESSARYTOANALYZEINDETAIL THE INTERACTIONSAMONGTHEPIGMENTSAND D THEIRCOUPLING TOTHEPROTEINMATRIX"EFOREDESCRIBINGOURCURRENT STATEOF KNOW K LEDGEONTHEENERGETICSAND FUNCT F IONAL PROPERTIESOFF THE03))2# ABRIEFSUMMARYO F F GENERAL PIGMENT PIGMENT AND PIGMENT PROTEIN INTERACTIONS ISPRESENTED *** 1IPUPQIZTJDBM
1SPQFSUJFT
PG
1JHNFOU 1SPUFJO $PNQMFYFT 4HESPECTRAL AND FUNCT F IONAL PROPERTIESOFF PIGMENTS BOUND INTOAPROTEINMATRIXAREESSENTIALLY DEPENDENT ONTHREEPARAMETERSI THECHEMICALNATUREOFTHE PIGMENT II THE STRENGTH OFF THE PIGMENT PIGMENT COUPLING AND III THE MODE OF PIGMENT PROTEIN INTERACTION " $IMPSPQIZMM
BOE
1IFPQIZUJO J 4HESTRUCTUREOF# F HL A AND ITSSPECTRAL PROPERTIES IN DILUTESOLUTIONSOFNUC F LEOPHILICANDORPOLARSOLVENTS W WHEREDIMERIZATIONORHIGHERAGGREGATIONDOESNOT D 0HEO A ARE OCCUR ARESUMMARIZED IN&IG#HL A AND BOTH CHARACTERIZED BYACARB R ONSKELETONTHATCONTAINS I AlVERINGCHLORINWITHACONJUGATED/ ELECTRON SYSTEM SYMB M OLIZED BYTHICK LINED BONDS AND II AN H DROPHOBICPHYTY H L CHAINTHATISCONNECTED EXTENDED HY VIAANESTERBONDWITHTHEPROPIONATEOFRING)6#HLA CONTAINS-G ASTHECENTRALATOMCOMPLEXED BYTHE

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI TETRAPYRROLE RING WHEREAS 0HEO A IS THE FREE F BASE WITHOUTACENTRALMETALION4HEABSORPTIONSPECTRAOF MONOMERIC#HL A EXHIBITSTRONG BANDSINTHEREDD AND BLUE3ORET BAND SPECTRAL REGIONS DUETOSP D IN AND SYMMETRY ALLOWED ELECTRONIC TRANSITIONS )N ADDI TION SYMMETRY FOR F RBIDDEN N/ TRANSITIONSWITHMUCH LOWEROSCILLATORSTRENGTH A FACTORnSMA F LLERAS COMPAREDTOTHE // TRANSITIONS AREPRESENTDUETO THECENTRAL PYRROLICNITROGENANDD THE KETOOXYGEN OFF RING6 WITH OCCUPIEDD NONBONDING ORBI R TALS4HE RELATIVE POSITIONS OF THE ENERGY LEVELS OF // AND NN TRANSITIONS DEPENDD STRONGLY ON THE POLARITY OF THEENVIRONMENT 0ETKEETAL   4HE TRANSITION FROM THE GROUND 3 TO THE lRST D L EXCITEDD SINGLETSTATE3 ISLOCATED INTHEREDSPECTRA RANGEANDD POLARIZED INTH T EY DIRECTIONOFT F THEMOLECULAR AXISSYSTEM&IG BOTTOMRIGHTSIDE !CCORDINGLY ITISSYMBOLIZED BY1Y IN0LATTSNOTATION4HESEC ONDD EXCITEDD SINGLET STATE 3 GIVINGRISETO THE 1X TRANSITIONISCOMPARATIVELYWEAKANDITSENERGETICS D Y   RESEMBLESTHATOFF THEVIBRONICALLY EXCITED1 TRANSITION!CTUALLY THERELATIVEPOSITIONOF 1X   AND1Y   DEPENDSSTRONGLYONTHESOLVENTPROPER TIES4HESTRONG ABSORPTION INTHE BLUEREGION 3ORET BAND ARISES FFROM TRANSITIONS INTO HIGHER EXCITED SINGLETSTATESANDISPREDOMINANTLYPOLARIZEDINTHE X DIRECTION"X TRANSITION FORAREV F IEW SEE3CHEER   4HE HIGHER EXCITED SINGLET STATES RAPIDLY RELAXWITHINAFEWPICOSECONDSDOWNTOTHELOWEST ELECTRONICALLYANDD VIBRONICALLY EXCITEDD STATE #HL A

3 )NDILUTE+- SOLUTIONSWITHOUTAGGREGATION  #HL A HASALIFETIMEOF›NS6ASILEVETAL F IALLY TWO DECAY A  4HISVALUEISTHERESULTOFESSENT PATHWAYSWITHRATECONSTANTSKKRAD 5 q Sn AND K)3# 5 q Sn FORRADIATIVEEMISSIONANDINTERSYSTEM CROSSING INTOTH T ETRIPLETSTATE 4 RESPECTIVELY 0ARK R ER ANDD *OYCE  $ISSIPATIONOFF EXCITATIONVIARADIA TIONLESS TRANSITION FROM #HL A INTOTHEGROUNDSTATE #HL A IS VIRTUALLY NEGLIGIBLE FFOR MONOMERIC #HL A IN PROTIC SOLVENTS BUT BECOMES DOMINANT IN #HL A AGGREGATES $UETOTHEVERY FAS F T INTERNALCONVERSIONPROCESSES SUB U PSTOPSTIMEDOMAIN FROM F HIGHEREXCITEDD SIN GLETSTATES THEREACTIONPROPERTIESOFELECTRONICALLY EXCITED# D HL A AREALMOSTEXCLUSIVELY DETERMINED BY THEBEHAVIOROFF THE3 STATE!CCORDINGLY REGARDLESS OFTHEEXCITATIONWAVELENGTH RADIATIVEEMISSIONGIVES RISETOACHARACTERISTICRED mUORESCENCE THERADIATIVE F HIGHEREXCITEDD SINGLETSTATESINTHE BLUE EMISSIONFROM ISWEAKERBYSEVERALORDERSOFMAGNITUDEANDDIFlCULT TODETECT SEE,EUPOLD D ETAL  !SACONSEQUENCE

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



&IG%NERGYLEVEL DIAGRAM LEFTS & F IDE MOLECULARSTRUCTURE BOTTOM RIGHTSIDE AND D ABSORPTIONSPECTRUMOF #HL A INETHANOL TAKENFROM F 0ETKEETAL  TOP RIGHTSIDE 4HECONJ N UGATED / ELECTRONSYSTEMISSYMBOLIZED BY THETHICK GREY LINES INTHE MOLECULAR STRUCTURE %NERGY LEVELSOFF ELECTRONICALLY EXCITEDD STATESARECHARACTERIZED BYTHICKAN K D VIBRATIONALSTATESOFT F HEMOLECULES BY THIN BARS0HONONS F F FORMAQUAS I CONTINUUMSYMBOLIZED BY GREYAREAONLY 3 ISCONSIDERED 2ATECONSTANTS WITHTHEIRMUCH SMALLERENERGY DIFFERENCES K& K|% K)# AND K)3# SYMBOLIZE mUORESCENCE PHOSPHORESCENCE INTERVAL CONVERSIONAND INTERSYSTEMCROSSING RESPECTIVELY&ORS Y IMPLICITY Y ONLYnTRANSITIONSAREEXPLICITLYSHOWN ANDRADIATIONLESSTRANSITIONSTOTHEGROUNDSTATEAREOMITTEDTHISPROCESSISNEGLIGIBLEFOR DILUTE#HL A SOLUTIONS SEETEXT 

ALL USEFU F LNATURAL PHOTOCHEMICALREACTIONSSTART FROM F  #HL A 3 4HELOWEST TRIPLETSTATE #HL A FORMED  F #HL A 3 PLAY A SAN VIA INTERSYSTEMCROSSING FROM IMPORTANTROLEFOR F PHOTOCHEMICALREACTIONSOFF #HL A INSOLUTIONBECAUSEOFITSMUCHLONGERLIFETIMEIN THE MS RANGE BUT IS NOT INVOLVED IN FORMAT F ION OF THE PRIMARYRADICALPAIR0s0HEOns3ECTION6!  )NCONTRAST HOWEVER #HLTRIGGERSUNDESIRABLESIDE REACTIONSTH T ATNATURETRIESTOMINIMIZE4HEPOPULATION OF #HL A INPHOTOSYNTHETICORGANISMSGIVESRISETO DELETERIOUSREACTIONSDUETOTHESENSITIZEDGENERATION F OF HIGHLY REACTIVE SINGLETOXYGEN 6G/4HEREFORE

SUITABLE MECHANISMS ARE REQUIREDD TO PROTECT THE APPARATUS FROM RAPID PHOTODAMAGE UNDER AEROBIC CONDITIONS3ECTION6)"  4HE COORDINATION OFF THE CENTRAL -G OF #HL IS Q MPLEXES UNSATURATED!SACONSEQUENCE #HLSFORMCOM D WITH NUCLEOPHILICLIGANDSANDD WITH EACHOTHER DUE TOTHEELECTRON DONATINGPROPERTIESOFT F HECARB R OXYLIC GROUPSANDTHEELECTRONACCEPTINGPROPERTIESOFTHE CENTRAL -G ION .ORRISETAL  )NTHEABSENCE F ITABLELIGANDSFROMT F HESOLVENTMOLECULES #HLS OFSU SELF AGGREGATE OFTEN THROUGH THE ACTION OF SMALL F IONSOFT F HEIR AMOUNTSOFF WATERANDORLIGANDING FUNCT

 OWNSIDECHAINS4HETYPEOFCOMP F LEXFORME F D DEPENDS ONTHENATUREOFTHESOLVENT)NGENERAL APOLARSOLVENTS F FAVORT HE FORMAT F ION OF LARGE #HLAGGREGATES OFTEN F EMPLOYINGANEQUIVALENTOFF ONEORTWOWATERMOL ECULESASABRIDGINGSTRUCTURE /NTHEOTHERHAND IN POLARNUCLEOPHILICANDORHYDRO D GEN BONDING SOLVENTS COMPLEXATION OCCURS BETWEEN MONOMERIC #HLAND THE SOLVENT NUCLEOPHILE !LTERNATIVELY OLIGOMERIC D F FORME D D COMPR ISING AGAINBIFUNCT F IONAL ADDUCTSARE POLARLIQUIDS EG (/ ASABRIDGE4HEROLEOFTHE F FUNCTIONALGROUPSOF#HLSANDTHECHARGEOFDETERGENT F GGREGATION INMICELLARSOLUTION HAS MOLECULESFORA BEENSUMMARIZED IN!GOSTIANOETAL 3ELF A F G GREGATIONOFPIGMENTSINPHOTOSYNTHETICORGANISMS ISADOMINATING FACTORON F LY FORT F HE FORMAT F IONOFF THE CHLOROSOMES THAT ACT AS ANTENNA SYSTEMS IN GREEN BACTERIA)NTHISCASE BACTERIOCHLOROPHYLL"#HL C D OR E MOLECULES UNDERGO SUPRAMOLECULAR SELF OR F GANIZATIONVIAANINTERNAL HY H DROXYGROUP "ALABAN ETAL  WHEREPROTEINSDONOTPLAYANYSTRUC TURAL ROLE)NOTHERANTENNASYSTEMSAND D ALL2#STHE " #HLSARENONCOVALENTLYBOUND D TOPROTEINS4HIS GIVES RISE TO SPECIAL COORDINATION WITH AMINO ACID D AND AXIAL LIGANDS  )NFORMAT F ION ON THE RESIDUES TYPEOFCOOR F DINATIONCAN BEOBTAINED FROMMO F DERN SPECTROSCOPICTECHNIQUESLIKERESONANCE2AMANO Q R &4)2 SPECTROSCOPY Y #HL A IN BOTH SOLVENTS AND IN PROTEINS ISTYPICALLYPENTACOORDINATED FORREV F IEWS SEE-ÊNTELE 2OBERT  4HISISFULLYSUP U PORTED BY THERECENTHIGHRESOLUTION8 RAY A STRUCTURE OFF 03) &ROMME ET AL   )NTERESTINGLY THE PENTACOORDINATIONDEPENDSONTHESTEREOISOMERYOF THE LIGATION AT ASYMMETRIC # ATOMS OF F THE CHLORIN RING3TUDIESONMODEL SYSTEMSREVEAL THATTHE BIND INGOFTHElFTHLIGANDISFAVOREDFROMTHESIDEOFTHE W ERETHEMETHOXYCARB R ONYL N MOIETY OF MACROCYCLEWH D 4AMIAKI  )N03) RING6PROTRUDES/BAAND OF THERMOPHILIC CYANOBACTERIA  #HLS OUT OF THE TOTALAMOUNTOFF #HLSAREPENTACOORDINATED FROM F THE@ANTISIDEWITHRESPECTTOTHESTEREOISOMERYOF THEPHYTYLESTERATTHEPROPRIONATEOFRING)6"ALABAN ETAL   D 0HEO A HAVEBEEN 4HEREDOX PROPERTIESOFF #HL A AND THOROUGHLY ANALYZED IN SOLUTION BY CYCLIC VOLTAM METRY 7ATANABEAND D +OBAY A ASHI  -ONOMERIC #HL A INSOLUTION ISCHARACTERIZED BYSTANDARD D REDOX POTENTIALSVERSUSNORMALHYDROGENELECTRODE.(% OFF ABOUT6AND D ABOUTn6FOR F ITSONE ELEC TRONOXIDATIONAND D REDUCT D ION RESPECTIVELY4HECOR RESPONDINGVALUESFOR0HEO A IN SOLUTION ARE ABOUT 6AND D ABOUTn6 RESPECTIVELY)T Y ISINTEREST

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI INGTONOTETHAT #HL As INSOLUTIONEXHIBITSMARK R EDLY LOWEROXIDATIONPOWERTHAN0s ANDTHATAGGREGATE F FORMAT ION IN SOLUTION LEADS TO EVEN LESS OXIDIZING CATION RADICALS 4HESE lNDINGS ILLUSTRATE THAT THE PROTEINENVIRONMENTPLAYSAKEYROLEINESTABLISHING THESPECIALPIGMENTCOMPLEX0WITH ITSSTRONGLY OXIDIZINGSPECIES0s 3ECTION)6$  !FUNCTIONALLYIMPORTANTSTRUCTURALPARAMETERISTHE PLANARITY OFF THECHLORINRING3TUDIESWITH CONFORMA F

TIONALLYDESIGNEDD NON PLANARMOLECULESREVEALEDD THAT DISTORTIONSOFRINGPLANARITYGIVESRISETOSIGNIlCANT CHANGES IN THE OPTICAL ANDD REDOX PROPERTIES FOR F A A ER   !CCORDING TO THEORETICAL REVIEW SEE &AJ CALCULATIONSTHESE DISTORTIONS LEAD D TO A DESTABILIZATION OFT F HE HIGHESTOCCUPIEDD MOLECULARORBI R TAL (/-/ WITH LITTLEEFFECTONT F HE LOWESTUNOCCUPI U EDD MOLECULAR ORBITAL,5-/ 3ENGEETAL   # 1JHNFOU J 1JHNFOU
J *OUFSBDUJ UJPOT 4HE STRENGTH OF F THE PIGMENT PIGMENT INTERACTION DEPENDS ON THE INTERPIGMENT DISTANCE MUTUAL ORI ENTATIONANDTHENATUREOFTHEINTERVENINGMEDIUM Y DIFFERENT F FFEATURES EMERGE DEPENDING !CCORDINGLY ONTHEMODEANDD STRENGTH OFCOUP F LINGBETWEENTHE PIGMENTS&ORTHESAKEOFSIMPLICITYTHEGENERALFEA TURESOFT F HEREACTIONBEHAVIORWILL BEILLUSTRATED FORA F SYSTEMCONSISTINGOFF ONLYTWOINTERACTINGPIGMENTS )NTHEELECTRONICGROUNDSTATE THEINTERACTIONBETWEEN TWOPIGMENTS ( GIVESRISETOANENERGY SHIFTO F F THE ENSEMBL M E DUETOT D THEVANDER7AA 7 LS INTERACTION!MORE COMPLEXPATTERNARISESWHENTHEPIGMENTENSEMBLE ISELECTRONICALLYEXCITED4HEPROPERTIESINTHISCASE AREDESCRIBED BYA (AMILTONIANOFT F HETYPE (  (  (  (



W ERE (I I  ARETHE(AMILTONIANSOFTHESEPA WH RATEDP D IGMENTSANDD WITHOUTINTERACTIONAND HAV A INGEIGENFUNCT F IONS  I KKI WITHKGORE WHICH DESCRIBESTHEGROUNDG ANDELECTRONICALLYEXCITED E STATES RESPECTIVELYAND ( ISTHEINTERACTION OPERATOR $EPENDINGONTHENATUREOF( THE INTERACTION CAN GIVE RISE TO TWO BASICALLY DIFFERENT F REACTION TYPES F IMPLE 4HISIS ILLUSTRATED IN&IGONTHEBASISOFAS (/-/,5-/ ANALYSISFORTHETWOIDENTICALPIG MENTS 0 ANDD 04HETWOTYPESOFF REACTIONSAREI EXCITEDSTATEENERGYTRANS D F %%4 AND II ELECTRON FER TRANSFER%4 FROMONE PIGMENTTOTHEOTHER4HESE F KINGPLACE INTHEPRIMARY ARETHETYPESOFPROCESSESTA

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



REACTIONSEQUENCES INPHOTOSYNTHETICANTENNAEAND IN2#SINGENERAL ASWILLBEOUTLINEDIN3ECTION)6 7HEN THE STRENGTH OF F ELECTRONIC COUPLING 6 IS WEAK K COMPARED TOTHEELECTRON VIBRATIONAL COUPLING THERATECONSTANTOFTHECORRESPONDINGREACTIONSCAN BECALCULATED BY QUANTUMMECHANICAL PERTURB R ATION THEORY WITHIN THE FRAMEWOR F K OF &ERMIS 'OLDEN 2ULE N

P  9 s )&:' 



WHERE6 ISTHEMATRIXELEMENTOFE F LECTRONICCOU PLING BETWEEN THE INITIAL I STATE 0 0 AND THE lNAL F F STATE 00 OR0s0ns IN &IGAND$IS THE&RANCK #ONDONWEIGHTED DENSITYOFF STATES4HE VALUEOF6 ISGIVENBY sF \(\sIœ INWHICH sI AND sF ARETHEWAVEFUNCTIONSOFTHEINITIAL0 0 AND lNAL 00 OR 0s0ns STATES RESPECTIVELY Y 
&YDJUFE
4UBUF
&OFSHZ S 5SBOTGFS 5 5S )NTHE CASE OFEXCITATIONENERGYTRANSFER%%4 TWO LIMITING DOMAINS CAN BE DISTINGUISHED WEAK AND STRONG COUPLING WH W ERE 6 ISMUCHSMALLERORLARGER RESPECTIVELY COMPAREDTOTHE &RANCK #ONDONWIDTH OFTHEOPTICALTRANSITIONSOFTHEINDIVIDUALPIGMENTS 3ECTION)))"  B
8FBL
$PVQMJOH 'ÕSTUFS5Z 5ZQF
&OFSSHZ 5 5SBOTGFS S

)N THE LIMITING CASE OF VERY WEAK COUPLING THE ELECTRONIC STRUCTURE OFF THE PARTICIPATING PIGMENTS REMAINS VIRTUALLY UNAFFECTED AND THE EXCITED STATE ISLOCALIZEDONONEOFTHEPIGMENTSANDTRANSFERRED F FROMT HE DONOR$ TOTHEACCEPTOR! PIGMENTVIA ANINCOHERENTHOPPINGTYPEPROCESS!NEVALUATIONOF SCHEME WITHINTHEFRAMEWORKOFTHEPOINT DIPOLE W ERETHECENTER TO CENTERDISTANCE APPROXIMATIONWH BETWEENTHEPIGMENTSISLARGEASCOMPAREDWITHTHE PIGMENT DIMENSION $AVYDOV  LEADS TO THE WIDELY USED D &ÚRSTEREQUATION FORT F HERATEOFF PAIRWISE %%4&ÚRSTER   N )|UVWHU  ((7

$ – K  )'  Q  5  T °

E$ 

GN N

&IG IG  %LECTRONICCOUPLING BETWEENTWOPIGMENTMOLECULES 0 AND 0 OFF THESAMEORBI R TAL STRUCTURE4HEINTERACTIONCANEITHER LEADTOEXCITEDSTATEEXCITONo SYMBOLIZEDBY j¡k LEFTSIDE OR ELECTRON RIGHT SIDE TRANSFER FROM PIGMENT MOLECULE 0 IN ITS LOWESTEXCITEDSINGLETSTATE0 TOPIGMENTMOLECULE0 INTHE ELECTRONICGROUNDSTATE&ORSIMPLICITY ONLYTHEHIGHESTOCCUPIED R TAL (/-/ AND LOWEST UNOCCUPIED D MOLECULAR MOLECULAR ORBI ORBI R TAL ,5-/ ARESHOWN4HELOWERPARTSHOWSASCHEMATIC D REAC REPRESENTATIONOFF THEPROCESSESINTHEANTENNASYSTEMAND TIONCENTERCORRESPONDING BACK K REACTIONSAREOMITTED WH W ERE0 ISTHEPHOTOCHEMICAL PIGMENTAND D ! ! THEPRIMARY SECON Y DARY ACCEPTOR RESPECTIVELY

TIONOFT F HETRANSITIONDIPOLEMOMENTSOFT F HEDONOR ANDACCEPTORPIGMENT ) g )  2ISTHEDISTANCE BETWEENTHEDONOR$ ANDD ACCEPTOR! POINTTRANSI TIONDIPOLEMOMENTS o ISTHERADIATIVE LIFET F IMEOF THEEXCITEDSTATEOFTHEDONOR &$i AND ¡! i ARE F THE NORMALIZED DONOR THE SPECTRAL DISTRIBUTIONS OF D BSORPTION RESPECTIVELY EMISSIONANDACCEPTORA 4HE&ÚRSTERTYPE%%4ACCORDINGTO%Q PROVIDES ONLY A PERTINENT DESCRIPTION IF F IN ADDITION TO THE VALIDITYOFF THEPOINTDIPOLEAPPROXIMATION THEPIG



WHERE! ISACONSTANTN ISTHEREFRACT F IVEINDEXOFT F HE F DESCRIBINGTHEMUTUAL ORIENTA MEDIUM g ISAFACTOR

o

)NPHOTOSYNTHESISRESEARCHTHEWORD D @EXCITON ISUSEDASANOP D

ERATIONAL TERMTODESCRIBEANEXCITED D ELECTRONICSTATETHATRESIDES ONMORETHANONEPIGMENTINA GROUPOFMO F LECULESDUR D ING ITS LIFETIME0EARLSTEIN  

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI

 MENT PIGMENTCOUPLINGISWEAKENOUG K HTOENSURETHAT THEINDIVIDUALPROPERTIESOFTHEDONORLIFETIMEAND LINESHAPEOF mUORESCENCE AND D ACCEPTOR LINESHAPE OFF ABSORPTION INCLUDINGOSCILLATORSTRENGTHS ARENOT PERTURBED(OWEVER INTHECASEOFSTATICDISORDER IT IS DIFlCULT TO OBTAIN THE HOMOGENEOUS LINE SHAPES &$i AND ¡!i IN%Q  &URTHERMORE APART FROM F THE NECESSITY OF HAVING STRUCTURAL INFORMATION FOR F HEREFRAC F OBTAINING g THEUSEOFF CORRECTVALUESFORT TIVE INDEX N IN BIOLOGICAL SYSTEMSISALSO IMPORTANT +NOX AND VAN!MERONGEN   )N THE CASE OF MORETHANTWOPIGMENTMOLECULESWITHINAPIGMENT PROTEIN COMPLEX THE PATTERN BECOMES EVEN MORE COMPLEXIFAGGREGATESOFSTRONGLYCOUPLEDPIGMENTS F D FORANEXTENS F IONOFF THE&ÚRSTERTHEORY AREFORME TOTHE%%4 DYNAMICS BETWEENAGGREGATES SEE3UMI 3CHOLESETAL   )NADDITIONTO&ÚRSTERTYPE%%4 WHI W CH ORIGINATES F FROM #OULOMBICINTERACTIONBETWEENTHETRANSITION DIPOLE MOMENTS WITHOUT EXCHANGE OF ELECTRONS BETWEEN THE INTERACTING PIGMENTS THE OVERLAP OF F IONSATCLOSE DISTANCEALSOGIVES ELECTRONICWAVEFUNCT RISE TO AN ELECTRON EXCHANGE)NTHISCASETHEELECTRON F FROMT HE,5-/OFF 0  SEE&IG ISTRANSFERRE F D TO THE,5-/OFF 0 AND D SIMULTANEOUSLYANELECTRONOF OPPOSITESPINFROMTHE(/-/OF0 TOTHE(/-/ F HE&ÚRSTER OFF 0 4HENETRESULTISTHESAMEAS FORT TYPE%%4 BUTTHEMECHANISMISQUITEDIFFERENT!S F ACONSEQUENCE ANOTHERTYPEOF%%4 REFERREDTOAS THE $EXTERMECHANISM $EXTER  EMERGESWITH ARATECONSTANTGIVEN BY N 'H[WHU (7

P H[FK  9''$ ° )'  

$  GN



EXCH WHERE6$! ISTHEELECTRONICMATRIXELEMENT FORT F HE EXCHANGEINTERACTIONBETWEENDONOR$ ANDACCEPTOR ! THATDECAY A SEXPONENTIALLY LY WITHDISTANCE2BETWEEN $AND D ! &$i AND ¡!i ARETHESAMEPARAMETERSAS IN%Q 4HECONTRIBUTIONOFEXCHANGEPROCESSESTO OVERALLSINGLET SINGLET%%4ISCOMPARATIVELY SMALLFOR " #HLSINPHOTOSYNTHETICORGANISMS%VENFORT F HE STRONGLYCOUPLEDPIGMENTSINTHEBACTERIALANTENNA COMPLEX,( VALUESOFONLYnWEREREPORTED INATHEORETICAL STUDY 4RETIAK K ETAL  (OWEVER THEYPLAYAKEYROLEFORTRIPLETSTATEENERGYTRANSFER F OM " #HLTOCAROTENOIDSINPHOTOSYNTHETICORGAN FR ISMSECTION6)" 

C
4USPOH
$PVQMJOH
&YDJUPO
$PODFQU

U )NTHECASEOFSTRON F GCOUPLING DELOCALIZEDD EXCITED STATESEXISTTHATCAN BEDESCRIBEDD ASA LINEARCOMBI NATIONOFLOCALLYEXCITEDSTATES&ORADIMEROFTWO PIGMENTS0 ANDD 0 ONEOBTAINS             sG  s G s G AND sE  Cs E s G  Cs G s E



  WHEREsI G W AND sI E  ARETHEEXACTNORMALIZEDD WAVEFUNC F TIONSOFTHENONINTERACTINGSUPERSCRIPT PIGMENTS I  OFT F HE GROUND G AND lRSTEXCITED E STATES ANDD C AND C ARECONSTANTS4HEENERGY DIFFERENCE F BETWEENTHEEXCITEDANDGROUNDSTATEOFTHEDIMER IS GIVENBY

6% DIMERRsE\(\sEœ ns n G\(\sGœ



4HEEVALUATIONFOR F A HOMODIMERWITH THTTHE (AMILTONIAN (  (  (  ( LEADSTOTHEEXPRESSION 6% HOMODIMERR  6% MONOMERR  67 › ¡EXCITON



WHERE 6% MONOMERR  ¡E n ¡G ¡G AND ¡E ARETHEELECTRONIC W F HESEPARATEDD MONOMERICPIGMENTMOL ENERGIESOFT ECULES WITH ¡G  ¡ GG  ¡ G AND ¡E  ¡ E  ¡ E  67            s E s G \(\s E s G œ ns n    Gs G \(\s   Gs   Gœ ISTHEDIF F F FERENCEO F THE VANDER7AALSENERGIESOFF THEINTERACTING PIGMENTSINTHEEXCITEDANDGROUNDSTATEOFTHEDIMER         s G \(\s G s E œ RESPECTIVELYANDD THETERM¡EXCITONN  s E ISREFERRE F D TOAS EXCITON INTERACTIONENERGY !SACONSEQUENCEOF%Q INSTEADOFASINGLE TRANSITION 6% MONOMERR INTHENONINTERACTINGMONOMERS TWODIFFERENTTRANS F ITIONSEMERGEFORT F HEEXCITONICALLY COUPLEDPIGMENTSINTHEHOMODIMERWITHANENERGY GAPOFF ¡EXCITON4HIS FEATURE F ISSCHEMATICALLYSHOWN IN &IG4HEEXTENTOFEXC F ITONSPLITTINGANDD THEOSCIL LATORSTRENGTHOFBOTHTRANSITIONS6%   6% MONOMER  67 ¡EXCITON AND 6% n  6% MONOMERR  67 n ¡EXCITON DEPENDD ONTHE DISTANCE BETWEENTHEPIGMENTS2 THE OSCILLATOR STRENGTHS OF THE OPTICAL TRANSITION OF THE ISOLATEDD MONOMERSAND THEMUTUALORIENTATIONOFF THE TRANSITIONDIPOLEMOMENTSWITHINTHE DIMER(OCH STRASSERAND+ASHA  4HEEXCITATIONENERGYIN THISCASEIS DELOCALIZEDD OVERTHETWOPIGMENTS WHI W CH ACTASAKINDD OFSU F UPERMOLECULE )F THE TWO PIGMENTS IN THE DIMER DIFFER IN THEIR TRANSITIONENERGIES 6% I I  BY66% EITHERDUE D TO DIFFERENT F CHEMICAL NATURE ˆ EG A #HL A#HL B HETERODIMER OR TO ENVIRONMENTAL DIFFERENCES THE

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



&IG  %LECTRONIC ENERGY LEVEL DIAGRAM OF A MONOMER AND A & STRONGLYCOUPLEDHOMODIMER2ATECONSTANTS K& K% K)# ANDKK))3# REPRESENTPROCESSESOF mUORESCENCE PHOSPHORESCENCE INTERSYSTEM CROSSING AND INTERNAL CONVERSION RESPECTIVELY Y INTHEMONOMER - AND DIMER$  K$)# ISTHE RATE CONSTANT FORINTERNAL CONVE N R SION FROM F HIGH TOLOWENERGYEXCITONLEVELS &ORSIMPLICITY T Y HE GROUND D STATEENERGYOFF THEMONOMERAND DIMERARESETATEQUAL LEVELSANDTHEPATHWAYSOFK$&ANDK$% ARENOTSHOWN,IKEWISE THERADIATIONLESSDECAYOFTHEMONOMERITSCONTRIBUTIONTOTHE O OVERALLDECAYOFTHELOWESTEXCITEDSINGLETSTATEISNEGLIGIBLE FOR DILUTESOLUTIONSOFMONOMERIC#HL A SEE3ECTION)))! ANDTHE L LLSPLITTINGOFT F THETRIPLETSTATEINTH T EDIMER3CHMIDT D VANISHINGLYSMA AND D 2EINECKER  AREOMITTED 4HEOSCILLATORSTRENGTHOFF THE TWOTRANSITIONS INTHE DIMERHi$ ANDHi$ DEPENDSONTHESTRENGTH AND D MUTUAL ORIENTATIONOFF THETRANSITION DIPOLEMOMENTSOFT F HE D L PIGMENTMOLECULES FORM F ING THE DIMERFOR F DETAILS TWO INDIVIDUA SEE(OCHSTRASSERAND+ASHHA  



TWO EXCITONTRANSITIONS 6%  AND 6% n AREGIVEN BY $(  p  ¥ $ §

PRQRPHU 

$( PRQRPHU    $:



$$  $$(

 E H[FLWRQ

´ ¶

 4HE EXCITON STATES ARE DESCRIBED BY NORMALIZED WAVEFUNCT F IONS sDIMER RC › WH W EREC SIN _ AND D C  COS _ IN%Q WITH

WDQ A 

$$( p







  E

 E H[FLWLRQ

 

!N INSPECTIONOFF %QS  AND  REVEALSTHAT SIGNIlCANTEXCITONICEFFECTSONTHESPECTRALPROPER TIES OF F THE DIMER CAN ONLY BE OBSERVED IF 66% IS

SMALL COMPAREDD WITH ¡EXCITON /N THE OTHER HAND IF 66%  ¡EXCITON ISSATISlED THETWOPIGMENTSRETAIN D L NONPERTURB R EDD OPTICALPROPERTIES)N THEIRINDIVIDUA ACTUALPIGMENTPROTEINCOMPLEXESCONTAININGMORE THANTWOSTRONGLYCOUPLEDPIGMENTS AMORECOMPLI CATEDD PATTERNOF EXCITONSTATESEMERGES0EARLSTEIN 4RETIAKETA K L  !DDITIONALLY THEABOVE EXCITONDESCRIPTIONASSUMESONLYATWO STATESYSTEM WHEREASINCLUSIONOF HIGHEREXCITEDD STATESAND CHARGE TRANSFER F #4 STATES WOULD FURT F HER COMPLICATE THE PICTURE4HISTREATMENTWOULDULTIMATELYBENECES SARYTOFU F LLY DESCRIBEEXCITONICEFFECTSONTRANS F IENT ABSORPTIONSPECTRA42ENGERANDD -AY   3OFARONLYTHEELECTRONICWAVEFUNCTIONSHAVEBEEN EXPLICITLYCONSIDERED4HEOVERALLPROPERTIESOFF APIG A A MENTMOLECULE HOWEVER AREDESCRIBED BY sI K R 2 A A W ERE R AND 2 ARE THE COORDINATES OF ELECTRONS AND WH Y HE "ORN /PPEN NUCLEI RESPECTIVELY)NMOSTCASES T HEIMERAPPROXIMATIONCANBEUSED A

A

A

A

s IKR 2 5 s IKR 1 IKV2



WHEREIANDKDESCRIBETHEPIGMENTNUMBERANDELEC TRONICEXCITATIONSTATE RESPECTIVELYANDD VSYMB M OLIZES THESTATEOFF NUCLEARVIBRATIONS!SACONSEQUENCEO Q F T THE"ORN / PPENHEIMERAPP A ROXIMATION THEELECTRONIC TRANSITIONDIPOLEMOMENTCAN BETREATEDD ASINDEPEN A DENTOFF COORDINATES 2 ANDD THEEFFECTO F F NUCLEARMO TIONSISGIVENBYTHE&RANCK #ONDON&# FACTOR A

A

 "1IEV E 2 \1IGV2 œ"



4HECOMPLEXITYOFT F HESYSTEM ISNICELY ILLUSTRATED FOR F #HL A INGLASSY DIETHERATLOWTEMPERATURES)NTHIS @SIMPLECASE VIBRATIONALMODESINTHERANGEOF n CMn WERESHOWNTOCONTRIBUTETOTHE&# F FACTOR !VARMAA ANDD 2EBANE   )N PIGMENT PROTEINCOMPLEXES THE PROTEINMATRIXMODULATESTHE SPECTRAL ANDD REACTIVEPROPERTIES!TTEMPTSHAVEBEEN REPORTEDD TODESCRIBETHE BANDSHAPEOFF #HL A ABSORP TION AT ROOM TEMPERATURE IN GLASSES AND A PROTEIN MATRIX :UCCHELLIETAL 3ECTION)))#  "ASEDD ONTHESEGENERALIZEDD CONSIDERATIONSWITHIN ASIMPLIlEDFRAMEWORK ITISOBVIOUSTHATTHEDETAILED DESCRIPTION ANDD UNDERSTANDING OFF THE OPTICAL AND %%4PROPERTIESOFPROTE F INSYSTEMSCONTAININGSEVERAL PIGMENTSISAHIGHLYCOMPLEXANDCHALLENGINGTASK !TTEMPTS AT THEORETICAL DESCRIPTIONS OFF A COUPLED MULTI PIGMENTPROTEINCOMPLEXWITH ITSENVIRONMENT HAVEBEENREPORTEDBY42ENGERAND-ARCUSA  !SUITABLETHEORY WOULD GREATLY BENElTTHE INTERPRE

 TATION OFF THE SPECTRAL PROPERTIES OF F PHOTOSYNTHETIC MULTI CHROMOPHORE PROTEINS 
&MFDUSP S O
5SB S OTGFS %LECTRONTRANSFER F %4 REACTIONS BETWEENMOLECULES TAKE PLACE VIA VIBRATIONALLY COUPLED D ELECTRON TUN NELING2EGARDLESSOFTHEELECTRONICSTATEGROUNDOR EXCITED D STATE OFT F HEREDOXPARTNERS THERATECONSTANT OFF THE REACTION CAN BE OBTAINED BY USING &ERMIS 'OLDEN 2ULE %Q  4HE INITIAL I AND lNAL F STATES INCLUDEAMULTIDIMENSIONALSYSTEMOFF VIBRA TIONS COMPRISING BOTH THE REDOX GROUPS AND D THEIR ENV N IRONMENT )THAS BEENTHE REMARKABLE ACHIEVEMENT OFF THE THEORY BY 2! -ARCUS -ARCUS AND D 3UTIN  TOPROJ O ECTAREACTIONPATHWAYONTOPARABOLIC F FREEENERGYCURVESOFSTATESIANDF4HISCONCEPTIS ILLUSTRATED IN&IG$EPENDINGONTHESTRENGTHOF ELECTRONICCOUPLINGBETWEEN$AND! TWO D LIMITING CASESCANBEDISTINGUISHEDI STRONGCOUPLINGAND II WEAK K COUPLING)NTHECASEOFF STRONG COUPLING A MARK R EDENERGYSP D LITTINGEMERGESATTHECROSSINGOF

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI THEPOTENTIAL CURVES FORSTATES F I AND F &IG RIGHT SIDE AND ASACONSEQUENCE THE%4OCCURSWITHINTHE SAMEPOTENTIALCURVEOFF THENUCLEI ˆ IE THEPROCESS ISADIAB A ATIC/NTH T EOTH T ERHAND D IFT F THECOUPLING ISRATH T ER WEAK THESPLITTINGISSOSMALLTHATTHE%4PROCESS INVOLVES A CROSSOVER BETWEEN THE POTENTIAL CURVES F FORSTATES I AND F ˆ IE THE PROCESSIS NON ADIABATIC &IG LEFTSIDE  &ORTHEVASTMAJ A ORITYOF%4REACT F IONS IN BIOLOGI CAL SYSTEMS WHI W CH ISACTUALLY @LONGRANGEELECTRON TRANSFER THENONADIABATICAPPROXIMATIONTURNSOUTTO BESUFlCIENTLY SATISlED -OSERAND$UTTON  D  (UPPMANNETAL  SHOWEDD THATEVENTHE FASTEST F %4REACTIONSOFTHELIGHTINDUCEDCHARGESEPARATION INTHEREACTIONCENTERSOFF ANOXYGENICPURPLEBACTERIA ARENONADIABATICATROOMTEMPERATUREANDD ATRANSI TIONTOTHEADIABATICREGIMEOCCURSONLYATRATHERLOW 7 THI T NTH T EFRAMEWOR F RK NONPHYSIOLOGICAL TEMPERATURES7I OF&ERM F IS'OLDEN2ULE%Q  THERATECONSTANT OF NONADIABATIC %4 CAN BE OBTAINED BY QUANTUM MECHANICALCALCULATIONSOFTHEMATRIXELEMENT\6 6F I\ 

OFE F LECTRONICCOUPLINGBETWEENSTATES I $!OR $ !

&IIG #OUPLING OFE & F LECTRONTUNNELING ANDD NUCLEARMOTION INREDOXREACTIONS4HEORDINATEREPRESENTSTHEENERGY OFF THENUCLEIOFF THE WHOLESYSTEMnIE DONORPLUSACCEPTORPLUSSURROUNDING4HE MULTIDIMENSIONALENERGYHYPERSURFACEISEXTREMELYSIMPLIlEDBY USING AONE DIMENSIONALHARMONICPOTENTIAL4HEABSCISSADESCRIBESTHENUCLEARGEOMETRYFORTHEINITIAL$! ANDTHElNAL$!n STATEOF THEWHOLESYSTEMDIFFERENTGEOMETRIESOFTHEINITIALANDlNAL STATEARESYMBOLIZEDBYASQUAREANDOVAL RESPECTIVELY ,EFTSIDENON ADIABATICELECTRONTRANSFERˆIE THEPROCESSOCCURSINTHEVICINITYOFTHECROSSOVEROFTHEPOTENTIALCURVESOFTHEINITIALI ANDlNALF STATESWITHACOMPARATIVELYLOWTRANSMISSIONCOEFlCIENT2IGHTSIDEADIABATICELECTRONTRANSFER F ˆ IE STRONGCOUPLINGLEADSTOSPLITTING OFF THEENERGY ITSMAGNITUDEISSYMBOLIZED BY 6 NEARTHECROSSOVERNUCLEARGEOMETRY SOTHATTHE INITIALSTATE $! PROCEEDSTOTHE lNAL STATE $!n WITHINTHESAMEPOTENTIAL SURFACE F

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



INTHECASEOFF PHOTOREACTIONS AND F $s!ns AND D US ING VARIOUS APPROACHES FOR DETERMINING THE FACTOR $OF%Q 4HEMAGNITUDEOF\6 6F I\ DEPENDS F IONS AND D THEREFORE F ON THE OVERLAP OFF THE WAVEFUNCT EXPONENTIALLY DECREASES WITH INCREASING DISTANCES BETWEENTHEINTERACTINGREDOX GROUPS \6 6F I 2$! \ \6 6F I  \ EXPn`2$! $



WHERE 2$! ISTHEEDGETOEDGEDISTANCEBETWEEN$AND ! AND ` ISACONSTANT&ORMANY N BIOLOGICALSYSTEMS INCLUDING "2#S THE LOGARITHMOFT F HERATECONSTANT LOGK%44 WASFOUNDTOBELINEARLYDEPENDENTON2 2$! WITHASLOPEOFABOUT ¯n THEACTUALVALUEDE PENDSONTHEPACKING DENSITYOFATOMS F INTHEPROTEIN D ! 0AGEETAL  ENVIRONMENTCONNECTING$AND +AWATSUETAL   D PROCEDURE D FFOR OBTAINING 4HE MOST WIDELY USED AN EXPRESSION OFF THE $ FACTOR F IS THE -ARCUS THEORY-ARCUSAND3UTIN  WHICHISBASEDON ACLASSICALTREATMENTOFNUC F LEARMOTION!SARESULT THEFO F LLOWINGEXPRESSIONEMERGES N (7 IL  P  9IL  

IL

N % 7

 

¨ s H[S © © ª



* IL 

I IL

 L IL N % 7





· ¸ ¸ ¹ 

WHEREK" ISTHE"OLTZMANNCONSTANT 6' F I AND hl ARE THESTANDARD'IBBSENERGYGAPANDTHEREORGANIZATION Y IVELY Y BETWEENSTATESFAN F D I ENERGY RESPECT T EORYWITH THA@CLASSICALREORGANIZATION 4HE-ARCUSTH ENERGYPARAMETERh FICL THATDESCRIBESTHECHANGESINTHE VIBRATIONAL MODESOFF THEREDOX GROUPSTH T EMSELVESAND THEIRMICROENVIRONMENTEG SOLVENTPOLARIZATION CANBEONLYSUCCESSFULLYAPPLIEDWHENTHEDOMINATING VIBRATIONSARETHERMALLY ACTIVATED)FF THISCONDITIONIS NOTSATISlED EG AT LOWTEMPERATURES THE$ F FACTORHASTOBECALCULATEDBYQUANTUMMECHANICAL TREATMENTOFT F HEVIBRATIONS4HIS ISESPECIALLY IMPOR TANT FFOR THE INTERPRETATION OF LOW TEMPERATURE DATA W WHERE%4CANBEASSOCIATEDTOASIGNIlCANTEXTENT WITH NUCLEAR TUNNELING FOR F ANALYSIS OF F "2#S SEE 7 HELETAL $8UAND3C 7ARS D HULTEN 6OS ETAL   )TSHOULD BEMENTIONED D THATTHE-ARCUSCONCEPT D STATEAT OF INTERSECTINGPARABOLASWITHTHEACTIVATED THECROSSINGPOINTHASTOBEMODIlEDWHENNONLINEAR SOLUTE SOLVENTCOUPLING ISPRESENT ˆ EG WH W ENTHE

ELECTRONICPOLARIZABILITYVARIESSIGNIlCANTLYBETWEEN STATES I AND F FOR FURTHER DISCUSSION SEE 3MALL ET AL   $ 1JH J NFOU1SPUFJO
*OUFSBDUJPOT )NTHEVISIBLESPECTRAL RANGE COUPLINGOFE F LECTRONIC STATESOFTHEPIGMENTSWITHTHOSEOFTHEPROTEINSCAN BENEGLECTED MATRIXISOLATEDD PIGMENTS ANDD ONLYTHE INTERACTIONWITH THENUCLEARMOTIONSOFT F HE PROTEIN PHONONS HAVE TO BE CONSIDERED )N THE CASE OF ELECTRONIC TRANSITIONS THE COUPLING WITH THE INTRA MOLECULARVIBRATIONSOFF THEPIGMENTMOLECULEHASTO BEEXTENDEDBYTHEINTERACTIONWITHTHEVIBRATIONAL MODESOFT F HEMATRIX,OW FREQUENC F Y i CM Mn INTERMOLECULARMODES PHONONS OFP F IGMENT PROTEIN COMPLEXESPLAYANIMPORTANTROLEINMEDIATING%%4 T ETICUNITS FORAREV F IEW ANDD %4PROCESSESINPHOTOSYNTH SEE/+àHNETAL   4HECOUPLINGSTRENGTHANDTHEAVERAGEFREQUENCY OFT F HEMODESCAN BEOBTAINED FROM F LOWTEMPERATURE LINENARROWINGSPECTROSCOPY mUORESCENCE LINENAR ROWING AND HOLE BURNING 0ETERMAN ET AL  0IEPER ET AL   #OMPLEMENTARY INFORMAT F ION ISGATHERED FROMMEASUREMENTSO F F INELASTICNEUTRON SCATTERING 0IEPER ET AL   )N ADDITION TO THE GENERAL PHENOMENONOFF ELECTRON PHONONCOUPLING THESPECIlC PROPERTIESOFF THE PROTEINMATRIXHAVETO BETAKENINTOACCOUNT%SSENTIALLYTWOPHENOMENA F YPEOF LIGATIONOF AREOFF RELEVANCE I THEDIFFERENTT #HLSAND II ENERGETIC DIFFERENCES F D DUETOVAR IATIONS OFCONFORMATIONALSTATESOFTHE PROTEININTHEMICRO ENVIRONMENTOFF THEPIGMENTS4HEFORMERE F FFECT F HAS BEEN ANALYZED BY QUANTUM MECHANICAL MOLECULAR ORBITAL -/ CALCULATIONS OF #HL MOLECULES USING VARIOUSAMINOACID D RESIDUESAS D LIGANDS6ARIATIONSOF THE1Y TRANSITIONENERGIESOFA F LMOSTCMn WERE FFOUND FOR #HL A 'UDOWSKA .OWAK ET AL  .ISHIGAKI ETAL  4HEREFORE T F HENATUREOFT F HE LIGANDD SIGNIlCANTLYMODU D LATESTHEOPTICALPROPERTIES 4HESECONDEFFECTGIVESRISETOINHOMOGENEOUSLINE BROADENING STATIC DISORDER IFF THE mUCTUATIONS OF F IONARECOMPARATIVELYSLOW4HIS ISTYPICAL CONFORMAT FORA PROTEINENVIRONMENT )T ISCLEARTHATTHEPROTEINMATRIXAND ITSDYNAMICS W CHCOVERWIDE DOMAINS INTIME n TO S WHI ANDSPACELOCAL LARGE SCALE ORCOLLECTIVE AREMOST D LATORSOFF THE KINETICSANDD ENERGETICSOF RELEVANTMODU %%4AND%4PROCESSES D FORAREV F IEW SEE+ARPLUSAND -C#AMMON  3INCEPROTEINSDONOTPOSSESSA UNIQUESTATEOFF MINIMUM'IBBSENERGY LIKEGLASSES

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI

 THEYATTAINALARGENUMB M EROFF CONFORMAT F IONAL SUB U STATES SEPARATEDBYENERGYBARRIERSOFDIFFERENTHEIGHTAND WIDTH 4HESE CHARACTERISTICS CAN LEAD D TO COMPLEX TEMPERATURE DEPENDENCIESOFT F HE KINETICSDUETOT D HE VARIETYOFACTIVATIONENERGIESFORTRANSITIONSBETWEEN CONFORMAT F IONAL SUB U STATES!SARESULT %4REACTIONS CANNOTSIMPLY BE DESCRIBED BYASINGLEDISCRETERATE CONSTANTK%4 BUTONLYBY ANAVERAGEPARAMETERK%4 AVE THAT ISGIVEN BY LOG K%4 AVE  EFK%4 LOG K%4D LOG K%4



WHEREFK K%%4 ISADISTRIBUTIONFUNCTIONOFRATECONSTANTS FOR INDIVIDUAL CONFO F RMATIONAL STATES %4 REACTIONS EXHIBITCOMPLEXNONEXPONENTIAL KINETICSWH W ENFK%4 SIGNIlCANTLY DEVIATES FROMA F b FUNCTIONANDTHERATE OF TRANSITION BETWEEN CONFO F RMATIONAL SUB STATES IS SLOWCOMPARED D WITH K%4 AV F IS ILLUSTRATED 4 E4HISEFFECT FFOR THE RECOMBINATION RATE AT LOW TEMPERATURE BE TWEEN 0s AND1!n s IN"2#S-C-AHONETAL   F HE PROTEIN DYNAMICSCAN BE !NANALOGOUSEFFECTONT D ATROOMTEMPERATUREWH W EN ISOLATED"2#S D ACHIEVED AREEMBEDDEDINTOSOLIDTREHALOSEWATERGLASSESOF HIGHVISCOSITY 0ALAZZOETAL   *7
/BUVSF
BOE
1SPQFSUJFT
PG
1
BOE
1IFP !DEEPERUNDERSTANDINGOFTHENATUREANDTHEPROPER TIESOFF THECOFACTORSREQU F IRESATHOROUGH KNOW K LEDGEOF THEIRARRANGEMENTAND D MODEOFCOOR F DINATIONWITHIN THE$$HETERODIMERICPROTEINMATRIX4HECURRENT K KNOW LEDGE ON THE STRUCTURAL ARRANGEMENT WILL BE BRIEmYSUMMARIZED BEFORE F DISCUSSINGIMPLICATIONSON THEPROPERTIESOF0AND0HEO&URTHERSTRUCTURAL DETAILSARE GIVENIN#HAPTERSn "
"SSBOHFNFOU
PG
UIF
$PGBDUPST
XJUIJO
UIF %%
)FUFSPEJNFS 4HE 8 RAYCRYSTALSTRUCTUREOFFUNCTIONALLYFULLYCOM PETENT03))CORECOMPLEXESFROMT F HE THERMOPHILIC 4 ERMOSYNECHOCOCCUS ELONGATUS CYANOBACTERIA 4H AND 4VULCANUS HASBEENREPORTEDATARESOLUTIONOF n ¯ :OUNIETAL 6ASILEVETAL  +AMIYA AND D 3HEN  AND D RECENTLY IMPROVED TO¯&ERREIRAETAL ANDFURTHERTO¯ "IESIADKA ET AL  FO F R4 4 ELONGATUS G  &IGURE  D 0HEO A MOL SHOWSTHEARRANGEMENTOFF THE#HL A AND ECULESANDTHECOFACTORS1! 9: AND D THETETRANUCLEAR MANGANESECLUSTEROFF THEWATEROXIDIZING COMPLEX 7/# TOGETHERWITH THENONHEME IRON.() CENTER

&IG IG  4O 4 P VIEW UPPERPART AND D SIDEVIEWBOTTOMPART OFF THE STRUCTURALARRAY A OFF THECOFACTORSO F F 03))WITHINTHEHETERODIMER OFF POLYPEPTIDES $ AND D $ #HL$ #HL$ 0$ AND 0$ ARE THE F FOUR# HL A AND D 0HEO$ AND D 0HEO$ THETWO0HEO A MOLECULESOF THEREACTIONCENTERPIGMENTCORE2# 0# #HL! AND#HL! THE DISTAL #HL A MOLECULES1! PLASTOQUINONEACCEPTOR-N  THE TETRANUCLEARMANGANESECLUSTEROFTHEWATEROXIDIZINGCOMPLEX AND&E THENONHEMEIRONCENTER&ORTHESAKEOFBETTERILLUSTRA TION THECOFACTORSO F F THEACTIVEBRANCHOF0 F #HL$ AND D 0$ AND D 0HEO$ AREMARK R ED BY BOLD BLACK K SYMBOLSWHIL W ETHEOTHER COMPONENTSAREMARK R ED IN GREY4 Y HE DIMENSIONSOFPO F LYPEPTIDES $AND D $ARESYMBOLIZED BY DOTTED D CONTOURLINES4HEDATAWAS KINDLY PROVIDED BY *AN+ERN3EETEXTFOR F DETAILS

NEAR1!4HEARRANGEMENTOFF THECHLORIN RINGSREADILY REVEALSTHATTHEREARETWOTYPESOF#HL A MOLECULES I A CORE OF FFOUR CLOSELY INTERACTING PIGMENTS RE F FERRE D TOAS#HL A AND II TWO#HL A MOLECULESAT THEPERIPHERYTHATARESPATIALLYWELLSEPARATEDFROM #HL A  WITH THESHORTESTCENTERTOCENTERDISTANCES OFABOUT¯:OUNIETAL  )NFORMATIONON

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS THEPUTATIVEORIENTATIONOFT F HETWO ` CAROTENESARE PRESENTEDIN+AMIYAAND3HEN AND&ERREIRA ETAL   "ASED D ON THE LARGE DISTANCE THE TWO WELL SEPA RATED #HL A MOLECULES DESIGNATED AS PERIPHERAL D HL: CANBE IGNORED D ASTHESE CHLOROPHYLLS#HL: AND# U STANTIALLYCONTRIBUTETOPIGMENT SPECIES DONOTSUB PIGMENTINTERACTIONTHEWIDELYUSEDSYMBOLS#HL$ AND D #HL: FORT F HEPERIPHERAL #HLSAREREPLACED HERE BECAUSETHEASSIGNMENTTO#HL: AND#HL: IN&IG ISSTILLNOTCLEAR 7ANGETAL  )NTERESTINGLY CORRESPONDING PERIPHERAL "#HL MOLECULES ARE NOT PRESENT IN THE "2#S WH W ERE ONLY FFOUR "#HLS AND TWO "0HEOS ARE BOUND D TO THE , AND D - HETERODIMER $EISENHOFERAN F D -ICHEL  4HEREFORE F ITSEEMS LIKELYTHAT#HL: AND#HL: AREOFSPECIALFUNCTIONAL RELEVANCEIN03))4HISIDEAWASCONlRMEDBYSTUD IESONMUTANTSOF #HL # AMYD M DOMONASREINHARD R TII WH W ERE THEAXIAL HISTIDINE LIGANDS(ISOFF $#HL: AND (ISOF$#HL: WEREREPLACEDWITHOTHERAMINO D 2UFmEETAL  !LTHOUGH H #HL: AND ACIDD RESIDUES #HL: WEREALSOSUGGESTED D TO BECONSTITUENTSOFF THE %%4PATHWAYFORM#0AND#0TOTHETETRAMERIC #HL A  *OHNSTONETAL  MODEL CALCULATIONS BASED D ONTHENEWSTRUCTURAL INFORMAT F IONREVEALED D THAT #HL: AND#HL: PLAYONLYAVERYMINORROLECOMPARED WITHTHEDIRECT%%4FROM# F HLSOFF #0AND #0TO THEREACTIONCENTERPIGMENTCORE 2# 0# TH T ATCONSISTS OF#HL A 0HEOX 3ECTION)6" 6ASILIEVETAL   )N$$#YTB PREPARATIONS #HL: AND# D HL: AFFECT F THE %%4 TO THE 2# 0# IN DIFFERENT F WAYS AS DEDUCEDFROMAKINETICANALYSISOF0HEOBLEACHINGIN THE 1Y BAND D ATDIFFERENTEXC F ITATIONWAVELENGTHS7ANG ETAL  4HEPERIPHERAL #HLS ISARE INFERRE F D TO PLAY A CRITICAL ROLE IN PROTECTION TO LIGHT STRESS 3CHWEITZERETAL ,INCEAND6ERMAAS  D  )TIS INTERESTINGTONOTETHATTHEEQUIVALENTAMINO ACID REPLACEMENTSAT$ (ISAND$ (ISDONOT RESULT INTHESAMEPHENOTYPE 2UFmEETAL   !NINDEPENDENT LINEOFEV F IDENCE FOR F HETEROGENEITY IN #HL: AND#HL: PROPERTIESISTHElNDINGTHATONLY D WHEN$$#YT B SAMPLES ONE#HL ISREMOVED H AREPREPARED BYUSINGMETALAFlNITYCHROMATOGRAPHY 6ACHAETAL  4HEMECHANISTICIMPLICATIONSOF THEFUNCT F IONALASYMMETRYOFF #HL: AND# D HL: MUST BE F DIES ANALYZED INFUTURESTU #
4USVDUVSBM
$PNQBSJTPOT
#FUXFFO
1 1
BOE
UIF
#BDUFSJBM
4QFDJBM
1BJS 4 )NTHElRSTPUBLISHED8 RAY A CRYSTALSTRUTUREAT ¯ RESOLUTION:OUNIETAL  THENEARESTNEIGHBOR

 F #HL A CENTER TO CENTER DISTANCES BETWEEN THE FOUR MOLECULESWITHINTHETETRAMER#HL A  COMPLEXWERE REPORTEDTOBEALMOSTIDENTICALAT›¯ WHILE THE CORRESPONDING DISTANCES BETWEEN THE NEAREST NEIGHBOR#HL A ANDD 0HEO A AREONLYSLIGHTLYLONGER ^ ¯  4HEREFORE EXCITONIC COUPLING BETWEEN THESIXPIGMENTSNEEDSTO BECONSIDERED"ASEDD ON ASIMPLIlEDMODELFORTHESITEENERGIESOFTHEPIG MENTSANDD THEIRVIBRATIONALCOUPLINGS ATHEORETICAL STUDYREPORTEDD VALUES INTHERANGEOFA F BOUTCMn n TOCM M FORTHEEXCITONICCOUPLING BETWEENEACH OFT F HE0$ 0$ ANDD THETWOOTHER#HL A MOLECULES IN THE #HL A  COMPLEXANDBETWEEN#HL$ n0HEO$ AND #HL$ n 0HEO$ 42ENGERAND-ARCUS B 4HE COUPLINGSOFF THESIXPIGMENTSGIVERISETOAREDISTRI BUTIONOFF THEOSCILLATORSTRENGTH TOWARDSTHE LOWER ENERGYEXCITONSTATES3IMILARCOUPLINGSTRENGTHSWERE REPORTED INANOTHERSTUDY "ARTERETAL  4HE NEW03))STRUCTUREAT¯RESOLUTION"IESIADKAET AL  HASBEENUSEDFORATHEORETICALANALYSISOF THESPECTRALPROPERTIES4HERESULTSOBTAINED BASICALLY CONlRMTHEEXCITONICCOUPLINGSOFT F HESIXPIGMENTS BUTTHEVALUESOFTHECOUPLINGSTRENGTHS COVERINGA RANGEOFABOUTnCM Mn DIFFER F FROMT F HEEARLIER REPORT 2ASZEWSKI ET AL   !NOTHER SPECTRO SCOPIC STUDY INCLUDING THEORETICAL CALCULATIONS LED TOTHECONCLUSIONTHAT0HEO$ ISPROBABLY DECOUPLED ANDD THAT A PENTAMER MODEL WITH lVE EXCITONICALLY COUPLED CHLORINS IE 0$ 0$ #HL$ #HL$ AND 0HEO$ MAY A BEAMOREREALISTIC DESCRIPTION*ANKO VIAKETA K L  4HESITUATIONBECOMESEVENMORE PUZZLINGASCIRCULARDICHROISM#$ MEASUREMENTS SEEMTOINDICATETHATNEITHEROFF THETWO0HEOS ISEX CITONICALLYCOUPLEDD WITH THE#HL A  UNIT6ACHAET AL  4HESECONCLUSIONSARENOTINAGREEMENT WITH THERECENT HIGH LEVELCALCULATIONS2ASZEWSKIET AL  4HE#$SPECTRAL DATAWEREOBTAINEDD WITH PREPARATIONSTHATMAYHAVEHADDIFFERENTDEGREESOF INTACTNESS ANDD AS A CONSEQUENCE MODIlEDD MODES F EXCITONIC COUPLING 3TUDIES USING INTACT 03)) OF PREPARATIONSWITHHIGH / EVOLUTIONCAPACITYWERE F F HECONCLUSIONTHATTHE FT ALSOINTERPRETED INFAVOURO 0HEOS ARE NOT EXCITONICALLY COUPLEDD WITH #HL A  0ETERSON !RSKÚLD  )NSUMMARY THECURRENTLY F HLS AVAILABLEDATASEEMSTO INDICATETHATTHEFOUR# 0$ 0$ #HL$ AND #HL$ AREEXCITONICALLYCOUPLED W WHILETHEEXTENTOFPHEOPHYTININTERACTIONREMAINSA CONTROVERSIAL ISSUE)NCONTRAST IT IS GENERALLYAGREED THATTH T EEXCITEDD STATESOFT F THEPERIPHERALPIGMENTS#HL: AND#HL: AREHIGHLYLOCALIZEDANDUNAFFECTEDINTHEIR 1Y TRANSITIONENERGIESBYEXCITONCOUPLING 42ENGER ANDD -ARCUS B2ASZEWSKIETAL  

 4HE ABOVE MENTIONED lNDINGS SUPPORT EARLIER PROPOSALSTHAT0ISAMULTIMERICPIGMENTCOM PLEX 4ETENKIN ET AL  $URRANT ET AL  0ROKHORENKOAND D (OLZWARTH  )NGENERAL THE PIGMENTARRANGEMENTOF#HL A  ANDTH D E TWO 0HEOS RESEMBLES THAT OF F THE FFOUR "#HLS AND D TWO "0HEOS WITHINTHE"2#S$EISENHOFERAN F D -ICHEL   )N THESE ORGANISMS THE PHOTOACTIVE PIGMENT 0 IS K KNOWNTO BEASTRONGLY EXCITONICALLYCOUPLEDSPEC D IAL PAIR+NAPPETAL  (OWEVER INSPITEOFT F HE BASICALLY SIMILAR PIGMENT ORGANIZATION SIGNIlCANT STRUCTURAL DIFFERENCESEX F IST BETWEEN #HL A  IN03)) D THEPHOTOACTIVESPECIAL PAIR0OF BOTH ANOXYGENIC AND BACTERIAAND0OF03)4HEMOSTSTRIKINGDIFFER ENCE ISTHECENTER TO CENTERDISTANCEBETWEEN0$ AND 0$ IN03)):OUNIETAL +AMIYAAND3HEN "IESIADKAETAL &EIRREIRAETAL WHICH IS LONGER THAN THE CORRESPONDING VALUES FO F R THETWO"#HLSINTHESPECIALPAIR0¯ -ICHEL AND $EISENHOFER  AND FOR THE HETERODIMERIC 0¯ &ROMMEETAL  4HE0CON SISTSOF #HL A AND ITS EPIMER#HL A| IN#HL A|THE POSITIONS OF n( AND n#//#( AT THE ASYMMETRIC # ATOM  OFF RING 6AREEXCHANGED SEE& D IG   EPIMERSAREAGENERAL FEATUREO F F 03) TYPE2#SIN ANOXYGENICANDOXYGENICPHOTOSYNTHESICORGANISMS FORAREV F IEW SEE!KIYAMAETAL  !LTHOUGH THEPHYS H IOLOGICAL SIGNIlCANCEOF F  EPIMERS INTHE PHOTOACTIVEPIGMENTISNOTYETCLEAR THEDESCRIPTION F R ASASPECIAL PAIRISAREASONABLEAPPROXIMATIONFO 0 BUTAPPEARSTO BE LESSAPPROPRIATE FOR04O F SUPP U ORTTHISIDEA 0CANBECOMPAREDWITH0OF THE"2#S4HESHORTERDISTANCEAND HIGHEROSCILLATOR F HLS INTHE"2#S LEADSTO LARGEEXCITONIC STRENGTH OF"# SPLITTINGWITHINTHESPECIALPAIR0nCM  +NAPPETAL  MARK R EDLYEXCEEDINGTHEVALUES D #HL A DIMER IN 0 CALCULATED FFOR THE PRESUMED 7HENTAKINGINTOACCOUNTTHEVALUEOFTHESHORTER DISTANCE BETWEEN0$ AND 0$ REPORTED BY "IESIADKA D &ERREIRAETAL THECALCULATED ETAL AND EXCITONIC COUPLING OF ABOUT  CM Mn 2ASZEWSKI ET AL  STILL REMAINS SMALLER THAN THAT OFF THE SPECIALPAIR0IN"2#S!SACONSEQUENCEOFTHISDIF F F FERENCE THEREISAMARKEDREDSHIFTOFTHETRANSITION INTOTHELOWERENERGY EXCITONSTATE 6%  3ECTION )))"B OFF 0 IN ANOXYGENIC BACTERIA COMPARED WITH THE ABSORPTION BANDS OF THE TWO MONOMERIC "#HLMOLECULES INTHE"2#AND INTHEANTENNA THAT ISABSENTIN03))!CCORDINGLY THEABSORPTION BAND OF0ISHIGHLYCONGESTEDANDSTRONGLYOVERLAPS WITHTHE 1Y TRANSITIONSOFF ANTENNA#HL A MOLECULES

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI #ONSEQUENTLY 0ACTSASAVERYSHALLOWTRAPFOR EXCITATION ENERGY (OLZWARTH  ' 2ENGER  &URTHERMORE THE LOWESTEXCITEDD SINGLETSTATE ISNOT LOCALIZEDD ONTHEELUSIVEDIMER0$ 0$ 4HIS IDEAISSUPPORTEDBYTHEORETICALCALCULATIONSBASED ONTHE03))STRUCTUREAT ¯RESOLUTION 2ASZEWSKI F BLETO ETAL  )TTHEREFOREAPPEARSMOREREASONA ASSIGN0TOA#HL A 0HEO A X UNITWITHX  ORVIDESUPRA ANDD TOCONSIDER 0 0AND 0s ASSTATESWITHSPECIlCELECTRONICCONlGURATIONS OFTHISENTITY&ORTHESAKEOFSIMPLICITY THEPIGMENT UNIT#HL A 0HEO A X WILL BE DESIGNATEDD ASREACTION CENTER PIGMENT COMPEX 2# 0#  4HE MOLECULAR ORBITAL-/ SCHEMESOF 2# 0# 2# 0# AND 2# 0# % DEPENDD NOT ONLY ON PIGMENT COUPLINGS BUTALSOONTHEMODEOFP F IGMENT PROTEIN INTERACTION WITHINTHE2# 0# /URCURRENTSTATEOFKNOWLEDGE IS LIMITED BY BOTHTHERESTRICTEDD STRUCTURAL RESOLUTION :OUNI ETAL +AMIYAAND3 D HEN "IESI ADKAETAL &ERREIRAETAL ANDBYTHELACK OFSU F FlCIENTLY DETAILEDSPECTRA D L INFORMAT F ION)TISSTILL AMATTEROF DEBATEASTOWH W ATEXTENTTHEEXPERIMENTAL DATAFROM$$#YT B PREPARATIONSREmECTTHETRUE PROPERTIES OFF 0 #OMPARATIVE MAGNETIC CICULAR DICHROISM-#$ MEASUREMENTSOF INTACT03))CORE COMPLEXESAND$$#YTB PREPARATIONSREVEALED F BETWEENTHETWOSAMPLETYPES SIGNIlCANTDIFFERENCES 3MITHETAL  CONlRMINGEARLIERSTUDIES BASED ON MEASUREMENTS OF DIFFERENCE ABSORPTION SPECTRA (ILLMANN ET AL   )N THIS RESPECT THE OFTEN F USED DESIGNATIONOFF $$#YT B PREPARATIONSAS 03)) 2#SISHIGHLYMISLEADINGWHENCOMPAREDWITH THEWELL DElNED ISOLATEDD "2#S4HE$$#YT B PREPARATIONSNOTONLYLACKCOMPONENTSO K F HE03)) FT ACCEPTORSIDE INCLUDING.()+URRECKETAL B BUTAREALSOUNABLETOOXIDIZETHETYROSINERESIDUES D 9: AND 9$ 4ELFERETA F L #HAPTER &URTHERMORE INMARKEDCONTRASTTO"2#S 03))ISCHARACTERIZED BY AN EXTRAORDINARY SUB U UNIT COMPOSITION 3HI AND 3CHRÚDER  THAT HASREMAINEDD VIRTUALLYINVARIANT DURINGEVOLUTIONFROMCYANOBACTERIATOHIGHERPLANTS 2AY A MONDD AND "LANKENSHIP   .EVERTHELESS RECENT DATASEEMTO INDICATETHATTHEINTRINSICPRIMARY ELECTRONTRANSFERRATESLEADINGTO0s0HEOns FO F RMA TION IN ISOLATEDD $$#YT B COMPLEXES ARE NOT SERIOUSLYAFFECTE F D COMPARED TOTHEIRVALUESIN INTACT 03))!2(OLZWARTH UNPUBLISHEDRESULTS  )NSUMMARY AN Y NYASSIGNMENTOFF 0TOASINGLE #HL AMOLECULEORA@SPECIALPAIRANALOGOUSTO0 IN "2#OR0IN03)ISINADEQUATEANDMISLEADING # 8IONG ET AL  PRESENTED IN A STUDY ON A #

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS RREINHARD R TII MUTANT WH W ERE 0HEO$ OFF THE @INACTIVE BRANCHISREPLACEDBY#HL A CLEAREVIDENCEFORTHE INTERDEPENDENCE OF F THE SIX PIGMENTS IN 2# 0#  4HEY FOUN F D THAT 03)) COMPLEXES ISOLATED FROM F THIS $ ,EU(IS MUTANT WERE VIRTUALLY UNABLE TOEVOLVE / OFT F HEWILDD TYPE74 CONTROL AND D TO PHOTOACCUMULATE 1!n s /N THE OTHER HAND THE KINETICS OF PRIMARY CHARGE SEPARATION ARE NOT SUB U STANTIALLY ALTERED IN$$#YT B PREPARATIONS F FROMT HISMUTANT4HESERESULTSWEREINTERPRETED BY THE ASSUMPTION THAT THE DISTRIBUTION OF THE EXCITED STATE ENERGY IN 2# 0# OF F THE $ ,EU(IS F FROMT F HATOFT F HE74 THUS LEADINGTO MUTANT DIFFERS DISSIPATIVEDECAYVIAmUORESCENCEATTHEEXPENSEOF CHARGESEPARATION8IONG ETAL   $
5IF
&MFDUSPOJD
4UBUF 1 BOE
UIF
-PXFTU
5 5SJQMFU
4UBUF 1 3EVERALATTEMP M TSHAVEBEENMADETOUNRAVELTHENATURE OFF THELOWESTEXCITED D SINGLETSTATE0 AND D THE LOWEST TRIPLET STATE 0 OF F 0 -ODIlCATION OFTHECOORDINATIONOFTHECENTRAL-G OFTHE#HL A MOLECULES 0$ AND 0$ IS ONE PROMISING APPROACH TOAVOID D PROBLEMSFROMT F HEUSEOFF $$#YT B PREPARATIONSANDTHEOVERLAPPINGSPECTRALPROPERTIES D DUETOEXC ITONICCOUPLING 3ECTION)6" "ASED D ON &4)2MEASUREMENTS.OGUCHI ETAL  INBOTH 0$ AND D 0$ THE-G CENTRALATOMSAREMOSTLIKELY PENTACOORDINATED D WITH (IS RESIDUES D $ (ISAND $ (IS RESPECTIVELY IN 3YNEC 3 HOCYSTIS SP0##  ASAXIALLIGANDS-ERRYETAL  2EPLACE MENTOFT F HE$ (ISAND D $ (ISBY'LNAND F IONALLYCOMPETENT !LALEADSTOTHEASSEMBLYOF FUNCT 03))COMPLEXESWHEREASREPLACEMENTWITHTHELARGER ,EU ISDISASTROUSANDPREVENTSAN D NY DETECTABLEASSEM BLYOFF STABLE03))COMPLEXES$INERETAL   3INCEBOTH!LAAND,EURESIDUESDONOTCOORDINATE TO-G OFF #HL A THEDRASTICALLY DIFFERENTPROPERT F IES F HE(IS LIGAND BYA AREEXPLAINED BYREPLACEMENTOFT WATER MOLECULEINTHE CASE OFTHE !LA MUTANT BUTNOT INTHE,EUMUTANTDUETOS D IZEEXCLUSION $INERAND 2APPAPORT  !THOROUGH SPECTRALANALYSISOF W WHOLECELLSANDOFMANGENESEDEPLETED03))CORE COMPLEXES ISOLATED FROMT F HESEMUTANTSINCOMPARI SONWITHTHECORRESPONDING74SAMPLESLED D TOTHE CONCLUSIONTHATWITHINTHE2# 0# THE1Y TRANSITION WITHTHE LOWESTENERGY IS LOCALIZED D PREFERENT F IALLY ON #HL$ AND ISCHARACTERIZED BYAPEAKPOS K ITIONOFF  NM AT  + AND n NM AT + (ILLMANN ET F HENATIVE AL  )NCONTRAST THE1Y TRANSITIONOFT

 0$ HAS BEENDETERMINEDD TOBEATNM$INERET AL  !LTERNATIVELYTHESESPECTRALFEATURESHAVE BEEN INTERPRETEDD AS EXCITON BANDS OF F THE 0$ 0$ DIMER3MITH ETAL  4AKINGINTOACCOUNTTHE VALUESCALCULATEDFORDIPOLE DIPOLECOUPLINGWITHIN THE 2# 0# THEABOVEMENTIONEDD ASSIGNMENTOF THE1Y TRANSITIONSATANDD nNMOF 0$ AND#HL$ RESPECTIVELY WOULDIMPL Y YTHATSIGNIlCANT HETEROGENEITYAMONGTHE FOUR# F HL A MOLECULESMAY A EXISTDUETO D DIFFERENCES F INTHEIRMICROENVIRONMENT 7 7ITHRESPECTTOTHEASSIGNMENTS ITISIMPORTANTTONOTE THAT BOTH SITE HETEROGENEITY ANDD EXCITONICCOUPLING CONTRIBUTETOTHEEXPERIMENTAL SPECTRA 42ENGERAND -ARCUS B2ASZEWSKIETAL   )FT F HEENERGY DIFFERENCECAUSE F D BY DISORDERISLARGE COMPAREDD WITH EXCITONICCOUPLING THEEXCITATIONBE COMESLOCALIZEDONINDIVIDUALPIGMENTS ASILLUSTRATED FFOR THE SIMPLEST CASE OF F A DIMERIC UNIT %Q   !TPHYS H IOLOGICAL TEMPERATURES THERMAL ENERGY K4 IS COMPARABLE TO THE SPECTRAL DIFFERENCES BETWEEN #HL AMOLECULESCAUSED BYTHE HETEROGENEITY INTHE  2# 0# 4HUS THEEXCITEDD STATEISEXPECTEDD TO BE DELOCALIZEDVIARAP A IDEQUILIBRATIONINTHESUB Q PICOSEC ONDD TIME DOMAIN -àLLERETAL  !TVERY LOW TEMPERATURES HOWEVER THEEXCITATION IS LIKELYTO BE TRAPPEDINANEXCITONSTATEWITHALARGECONTRIBUTION OF# F HLA$ 0ROKHORENKOANDD (OLZWARTH  AND F HARGESEPARATIONAPPEARSTOTAKE THEPRIMARYSTEPOFC PLACEFROM #HL $ TO0HEO3ECTION6 !NALYSISBY *ANKOWIAKK ETAL  LEDD TOTHECONCLUSIONTHATA DISTRIBUTIONOF DIFFERENTSTATESW F ITH LOWESTEXCITATION ENERGYEXISTSWITHINTHEMACROSCOPICENSEMBLEDE PENDINGONTHENATUREOFT F HE03))SAMPLE!NOTHER INTERESTINGIMPLICATIONOFF THEMULTIMERMODEL ISTHE POSSIBLE MIXING OF SINGLET AND RADICAL PAIR STATES !S A CONSEQUENCE THE 2# 0# SINGLET STATES COMPRISESOME DEGREEOFC F HARGETRANSFERC F HARACTER "ARTERETAL  #ALCULATIONSBASEDONTHENEW 03))STRUCTURE"IESIADKAETAL  SUGGESTTHAT THELOWESTEXCITONSTATEOF 2# 0# IS DOMINATED BY#HL$ 2ASZEWSKI  !DIFFERENTS F ITUATIONARISESFORT F HETRIPLETSTATE)N THISCASE THEEXCITONICSPLITTINGISDRASTICALLYSMALLER BECAUSE THE TRANSITIONS ARE SPIN FORBIDDEN ACCORD INGTO lRSTORDERPERTURB R ATIONTHEORY 3CHMIDTAND 2EINECKER  4HEREFORE T F HISEFFECT F ISNEGLIGIBLY SMALLANDCANBEIGNORED4HELOCALIZATIONOFTHETRIP LETSTATEWASlRSTOBTAINED FROMOR F IENTATIONSTUDIES F D  SHOWEDD THAT 6AN -IEGHEM ANDD 2UTHERFOR THEPLANEOFTHECHLORINRINGWHERETHETRIPLETRESIDES MARK R EDLY DIFFERS F F FROMT HENORMALOFF THETHYLAKOID

 MEMB M RANE4HISGEOMETRYISDRAST D ICALLY L DIFFERENT F F FROM THEPARALLELORIENTATIONWITHTHEMEMBRANENORMAL D AND D RAISED OF 0 IN"2#S 4IEDEAND$UTTON  F IONIN03)) QUESTIONSONTHEORIGINOFF TRIPLETFORMAT AND THE POSSIBILITY OF MIGRATION WITHIN THE #HL A ENSEMBLE4HE LATTERPROCESSWASSHOWNTO INDEED TAKEPLACEAND D THEENERGYGAP FORTR F IPLETDELOCALIZA TION WAS ESTIMATED TO BE OF THE ORDER OF  ME6 .OGUCHIETAL  3TUDIESUSING 3YNECHOCY CYSTIS D $$#YT B COMPLEXESLED MUTANTSAND ISOLATED TOTHECONCLUSIONTHATATTEMPERATURESBELOWLIQUID Q NITROGEN TEMPERATURE THE TRIPLET STATE IS LOCALIZED AT #HL$ AND THAT IT BECOMES DELOCALIZED AT HIGHER TEMPERATURES+AMLOWSKIETAL $INERETAL  4HISCONCLUSION ISSUPORTED BY ATHEORETICAL ANALYSIS2ASZEWSKI ETAL   %
5IF $BUJPO
3BEJDBM 1 r 4HEUNUSUALLYHIGHOXIDIZINGPOWEROF0s ISTHE F IDATIVEWATERCLEAVAGE ENERGETICPREREQUISITEFOROX &ORANUNDERSTANDINGOFF THISUNIQUEPROPERTYSEVERAL QUESTIONSNEEDTOBEADDRESSEDI HOWISTHECATION F FORME D AND WHEREISTHEHOLEPREDOMINANTLY LOCATED II HOW ISELECTRONTRANSFER F FROM F 9:TO0s EFl CIENTLYACHIEVEDANDHOWAREDISSIPATIVESIDEREAC TIONS MINIMIZED III WH W AT ARE THE MECHANISMS TO DESTABILIZE0s ASCOMPAREDTO# D HL As INSOLUTION ANDIV WHATISTHEROLEOFPIGMENT PROTEININTERAC TIONS INTUNINGTHEENERGETICAND KINETICPROPERTIES 7ITH OURCURRENTSTATEOF KNOW K LEDGE ASTRAIGHT F FORWARDANSWERTOTHESEQUESTIONSCANNOTBEGIVEN BECAUSE0CANATTAINDIFFERENTE F LECTRONICCONlGU RATIONS DEPENDINGONTEMPERATURE DETERGENTCONTENT ALLOSTERICEFFECTSWITHOTHERPROTEINSUBUNITSWITHIN THE 03)) CORE COMPLEX AMONG OTHER FFACTORS !T PRESENTTHESYSTEM ISFARTOOCOMP F LEX FORACOMP F LETE QUANTUMMECHANICALANDMOLECULARDYNAMICSTREAT MENT4HUS THE FO F LLOWING SECTIONS SUMMARIZE OUR F FRAGMENTARY K KNOW LEDGE INANATTEMPTTOADDRESSTHE Q ABOVE QUESTIONS  
&MFDUSPO
&KFDUJPO
GSPN G 1 BOE
%JTUSJCV UJPO
PG
UIF
)PMF
XJUIJO G 1 r

#OHERENT PHOTON ECHO SPECTROSCOPY IS A SUITABLE METHODTHATCANDETECTTHElRSTSTEPOFEXCITATIONOR F FROMT F HE INITIALLY EXCITED D PIGMENT ELECTRONTRANSFER MOLECULE!PPLICATIONOFT F HISTECHNIQUETO$ Q $ #YT B PREPARATIONSAT+REVEALEDTHATANELEC  TRONFROM F #HL $ ISTRANSFERRE F D TO0HEO$ FORM F INGTHE

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI s ns PRIMARYRADICALPAIR#HL$ 0HEO$  0ROKHORENKOAND (OLZWARTH  !TPRESENT THISDATAISTHEONLY DIRECTEXPERIMENTALEVIDENCE FO F R #HL $ ACTINGASTHE PRIMARY ELECTRON DONOR .O CORRESPONDING EXPERI MENTALRESULTSAREAVAILABLEFORHIGHERTEMPERATURES ORINTACT03))COMPLEXES4HE INITIAL LOCATIONOFF THE HOLE ON #HLs$ IS SUPPORTED BY THEORETICAL ANALYSIS 2ASZEWSKIETAL  ANDBYRESULTSOBTAINEDUS ING INCOHERENTSPECTROSCOPICTECHNIQUESON74AND F 3 3YNEC HOCYSTIS $INERET MUTANT03)) PARTICLESFROM AL  &ORSTRUCTURALREASONS HOWEVER THEPOSI TIONOF #HL $ ISNOT FAVORA F BLEFORANE F FlCIENTTRANSFER F F HEOXIDIZINGREDOXEQUIVALENTTO9: ANDTHE 7/# OFT SEE&IG !MORESUITABLELOCATIONWOULDBE0$ )T ISTHEREFOREASSUME F D THAT#HLs$ RAPIDLYOXIDIZES s ns 0$ LEADING TO THE RADICAL PAIR 0$ 0HEO$  AND THE CHARGESEPARATIONISINFERREDTOBEATWOSTEPPROCESS 0ROKHORENK N OANDD (OLZWARTH T 3ECTION6! 4HE s LIFET F IMEOF0 F CAN BEAS LONGASSEVERAL HUNDREDS OF MICROSECONDS (AVEMAN AND -ATHIS  ' 2ENGER AND D 7OLF F  4HE LONG LIFET F IME RAISES THEQUEST Q IONASTOWH W ERETHE HOLEIS LOCATEDD WITHIN 0s4HEPARTICIPATIONOF0HEOSCANBEEXCLUDED F FORENER GETICREASONSITSREDUCT D IONPOTENTIAL IS 6INSOLUTION 3ECTION)))! ANDD OF #HL: AND #HL: FORKINETICREASONSTHELARGEDISTANCESBETWEENTHESE SPECIES ANDD THE 2# 0# PIGMENTS RESULT IN RATHER F REACTIONS SEE 3ECTIONS ))) SLOW ELECTRON TRANSFER "AND)6!  %LECTRON NUCLEAR DOUBLE RESONANCE %.$/2 SPECTROSCOPYCANBEUSEDD TODETERMINESPINDENSITY DISTRIBUTIONS,UBITZAND,ENDZIAN  /NEOF THEMOST IMPORTANTRESULTSUSING THISTECHNIQUEISTHE lNDINGTHAT IN"2#S THESHARINGOFT F HEELECTRONHOLE BETWEENTHETWO"#HLSOFTHESPECIALPAIRDEPENDS NOTONLY ONTHESPECIES BUTALSOONTHETYPEOF DETER GENTUSED FORSAMP F LEPREPARATION-àH ETAL   !NALOGOUS%.$/2MEASUREMENTSOF$$#YT B PREPARATIONSREVEAL ANPROBABILITYTHATTHEHOLE IS LOCALIZEDD TO ONE SPECIlC #HL A MOST LIKELY 0$ 2IGBY ET AL   4HIS RESULT HOWEVER SHOULD BECONSIDEREDD WITH CAUTIONINTHELIGHTOFT F HEPOS SIBLEPROBLEMS INUSING$$#YT B PREPARATIONS VIDESUPRA 3TRICTLYSPEAKING SOUNDEXPERIMENTAL F HEDISTRIBUTIONPROBABILITY EVIDENCE ISLACKING FORT OFT F HEHOLEWITHIN0s(OWEVER FORP F H IOLOGI HYS CALREASONSIE THEHIGHLYEFlCIENTOXIDATIONOF9: ITSEEMS LIKELY THATIN03))SAMPLES WITH ANINTACT 7 7/# T HE HOLE IS LOCATEDD WITH AHIGHPROBABILITYON 0$ 4HIS CONCLUSION IS SUPPORTED BY COMPARATIVE 3 NECHOCY CYSTIS MEASUREMENTSIN74ANDD MUTANTSOF 3Y

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS WHERE THE AXIAL 0$ LIGAND D $ (IS IS REPLACED BY 'LU $INER ET AL  $INER AND 2APPAPORT F IAL PROBABILITY OF LOCALIZATION  4HE PREFERENT D TOORIGINATE FROMANASYMMETR F IC ON0$ ISEXPECTED PROTEINENVIRONMENTASWASPREVIOUSLYDISCUSSEDFOR THE ASYMMETRY OF F THE REDOX PROPERTIES OF F MANGA NESEWITHINTHETETRANULEARCLUSTEROFT F HE7/# ' 2ENGER  !NELECTRICALPOTENTIALGRADIENTOFTHE F NMAROUND D THE2# 0#UNITWOULD ORDEROFM6 BESUFlCIENT FORARAT F HERPRONOUNCED LOCALIZATIONOF THE HOLE ON 0$ ATROOMTEMPERATURE ATLOWERTEM PERATURESTHEEFFECT F ISEVENSTRONGER 4HEEXISTENCE OFF STRONG LOCAL ELECTROSTATIClELDSNEAR0sHAVE BEENINFERREDFROMMEASUREMENTSOFLIGHTINDUCED  #.-2SIGNALSIN$$#YTB PREPARATIONS4HE DATAOBTAINED D WEREINTERPRETED BYANASYMMETRICSPIN DENSITYDISTRIBUTIONWITHINTHECHLORINRINGSYSTEMOF 0s THAT ISMARK R EDLY DIFFERENT F F FROMT HESYMMETRIC DISTRIBUTIONIN0s OF PURPLEBACTERIA! PROTONATIONOF THEKETOGROUPATRING6ISDISCUSSEDASONEPOSSIBIL ITYOF GENERATING THESTRONG LOCALELECTRIC lELD D THAT ISRESPONSIBLE FORT F HESHIFTO F F THESPIN DENSITYTORING )))OFTHE#HL A MOIETYWITHIN0s -ATYSIKETAL  )TWASSUGGESTED D THATTHEASYMMETRYWITHIN THE0$ n 0$ PAIRPROBABLYARISES FROMT F HEPOSITIVE CHARGEOFTHEPROTONTRAPPEDINTHEENVIRONMENTOF 9$OX4HEROLEOFF 9$ ASAPOSITIVECHARGE GENERATORIN H DROPHOBICENVIRONMENTAND ITS KEYFUNCT F IONFOR F A HY  D IN SPINDISTRIBUTIONIN0 HAS BEEN SUMMARIZED AREVIEWBY2UTHERFOR F D AND &ALLER  
1PTTJCMF
'BDUPST
GPS
UIF
)JH J I
0YJEJ[JOH 1 1PXFS
PG
1 r "ASED ONTHE EARLIER STRUCTURE MODEL OF :OUNI ETAL  THE@LARGEDISTANCEOFABOUT¯ BETWEEN THE CHLOROPHY H LLS 0$ AND 0$ WAS CONSIDERED D AS A PREREQUISITE IN ESTABLISHING A STRONGLY OXIDIZING #HL As SPECIES#ONSEQUENTLY T Y HEKEY EVENTINTHE D TO BEA EVOLUTIONOFF THE / EVOLVING03)) ISPROPOSED MUTATIONOFTHEPROTEINTHATRESULTEDINTHESEPARATION OFF THETWOPIGMENTSINTHE@SPECIALCHLOROPHYLLPAIR 2UTHERFOR F D AND &ALLER  .EWINFORMAT F IONON THE03))STRUCTURE HOWEVER REVEALSTHATTHEDISTANCE BETWEEN 0$ AND 0$ ISSHORTER"IESIADKAETAL  &ERREIRAETAL  THANORIGINALLYREPORTED :OUNI ETAL  4HEREFORE THEHYPOTHESISOF2UTHERFORD D &ALLER APPEARSTO BELESSATTRACTIVE!PART AND F FROMT HIS PARTICULARPOINTTHEREEXISTSLITTLEDOUB U TTHAT THEREDOX PROPERTIESOFTHE#HL A MOLECULES WITHINTHE 2# 0#UNITAREESSENTIALLY DETERMINED BY THE BINDING

 ENVIRONMENT OFF THE $$ HETERODIMER INCLUDING THEBINDINGDOMAINSFORCATIONICCOFACTORSANDTHE U UNITS INTHE PROTEIN PROTEIN INTERACTIONSWITHOTHERSUB 03))COMPLEX &OREXAMPLE SYSTEMATICSTUDIESHAVE SHOWNTHATTHEPIGMENT PROTEININTERACTIONSMODULATE THEREDUCT D IONPOTENTIAL OFT F HESPECIAL PAIR0 IN"2#S #HAPTER +ÈLMÈNETAL 4HEREDUCT D IONPOTENTIAL WASFOUNDTODEPENDSTRONGLYONTHEHYDROGENBOND INGTOTH T E"#HLCARB R ONYL N GROUPS!DDITIONAL HYDRO D GEN BONDSTOTH T ESECARB R ONYL GROUPS INTRODUCE D D BYGENETIC ENGINEERING CAUSESANINCREMENTALINCREASEOFABOUT M6PERBONDD WHILEADECREASEISOBTAINEDD AFTER F REMOVAL OFF THE SINGLE HY H DROGEN BONDD EXISTING IN THE74,IKEWISE ACORRELATIONWASFOUNDBETWEEN THEINCREASEOFT F HEREDOXPOTENTIALOFF 00s ANDD THE STRENGTHOFF THE HY H DROGEN BONDD TOTHE# KETOCAR BONYLGROUPOFRING6OFTHE"#HLMOLECULESATTHE 0- POSITIONOFT F HESPECIALPAIR)VANCICHETAL   &URTHERMORE THEINmUENCEOFF ANADDITIONAL NEGATIVE CHARGEINTHEPROTEINMATRIXONTHEDECREASEOFTHE REDUCT D IONPOTENTIAL DUETOSTA D ABILIZATIONOFF 0s HAS BEEN ILLUSTRATED BYREPLACINGPARTICULARRESIDUES D !SN A !SP ATANAVERAGEDISTANCE OF¯TOTHEATOMSOF N UGATED / ELECTRONSYSTEMOFF 0)TWASSHOWN THECONJ THAT THE INSERTION OFF AN ADDITIONAL NEGATIVE CHARGE GIVESRISETOASHIFTOFnUPTOnM6DEPENDING ONTHEPOSITION ANDD THATTHEEFFECTO F F TWONEGATIVE CHARGES ISADDITIVE 7ILLI 7 AMSETAL   3PIEDELETAL ANALYZEDTHEROLEOFCHARGE DIPOLE INTERACTIONS IN MUTANTS OFF (IS IN THE , ANDD 0HE IN THE - SUB U UNIT THESE RESIDUES D AREINCONTACTWITHRING)OFEACHOFTHETWO"#HLS IN 0 4HE %M VALUE COULD BE MODU D LATEDD WITHIN A RANGEOFF M6WITH ASTRONGCORRELATIONBETWEEN 6%M ANDTHEDIPOLESTRENGTH!NIMPORTANTFEATURE EMERGING FFROM A DETAILEDD ANALYSIS OFF ELECTROSTATIC INTERACTIONSIN2#S ISOLATED FROM F DIFFERENTMUTANTS F OF 2BSP S HAEROIDES ISTHECONCLUSIONTHATTHEEFFECTS D RESIDUESONT D HEPROPERTIES OF IONIZABLEAMINOACID F LYSCREENED BYCOUNTERIONS *OHNSON OF0ARESTRONG ETAL  3LIGHTUPSHIFTSOF%MBYnM6ARE D D BY ADDITIONOFSO F LVENTSLIKE GLYCEROLOR ALSOINDUCE DIMETHY H LSULFOX F IDE$-3/ TOSUSPENSIONSOF"2#S F 0ASCHENKOETAL   7HENTAKING INTOACCOUNTTHESElNDINGS HYDRO D GEN BONDINGAPPEARSTO BEANATTRACTIVEMECHANISM FOR F MODULATINGTHEREDUCTIONPOTENTIALOF0(OW EVER THISPOSSIBILITY ISOF LIMITEDPOTENT D IAL FOR# F HL A BECAUSETHISMOLECULE LACKSTHEACETYLGROUPATRING) ITISREPLACEDBYAVINYLGROUP SEE&IG THATWAS F FFECT F ING TH T EREDOXPOTENTIAL SHOWNTO BE IMPORTANTFORA

 OFF 00s IN"2#S4HEREFORE T F HEINSERTIONOF POSITIVE CHARGESAMINOACIDRESIDUESORINERTCATIONS ˆ EG #A INTOTHE HETERODIMERIC$$PROTEINMATRIX OFF 03))SEEMSTOBEAMORERELEVANT@TOOLTO DESTA BILIZETHE RADICAL STATE 0s)NLINEWITHTHISIDEA THEPOSITIVECHARGEOF 9$OX ISASSUMED D TOENHANCETHE D 2APPAPORT OXIDIZING POWER OF 0s $INER AND  (OWEVER THEEFFECTON %M ISCOMPARATIVELY D D FROM F KINETICSTUDIESON 0s RE SMALLASDEDUCE D ION BY9: INTHENANOSECONDTIMEDOMAIN*EANS DUCT ETAL  ANDTHEMAJORROLEOF 9$OX APPEARSTOBE THE MODU D LATION OFF SPIN DISTRIBUTION WITHIN 0s 3ECTION)6$ RATHERTHANASIGNIlCANTCONTRIBUTION TOITSUNIQUEOXIDIZINGPOWER!COMPARATIVESTUDY F 3YNECHOCY CYSTIS 74 USING03))CORECOMPLEXESFROM AND D A MUTANT WHERE TYROSINE9$ WAS REPLACED BY PHENYLALAMINE CONlRMSTHATTHEEFFECTOF9$OX ONTHE %M ISVERY SMALL )M6 $INERETAL   !LTERNATIVE POSSIBILITIES FFOR TUNING THE MIDPO D INT POTENTIAL ARE THE PREVENTION OF @PUCKERING OF THE R AAND D &AAJER &AJ A ER  CHLORINRING "ARKIGI F PROTEIN CONFORMAT F IONS THAT  ANDOR @FROZEN BLOCK RELAXATION OF THE PROTEIN ENVIRONMENT IN RE SPONSETO FORMAT F IONOFF THEPOSITIVECHARGEAT0s -ULKIDJANIAN  4HE LATTERSUGGESTIONSEEMS TO BE AT VARIANCE WITH THE lNDING OF A SUBSTANTIAL F HE FREEENER F GY OFT F HERADICAL PAIRAFTER F RELAXATIONOFT CHARGESEPARATIONONTHENANOSECOND D TIMESCALE3EC TION6#B &LETCHERAND4HERIEN SHOWEDTHAT THECOFAC F IALPORPHYRINMOTIFF CAN BELARGELY MODIlED INITSELECTRONICSTRUCTUREAND D REDOXPROPERTIES4HIS @TOOLOFTUNINGTHE / / INTERACTION BETWEEN MACRO CYCLESCOULD BEALSOCONSIDERED FORTA F ILORING TH T EREDOX PROPERTIESOF0 F !NOTHERIMPORTANTFACTORTHATHASSOFARESCAPED DETAILED D ANALYSISISTHEROLEOF LIPIDS4HESTRUCTURAL F HREE LIPID D MOLECULES ARRANGEMENTWASRESOLVED FORT INTHE"2#FROM 2BSP S HAEROIDES "ASED ONTHESE RESULTS ITWASCONCLUDED D THATLIPID COFACTOR F INTERAC TIONS MAY HAVE A SIGNIlCANT ENERGETIC EFFECT F AND CONTRIBUT U ETO FU FUNCTIONAL DIFFE F RENCES BETWEE T NTHE ACTIVE I AND INACTIVE BRANCH #AMARA !RTIGASETAL   2INYUETAL  FOUN F D THATTHENEGATIVELYCHARGED CARDIOLIPINCAUSESASHIFTOFnM6TOWARDSMORE NEGATIVEVALUESOFT F HEMIDPO D INTPOTENTIALSOFF 1! IN "2#S#HARGED LIPIDSCOULD D ALSO BEUSED D TOMODU D LATE THEREDOXPROPERTIESANDHOLEDISTRIBUTIONWITHINTHE #HL A  UNITOF04 F HISQUESTIONREMAINSTOBE ANSWERED !VERYINTERESTINGRESULTWASOBTAINEDBYGLOBAL T TREATMENTO F ENVIRONMENTAL EFFECTS$ENS F ITY T FUNCT F IONAL

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI CALCULATIONS REVEALEDD THAT THE REDUCT D ION POTENTIAL OF #HL As REACHESVALUESOF6WITHINA HY DROPHOBIC ENVIRONMENT OF F A DIELECTRIC CONSTANT OF ABOUT(ASEGAWAAND.OGUC D HI  "ASEDD ON NEW03))STRUCTURES&ERREIRAETAL "IESIADKA F HESIX#HLSBOUNDD TO ETAL  THE %M VALUESOFT THE$$ HETERODIMERWERECALCULATED BYUSINGAN ELECTROSTATICCONTINUUMMODEL)SHIKITAETAL   6ALUESINTHERANGEOFF TO6WEREOBTAINED F 0$ 0$ #HL$ AND#HL$ WHILETHOSEOF#HL: AND FOR #HL: WEREMUCHLOWERABOUT6 ANDNOTFARFROM F #HL A IN SOLUTION 3ECTION )))!  THE %M VALUE OF 4HISSTUDYSHOWSTHATTHEESSENTIALCONTRIBUTIONSTO THE DRASTIC %M UPSHIFTOFTHE2# 0# #HLSORIGINATE F FROMT HE$$PROTEINMATRIX ABOUTOFT F HE F ANDD ONLYTOAMINOREXTENT FROMT F HEOTHER TOTAL EFFECT SUBUNITS3PECIlCHYDROGENBONDSAREINFERREDTOBE NOTESSENTIAL4HEDIFFERENTVA F LUESOBTAINEDD ONTHE BASISOFT F HESTRUCTUREMODELSOF&ERRE F IRAETAL AND "IESIADKA ET AL  REVEAL THAT RESOLUTIONS OFAND¯ RESPECTIVELY ARENOTSUFlCIENTFOR Y DETAILEDD CALCULATIONS&URTHERMORE THEREPORTED DATA OF)SHIKITAETAL IMPLYTHATTHEHOLEOF0s SHOULD BETRAPPEDAT# D HL$ AT LOWTEMPERATURES4HIS PREDICTION RAISES QUESTIONS ON THE PATHWAY OFF %4 FROM 9: TO 0s INPARTICULARWITHRESPECTTOTHE F IONAT+IN EXPERIMENTAL lNDINGSOFF A9:OX FORMAT M EXESOF 4E 4 LONGATUS A OUTOFF THE03))CORECOMPL AB :HANGETAL  !CCORDINGLY FURTHERSTUDIESARE REQUIREDD TORESOLVETHE DETAILSOFF THEREDOXTUNING OF THE#HLSIN2# 0# 2EGARDLESSOFF THESEDETAILS IT ISCLEARTHATTHENATUREOFTHEPROTEINMATRIXISTHE KEY FACTOR F F HEUNUSUALLY HIGH OXIDIZING POWEROF FORT 0s&URTHERMORE THE DISTANCETOTHEOTHER#HLS WITH LOWER %M VALUES IN THE 03)) COMPLEX EG #HL: ANDD #HL: ANDD TOTHECAROTENOIDSMUST BELARGE ENOUGH TOPREVENTUNDESIRED DISSIPATIVEAND DESTRUC TIVE SIDE REACTIONS $YNAMICSTRUCTURALCHANGESOFT F HEPROTEINENVI RONMENTCAN LEADD TOTIME DEPENDENTVARIATIONSOFF THE LOCALCHARGEDISTRIBUTION THUSAFFECTINGTHEENERGET ICSAND KINETICSOFF CHARGESEPARATION4HERELEVANCE F THIS IMPORTANT PHENOMENON WILL BE OUTLINED IN OF 3ECTION 6 # 5NTIL RECENTLY 0 HAS BEEN ENVISAGEDD AS A #HL A COMPLEX (OWEVER WITH THE DISCOVERY OF / EVOLVINGCYANOBACTERIACONTAININGMAINLY#HL D !CARYOCHLORIS ! ! MARINA ANDD THE lNDING THAT THE D PHOTOACTIVEPIGMENTOFF 03) ISASPECIAL#HL DDIMER IN THESE SPECIES !KIYAMA ET AL  QUESTIONS ARISEASTOTHENATUREOFF THECORRESPONDINGPHOTOAC

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS TIVEPIGMENTS IN03))OFF THISSPECIES-IMUROET AL SUGGESTTHATINTHESEORGANISMS03))STILL CONTAINS 0 WITH #HL A AS ESSENTIAL CONSTITUENT BUTTHEEXPERIMENTAL EVIDENCEFORT F HISCONCLUSIONIS LIMITED&URTHERSTUDIESAREREQUIREDTOCLARIFYTHIS VERY IMPORTANTPOINT)NTHISRESPECTIT ISINTERESTING TOMENTIONTHATMUTANTSOF 3YNECHOCYSTIS HAVEBEEN CONSTRUCTEDBYGENETICENGINEERINGWHERE#HL A AND 0HEO A AREPARTLYREPLACED BY #HL B AND D 0HEO B INTHE 03))CORE 6AVILINETAL  4HESElNDINGSSHOW THAT #HL A ANDD 0HEO AARENOTNECESSARILYTHEONLY CHLORINMOLECULESTHATCANCONSTITUTEAFUNCT F IONALLY COMPETENT03))WITH INTACT7/#ACTIVITY & 5IF "OJPO 3BEJDBM 1IFPmr & )NCONTRASTTOTHEMODIlCATIONOFT F HEPROPERTIESOF #HL A IN ORDER TO ACHIEVE THE UNIQUE Q PHOTOACTIVE COMPLEX03ECTION)6$ THE0HEO A MOLECULES IN03))AREMUCH LESSAFFECTE F D4HEMIDPO D INTPOTEN TIAL%M OFF 0HEO0HEOns IN 03 ))WASDETERMINED D TO BE nM6+LIMOVETAL  4HISVALUEISALMOST THESAMEASTHATOFF 0HEO A INSOLUTION3ECTION))) ! (OWEVER WH W ENTAKING INTOACCOUNTELECTROSTATIC EFFECTSDUETOTHE PRESENCEOF1!ns DURINGTHEREDOX TITRATION TH T EACTUAL @WORKI R NGPOTENTIALOFF 0HEO0HEOns ISEXPECTED D TO BESHIFTE F D TOWARDSLESSNEGATIVEVALUES %XPERIMENTALSTUDIESREVEALTHATTHISEFFECTISABOUT M6 'IBASIEWICZETAL  SOTHATAN%M VALUE OFF ABOUT n M6 SEEMS TO BE MORE REALISTIC FO F R F FUNCTIONALCOM PLEXESWITHANOXIDIZED1! 03))CONTAINSTWO0HEOMOLECULESSEE&IG IN ANALOGYTOTHETWO"0HEOSIN"2#S!CCORDINGLY F FORANUNDERSTANDINGOFTHECHARACTERISTICSOFTHETWO 0HEOSPECIESIN03))ITISMOSTSUITABLETOUSEASA D IESOFT F HECORRESPOND GUIDETHEWELL RESOLVEDPROPERT ING"0HEOSIN"2#S!SPECTACULARlNDINGINTERMS F ION ISTHAT INSPITEOFANA F LMOSTSYMMETRIC OF FUNCT F HECHARGESEPARATION ARRANGEMENTOFF THECOFACTORS T INTHE"2#ISAUNIDIRECTIONALPROCESSINVOLVINGONLY "#HL! "0HEO! AND 1! "YLINAETAL -ICHEL "EYERLE ET AL  :INTH AND D +AISER   ! SWITCHOFTHEELECTRONTRANSFERPATHWAYINTOTHEOTHER DIRECTIONCANONLY BEACHIEVED IFF THEPROTEINMATRIXIS SPECIlCALLYMO L DIlED BYGENETICENGINEERING-UTANTS WITH"2#SOFREVERSED@SIDEDNESSOFTHE%4 PATHWAY LEADING TO SIGNIlCANT 0s1"ns FORMAT F ION HAVE BEEN GENERATED ,AIBLEETAL 7AKEHAMETAL   )N ANALOGY TO THE WILD TYPE "2# THE TWO 0HEO A D TO BE FUNCT F IONALLY MOLECULES IN 03)) ARE ASSUMED

 HETEROGENEOUS ˆ IE ONLYONE 0HEO$ PARTICIPATES F RMATION OF 0s0HEOns)TISINTERESTINGTO INTHE FO F IONAL BRANCHINGNOTONLY NOTETHAT IN03))THEFUNCT COMPRISESTHESTEPS LEADINGTO0s1!ns FORMAT F ION Q SITE BUTALSO ANDSUBSEQUENT01REDUCTIONATTHE1 " THE PATHWAY A FFOR WATER OXIDATION VIA9: IN THE $ POLYPEPTIDE$EXHIBITSASTRIKING@SIDEDNESS 4HE FU F NCTIONAL DIFFE F RENCES OFTHE TWO "0HEOS ARE REmECTED INTHEIRSPECTRALPROPERTIES)NTHEVISIBLE NEARINFRARE F D 6)3.)2  REGIONOFF "2#S THE 1Y TRAN SITION OFTHE @ACTIVE "0HEO! ATABOUT  NM IS RED SHIFTE F D BYALMOSTNMRELATIVETO"0HEO" 2OBERT ETAL  ,IKEWISE THEPEAKK POSITIONSINTHE1X REGIONEXHIBITASIMILARDIFFERENCEWITHNMAND NM FOR"0 F HEO" ANDD "0HEO! RESPECTIVELY3TU Y D IESONSPECTRALPROPERTIESOFF 0HEOIN03)) MAINLY ANALYZEDIN$$#YTB SAMPLES GAVECONmICTING RESULTS -IMUROETAL +ONERMANNANDD (OLZ WARTH 3HKURA K PATOVETAL 9RUELAETAL  !PARTFROMTHEAMBIGUITIESDUETOTHESAMPLE TYPE A FURT F HERCOMPLICATIONEMERGESFROMT F HE FAC F T THATTHESPECTRAOFSAM F PLESWITH PHOTOACCUMULATED 0HEOns AREINmUENCEDBYBOTHACHANGEOFTHEEXCI TONIC INTERACTIONS ANDD THE ELECTROCHROMIC EFFECT F OF THENEGATIVELYCHARGEDD 0HEOn s ONTHE2# 0# PIG MENTS!RELIABLEESTIMATIONOFTHESPECTRALEFFECTS D DUETOEXC ITONIC INTERACTIONREQUIRESCALCULATIONSON THE BASIS OF DETAILED INFORMAT F IONONTHEGEOMETRI CAL ARRANGEMENT OF THE PIGMENTS IN PARTICULAR THE MUTUALORIENTATIONOFF THETRANSITION DIPOLEMOMENTS 2ASZEWSKI ET AL  %XPERIMENTAL DATA FOR F MORENATIVE03))COMPLEXESHASBEENOBTAINEDBY USINGSAMPLES ISOLATED FROM F 3 NECHOCY 3Y CYSTIS 74AND MUTANTCELLS4HESEPREPARATIONSCONTAIN1! ANDD THE ELECTROCHROMICEFFECTOFTHENEGATIVECHARGEOF1!ns INITSREDUCE D D SEMIQUINONE FORM F 3ECTION6# ON 0HEO$ PROVIDESASPECIlC lNGERPRINT FOR F ITSSPECTRAL IDENTIlCATION)NTHESESAMPLES hMAX VALUES OF  F HE@ACTIVE ANDD NMWEREMEASURED AT+FORT ANDD @INACTIVE 0HEO RESPECTIVELY 3TEWART ET AL  4HESEVALUES HOWEVER HAVETOBECORRECTED FFOR EFFECTS F DDUE TO EXCITONIC INTERACTIONS "ASEDD ON F PTICALSPECTRAAT+ VALUESOF MEASUREMENTSOFO  NM AND  NM WERE OBTAINEDD FOR 0HEO$ IN / EVOLVING 03))CORECOMPLEXESANDD 03))MEM BRANE FRAGMENTS F F FROMSP INACH RESPECTIVELY WHIL W EA SLIGHTLYSHORTERWAVELENGTHOFNMWASREPORTED F FOR0 HEO$ IN/ EVOLVING03))CORECOMPLEXESFROM F 3 3YNEC HOCYSTIS 0ETERSON !RSKÚLD D ETAL  4WO CONCLUSIONSEMERGEFROMTHEDATAI THE 1Y PEAKOF THEACTIVE0HEO IN03))EXHIBITSASIMILARREDD SHIFT

 RELATIVETOTHATOFF THEINACTIVE0HEOASOBSERVED IN "2#SVIDESUPRA ANDII $$#YT B PREPARA TIONSMAY A BEOF LIMITED D VALUEFOR F DETAILED D SPECTRAL ANALYSESSEE3ECTION)6" &ORTHE1X REGION DATA WEREREPORTEDFORTHE@ACTIVE0HEOINISOLATED$$ #YT B PREPARATIONSFROM F DIFFERENTOR F GANISMS4HE ns DIFFERENCES F PECTRAFOR0 F HEO$  FORMATIONWEREFOUND TOBESLIGHTLYREDSHIFTEDBYnNMINPLANTMATERIAL PEAKAROUN K D NM COMPARED D WITH SAMPLES FROM F CYANOBACTERIA 'IORGI ET AL   #OMPARATIVE MEASUREMENTSOFTIME RESOLVEDABSORPTIONCHANGES F #REINHARD R TII ANDD A OFF 03))PREPARATIONSFROM74 W ERE 0HEO$ IS REPLACED BY #HL A REVEAL MUTANT WH THATTHEREDSHIFTEDABSORPTIONBANDINTHE1X REGION NM BELONGSTO0HEO$ OFT F HE@ACTIVEBRANCH 8IONGETAL   ! WELL RESOLVED CHARACTERISTIC OF "0HEO! IS THE HYDROGEN BONDINGOF ITSRING 6 KETOGROUP BY'LU U UNITOFF "2#S &EHERETAL -ICHEL OFF THE, SUB AND$EISENHOFER  !NANALOGOUSFEATUREWAS F D FORT FOUN F THE@ACTIVE0HEO IN03))WH W ERETH T EHYDRO D GEN BOND D WASASSUMED TOBE FORME F D BETWEENTHERING6 KETOGROUPANDTHECORRESPONDING'LUOFPOLY PEPTIDE$ ,UBI U TZETAL -OÑNNE ,OCCOZET AL .AB A EDRY D KETA K L  $IRECTEXPERIMENTAL EVIDENCEFORTHISASSIGNMENTWASOBTAINEDFROMHIGH ns lELD D %02STUDIESOFF 0HEO$ R TII MUTANTS  IN #REINHARD WHERE'LU ISREPLACED BY(IS 'LNOR,EU $ORLET ETAL  /PTICALDIFFERENCESPECTRAOF0HEOns0HEO AREALSOAFFECTE F D BYCHANGESOFT F HE HYDROGEN BOND 'IORGIETAL  FOUN F D THATTHEPEAK K POSITIONIN THE18 REGIONSHIFTSBYnNMIN$$#YT B PREPARATIONS ISOLATED FROM F 3 NECHOCY 3Y CYSTIS MUTANTS WHERE'LNISCHANGEDTO' D LUOR,EU INCONTRAST TOPLANTS THE74CELLSCONTAIN'LNATPOSITION INSTEAD D OF 'LU  "ASED D ON HIGH lELD D %02 STUDIES $ORLET ET AL  INFERRED THAT THE DIFFERENCES IN G VALUES ns BETWEEN "0HEO!ns IN 2 VIRIDIS AND D 0HEO$  IN 03)) FFROM SPINACH ARE PREDOMINANTLY D DUE TO STRUCTURAL DIFFERENCESBETWEENTHETWOTYPESOFREACTIONCENTERS F F ACONSERVED HYDROGENBOND 4HESTRIKING FEATUREO RAISESA QUEST Q IONONITSPOSSIBLEFUNCT F IONALRELEVANCE !COMPARISONOFTHE%.$/2 SPECTRAOF0HEOns IN SOLUTIONWITHTHATOFF THEPHOTOACCUMULATED D SPECIES IN$$#YT B PREPARATIONS LED D TOTHECONCLUSION THATTHEPROTEINMATRIXEXERTSCONSTRAINTSTHATLEADTO STRUCTURAL CHANGESOFT F HE0HEO MOST LIKELYAROTATION U TZETAL  4HESEAUTHORS OFF THEVINYLGROUP ,UBI SPECULATED THAT THIS CHANGE COULD GIVE RISE TO AN

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI OPTIMAL ELECTRONTRANSFERRATEV F IAORBI R TAL OVERLAP/N THEOTHERHAND REPLACEMENTBYSITEDIRECTEDMUTAGEN ESISOFF 'LU INTHE, SUB U UNITINPURPLE BACTERIAWAS SHOWNTOEXERTONLYMINOREFFECTSONT F HEKINETICSOF T THE%4STEPSLEADINGTO 0s1!ns FORMATION"YLINAETAL F D  !NALOGOUSEXPERIMENTSCANNOT BEPERFORME IN$$#YT B PREPARATIONSDUETOT D HE LACKO K F 1! F RMATION 4HERESULTSREPORTEDSOFARFOR0s0HEOns FO INDICATETHATTHEDETECTEDD nPS KINETICSARENOT CHANGED INTHESAMPLESISOLATED FROMT F HEABOVEMEN TIONEDD MUTANTS OF 3YNECHOCYSTIS 3 WHERE$ 'LN ISREPLACED BY'LUOR,EU 'IORGIETAL  )N F HE nPS CONTRASTTOTHEVIRTUAL INVARIANCEOFT KINETICSTOCHANGESINTHEHYDROGENBONDINGTOTHE KETOGROUPOFF RING6 THEMIDPO D INTPOTENTIAL OFF 0HEO VARIESASDEDUCE D D FROM0 F s0HEOns RECOMBINATION RATES-ERRYETAL CONCLUDEDTHATREPLACEMENT OF$ ' F LN BY 'LUCAUSESANUPSHIFT F BY ABOUT M6 WH W EREAS,EUGIVESRISETOA DOWNSHIFT F BYABOUT nM6!LTHOUGHTHESECHANGESARECOMPARATIVELY SMALL THEY COULD BEOFRE F LEVANCEFORT F HEPATHWAY A OF 0s1!ns RECOMBI M NATIONANDD THEPROBABILI A TYOF 0 F FORMATION3ECTION6)! 4HISRAISESAQUESTIONON THE EVOLUTIONARY ADVANTAGE OF F THE REPLACEMENT OF 'LN IN03))OFF CYANOBACTERIA BY'LU INPLANTS BECAUSEANUP U SHIFTOFTHEMIDP D OINT POTENTIALOF0HEO IS EXPECTEDD TO ENHANCE THE PROBABILITY OF DANGER OUS 0 FORMAT F ION PROVIDING THAT THE MIDPO D INT POTENTIALOF1!ns1! ISTHESAMEASINCYANOBACTERIA  3INCEDETAILED INFORMAT F IONONTHISPOINT ISLACKING ITAPPEARS PREMATURETOSPECULATEONTHEREASONFOR F THEEXCHANGEOF'LN'LUDURINGEVOLUTION 7
,JOFUJDT
BOE
&OFSHFUJDT
PG
$IBSHF 7 4FQBSBUJPO 4HE LIGHT INDUCED CHARGE SEPARATION IN 03)) VIA OVERALL REACTION SEQUENCE %Q  COMPRISES BASI CALLYTWOSTEPS I FORMAT F IONOFT F HERADICAL IONPAIR 0s0HEOns AND II STABILIZATION OF THE PRIMARY D ELECTRON TRANSFER F FROM F CHARGE SEPARATION BY RAPID 0HEOnsTO 1! 4HE UNIQUE Q PROPERTIES OFF 03)) ARE CONNECTEDWITHTHElRSTSTEP3INCE0ACTSASA D HENATUREOF #HL A  0HEO8 SHALLOWTRAPAND DUETOT 3ECTION)6" THEOVERALL KINETICSOF0 F s0HEOns FORMATIONARECOMPLEX

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS "
.FDIBOJT J N BOE ,JOFUJDT J PGG 'PSNBUJP J O PG UIF
3BEJDBM
*PO
1BJS
1 r 1IFPmr 4HE KINETICS OFF 0s 0HEOns FORMAT F ION COMPRISE Q PSBETWEENTHEELECTRONI DIFFERENTEQUILIBRATIONSTE CALLY EXCITED D ANTENNA #HLS AND D 0 0 AND 0s 0HEOns AND D THEMOLECULESWITHINTHE #HL A  UNIT$EPENDINGONTHEKINETICSOFTHESEPROCESSES TWO LIMITINGCASESCANARISEFORT F HEOVERALL REACTION I AN%%4 LIMITEDPROCESS OR D II ATRAPPING LIMITED PROCESS#HAPTER "ARTERETAL  4HE@EXCITON RADICAL PAIREQUILIBRIUMMODEL WAS DEVELOPED BY(OLZWARTH AND D COWORRKERS BASED D ONTHE ASSUMPTIONOFRAPIDEXCITEDSTATEEQUILIBRATION3CHATZ ETAL  (OLZWARTH  !CCORDING TO THISMODEL THEOVERALL REACTION ISTRAPPING LIMITED Q THE MEASURED RATE CONSTANT FOR !S A CONSEQUENCE TRAP 0s 0HEOns FORMAT F ION K0# CANBEEXPRESSED D AS INT THEPRODUCTO D F HE INTRINSICRATECONSTANTK0# FT AND D A CONSTANT+ EQQ THATSUMMARIZESALL%%4EQUILIBRATION Q STEPSBETWEENEXCITED D SINGLETSTATESWITHI T NTH T EANTENNA ANDAT0 ˆ IE + EQQ ISTHEPOPULATIONPROBABILITY OF 0 PROVIDEDTHATTHEEQUILIBRATIONISSUFlCIENTLY TRAP F FAST"ASE D ONTHISMODEL THERATECONSTANT K0# IS D TO DEPEND D ONTHE@SPECTRALLYWEIGHTEDAN EXPECTED TENNASIZETHATISATEMPERATUREDEPENDENTNUMBER D DUETOT HETHERMAL POPULATIONOFF STATES '2ENGER ETAL  4HISGENERALCONCLUSIONWAS BASICALLY CONlRMEDBYMEASUREMENTSOFEXCITEDSTATETRAPPING D VIA mUORESCENCELIFET F IMEMEASUREMENTS MONITORED INSAMPLESWITH FUNCT F IONALLYCOMPETENT03))COM PLEXESOFDIFFERENTANTENNASIZE3CHATZETAL  2OELOFSETA F L '2ENGERETAL 4RISSL AND D ,AVERGNE  !S A RESULT OFF THESE STUDIES THE INTRINSIC RATE CONSTANT WAS ESTIMATED D TO BE ABOUT PS n ATROOMTEMPERATURE4HISVALUEEXHIBITSA STRIKINGSIMILARITYWITH THECORRESPONDINGMOLECULAR RATE CONSTANTFO F RTHE FO F RMATION OF 0s"0HEO!nsINTHE "2#S :INTH AND D +AISER  )TISAREmECTIONOF THECLOSESTRUCTURALAND FUNCT F IONAL SIMILARITIESOFT F HE CHARGESEPARATIONINTHE "2#SAND03))-ICHELAND $EISENHOFER  F (OWEVER INTHECASEOFF PURPLE BACTERIA THESPECTRALPROPERTIESOFF 03ECTION)6" AND D THE ISOLATION OF INTACTT 2#SPERMITASTRAIGHTFOR WARD D ANALYSISOFT F HE KINETICS4HESECONDITIONSARE F 3)))NORDERTOMINIMIZETHEEFFECTS F NOTSATISlED FOR0 DUETOTHEEQUILIBRATION Q PROCESSESBETWEENANTENNA AND D 0 NUMEROUS EXPERIMENTS WERE PERFORME F D WITH$$#YT B PREPARATIONS!PARTFROMST F ILL UNRESOLVEDQUESTIONSONTHISSAMPLETYPE3ECTION )6" THE DATA OF DIFFERENT F RESEARCH GROUPS LED D TO

 INT  6A 6 LUES SEEMINGLY CONmICTING CONCLUSIONS ON K0# RANGINGFROMABOUTPS n UPTOPS n ATROOM TEMPERATUREWEREREPORTED 7ASIELEWSKIETAL  +LUGETAL -àLLERETAL 'REENlELDD ETAL 'IORGIETAL 3EIBERTAND7ASIELEWSKI W EREASATLOWTEMPERATURESEVENMARK R EDLY  WH HIGHERRATECONSTANTSWEREOBTAINED BYSOMEAUTHORS 4ANGETAL 'ROOTETAL   4HE IDEA OFF A TRAPPING LIMITEDD PROCESS IN INTACT 03)) CORES HAS BEEN QUEST Q IONED "ASEDD ON STRUC TURALDATAOFTHEPIGMENTARRANGEMENTIN03)) THE F PHOTOCHEMICAL LIGHT CAPTURE IN OVERALL REACTION OF 03))WASINFERRE F D TO BE LIMITED BY%%4OFF THEORDER OFPSBETWEENTHEANTENNAANDTHE2#6ASILIEV INT ETAL  3IMILARLY AMUC Y H LARGERVALUEFOR F K0# n OF *FS WASALSOOBTAINEDFROMTHISANALYSIS "ASED ON TRANSIENT ABSORPTION AND TIME RESOLVED mUORESCENCESPECTROSCOPYTHETRAPPING HASRECENTLY BEEN INFERRE F D TOBENEITHERDIFFUS F ION NORTRAPPING LIMITEDIN#0 $$PARTICLESBUTSOMEWHEREIN BETWEEN!NDRIZHIYEVSKAY A AETAL  )NVIVOTHE 03))CORE ISCOUPL U EDD WITH PROXIMAL AND PERIPHERAL ANTENNA COMPLEXES 6AN !MERONGEN AND $EKKER  CONCLUDEDD THATASTRAIGHTFORWAR F D DISTINCTION BETWEENTRAPANDD EXCITONDIFFUS F ION LIMITEDD TRAPPING CANNOTBEMADEFOR03))INNATIVETHYLAKOIDMEM BRANES4HETHYLAKOIDD STRUCTUREAND ORGANIZATIONOF THEANTENNASYSTEMS INVARIOUSORGANISMSCANAFFECT F THEOVERALLTRAPPINGINVARIOUSWAYS ALSODEPENDING ONENVIRONMENTAL CONDITIONS )N A DETAILEDD STUDY USING COHERENT PHOTON ECHO SPECTROSCOPYAT+AND$$#YT B PREPARA TIONSASTHESAMPLEMATERIAL THECHARGESEPARATION LEADING TO 0s0HEOns FORMAT F ION WAS INFERRE F D TO OCCURVIAATWO STEPELECTRONTRANSFERAFTERFAST%%4 EQUILIBRATIONWITHINTHE 2# 0# UNIT4HE DATAARE INTERPRETED BYTHEFO F LLOWINGSCHEME 0ROKHORENKO AND(OLZWARTH  

;2#n0# =INITIAL n FS F ;2#n0# =EQUIL Q



KINT 0#  s ns ;#HL$ 0$ 0$ #HL$=0HEO$

KINT 0#  ns

s ;#HL$ 0$ 0$ #HL$= 0HEO$

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI

 WHERE;2#n0# =INITIAL DESCRIBESTHEINITIAL FORM F A TIONOFANELECTRONICALLYEXCITEDSINGLETSTATEINTHE D SINGLETSTATE UNITAND ;2#n0# =EQUIL ISTHEEXCITED POPULATIONREACHED D AFTERRA F APID D EXCITED D STATEEQU Q ILIBRA TION 4HE VALUES OFTHE RATE CONSTANTS KINT D KINT 0#  AND 0#  n n AREABOUT PS AND PS RESPECTIVELY AT Y +4HEYAREMOSTLIKELYSOMEWH W AT DIFFERENTATROOM F TEMPERATURE WHICH COULD NOT BE TESTED USING THE Y IS IMPORTANT HOWEVER TONOTE SAMEMETHODOLOGY)T THATTHEEXPERIMENTALLYOBSERVED D RATES INCONTRASTTO THE DESCRIBEDINTRIN D SIC RATES KINT AREHIGHLYDISPERSIVE D DUETOT HEVARIABLEMIXINGOFF THEEXCITONSTATES INTO THEWAVEFUNCT F IONSOFF THEELECTRONDONORAND D ACCEPTOR ANDSAMPLEINHOMOGENEITIES4HUS THELONG STAND ING DISCUSSIONOFF ASPECIlCELECTRONTRANSFERRATE F IN 03)) 2#S BECOMES MEANINGLESS 4HE OBSERVABLE ELECTRONTRANSFERRATESATLEASTATLOWTEMPERATURE SPANARANGEOFF nORDERSOFF MAGNITUDE DESPITEA SINGLE INTRINSICELECTRONTRANSFERRATE%AC F H 2# HAS ITS OWNELECTRONTRANSFERRATE DEPENDINGONTHESPECIlC NUCLEARARRANGEMENTANDCOUP D LING BETWEENPIGMENT ANDPROTE D IN0ROKHORENKOAND D (OLZWARTH T  4HIS CONCLUSION IS CONlRMED BY LOW TEMPERATURE HOLE BURNING EXPERIMENTSON$$#YT B PREPARATION CONTAININGlVE#HLS2ILEYETAL  !THIGHER TEMPERATURES THIS PICTURE WILL BE FURTHER MODIlED BY ADDITIONAL TIME DEPENDENTEFFECTS F D DUETONUC LEAR MOTIONOFF THEPROTEIN!T PRESENT ITISNOTCLEARASTO W WHATEXTENTTHEKINETICSOFPRIMARYCHARGESEPARATION AREDISPERSIVEATROOMTEMPERATURE 2EGARDLESS OF F THE EXACT NUMBERS FFOR THE RATE CONSTANTS THESCHEMEINCLUDINGAFASTINTRINSICHOLE T TRANS F FERSEEMSTORE mECTTH T E GENERAL PATTERNOFPR F IMARY CHARGESEPARATIONIN03))4HISCONCLUSIONISSUPPORT EDBYTWODIFFERENTLINESOFEXPERIMENTALEVIDENCE I SPECTROSCOPICSTUDIESATDIFFERENTTEMPERATURESON F 03))CORECOMPLEXESFROM F 3YNECHOCOCCUS MUTANTS W WHERETHE(IS LIGANDSOF0 $ $ (IS AND0$ $ (IS AREREPLACED BY DIFFERENTRES F IDUES D 'LU 'LN !SN !LA 4HR #YS ,YS 6AL $INERETAL   ANDII MEASUREMENTSOFABSORPTIONCHANGESWITH F FEMTOSECON D TIMERESOLUTIONAT+ USING$$ #YT B PREPARATIONSWH W ERETHE@INACTIVE0HEOWAS SUBSTITUTED BY | DEOXY  HYDROXYPHEOPHYTIN 'ERMANOETAL  !CCORDINGLY T Y HE FORMAT F ION D AS OFF 0s0HEOns IN 03 ))CAN BESUMMARIZED 



 5& 3& LQLWLDO k  m  5& 3& HTXLO k N

 

‡ 

‡

3KHR k

N LQW 

m 

‡ 

3K 3 HR

‡

LQW Q 3& 3&

m 

WITH 2# 0# 0 s 0HEOns AND 0 s 0HEOns SYMBOLIZING ;#HL A 0HEOX= ;#HL$s0$0$#HL$= ns s ns 0HEO$ AND ;#HL$0$ 0$#HL$=0HEO $ RESPECTIVELY Y F ION))) ANDD j¡kASINGLETEXCITON SEEFOOTNOTE3ECT "A  !COMPARISONOFT F HISSCHEMEFOR03))W F ITHTHE F ION IN PURPLE REACTION PATTERNOF 0s "0HEOns FORMAT BACTERIAREVEALSSTRIKINGMECHANISTICDIFFERENCESWITH F HL$ ANDD "#HL! THATARELOCATED RESPECTTOTHEROLEOF# ATEQUIVALENTPOSITIONSWITHINTHESIXPIGMENTCORESOF BOTHTYPESOFORGANISMS)NTHEFORMERCASE #HL$ IS ASSUMEDD TO BETHEPHOTOACTIVECONSTITUENTnIE THE  F D FROM F #HL $ TO0HEO A FOLLOWED ELECTRON ISTRANSFERRE s BYRE REDUCTIONOF #HL $ WITH0$ASDONOR WHILEIN THELATTERCASE "#HL! SERVESASELECTRONACCEPTORFO F R 

0 AND"#HL!ns BECOMESRAPIDLYREOXIDIZEDBY"0HEO &URTHERMORE THEDECAYOF"#HL!ns IS FAS F TERTHANTHE F ION:INTH THAND +AISER  WH W EREAS RATEOF ITS FORMAT #HLs$ IS INFERRE F D TO BE COMPARATIVELY LONG LIVED RELATIVETOITSFORMATION ATLEASTATLOWTEMPERATURES 0ROKHORENKOANDD (OLZWARTH   4HE PECULIARITIES OFF CHARGE SEPARATION IN 03)) SATISFYTHEENERGETICCONSTRAINTS FORTHE GENERATION F A STRONGLY OXIDIZING SPECIES BY DISSOCIATION OF OF THESINGLETEXCITONINTOARADICAL IONPAIRATTHEDI ns MERICUNIT#HL$0HEO$ LEADINGTO #HLs$0HEO$  4HE s ns F IONIN ALTERNATIVEOFF A 0 $ #HL$ RADICAL PAIRFORMAT THEPRIMARYSTEPOFF CHARGESEPARATION $INERETAL  APPEARSTOBEHIGHLYUNLIKELY!COMPARISON F HEENERGETICS FORONE E F LECTRONREDUCT D IONOF# F HL A OFT ANDD 0HEO A 3ECTION)))! READILYSHOWSTHATA 0s$ ns #HL$ PAIRWITHASTRONGLYOXIDIZINGCATIONRADICALIS F BLEATANEXCITONENERGYOFA F BOUT MUCH LESSFAVORA ns ANIONRADICAL E6 UNLESSTHEENERGETICSOF #HL$ FORMATION ARE NOT UNCONVENTIONALLY AND STRONGLY D LATED BYTHEPROTEINENVIRONMENT MODU 5NTIL NOW W CONSIDERATIONSONTHEMECHANISMOFT F HE PRIMARYCHARGESEPARATIONINPHOTOSYNTHETICREACTION CENTERS HAVE BEENRESTRICTEDD TOTHECHLORINS2ECENT STUDIESONNUCLEARWAVE PACKETMOTION BETWEENTHE POTENTIAL SURFACES OF STATES 0 AND 0s"#HL!ns IN "2#S HOWEVER SUGGESTTHATA BRIDGING WATERMOL ECULEBETWEENONE"#HLMOIETYOFT F HESPECIALPAIR 0" AND THE MONOMERIC "#HL! MIGHT BE INVOLVED INTHEPATHWAY A OFF THEPRIMARY ELECTRONTRANSFERSTEP F 9AKOVLEVETAL  4HISISANATTRACTIVE IDEATHAT SHOULD BETAKENINTO ACCOUNTINFU F TURE CONSIDERATIONS ONCHARGESEPARATION IN03))

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS #
.FDIBOJT J N BOE ,JOFUJDT J PGG 'PSNBUJP J O PG UIF
3BEJDBM
*PO
1BJS
1 r 2"mr 4HE LIGHT INDUCED CHARGE SEPARATION PRODUCT 0s0HEOns MUSTBE@STABILIZEDBYRAPIDELECTRON ns TRANSFER F FROM0 F HEO$  TO1! &IG INORDERTOMINI MIZETHE DISSIPATIVERECOMBINATIONOFT F HERADICAL ION PAIR0s 0HEOns 3ECTION6)! 4HEKINETICSOFTHE @STABILIZATIONREACTIONHAVEBEENRESOLVED BY MEA SUREMENTSOF mASH INDUCE D D ABSORPTIONCHANGESTHAT ARECHARACTERISTICFORTRANSIENT POPULATIONSOF0HEOns ns AND 1!ns &IG   0HEO$  REOXIDATION .UIJS ET AL 3CHATZETAL   AND 1!ns FORMAT F ION %CKERTETAL "ERNARDINGETAL  EXHIBIT VIRTUALLYTHESAMEKINETICSWITH LIFET F IMESOFF ABOUT PS!NALYSISOFF THE mUORESCENCEDECAY KINETICS WITHIN THE FRAMEWORK OF THE @EXCITON RADICAL PAIR F L 6ASILEVET EQUILIBRIUMMODEL 2OELOFSETA AL  AND D CURVElTTINGOFF PHOTOVOLTAGEMEASURE MENTS,EIBLETAL  LEDTOCOMPARABLEVALUES OFF nPS)NTERESTINGLY ARATECONSTANTO Y F ABOUT PS n DIFFERSON F L LIGHTLY LYS L FROMT F THECORRESPONDING F RTHE ELECTRONTRANSFE F R FROM VALUEOFABOUTPS n FO "0HEO!ns TO 1! IN"2#S :INTHAND D +AISER  4HIS ISANOTHERREmECTIONOFF THECLOSESIMILARITYBETWEEN THEACCEPTORSIDEOF03))AND"2#S3ECTION6"  $IFFERENCES F BETWEENTHETWOTYPESOFF 2#S HOWEVER F INGTOREPLACEMENTOF EXIST INTERMSOFF THEEFFECTSOW EXCHANGEABLEPROTONSBYDEUTERONSANDTHEREMOVAL OFF THE.()&IG 4HEKINETIC($EFFECT F ISMORE PRONOUNCED IN"2#S0ASCHENKOETAL  THAN IN 03))MEMBRANEFRAGMENTS6ASILEVETAL  ! GENTLEEXTRACTIONOFF THE.() -AC-ILLANETAL  + +URREC K ETAL  ISVIRTUALLYWITHOUTEFFECTON F ns THE RATE OF 0HEO$  REOXIDATIONBY1! "ERNARDINGET D THEPROPERTIESOF 1!ns IN03)).OGUCHI AL  AND W ETHECORRESPONDING%4STEPFROM F ETAL  WHIL "0HEO!ns TO1! IN"2#SBECOMESRETARDEDBYAFACTOR OFF ABOUT/NTHEOTHERHAND T D HEKINETICSREMAIN F D WHEN.() DEPLETED D "2#SAREADDITION UNAFFECTE ALLYDEPRIVEDOFTHE( SUBUNIT,IUETAL  4HIS ILLUSTRATES BOTH THE DIFFERENCES F BETWEEN 03)) AND "2#SAND D THEREGULATORYROLEOFF PROTEINSUB U UNITSTHAT DONOTCONTAINCOFACTORS!NOTHERIMPORTANTDIFFER ENCE BETWEEN"2#SAND03)) D ISTHEMODEOFF AXIAL COORDINATIONOFF THE.())NADDITIONTOTHEFOUR( F IS LIGANDSINTHETWOTYPESOFORGANISMS THE.()OF "2#S ISCOORDINATED BY A'LURESIDUE D $EISENHOFE F R D -ICHEL  WHIL W E IN03))THEEQUIVALENTSITE AND ISMOSTLIKELYOCCUPIEDBYBICARBONATE&ERREIRAET AL  !SACONSEQUENCE THE03))ISCHARACTER

 IZEDBYASTRIKINGBICARBONATEEFFECTTHATISMISSING IN"2#SVAN2ENSENETAL #HAPTER VAN 2ENSENANDD +LIMOV  ns 4HEELECTRONTRANSFER F FROM0 F HEO$ TO1! ISTHEES SENTIALSTEPINORDERTOACHIEVEACHARGESEPARATION THAT ISSUFlCIENTLY STABLETOPROVIDETHEDRIVING FORCE F F HEREACTIONSEQUENCESSUMMARIZED BY%QS FORT AND 4HEREFORE ONLY03))COMPLEXESCONTAINING 1! INTHEIROXIDIZED FORMARE F F FUNCT IONALLYCOMPETENT F FORE FlCIENTTRAPPINGOFE F LECTRONICALLYEXCITEDD STATES INFORMOFAUSEFULELECTROCHEMICALPOTENTIALDIFFER ENCE!CCORDINGLY T Y HIS FUNCT F IONALLYCOMPETENTSTATE ISREFERRE F D TOAS@OPENREACTIONCENTER WHIL W E03)) IS@CLOSEDWHENLACKING1! ORCONTAININGREDUCED F F HESEMIQUINONE 1!ns ORTHE FT PLASTOQUINONE IN FORMO DOUBL U EREDUCE D D QU Q INOL 1!( !SACONSEQUENCE Q 1! ACTSAS PHOTOCHEMICAL QUENCHEROF#HL Q A mUORESCENCE $UY U SENSANDD 3WEERS  4HISPROPERTY T ISTH T E BASIS F IDELYUSED mUOROMETRICMETH T ODSFORNON F INVASIVE OFW MONITORINGOFTHEFUNCTIONALSTATEOFPHOTOSYNTHETIC D INDJEE  4HE APPARATUS0APAGEORGIOUAND'OV PROPERTIESOFF 1! AREDESCRIBED IN DETAIL IN#HAPTER 0ETROULEASAND#ROFTS  $
&OFSHFUJDT
PG 1 r 2"mr 'PSNBUJP U O BOE 1SPUFJO
3FMBYBUJPO
1SPDFTTFT 1 !THOROUGHANALYSISOFT F HEPHOTOSYNTHETICORGANISMS AS CONVERTERS OF LIGHT INTO 'IBBS ENERGY HAS TO BE PERFORME F D WITHINTHEFRAMEWOR F RKOF IRREVERSIBLETHER MODYNAMICS "ELL AND'U D DKOV  FORAREV F IEW SEE7ALZ  4HISCHALLENGINGPROBLEMSHOULD F D WITH ANALYSISOFF THEENERGETICS FOR F NOTTOBECONFUSE CHARGESEPARATIONWITHINTHE2#S 0ARSON  4HE F FORMERTOPICISBEYONDTHESCOPEOFTHISCHAPTERAN D THEREFOREON F LY THELATTERONEWILL BEADDRESSED 
&OFSH S JFT
PG
UIF
*OJUJBM
1
BOE
A'JOBM r 1 2"mrr


4UBUFT 4HE ENERGETICSOFEACHPROCESSISDETERMINEDBYTHE INITIAL ANDD THE@lNALSTATES4HE INITIALSTATEOFF @STABLE CHARGESEPARATION IN03))ISTHE LOWESTELECTRONICALLY EXCITEDSINGLETSTATEFORMEDAT0AFTERPHOTONAB SORPTIONANDD SUUBSEQUENTRAPIDD RADIATIONLESSEQUILIBRA TIONVIAA VIBRATIONALRELAXATION4HE 1Y TRANSITIONAT NMCORRESPONDSWITHANENERGYOFE64HERAPID DISTRIBUTIONOFF THEEXCITEDD STATEBETWEENTHEENERGETIC LEVELSAVAILABLE BYA BEDD OFP F IGMENTSWITH VIRTUALLY ISOENERGETIC1Y TRANSITIONSATROOMTEMPERATURETHE F G THE#HL AMOLECULESARE ENERGY DIFFERENCESAMON

 COMPARABLEWITH K4 GIVESRISETOANENTROPYTERM K"LN7 WHEREK" ISTHE"OLTZMANNCONSTANTAND7 IS THE THERMODYNAMIC PROBABILITY Y )N THE SIMPLEST CASE OFF AN ARRAY OF IDENTICAL PIGMENT MOLECULES WITHNEGLIGIBLYSMALLEXCITONSPLITTINGBUTCONNECTED VIARAPID INCOHERENT&ÚRSTER TYPE%%43ECTION))) "A THEVALUEOF7CORRESPON F DSTOTHENUMBEROF PIGMENTS .#HL)NREALITY HOWEVER THELEVELSARENO Y T ISOENERGETIC INTHE03))ANTENNAAND D 7 ISSMALLER THAN.#HL%FFECTSDUETODIFFERENTSITEENERGIESCAN BE EXPRESSED TO A lRST APPROXIMATION BY USING AN @EFFECT F IVEPIGMENTNUMBER '2ENGERETAL   F HE'IBBSENERGYTHATARISESFROM F 4HE DECREASEOFT EXCITEDSTATEEQUILIBRATIONIN03))WITHANANTENNA Q F #HL 5  ISESTIMATED D TOBEABOUTnME6 SIZEOF. 'IBASIEWICZETAL  (OWEVER AS DISCUSSED BY + +RISHTALIK THECONlGURATIONALRATHERTHANTHE 'IBBSENERGY ISTHE KEYPARAMETERFORT F HEENERGETICS OFF CHARGESEPARATION4HE CONlGURATIONAL ENERGYOF  0 0ISE6 6 4HE @lNAL STATE 0s 1!ns IS LESS WELL DElNED BECAUSETHEFREEENERGYO F F THEREDOXCOUPLES0 0s AND 1!ns1! DEPENDSNOTONLYONTHECHEMI CALNATUREOFF THECOFACTORS F BUTALSOONEFFECTS F D DUETO THE PROTEIN ENVIRONMENT %Q  3ECTION6#  )N PRINCIPLE REDUCTION POTENTIALS ARE THERMODY NAMIC PARAMETERS THAT ARE DElNED BY EQUILIBRIUM CONDITIONS(OWEVER THESTATE 0s 1!ns DOESNOT ATTAINATRUEEQUILIBRIUMANDEVENTHEVALUESOFTHE Q THERMODYNAMICALLY WELL DElNED D REDOX POTENTIALS ARENOTPRECISELY KNOWN K F FOR0 0s AND 1!ns1! 3OFAR NOSTRAIGHTFORWARD REDOXTITRATIONS COULD BE PERFORME F D FOR0 F 0s AND D ONLYESTIMATIONSOF 6UPTO6AREREPORTED FORT F HISSYSTEM *URSINICAND'OVINDJEE +LIMOVETAL  2APPAPORTETAL  )NCONTRAST NUMEROUSREDOX TITRATIONEXPERIMENTS HAVE BEEN PERFORME F D OVERTHE LASTTHREEDECADESTODETERMINETHEREDOX POTENTIAL F HESE OF 1!ns1!!COLLECTIONOF DATA GATHERED FROMT F HEVALUEAROUND n STUDIESSHOWSACLUSTERINGOFT M6 nM6 M6ANDTOM6FORILLUS TRATION SEE&IG IN+RIEGERETAL  3OMEOF THESELARGEVARIATIONSCAN BEEXPLAINED BYTHElNDING THATTHEREDUCTION POTENTIALDEPENDSONTHEFUNCTIONAL INTEGRITY OFF THE7/# +RIEGERETAL  &ORA F BOUTnM6SEEMSTO ROUGH ESTIMATION AVALUEOFA BEAREASONABLEASSUMPTION)NTHISCASE AFREEENERGY F BOUTE6ISOBTAINED FORT F HE RADICAL CONTENTOFA PAIR0s 1!ns/NTHEBASISOFF THESEESTIMATES THE EFlCIENCYOFTHE@USEFULCHARGESEPARATIONIN03)) ISCALCULATED D TO BEABOUT4HISNUMBERISRATHER

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI LARGECOMPAREDD WITH THEEFlCIENCYOFC F HARGESEPARA TIONINANOXYGENICPURPLEBACTERIATHATISREPORTEDTO BEABOUTIN 2HODO D BACTERSPHAEROIDES D FORT F HE 0s 1!nsRADICAL PAIR 7ARNCKEAND$UTTON  D  4HEDIFFERENCEBETWEENTHETWOORGANISMSISALMOST ENTIRELY DUETOT D HENECESSITY OF DEVELOPINGASTRONGLY OXIDIZINGSPECIES 0s ASANENERGETICPREREQUISITE F R WATER OXIDATION FO 
&GGFD & U PGG UIF
1SP UT S UF U JO
&OWJSP S ONFOU 4HEPROCESSOFC F HARGESEPARATIONTAKESPLACEWITH F THATAREEMB M EDDED INTOATAILORED PROTEINMA COFACTORST TRIX 4HE FO F RMATION OF DIFFE F RENT STATES OFTHE REACTION SEQUENCE GIVESRISETORESPONSEOFF THEPROTEINMATRIX INATIMEDEPENDENTMANNER!NY CHARGEDENSITYRE DISTRIBUTIONWITHINTHECOFACTORS DUETOPOPULATION OFAPART F ICULARSTATENECESSARILY LEADSTOCHANGES IN THEINTERACTIONWITH THEATOMSOFF OTHERCOFACTORSAN F D THEPROTEINENVIRONMENT4HEGENERALPRINCIPLESOF F IONAL ADAPTATIONINDUCE D D BYSPATIALSHIFT F CONFORMAT OFC F HARGESEG ELECTRONANDD PROTONMOVEMENT AND THEIMPLICATIONSTOBIOLOGICALPROCESSESHAVEBEEN OUTLINEDD EARLIERBY#HRISTOPHOROVETAL !T THISSTAGE ONLYTHESTATESWILL BE DISCUSSEDD THATARE RELEVANTFORCHARGESEPARATIONBYLEADINGTO 0s 0HEOn s FORMAT F ION "ASEDD ONTHESCHEMEOFF %Q  ATHOROUGH CAL CULATIONOFTHEENERGETICSREQUIRESDETAILEDINFORMA TIONONTHEELECTRONICCONlGURATIONOFT F HE DIFFEREN F T STATESANDD THEIRINTERACTIONWITHTHEENVIRONMENT!N INDISPENSABLE ˆ BUT NOT SUFlCIENT ˆ PREREQUISITE F FORASTRA IGHTFORWAR F D ANALYSISISTHEKNOW K LEDGEOFA F HIGH RESOLUTIONSTRUCTUREOFT F HEWH W OLESYSTEM4HIS CONDITIONISNOTSATISlEDAT PRESENTFOR03))WITHA CRYSTALSTRUCTUREOFn¯RESOLUTION:OUNIET AL +AMIYAANDD 3HEN "IESIADKAETAL &ERREIRAETAL  ANDTHEREFOREONLYSOME ROUGH ESTIMATIONS CAN BE PRESENTED 4O ILLUSTRATE THECOMPLEXITYOFF THEPROBLEM INFORMAT F IONONTHE F FUNCTIONALLYANDSTRUCTURALLYMUCHBETTERCHARACTE R IZEDD "2#SWILL BEUSED FORCOMPAR F ISON 
&OFSH S FUJDT
BOE
3FMBYBUJPO
1SPDFTTFT %VSJ SJOH
UUIF -JGFUJNF
PG
1 )NGENERAL THE DIPOLEMOMENTAND NUCLEARCONlGU RATIONOFF ELECTRONICALLY EXCITEDD PIGMENTSDIFFER F FROM F THOSE OF F THE GROUNDD STATE AND THEREFORE F RELAXATION PROCESSES THROUGH THE ENVIRONMENT ARE INDUCED &URTHERMORE CHARGETRANSFERSTATESCAN F BEPOPULATED

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



INCOUPLED D PIGMENTS LIKETHESPECIAL PAIR IN"2#S (ARANETAL (8UETAL  ANDPROBABLY F "ARTER ALSOINTHEMULTIMERIC 2# 0# OF03)) ETAL 2ASZEWSKIETAL  4HERELAXATION PROCESSESWITHINTHE EXCITED STATE 0 WERE FOU F ND D TO BEULTRAFAST F (ARANETAL 9AKOVLEVETAL   )N03))THEINITIALLY FORME F D EXCITED D STATE 2# 0#

EQUILIBRATESINTHESUB PSTIMEDOMAIN$URRANTETAL F LLOWED -àLLERETAL  THAT ISMOSTLIKELY FO BYARESPONSEOFT F HEPROTEINMATRIX4HEENERGETICS OFTHISVERYFASTENVIRONMENTALRESPONSEISEXPECTED TOBE INTHERANGEOFTENSO F F ME6BUTPRECISEVALUES K 0ASCHENKOETAL  AREUNKNOWN  &OFSHFU & UJDT
BOE 3FMBYBUJUJPO 1SPDFTTFT 1 r %VSJOH UIF
-JG JGFUJNF
PG
1 G 1IFPmr !S OUTLINED IN 3ECTION 6! THE RADICAL PAIR 0s 0HEOns CANATTAINTWODIFFERENTSTATES 0s 0HEOn AND 0s 0HEOns THATARE DISTINGUISHED BY THEIRMODEOF HOLE LOCALIZATION)FF ONEACCEPTSTHE SCHEME PROPOSED 0ROKHORENKO AND (OLZWARTH D SUMMARIZED BY%Q ANENERGETIC GAP  AND OFF ABOUTME6WOULD BESUFlCIENTTOASSURETHAT THESPINDENSITYOFTHECATIONRADICALISPREDOMINANTLY LOCATED D AT0$ 3ECTION)6$  F HISPARTICULARNATUREOFF THESTATESOF !PART FROMT 0s THE DISSOCIATIONOFANEXCITONSEEFOOTNOTTE IN3ECTION)))"A INTOACATION ANIONRADICALPAIR INHERENTLYLEADSTOGENERATIONOFF SPATIALLYSEPARATED ELECTRICALCHARGES!GENERALMETHODFORTHECALCU LATIONOFF THE 'IBBSENERGYOFRA F DICALPAIRFORMAT F ION 0s0HEOns IN "2#S 6' 20 HAS BEEN PRESENTED BY 0ARSONETAL )NANALOGYTOTHISAPPROACH THE EXPRESSION FOR F 6' 20 OFF 0s0HEOns ISGIVENBYTHE SUMOFSEVERA F LCONTRIBUTIONS 6' 20  )0 n !0HEO  6% 20  6%20 —  6%IND —  6'BULK



WHERETHESUMOFF THE lRSTTWOPARAMETERS THEIONIZA TION POTENTIAL OF0 ) F D THEELECTRONAFlNITY 0 AND OF0HEO !0HEO ISTHEENERGYOFFORMATIONOFTHEIN DIVIDUA D LRADICALS 0s AND 0HEOns INTHEVACUUM D THESUMOFF THE LAST AT INlNITELARGESEPARATIONAND F FOURTERMSGIVESTHE 6'OFTHEELECTROSTATICINTERAC TION 6% 20 ISTHECHANGEOFE F LECTROSTATICENERGY WH W EN THERADICALSAREPUTTOGETHERATEQUILIBRIUMDISTANCE 6%20 — ISTHEINTERACTIONENERGYOFTHECHARGESOFTHE RADICAL PAIRWITHTHECHARGEOFF EACHATOM + OFF THEPRO

TEINMATRIX BOUNDSOLVENTWATERMOLECULESANDOTHER COFACTORS 6%IND —ISTHEINDUCEDDIPOLEINTERACTIONDUE TOPOLARIZATIONOFF ATOMS+AND 6'BULKISTHECONTRIBU TIONDUETO D INTERACTIONWITH THE BULKK SOLUTION   IS A TIME !NINSPECTIONOF%Q SHOWSTHAT 6'20 DEPENDENTQUANTITY BECAUSETHELASTTHREEPARAMETERS AREFUNCT F IONSOFF THENUCLEARCOORDINATESOFATOMS F + OFTHEPROTEINMATRIXANDTHESOLVENTMOLECULESTHAT VARY WITH TIME DUETORE D LAXATIONPROCESSES!SACON SEQUENCE MULTIPHASICKINETICSEMERGE VIDEINFRA F )N PRINCIPLE CALCULATIONSONTHEBASISOF%Q CANBE PERFORME F D BYUSING QUANTUMMECHANICALMETHODS IN COMBINATIONWITH MOLECULARDYNAMICSSIMULATIONS F RMATION WITHTHIS 4 ACHIEVE DETAILED ANDD RELIABLE INFO 4O APPROACH ISAGREATSCIENTIlCCHALLENGE)TISCLEARTHAT ATPRESENTTHISGOALCANNOT BEREACHED FOR03))PART F LY BECAUSEOFOURRATHERLIMITEDKNOWLEDGEONTHESTATIC D HAPTERS STRUCTUREOFF THISCOMPLEX 3ECTION)6!AND# n COUPLEDD WITHTHECOMPLEXNATUREOFF 03))/N THEOTHERHAND FOR"2#SSUFlCIENTDATAISAVAILABLE F FORSUCCESS F LATTEMPTSTOCALCULATE 6' 200 FU "ASEDD ONTHESTATICCRYSTALLOGRAPHICSTRUCTURE VAL UESOFABOUTnME6WERECALCULATEDFORTHEENERGY GAP BETWEEN0 ANDD THE RADICALPAIR0s"0HEO!ns AND F HESUB U SEQUENTRELAXATION 0AR ABOUTnME6 FORT SONETAL  4HESEVALUESHAVETOBECOMPARED WITH ESTIMATES GATHERED FFROM EXPERIMENTAL RESULTS OF DELAYED mUORESCENCETHATLEADD TOCORRESPONDING 6'  VALUESOFnME6ANDnME67OODBURYET AL /GRODNIK K ETAL  !RATHERSATISFYING F THEOVERALL 6'  BETWEEN0

CORRESPONDENCEEXISTSFORT ANDTHE@RELAXEDRADICAL PAIR0s"0HEO!ns (OWEVER F THEENERG R ETICSOFF THERELAXATION T ERESULTSOBTAINED FORT TH F IONALIZE PROCESSESEXHIBITSTRIKING DIFFERENCES4ORAT THEDISCREPANCIES POSSIBLELIMITATIONSOFTHEORETICAL ANDD EXPERIMENTAL APPROACHES HAVETO BETAKENINTO CONSIDERATION4HEORETICALCALCULATIONSAREBASEDD ON THESTRUCTUREDERIVEDFROM8 RAYDIFFRACTIONPATTERN ANALYSESTHATARENOTPRECISEENOUGHASSHOWNBY A COMBINED QUANTUMMEC Q HANICALMOLECULARMECHANI CALMETHODPROPOSEDFORRElNEMENTOFTHECOFACTOR GEOMETRY (UNTERETAL  &URTHERMORE CALCULA TIONONTHEROLEOFNUC F LEARMOTIONSFORT F HEELECTRON TRANSFER REACTION ARE MAINLY BASED ON SPIN BOSON DISPERSED POLARON MODELS FOR F A RECENT DETAILED STUDYONTHEPRIMARY%4STEPS IN"2# SEE0ARSON AND7ARSHEL  ANDREFERENCESTHEREIN 4HEREFORE THEDATAOBTAINEDD CRITICALLY DEPENDD ONTHERELIABILITY OF F THE UNDERLYING MODEL /N THE OTHER HAND D THE RELEVANCEOFVALUESBASEDONDELAYEDmUORESCENCE MEASUREMENTSARERESTRICTED BY THELIMITATIONSINTHE

 INHERENTTIMESCALEOFT F HEEXPERIMENT)NTHISCASE THETIMESCALEISLONGERTHANTHECHARGERECOMBINA TIONSTEP WHI W CH INTURN ISSUB U STANTIALLY LONGERTHAN THE CHARGE SEPARATION STEP &URTHERMORE IN "2#S THEPRIMARYCHARGESEPARATIONGENERATESTHERADICAL PAIR0s"#HL!ns THATSUB U SEQUENTLY LEADSVIAAVERY FAS F T REACTIONTO0s"0HEOns4HE 6'VALUE FORT F HISREACTION WAS SHOWNTO BE MUCH SMALLER WITHVALUES OFTENS OFF ME6(UPPMANNETAL  ,IMITATIONSOFT F HE TIMERESOLUTIONARELESSCRITICAL FOR F DATAGATHERED FROM F PROMPTmUORESCENCE-àLLERETAL  6ARIATIONS OFF THE 6' 20 VALUESCOULD D ALSOORIGINATE FROMT F HEUSE F PLEMATERIAL)TREMAINSTOBECLARI OF DIFFERENTSAM lEDASTOWHATEXTENTTHECHROMATOPHOREMEMBRANE AFFECTST F HEENERGETICSOFT F HERELAXATIONPROCESSES IN THE"2#S !PARTFROMTHEPROBLEMSINOBTAININGPRECISEDATA R ETICSOFF RELAXATIONPROCESSES ANOTH T ERWAY A ONTH T EENERG TODISCUSSTHISTOPICAPPEARSTOPROVIDEINTERESTING CONCLUSIONS &OR AN OPTIMAL ELECTRON TRANSFER RATE THE REORGANIZATION ENERGY NEEDS TO MATCH THE FREE F ENERGYGAP SEE%Q 4HE FREEENERGY F LOSS DUETO D NUCLEARREARRANGEMENTONTHEULTRAFASTTIMESCALEOF THEINITIALELECTRONTRANSFERSTEPMUST F BEVERYSMALL BOTH DUETOT D HE LIMITED D NUCLEARRELAXATIONRATESAND THELOWPOLARITYENVIRONMENT4HUS ANULTRAFASTRATE F HEPRIMARYPHOTOSYNTHETICELECTRONTRANSFERSTEP FORT F F HEFREEENERGY F LOSS ISVERY CANONLYBEACHIEVED IFT SMALL!CCORDINGLY THEACTUALFREEENERGYDIFFERENCE BETWEENTHEEXCITED D STATEAND THE INITIAL UNRELAXED RADICAL PAIRMAYBEARATHERNARROWGAP4HIS IDEAIS SUPPORTEDBYEARLYEXPERIMENTS WHICHSHOWASEEM INGLYY CONTRADICTORYRESU Y LT ˆ IE UPONLOWERINGOFT F THE TEMPERATURETHEFREEENERGY F LOSSAPPROACHESA LIMIT OFCLOSETOZERO7OODBURYAND0ARSON   #OMPARED D WITHTHESTUDIESON"2#S SUB U STANTIALLY LESS INFORMAT F IONISAVAILABL A E FOR0 F 3))6ALUESOFF ABOUT nM6WEREGATHEREDFROMANALYSESOFmUORESCENCE DECAY A KINETICSOF INTACT03))CORECOMPLEXES6ASILEV ETAL  4IME RESOLVEDP D HOTOVOLTAGEMEASURE MENTSWITHUNSTACKED03))MEMBRANEFRAGMENTSAND DATAEVALUATIONWITHINTHEFRAMEWOR F RK OFT F HEEXCITON RADICAL PAIREQUILIBRIUMMODEL LEAD D TOAVERYSIMILAR 6' 20VALUEOFABOUTnME64HISENERGYGAPWAS SHOWNTOBEMARK R EDLY DIMINISHED BY ABOUTME6 BYTHENEGATIVECHARGEON1!ns WH W EREAS FORMAT F IONOF 1!( LEADSTOMARGINALEFFECTSATMOST'IBASIEVICZ ETAL  4HE INmUENCEOFT F HENEGATIVECHARGE ns OF 1!ns ONTHEPROPERTIESOFF 0HEO$  ALSOGIVESRISETO A PRONOUNCEDELECTROCHROMIC BANDSHIFTTHATIS WELL K KNOWNAS#RE mECTING THEPOPULATIONOFT F HE1!ns

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI REDOXSTATEVAN'ORKOM   "ASEDONmUORESCENCEQUANTUMYIELDANDLIFETIME MEASUREMENTSOFF $$#YT B PREPARATIONS A 6'  VALUEOFnME6AT+WASOBTAINEDFORTHEENER R GYGAPBETWEEN 2#nCORE AND0s 0HEOns RELAXED X W E FROMT WHIL F HENSLIFET F IMECOMPONENTA 6' 20 OF nME6WASESTIMATED "OOTH ETAL  ! DE TAILEDLOWTEMPERATURESTUDYOF CHARGERECOMBINATION mUORESCENCEANDD RADICAL PAIRRELAXATIONREVEALEDD THAT lVEDIFFERENTRE F LAXATIONSTEPSCANBEDISTINGUISHED WITHINCREASINGGAPSOFFREEENERGY+ONERMANNET AL   4HE DIFFERENCE F OF FFREE ENERGY BETWEEN THEEXCITEDD STATE 0 ANDTHElRSTRADICAL PAIR20 6'  WASSHOWNTOBEONLY nME6AT+AND D 6' TOTAL BETWEEN 0 ANDD THE THETOTALENERGY DROP @RELAXEDRADICAL PAIRWASABOUTME6WITHINTHE AVAILABLETIMESCALEOFMEASUREMENT!NANALOGOUS F D ATROOMTEMPERATUREBYUSING ANALYSISWASPERFORME (IS TAGGED03))CORECOMP D LEXESFROM F 3 3YNEC HOCYSTIS WITHHIGH/ EVOLVINGCAPACITY6ASILEVETAL  )NPERFECTA F GREEMENTWITH THERESULTSOF+ONERMANET F F DICAL AL  RELAXATIONSTEPSWITHINA LADDEROFRA PAIRSTATESWEREINFERREDTOTAKEPLACEBUTWITHATOTAL 6' OFF nM6 THISVALUENICELY lTSWITH EARLIER DATA VIDE SUPRA 4HE QUANTITATIVE 6'O DIFFERENCE F BETWEEN THE TWO REPORTS IS NOT SURPRISING BECAUSE F HE SAMPLEMATERIALANDD TEMPERATUREWEREDIFFERENT4 KEYPOINTOFF THESESTUDIES ISTHECLEARDEMONSTRATION OFTHEIMPORTANCEOF PROTEINRELAXATIONMODESFORTHE STABILIZATIONOFF THERADICAL PAIR!T LOWTEMPERATURE U STANTIALLYS L LOWERDUE D T ESERELAXATIONPROCESSESARESUB TH TOTHEDECREASEDMOBILITYOFTHEPROTEIN/FCENTRAL RELEVANCE ISTHEDEMONSTRATIONTHATTHERADICALPAIR ENERGETICSISCHARACTERIZED BYASEQUENTIALRELAXATION VIAASERIESOFRADICALPAIRS ANDNOTBYSAMPLEHET EROGENEITY WITHAPOPULATION DISTRIBUTIONOFREACT F ION F F FREE CENTERS CHARACTERIZED BY INHERENTLY DIFFERENT ENERGYGAPS+ONERMANNETAL 6ASILEVETAL F RGY  4HEAPPARENTDISCREPANCY BETWEENFREEENER GAPSOFT F HEINITIALAND lNAL STATEOFT F HERADICAL PAIR AT ROOM AND LOW TEMPERATURES ORIGINATES FROM THE LARGELY DIFFERENT F PROTEIN CONFORMAT F IONAL RELAXATION RATESATTHEDIFFERENTTEM F PERATURE4HUS ITIS DIFlCULT TOMARKTHEINITIALRADICAL PAIRSTATEANDDETERMINEITS F FREEENER GY ATROOMTEMPERATURE2EGARDLESSOFT F HIS PROBLEM TH T ERESULTSSUGGESTTH T ATTH T E INITIAL FREEENERGY F GAPISLIKELYTOBESMALLANDNOTSTRONGLYDEPENDENT ONTEMPERATURE4HISSUPPORTSTHENOTIONTHATCHARGE SEPARATION IN03))MOSTLIKELY INALL 2#S COMPRISES ARATHERSMALLINITIAL'IBBSENERGYGAPPERHAPSONLYA F FEWME6 INORDERTOOBTAINTHEHIGH RATESOFF ELECTRON

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS TRANSFERACCOR F DINGTO-ARCUSTHEORY INAMEDIUMOF LOWPOLARITYVIDESUPRA 4HISLEAVESTHEINITIALRADI CALPAIR@VULNERABLEFORC F HARGERECOMBINATION4HUS A FUN F DAMENTAL FUNCT F IONOFF THEPROTEIN ISTO PROVIDE RAPIDANDEFlCIENT RELAXATION CHANNELS TO LOWERTHE F FREEENER GY GAPONCETHERADICALPAIRISFORME F D IN K REACTIONS )T IS ATTRACTIVE TO ORDER TO PREVENT BACK HYPOTHESIZETHATPROBABLYTHEPROTEINENVIRONMENT AROUND D THEREACTIONCENTERHASBEENOPTIMIZED DUR D ING EVOLUTIONTOFU F LlLL THISACTIVEROLE Q RADICAL PAIRRELAXATION 4HEMODELOFASEQUENTIAL F HARG R ESEPARATIONCANBE GENERALIZED INASCHEME AFTERC WHERE THE THERMODYNAMIC PARAMETERS BECOME TIME DEPENDENT 0ELOQUIN ET AL  (OLZWARTH AND D -àLLER  4HISBEHAVIORISFUN F DAMENTALLY DIFFERENTFROMSTATICDISTRIBUTIONSOFFREEENERGYDIF F F FERENCES)TREmECTSTHEESSENTIALTIME DE PENDENTROLE F IONAL RELAXATIONINSTABILI A ZING TH T E OF PROTEINCONFORMAT INTERMEDIATERADICAL PAIRSTATES!CLEARILLUSTRATIONOF THISPRINCIPLEISPRESENTEDINASTUDYONTIME RESOLVED mUORESCENCE IN CHROMATOPHORES FFROM ANOXYGENIC PURPLEBACTERIA-àLLERETAL  $ATAGATHERED OVERAWIDERANGEOFTEMPERATURESn+ AND F FROMMUTANTSW WHERETHEREDOXPOTENTIAL OFF 00 IS F D BYUPTOM6 AREWELL DESCRIBED BYASE SHIFTE QUENCEOFRELAXATIONSOFTHE RADICAL PAIR0s"0HEOns Q INCLUDING THREE DIFFERENT F STATES +ATILIENE ET AL  )NSUMMARY PROTEINSPLAYANACTIVEROLE IN SUPPORTINGCHARGESEPARATIONBYPROVIDINGEFlCIENT RELAXATION CHANNELS THAT SPAN A WIDE TIME DOMAIN F FROM PICOSECONDS UP U TO NANOSECONDS AND PERH R APS EXTENDINGINTOTHEMICROSECONDRANGE 
&OFSHFUJDT
BOE
5VSOPWFS
PG 1 r 2"mr 4HE RADICAL PAIR 0s 1!ns ISSUFlCIENTLY STABLETO PERMITEFlCIENT 0s REDUCT D ION BY9: FO F LLOWED BY OXIDATION OF THE7/# &OR A CONTINUOUS TURNOVER 03)) HASTOBEREOPENED BYOXIDATIONOF 1!ns4HIS ISACHIEVED BYSTEPWISEELECTRONTRANSFERTOANOT F HER PLASTOQUINONE Q MOLECULE 1" THAT TRANSIENTLY BINDS TOASPECIALPOCKET IE THE 1" SITE SEE#HAPTER 0ETROULEASANDD #ROFTS F 4HEREEXISTSEVERAL LINESOF EVIDENCETHATTHEELECTRONTRANSFERSTEP FROM 1!ns TO 1" REQUIRESSTRUCTURAL mEXIBILITY OFT F HEPROTEINMATRIX H STUDIES OFF REACTION CENTERS 8 RAY CRYSTALLOGRAPHY FFROM THE PURPLE BACTERIUM 2B SP S HAEROIDES LED D TO THEPROPOSAL THAT COMPARED D TOTHEPOSITIONOF1 F " IN THEDARK R ADAPTEDSTATE T D HEUBI U QUINONE HEADGROUP D IS DISPLACEDBYABOUT¯ANDROTATEDBY AROUND THEISOPRENOIDD SIDECHAINWH W ENTHESAMPLESAREIL

 LUMINATED 3TOWELL ET AL  4HIS PRONOUNCED STRUCTURALCHANGEWASSUGGESTEDTOLIMITTHERATEOF 1!ns REOXIDATION BY1"(OWEVER THIS IDEAHASBEEN QUESTIONED 18UETAL  ANDD SEEMSTOBEOB SOLETE ACCORDING TO STEP SCAN &4)2 MEASUREMENTS 2EMY M AND'ERWERT  D ANDD TIMERESOLVEDD 8 RAAY H LYSIS"AXTERETAL  7I 7 TH CRYSTALLOGRAPHYANA RESPECTTOTHECORRESPONDINGREACTIONIN03)) INDIRECT LINESOFF EVIDENCESHOWTHATTHEFORMAT F IONOF 1"ns IN 03))ALSOSTRONGLY DEPENDSONTHE CONFORMAT F IONAL mEXIBILITY OF THE PROTEIN ' 2ENGER ET AL  'ARB R ERSETAL 2EIFART F H AND'2EN D GER 0 + HNETAL  "ULKWATERMO +à K LECULESPLAYA KEY ROLEAS@PLASTICIZERSASILLUSTRATEDIN03))MEMBRANE F GMENTSOF DIFFERENTEXTENTO FRA F F DEHYDRAT D ION+AMIN SKAYAETAL 0+àHNETAL  4HENATUREOF THESTRUCTURALTRIGGERINGOF1!n REOXIDATION REMAINSTO BECLARIlED FOR F BOTHTHE"2#AND03)) D F STEPWISE ELECTRON REDUCT D ION WITH 1!ns AS !FTER REDUCTANT 01( IS RELEASED FROM THE 1" SITE AND BECOMESREPLACED BY ANOTHERMOLECULE FROMT F HEPLAS TOQUINONEPOOL #ROFTSAN F D 7RAIGHT #HAPTER  0ETROULEASAND#ROFTS &ORA HIGHEFlCIENCYOF D IONPOTENTIALOF 1!ns HASTOBE THISPROCESS THEREDUCT SUFlCIENTLYLOW)N03))COMPLEXESWITH INTACT7/# AVALUEOFABOUTnM6 SEEMSTOBEREASONABLE 3ECTION6# (OWEVER ASIGNIlCANTUPSHIFTO F FT F HE REDUCT D IONPOTENTIAL BYABOUTM6WASFOUN F D IN SYSTEMSLACKINGTHE7/#+RIEGERETAL  !SA CONSEQUENCE THEDRIVING FORCE F F 1"1"ns REDUCT FOR D ION BY 1!ns ISDRASTICALLYDIMINISHEDD AND A LARGEFRACT F ION OF 03)) CENTERS BECOMES @CLOSED 4HIS ENERGETIC F IS ASSUMEDD TO BE OF F PHYSIOLOGICAL RELEVANCE SHIFT F FORNEW LYSYNTHESIZEDD 03))COMPLEXESTHATDONOT CONTAIN A FUNCTIONALLY COMPETENT 7/# 3TRASSER K ION ANDD 3IRONVAL  )NTHISCASE THEBACKREACT BETWEEN 0s AND 1!ns ISASSUMEDD TOPROTECTAGAINST DESTRUCTIVEACTIONBYTHESTRONGLYOXIDIZING 0s RADICAL '2ENGERANDD 7OLFF  F /FT F HE DIFFE F R ENTPATHWAYSFORT F HEBACKREACT K ION3ECTION6) ONE W CH ACTS AS SENSITIZER FO F R SINGLET LEADS TO 0 WHI OXYGENFORMAT F ION!SHIFTO F F THEREDUCT D IONPOTENTIAL OF 1!ns TOWARDSAMOREPOSITIVEVALUEDIMINISHESTHE PROBABILITYOF 0 FO F RMATION ANDTH D EREFO F RE LEADS TO FURT F HER PROTECTION OFF 03)) COMPLEXES WITHOUT 7/# TO RAPID 7 D PHOTODEGRADATION +RIEGER ,ISZKAY AND2UTHERFORD  

 7*
'PSXBSE
#BDL
BOE 4JEF
3FBDUJPOT
PG 3BEJDBM *PO 1BJS 1 r 2"mr )N3ECTIONS6!AN 6 D 6"T 6 HE KINETICSOFF THEFORWAR F D REACTIONS LEADING TO 0s 1!ns WERE DESCRIBED (OWEVER ALL OF F THESE STEPS CAN BE REVERSED &UR THERMORE THE POPULATIONOFSTATES F LIKE 0ORTHE EXTENT OF COMPETING REACTIONS EG OXIDATION OF COMPONENTS LIKE #HL: #AR #YT B SEE #HAPTER D ONTHEFUNCT F IONAL STATEOF  &ALLERETAL DEPEND THE03))COMPLEX "
1 r 2"mr 3FDPNCJO J BUJP U O BOE 1 ' 'PSNBUJP O 4HE LIFET F IMEOFT F HERADICAL PAIRR 0s 1!ns IS LIMITED T T BYTHE TUNNELINGRATEOFTHEDIRECTELECTRONTRANSFERTHA LEADSTO01! IN ITSGROUND D STATE4HERATECONSTANT OFF THISREACTIONDEPENDSON 6'  h AND D THEEDGE TO EDGEDISTANCE20 1 %Q 4HEDISTANCEBETWEEN THE SPIN DENSITY CENTERSOFT F HERADICALS 0s AND 1!ns HAVE BEENDETERMINED BYUSINGPULSED%02SPECTRO D S COPYANDAVALUEOF¯WASOBTAINED:ECHET D ONTHESE lNDINGSAND D RECENTCRYSTAL AL  "ASED STRUCTUREDATA :OUNIETAL+AMIYAAND3 D HEN "IESIADKAETAL &ERREIRAETAL  AREALISTICEDGE TO EDGEDISTANCEOFABOUT¯ CAN BE DERIVED SEEALSO&IG 7I 7 THINTHEFRAMEWOR F RK OFTHEEMPIRICAL RATECONSTANT DISTANCERELATIONSHIP -OSERAND D $UTTON  ARATECONSTANTOFT F HEORDER OF MS n ISOBTAINED D THATREPRESENTSTHEMAXIMUM VALUE FO F RTHE CASE OF n6'   h 4HE ACTUAL VALUE OF THEMAXIMUMRATECONSTANTDEPENDSONTHEPRECISE D OF PARAMETER ` %Q  VALUESOFF THE DISTANCEAND !NESTIMATIONOFABOUTMS n CORRESPONDSWITHA D EXPERIMENTAL VALUEOF MS n 2EINMAN REPORTED D -ATHIS  )NSYSTEMSLACKINGA FUNCT F ION AND ALLYCOMPETENT7/#THEOXIDIZEDCOMPONENT9:OX IS SLOWLY RE REDUCE D D)F 0s CANNOTBEREDUCE D D BY9: ITRECOMBINESWITH 1!ns WITH A DOMINATING KINETICSOF n +S (AVEMANAND-ATHIS '2ENGER AND D 7OLFF  F 4HISREACTIONISABOUTONEORDER F HANTHE DIRECTELECTRONTRANSFE F R OFF MAGNITUDE FASTERT F OM 1!ns TO 0s4HEREFORE An +S KINETICS FR INDICATETHAT INTHISCASE THE DOMINATINGPATHWAY A OFF THE RECOMBINATION REACTION BETWEEN 0s AND 1!ns ISTHE THERMALLYACTIVATEDUPHILLREVERSALOFTHE F FORWAR D REACTION LEADING TO INTERMEDIARYPOPULATION F IMEOFF n +S OFF STATE0s 0HEOns4HELIFET ISNOTMARKEDLYAFFECTEDBYTHEREDOXSTATEOF 9: ISVIRTUALLY INDEPENDENTOFP( F IN ) P( ) AND IS

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI INVARIANTTOREPLACEMENTOFEXCHANGEABLEPROTONSBY DEUTERONS#HRISTENETAL  "ASEDONANANALY SISOFT F HE($EXCHANGEEFFECTONT F HEPROBABILITYOF MISSESINTHE PERIOD FOUROSC F ILLATION PATTERNOF mASH INDUCED/ EVOLUTION#HRISTENETAL  ITWAS F D THAT FFOR 03)) COMPLEXES WITH INTACT ALSO INFERRE 7 7/# AT LEAST IN PLANTS THE n +S KINETICS DOMINATETHEPATHWAYOF0s1!ns RECOMBINATION F HEFORWAR F D REACTION WITH 0s VIATHEREVERSEOFT 0HEOns ASINTERMEDIATE4HECONTRIBUTIONOFTHEINDI RECTPATHWAYVIA 0s 0HEOns HAS BEEN ESTIMATEDD TO BEALMOSTOFT F HETOTAL RECOMBINATIONOF 0s 1!ns IN 74CELLSOF 3YNECHOCYSTIS 2APPAPORTETAL  4HISROUTETHATALSODOMINATESTHERECOMBI NATION REACTION IN74 CELLS OFF THE GREEN ALGAE # # R HARD REIN R TII #UNI ETAL  CAN LEADD TOSIGNIlCANT POPULATIONOFTHETRIPLETSTATE 0 ! mUOROMETRIC STUDY ON LEAVES FROM F !RABIDOPSIS D THALIANA WITH F HE MODIlED LIPIDD CONTENTREVEALEDD THATTHEEXTENTOFT RECOMBINATIONREACTIONINCREASESINMUTANTSLACKING F ET AL THE LIPID DIGALACTOSYLDIACYLGLYCEROL 3TEFFEN  4HIS lNDING INDICATESTHATSPECIlC LIPIDSARE OFFUNCTIONALSTRUCTURALRELEVANCEFOR03 )) # 1 r 1IFPmr 3FDPNCJO J BUJPO U 4HECHARGESEPARATIONFROMT F HEELECTRONICALLYEXCITED T E SPINMULTIPLICITYAND SINGLETSTATE 0 PRESERVESTH GENERATESA SINGLETSTATERADICALPAIR ;0s 0HEOns= 4HETWOSTATESEQUILIBRATEVIAANEXCITON RADICALPAIR EQUILIBRIUM 3ECTION6! BUTTHETOTAL POPULATIONOF T THESESTATESRAPIDLYDECAYSDUETOCOMPETINGREACTIONS 5NDER PHYSIOLOGICAL CONDITIONS THE MOST EFlCIENT REACTIONISTHEREOXIDATIONOF 0HEOns BY1! VIAAPS KINETICS3ECTION6" (OWEVER IN03))COMPLEXES F IONAL EITHERDEPRIVEDD OF 1! ORWITH 1! INANONFUNCT REDUCE D D STATE THE MOST EFlCIENT REACTION CHANNEL IS BLOCKED AND ;0s 0HEOns= DECAYSVIAALTERNA TIVEPATHWAY A S)NTHISCASETHE LIFET F IMEOF ;0s 0HEOns= ISSIGNIlCANTLYLENGTHENED2ECOMBINATION D FOR 0s TIMES OFTHE ORDER OF  NS WERE OBTAINED 0HEOns IN $$#YT B PREPARATIONS $ANIELUS K L ETAL 4AKAHASHI ETAL +URRECKETA A 4HESESAMPLESLACK1! )N03))COMPLEXES CLOSED BY FORMAT F IONOF 1!ns THERECOMBINATIONTIME ISSIGNIlCANTLYSHORTER%CKERTETAL 3CHATZET AL  ,EIBLETAL 2OELOFSAND(OLZ WARTH  %SSENTIALLY THREEREACTIONPATHWAY A SOF 0s 0HEOns DECAY HAVETOBETAKENINTOCONSIDER ATIONI BACKREACTIONTOTHEELECTRONICALLYEXCITED F LLOWED BYRADIATIVEEMISSION SINGLETSTATE 0 FO

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS



AND D NONRADIATIVEDISSIPATION II SPIN DEPHASINGIN  ;0s 1!ns= UNDERFORMATIONOFTHE TRIPLETRADICAL PAIR ;0s 1!ns= AND D SUUBSEQUENTRECOMBINATIONINTO THETRIPLETSTATE 0AND III DIRECTRECOMBINATION OF ;0s 0HEOns= INTOTHEGROUNDSTATE A SONLYTHE 0 !MONGTHESECOMPETINGPATHWAY F FORMAT ION3ECTION)6# ISOFF PHYS H IOLOGICAL RELEVANCE BECAUSE#HLTRIPLETSGIVERISETOSINGLETOXYGEN6G/ VIATHESENSITIZED D REACTION 

#HL  YG/ A #HL3 6G/



4HESPECIES 6G/ ISVERYREACTIVEANDLEADSTOOXIDA TIVEDEGRADATIONREACTIONS )T ISSTILL AMATTEROF DISCUSSIONASTOWH W ATEXTENT THENATUREOFF A@CLOSED03)) BYEITHERLACKING1! OR POPULATIONOFSTATES 1!ns OR1( AFFECTTHEEXTENTOF  0FORMAT F ION4HISPROBLEMWILLNOT BEDISCUSSED HEREHOWEVER SEE$INERETAL  #HLOROPHY H LL TRIPLETSINTHEANTENNAAREMOSTEFlCIENTLYQUENCHED BY RAPIDD TRANSFERTO#ARW F ITHARATETHATISMUCH FASTE F R THAN#HL FORMAT F ION3CHÚDELETAL  )NCONTRAST INTHE CASE OF 0THISPROCESSISINEFlCIENTDUETO THEREQUIRED LARGE DISTANCESBETWEEN0AND#AR D 3ECTION)6$ AND D THEPHYS H IOLOGICALLYRELEVANTROLE OFTHE TWO ` CAROTENESINTHE $$ HETERODIMER IS PREDOMINANTLYTHEQUENCHINGOF 6G/ GENERATED D VIA POPULATIONOF 04ELFER  F )NADDITION THE ` CAROTENES ALSO PARTICIPATE AS ELECTRON SHUTTLES IN PROTECTIVEELECTRONTRANSPORTPATHWAY A SINTHE03)) F IONOFWATERC F LEAVAGE IS COREWH W ENTHENORMAL FUNCT DISTURBED4ELFER 6ASILEVETAL $IRECT MEASUREMENTS OF F THE ELECTRONIC DECAY A CONSTANT OF D POLYENES` ¯n SEE3ECTION))) CAROTENOID " %Q (EETAL  SUPPORTTHEIDEATHAT ` CAROTENEACTSASAN@ELECTRONICWIRE4HE IMPLICA TIONOF 0FORMAT F IONFOR F PHOTODAMAGEAREBEYOND THESCOPEOFTHISCHAPTERANDWILLNOTBEDISCUSSED HEREHOWEVER SEE+RIEGER ,ISZKAY A AND D 2UTHERFOR F D D D  #HOWAND #HAPTERS 0OGSONETALAND !RO  $ 3FEVDUJP J O
PG 1 r 4HEEFlCIENTEXPLOITATIONOFF THERADICAL PAIR0s 1!ns ASTHEDRIVINGFORCE F F FORWATERC LEAVAGECAN BEONLY ACHIEVEDBYFASTELECTRONTRANSFERFROM9: TO 0s )N SYSTEMSWITH INTACT7/#THEKINETICSOFT F HISREACTION ARESURPRISINGLYCOMPLEXWITHATLEASTTHREECOMPO NENTSWITHRATECONSTANTSOFnNS n ABOUT D TENSOF MICROSECONDS 3CHILSTRAETAL  NS n AND

#HRISTENETAL  !SINTHECASEOFLIGHT INDUCED CHARGESEPARATION3ECTION6# THESEMULTIPHASIC KINETICSAREPOSSIBLYY AREmECTIONOFF THEEFFECTO F F PROTEIN RELAXATIONONELECTRONTRANSFERREACT F ION IN BIOLOGICAL SYSTEMS#HEREPANOVETAL '2ENGER  F HERDETAILSOFT F HE 0+àHNETAL  &ORFURT PROPERTIESANDD REACTIVITYOFF 9: SEE#HAPTER 7** $PODMVEJOH
3FNBSLT
BOE
'VUVSF 1FSTQFDUJWFT )NTHISCHAPTERTHE PRINCIPLESOF%%4AN F D %4 HAVE BEENSUMMARIZEDDESCRIBINGOURCURRENTKNOWLEDGE ONTHEENERGETICS KINETICSANDD MECHANISMOF LIGHT INDUCE D D CHARGESEPARATIONIN03))4HESIMILARITIES IN THEGENERALFUNCTIONALANDSTRUCTURALORGANIZATIONOF THISPROCESSWITH THE2#SOFANOX F YGENICNON OXYGEN EVOLVING PURPLEBACTERIAAREOUTLINED/NTHEOTHER HAND STRIKING DIFFERENCES EMERGE FROM THE INDIS PENSABLEPREREQUISITEOF GENERATINGELECTRONHOLESOF SUFlCIENTLYSTRONGOXIDIZINGPOWERTOPERMITWATER OXIDATION4HEREFORE THEUNDERSTANDINGOFTHENATURE ANDD THEPROPERTIESOFT F THEPHOTOACTIVECOMPONENT0 ISOFF CENTRALRELEVANCE4HEMOSTIMP M ORTANTCONCLUSION ISTHEIDEATHAT0IS NOTASINGLECHROMOPHOREOR SPECIALPAIRBUTA MULTIMERICPIGMENTCOMPLEXWITH THESURROUNDINGPROTEINTHAT FORMSAN F INTEGRAL AND DECISIVEPARTFORITSFUNCTIONALPROPERTIES4HUS THE F BYPROTEIN REGULATIONOFF ELECTRONTRANSFERPROCESSES DYNAMICS ISOF FUN F DAMENTAL RELEVANCEFORA F DEEPER UNDERSTANDING OF STRUCTURE FUNCTION RELATIONS NOT ONLY INPHOTOSYNTHETICREACTIONCENTERS BUTBIOLOGICAL SYSTEMSINGENERAL4HE DISTINCTIONBETWEENCOFAC F TORSANDPROTEINMATRIXAPOPROTEIN APPEARSTOBE A FUT F ILE CONCEPT FFOR A DEEPER UNDERSTANDING!S A Q ITSEEMSMOREAPPROPRIATETOCONSIDER CONSEQUENCE ENZYMES IN GENERAL AND THE 0HOTOSYSTEMS IN PAR TICULARASSPECIALLY TAILOREDD MOLECULARMACHINES' 2ENGER  4HISCONCLUSION ISCLEARLY ILLUSTRATED BY THE DRAMATIC EVOLUTIONARY JUMP THAT OCCURRED WITHINARATHERNARROW GEOLOGICTIMEWINDOWINTHE TRANSITION FFROM THE 2#S OFF ANOXYGENIC BACTERIA TO THE OXYGEN EVOLVING 03))!S A RESULT COMPARED WITH THE"2#S THECOMPLEXITY OF03)) F DRASTICALLY INCREASEDD ASREmECTED BYTHENUMBEROFAT F LEAST ANDPOSSIBLYMORETHAN DISTINCTSUBUNITSWITH F DEVELOPMENT ONLY MINORCHANGESATALL LEVELSOF03)) F FROMCYANO BACTERIATOHIGHERPLANTS 2AYMONDD AND "LANKENSHIP  4HEUNRAVELINGOFTHEPRINCIPLES OF FUNCT F IONAL @GIANTMOLECULESANDD THEIRINTERACTION

 WITHREGULATORYSUB U UNITS ISAGREATAND FASC F INATING CHALLENGEFORFUTURERESEARCH "DLOPXMFEHNFOUT 7E WOULD LIKE TO THANK4 .OGUCHI FOR PROVIDING 7 UNPUBLISHEDRESULTSONCALCULATIONSOFTHEREDOX PO TENTIAL OF0 F 0 *+ERNFOR& F IG 42ENGER F HELPFU FOR F L DISCUSSION 0 +àHN FFOR PREPARING THE ELECTRONICVERSIONSOF&IGURESnAND'OVINDJEEAND 47YD 7 RZYNSKI FORCR F ITICALREADING OFF THEMANUSCRIPT AND INVALUABLEHELP INIMPROVINGTHELANGUAGE 3FGFSFODFT !GOSTIANO! #ATUCCI, #OLAFEMMINA'AND3CHEER( F GROUPSAND SURFACTANT CHARGEIN REGULATING 2OLEOFFUNCTIONAL CHLOROPHYLL AGGREGATION IN MICELLAR SOLUTIONS * 0HYS #HEM  n !KIYAMA - -IYASHITA ( +ISE ( 7ATANABE 4 -IMURO - -IYACHI 3 ANDD +OBAY A ASHI -  1UEST FFOR MINOR BUT KEY CHLOROPHYLL MOLECULES IN PHOTOSYNTHETIC REACTION CEN TERSˆUNUSUALPIGMENTCOMPOSITIONINTHEREACTIONCENTERSOF THE CHLOROPHYLL DD DOMINATED CYANOBACTERIUM !CARYOCHLORIS MARINA 0HOTOSYNTH2ESn !NDRIZHIYEVSKAYA%' &ROLOV$ VAN'RONDELLE2AND$EKKER *0 /NTHEROLEOFTHE#0COREANTENNAINTHEENERGY F D DTRAPP INGDYNAMICSOFF 0HOTOSYSTEM))0HYS# H HEM TRANSFERAN #HEM0HYSn D 2EBANE ++  (IGH RESOLUTION OPTICAL !VARMAA 2! AND SPECTRA OFF CHLOROPHYLL MOLECULES 3PECTROCHIM !CTA ! n "ALABAN43 ,EITICH* (OLZWARTH!2AND3CHAFFNER+ !UTO CATALYZEDSELF AGGREGATIONOF2 ;%T %T=BACTERIOCHLOROPHYLL CFMOLECULESINNONPOLARSOLVENTS!NALYSISOFTHEKINETICS* 0HYS#HEM"n "ALABAN43 &ROMME0 (OLZWARTH !2 +RAU” .ANDD 0ROKHO RENKO6)  2ELEVANCE OF F THE DIASTEREOTOPIC LIGATION OF F HLOROPHYLLSIN0HOTOSYSTEM)"IOCHIM MAGNESIUMATOMSOFC "IOPHYS!CTAn A+-AND D &AAJER* -ODELSOFP F HOTOSYNTHETICCHRO "ARKIGI R MOPHORES-OLECULARSTRUCTURESOFCHLORINSANDBACTERIOCHLO RINS)N$EISENHOFER*AND.ORRIS*2EDS 4HE0HOTOSYNTHETIC 2EACTION #ENTER 6OL)) PPn!CADEMIC0RESS .EW 9ORK "ARTER ,-# $URRANT *2 ANDD +LUG $2  ! QUANTITATIVE STRUCTURE FUNCT F IONRELATIONSHIP FORT F HE0HOTOSYSTEM))REACTION CENTER 3UPERMOLECULAR BEHAVIOUR IN NATURAL PHOTOSYNTHESIS 0ROC.ATL !CAD 3CI 53!n A ER6 0AHL2 -OFFAT+AN F D "AXTER2(' 0ONOMARENKO. 3RAJ .ORRIS *2  4IME RESOLVED CRYSTALLOGRAPHIC STUDIES OF LIGHT INDUCEDSTRUCTURALCHANGESINTHEPHOTOSYNTHETICREACTION CENTER0ROC.ATL!CAD3CI53!n "ELL,.AND'UDKOV.$ 4HERMODYNAMICSOFLIGHTENERGY CONVERSION )N "ARB R ER* ED 4OPICS IN 0HOTOSYNTHESIS 4HE 0HOTOSYSTEMS3TRUCTURE &UNCTIONAND-OLECULAR"IOLOGY Y PP

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI R $ORDRECHT n +LUWER!CADEMIC0UBLISHERS "ERNARDING* %CKERT(* %ICHLER(* .APIWOTZKI!AND2ENGER'  +INETICSTUDIESONTHESTABILISATIONOFTHEPRIMARYRADICAL PAIR00HEOn INDIFFERENT0HOTOSYSTEM))PREPARATIONSFROM HIGHERPLANTS0HOTOCHEM0HOTOBIOLn "IESIADKA* ,OLL " +ERN* )RRGANG + $AND D :OUNI !  #RYSTAL STRUCTURE OF CYANOBACTERIAL 0HOTOSYSTEM )) AT  ¯ K HE-N CLUSTER0HYS#HEM#HEM RESOLUTION!CLOSERLOOKATT 0HYSn "OOTH0* #RYSTALL" 'IORGI ," "ARB R ER* +LUG$2AND D 0ORTER'  4HERMODYNAMICPROPERTIESOF$$CYTOCHROME B  REACTION CENTRES INVESTIGATED BY TIME RESOLVED mUORESCENCE MEASUREMENTS"IOCHIM"IOPHYS!CTAn "YLINA %* +IRMAIER # -C$OWELL , (OLTEN $ AND 9OUVAN D HEOPTICAL $# )NmUENCEOFF ANAMINO ACIDD RESIDUEONT PROPERTIESAND D ELECTRONTRANSFER F DYNAMICSOFAP F HOTOSYNTHETIC REACTIONCENTRECOMPLEX.ATUREn #AMARA !RTIGAS! "RUNE$AND D !LLEN*0  )NTERACTIONSBE TWEENLIPIDSAND BACTERIAL REACTIONCENTERSDETERMINED BYY PROTEIN CRYSTALLOGRAPHY0ROC.ATL!CAD3CI53!n #HEREPANOV$! +RISHTALIK,)AND-ULKIDJANIAN!9 0HOTO SYNTHETIC ELECTRON TRANSFER CONTROLLED BY PROTEIN RELAXATION !NALYSIS BY ,ANGEVIN STOCHASTIC APPROACH "IOPHYS *  n #HRISTEN' +ARGE- %CKERT(*AND D 2ENGER' 4HEROLEOF PROTONATIONSTEPSINELECTRONTRANSFERREACT F IONSIN4RIS TREATED F GMENTS0HOTOSYNTHETICAn 03))MEMBRANE FRA #HRISTEN ' 3EELIGER ! AND D 2ENGER '  0 REDUCT D ION KINETICSANDREDOXTRANSITIONPROBABILITYOFTHEWATEROXIDIS INGCOMPLEXASAFUNCTIONOFP(AND($ISOTOPEEXCHANGEIN SPINACHTHYLAKOIDS"IOCHEMISTRYn #HRISTOPHOROV ,. (OLZWARTH !2 +HARKYANEN 6. AND VAN -OURIK &  3TRUCTURE FUNCT F ION SELF ORGAN F IZATION IN NONEQUILIBRIUM MACROMOLECULAR SYSTEMS #HEM 0HYS  n #ROFTS!2AN F D D7RA IGHT#! 4HEELECTROCHEMICAL DOMAIN OFF PHOTOSYNTHESIS"IOCHIM"IOPHYS!CTAn #UNI! 8IONG, 3AYRE2 2APPAPORT&AND,AVERGNE* -ODIlCATIONOFTHEPHEOPHYTINMIDPOINTPOTENTIALIN0HOTO SYSTEM))-ODULATIONOFTHEQUANTUMYIELDOFCHARGESEPARA TIONANDOFCHARGERECOMBINATIONPATHWAYS#HEM0HYS0HYS #HEMn $ANIELIUS26 3ATOH+ VAN+AN0*- 0LIJTER** .UIJS!-AND VAN'ORK R OM(* 4HEPRIMARYREACTIONOFF 0HOTOSYSTEM)) IN THE $ $ CYTOCHROME B  COMPLEX &%"3 ,ETT  n $AVYDOV!3  4HEORY OF -OLECULAR %XCITONS -C'RAW R (ILL .EW9ORK $EISENHOFER*AND-ICHEL( 4HREE DIMENSIONALSTRUCTURE OFTHE REACTION CENTER OF 2HODOP O SEUDOMONASVIRIDIS )N $EISEN HOFER F * AND .ORRIS *2 EDS 4HE 0HOTOSYNTH T ETIC 2EACTION #ENTER R 6OL )) PP n !CADEMIC0RESS .EW9ORK $EXTER$) !THEORY OFSENS F ITISED LUMINESCENCE INSOLIDS *#HEM0HYSn $INER "! AND D 2APPAPORT &  3TRUCTURE DYNAMICS AND ENERGETICSOFTHEPRIMARYPHOTOCHEMISTRYOF0HOTOSYSTEM))OF OXYGENICPHOTOSYNTHESIS!NNU2EV0LANT"IOLn $INER"! 3CHLODDER% .IXON0* #OLEMAN7* 2APPAPORT& ,AVERGNE* 6ERMAAS7&*AND#HISHOLM$! 3ITE DI RECTED D MUTATIONSAT$ (ISAND D $ (ISOFF 0HOTOSYS TEM )) IN 3Y 3 NECHOCY CYSTIS 0##  3ITES OFF PRIMARY CHARGE

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS SEPARATION AND D CATION TRIPLET STABILIZATION "IOCHEMISTRY  n * "ERTHOMIEU# (IENERWADEL2 $INER"! "AUTISTA*! .IXON0* "RITT2$ 6ERMAAS7&*AND#HISHOLM$! #OORDINATION OFPROTONANDELECTRONTRANSFERFORMTHEREDOX ACTIVETYROSINE 9: OFF 0HOTOSYSTEM )) ANDD EXAMINATION OFF THE ELECTROSTATIC INmUENCE OF F OXIDIZEDD TYROSINE 9$ (  0HYS #HEM #HEM 0HYSn $ÚRING ' 2ENGER' 6ATER*ANDD 7ITT(4 0ROPERTIESOFF THE PHOTOACTIVECHLOROPHYLLA))INPHOTOSYNTHESIS:.ATURFORSC F H Bn $ORLET0 8IONG, 3AYRE24AND5N3 (IGHlELD%02 STUDYOFTHEPHEOPHYTINANIONRADICALINWILDTYPEAND$ % #HLAMYDOMONAS REINHARDTII MUTANTS OF 0HOTOSYSTEM )) IN # * "IOL #HEMn R ER * 0ORTER ' AND $URRANT *2 (ASTINGS ' *OSEPH $- "ARB U PICOSECONDD EQUILIBRATION OF F EXCITATION +LUG $2  3UB ENERGY IN ISOLATEDD 0HOTOSYSTEM))REACTIONCENTERS0ROC.ATL !CAD D 3CI 53!n $URRANT*2 +LUG$2 +WA3,3 VAN'RONDELLE26 0ORTER'AND $EKKER *0  ! MULTIMER MODEL FOR 0 THE PRIMARY ELECTRON DONOROF0HOTOSYSTEM))0ROC .ATL !CAD3CI53 D !  n F TWO $UYSENS ,-. AND 3WEERS (%  -ECHANISM OF PHOTOCHEMICAL REACTIONS IN ALGAE AS STUDIED BY MEANS OF mUORESCENCE)N 4A 4 KAMIYA!ANDD 3HIBATA + EDS 3TUDIESON D 0HOTOSYNTHETIC "ACTERIA PP n 5NIV -ICROALGAE AND 4 KYO0RESS 4OKYO 4O %CKERT(* 2ENGER' "ERNARDING* &AUST0 %ICHLER(*AND3ALK * %XAMINATIONOFmUORESCENCELIFETIMEANDRADICALPAIR DECAYIN03))MEMBRANEFRAGMENTSFROMSPINACH"IOCHIM "IOPHYS!CTAn %CKERT (* 7 7IESE . "ERNARDING * %ICHLER (* ANDD 2ENGER '  !NALYSISOFF THEELECTRONTRANSFER F FROM0 F HEO< TO 1! IN 03)) MEMBRANE FRA F GMENTS FFROM SPINACH BY TIME RESOLVED NMABSORPTIONCHANGES INTHEPICOSECOND DOMAIN&%"3 ,ETT n %IJCKELHOFF # VAN 2OON ( 'ROOT -, VAN 'RONDELLE 2 AND $EKKER*0  0URIF ICATION AND SPECTROSCOPIC CHARACTERIZATIONOF0HOTOSYSTEM)) REACTIONCENTER COMPLEXES ISOLATED WITH OR WITHOUT 4RITON 8  "IOCHEMISTRY  n &AJ A ER *  3TRUCTURAL EFFECTS F IN CHEMISTRY AND BIOLOGY Y *0ORPHYRINS0HTHALOCYANINESn A ER *  #HLOROPHYLL CHEMISTRY BEFORE F AND AFFTER CRYS &AJ TALS OFF PHOTOSYNTHETIC REACTION CENTERS 0HOTOSYNTH 2ES  n &EHER ' )SAACSON 2! /KAMURA -9 AND ,UBITZ 7  %.$/2OFEXCHANGEABLEPROTONSOFTHEREDUCEDINTERMEDIATE ACCEPTORINREACTIONCENTERSFROM 2HODOBACTER3P 3 HAEROIDES 2 )N"RETON*ANDD 6ERMEGLIO! EDS 4HE0HOTOSYNTHETIC"AC TERIAL 2EACTION#ENTER3TRUCTUREANDD $YNAMICS PPn 0LENUM0RESS .EW9ORK R &ERREIRA + )VERSON4- -AGHLOUNI + "ARB R ER * ANDD )WATA 3  !RCHITECTURE OF F THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn &LETCHER*4AND4HERIEN-* %XTREME ELECTRONICMODULATION OF THE COFACIAL PORPHYRIN STRUCTURAL MOTIF *!M #HEM 3OC  n &ÚRSTER4H  $ELOCALIZED EXCITATION AND EXCITATIONTRANSFER )N3INANOGLU/ED -ODERN1UANTUM#HEMISTRY6OL ))) PP

 R n !CADEMIC0RESS .EW9ORK &ROMME 0 *ORDAN 0 AND +RAU” .  3TRUCTURE OF 0HOTO SYSTEM)"IOCHIM"IOPHYS!CTAn 'ARBERS! +URRECK* 2EIFARTH& 2ENGER'AND0ARAK& #ORRELATION BETWEEN PROTEIN mEXIBILITY AND ELECTRON TRANSFER F FROM 1!n TO1" IN03))MEMBRANE FRA F GMENTS FROMSP F INACH "IOCHEMISTRY n 'ERMANO- 'RADINARU## 3HKURO K PATOV!9 VAN3TOKKUM)(- K 3HUVALOV6! $EKKER*0 0 VAN'RONDELLE2ANDVAN'OR D RKOM(*  %NERGYAND D ELECTRONTRANSFER F IN0HOTOSYSTEM))REAC TIONCENTERSWITHMODIlEDPHEOPHYTINCOMPOSITION"IOPHYS *n 'IBASIEWICZ+ $OBEK! "RETON*AND,EIBL7 -ODULATION OFPRIMARYRADICALPAIRKINETICSANDENERGETICSIN0HOTOSYSTEM)) BY THEREDOXSTATEOFT F HEQUINONEELECTRONACCEPTOR1! "IOPHYS * n 'IORGI ," .IXON0* -ERRY 3!0 *OSEPH $- $URRANT*2 $E,AS R ER* 0ORTER'AND D +LUG $2 #OMPARISON 2IVAS* "ARB OFF PRIMARYCHARGESEPARATION INTHE0HOTOSYSTEM))REACTION CENTERCOMPLEXISOLATEDFROMWILD TYPEAND$ MUTANTS OFTHECYANOBACTERIUM 3YNECHOCYSTIS 0##*"IOL#HEM n 'REENlELD32 3EIBERT- 'OVINDJEEAND7ASIELEWSKI-2* F HEEFFECT F IVERATECONSTANT FORPR F IMARY $IRECTMEASUREMENTOFT CHARGESEPARATION IN ISOLATED D 0HOTOSYSTEM))REACTIONCENTERS *0HYS#HEM"n 'ROOT - , VAN -OURIK K & %IJCKELHOFF F # VAN 3TOKKUM K )(- D VAN'RONDELLE2 #HARGESEPARATIONIN $EKKER*0AND THEREACTIONCENTEROF0HOTOSYSTEM))STUDIEDASAFUNCTIONOF TEMPERATURE0ROC.ATL!CAD3CI53!n 'UDOWSKA .OWAK% .EWTON-$AND&AJER* #ONFORMA TIONALANDENVIRONMENTALEFFECTSONBACTERIOCHLOROPHYLLOPTI CALSPECTRA#ORRELATIONSOFCA F LCULATED D SPECTRAWITH STRUCTURAL RESULTS*0HYS#HEMn (ARAN' 7Y 7 NNE+ -OSER## $UTTON0,AND D (OCHSTRASSER2 &EMTOSECOND INFRARE F D STUDIESOFP F HOTOSYNTHETICREAC TIONCENTERS.EWCHARGETRANSFER F BANDSAND D ULTRAFASTENER F GY REDISTRIBUTION )N "ARBARA 0& &UJIMOTO *' +NOX7( AND :INTH : EDS 5LTRAFAST0HENOMENA8 PPn 3PRINGER 6ERLAG "ERLIN (ASEGAWA+AND.OGUCHI4 $ENSITYFUNCTIONALTHEORY CALCULATIONSONTHEDIELECTRIC CONSTANTDEPENDENCEOFT F HEOXIDA TIONPOTENTIAL OFF CHLOROPHYLL)MPLICATION FORT F HEHIGHPOTENTIAL OFF 0IN0HOTOSYSTEM))"IOCHEMISTRY n (AVEMAN * AND -ATHIS 0  &LASH INDUCED ABSORPTION CHANGESOFT F HEPRIMARY DONOROFF 0HOTOSYSTEM))ATNM IN CHLOROPLASTSINHIBITEDBYLOWP(OR4RIS TREATMENT"IOCHIM "IOPHYS!CTAn (E * #HEN & ,I * 3ANKEY /& 4ERAZONO9 (ERRERO #H 'UST $ -OORE4! -OORE!,AND,INDSAY3- %LECTRONIC DECAY A CONSTANTOFCAROTENO F IDPO D LYENESFROMS F INGLE MOLECULE MEASUREMENTS * !M #HEM 3OCn (ILLMANN" "RETTEL + VAN-IEGHEM&*% +AMLOWSKI! 2UTH ERFOR F D!7AN D D HLODDER% #HARGERECOMBINATIONIN D3C 0HOTOSYSTEM))4RANSIENTABSORB R ANCEDIFFERENCESPECTRAAN F D THEIRTEMPERATUREDEPENDENCE"IOCHEMISTRYn (OCHSTRASSER2-AND+ASHA- !PPLICATIONOFTHEEXCITON MODELTOMONONUCLEARLAMELLARSYSTEMS0HOTOCHEM0HOTOBIOL  n (OLZWARTH !2  !PPLICATIONS OF F ULTRAFAST F LASER SPECTROS COPY FORT F HESTUDY OF BIOLOGICAL SYSTEMS1UART2EV"IOPHYS

 n (OLZWARTH!2AND-àLLER-' %NERGETICSANDKINETICSOF RADICALPAIRSINREACTIONCENTERSFROM 2HODOBACTERSPHAEROI DES!FEMTOSECONDTRANSIENTABSORPTIONSTUDY "IOCHEMISTRY n (UNTER#. (UGHES*- 2EIMER*2ANDD (USH.3 -ODEL LING THE BACTERIAL PHOTOSYNTHETICREACTIONCENTER!COMBINED Q QUANTUMMEC HANICALMOLECULARMECHANICALSTUDY OFF THESTRUC TUREOFF THECOFACTORS F INTHEREACTIONCENTERSOF PURPLEBACTERIA *0HYS#HEM"n (UPPMAN0 !RLT4 0ENZKOFER( 3CHMIDT3 "IBIKOVA- $OHSE " /ESTERHELT$ 7ACHTVEITL*AND:INTH7 +INETICS ENERGETICS AND ELECTRONIC COUPLING OF THE PRIMARY ELECTRON F R REACTIONS IN MUTATED D REACTION CENTERS OF "LASTOCHLORIS TRANSFE VIRIDIS "IOPHYS*n (UPP U MAN0 3PÚRLEIN 3 "IBIKOVA- /ESTERH R ELT$ 7ACHTVEITL *AND D :INTH7  %LECTRONTRANSFER F INREACTIONCENTERSOF "LASTOCHLORISVIRIDIS 0HOTOSYNTHETICREACTIONSAPPROXIMATING THEADIABATICREGIME*0HYS#HEM!n )SHIKITA( ,OLL" "IESIADKA* 3AENGER7 AND+NAPP% 7 2EDOXPOTENTIALSOFCHLOROPHYLLSINTHE0HOTOSYSTEM))REACTION CENTER"IOCHEMISTRYn )VANCICH! !RLTZ+ 7ILLIAMS*# !LLEN*0 -ATTIOLI4! F F HYDRO D GEN BONDSONTHEREDOXPOTENTIALANDD ELECTRONIC %FFECTSO STRUCTUREOFF THEBACTERIALPRIMARY ELECTRON DONOR"IOCHEMISTRY  n *ANKOWIAK2 (A K AYES*-AND3MA D LL '* !NEXCITONICPEN TAMERMODEL FORT F THECORE 1Y STATESOFTHEISOLATED0HOTOSYSTEM)) REACTIONCENTER*0HYS#HEM"n *ANKOWIAK2 2ÊTSEP- (AYES* :AZUBOVICH6 0ICOREL2 3EIBERT -AND3MALL'* 0RIMARYCHARGE SEPARATIONRATEAT+IN ISOLATED0HOTOSYSTEM))REACTIONCENTERSCONTAININGlVEANDSIX CHLOROPHY H LL A MOLECULES*0HYS# H HEM"n *EANS# 3CHILSTRA-* 2AY A . (USAIN3 -INAGAWA* .UGENT *(! AND D +LUG $2  2EPLACEMENT OF TYROSINE $ WITH PHENYL N ALANINEAFFECTST F HENORMAL PROTONTRANSFERPAT F HWAY A S FO F R D IONOFF 0 INOXYGEN EVOLVING 0HOTOSYSTEM))PARTI THEREDUCT CLESFROM #HLAMYDOMONAS # "IOCHEMISTRYn *OHNSON%4 .AGARAJAN6 :AZUBOVICH6 2ILEY+ 3MALL'*AND 0ARSON77  %FFECTS OF IONISABLE RESIDUES ON THE AB SORPTIONSPECTRUMANDINITIALELECTRON TRANSFERKINETICSINTHE PHOTOSYNTHETIC REACTION CENTER OF 2HODO D BACTER SPH S AEROIDES D  "IOCHEMISTRYn *OHNSTON( 7ANG * 2UFmE36 3AY A RE24ANDD 'USTAFFSON4,  A KINETICSOFF WILDD TYPE$ (.MUTANT &LUORESCENCEDECAY 0HOTOSYSTEM))REACTIONCENTERSISOLATED FROM F #HLAMYD M DOMONAS REINHARDTII*0HYS#HEM"n *URSINIC 0 AND 'OVINDJEE  4EMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE n MICROSECOND RANGE AF F F TER A SINGLE mASH IN CHLOROPLASTS 0HOTOCHEM 0HOTOBIOL  n +AMINSKAY A A/ 2ENGER'AND3 D HUVALOV6!  %FFECTO F F DEHY DRATIONON LIGHT INDUCE D D REACTIONS IN0HOTOSYSTEM))%VIDENCE FFOR THE PRESENCE OF F TWO FUNCT F IONALLY DIFFERENT F #YTOCHROMES B"IOCHEMISTRY n +AMIYA.AND3HEN* 2 #RYSTALSTRUCTUREOFOXYGEN EVOLV ING0HOTOSYSTEM))FROM 4HERMOSYNECHOCOCCUSVULCANU 4 S AT  ¯ RESOLUTION0ROC.ATL!CAD3CI53!n +AMLOWSKI! &RANKEMÚLLER , 6AN DER %ST! 3TEHLIK $ AND F DELOCALIZATIONOFF THETRIPLET (OLZWARTH !2  %VIDENCEFOR STATE 0 INTHE$$#YT B COMPLEXOFF 0HOTOSYSTEM))

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI "ER"UNSENGES0HYS#HEMn +ARPLUS-AND-C#AMMON*- $YNAMICSOFPROTEINS %LEMENTSANDFUNCTION!NNU2EV"IOCHEMn +ATILIENE: +ATILIUS%AND7OODBURY.7 %NERGYTRAPPING ANDDETRAPPINGINREACTIONCENTERMUTANTSFROM 2HODOBACTER SPHAEROIDES D  "IOPHYSn +AWATSU4 +AKITANI 4AND D 9AMATO4  7ORM MODEL FOR ELECTRON TUNNELING IN PROTEINS#ONSOLIDATIONOFF THE PATHWAY A MODELAND D THE$UTTON PLOT*0HYS#HEM"n + +E"  0HOTOSYNTHESIS0HOTOBIOCHEMISTRYAND D 0HOTOBIO PHYSICS+LUWER!CADEMIC0UBLISHERS $ORDRECHT +LIMOV 66 +LEVANIK !6 3HUVALOV 6! AND +RASNOVSKY !!  2EDUCTIONOFPHEOPHYTININTHEPRIMARYLIGHTREACTION OF0HOTOSYSTEM))&%"3,ETTn +LIMOV66 !LLAKHVERDIEV3) $EMETER3AND D +RASNOVSKY !!  0HOTOREDUCT D ION OFF PHEOPHYTIN IN 0HOTOSYSTEM )) OF CHLOROPLASTSASA FUNCT F IONOFRE F DOX POTENTIAL OFT F HEMEDIUM D .AUK 3332n $OKL !KAD +LUG$2 2ECH 4 *OSEF$- "AR F RBER* $URRANT*2AN0ORTER'  0RIMARYPROCESSESINISOLATED0HOTOSYSTEM))REACTION CENTERSPROBEDBYMAGICANGLETRANSIENTABSORPTIONSPECTROS COPY#HEM0HYSn + APP%7 &ISCHER3& :INTH7 3ANDER- +AISER7 $EISENHOFER +NA D -ICHEL (  !NALYSISOFOPT F ICAL SPECTRA FROMS F INGLE *AND CRYSTALSOF 2HODO DOPSEUDOMONASVIRI D DIS REACTION CENTERS 0ROC .ATL!CAD 3CI 53!n + +NOX23AN D VAN!MERONGEN(  2EFRACT F IVEINDEX DEPEN DENCEOFF THE&ÚRSTERRESONANCEEXCITATIONTRANSFERRATE*0 F HYS #HEM"n + +OBAYASHI- -AEDA( 7ATANABE4 .AKANE(AND3ATOH+  #HLOROPHYLL A AND` CAROTENECONTENTINTHE$$ CYTOCHROME B REACTIONCENTERCOMPLEXFROMSPINACH&%"3 ,ETT n + +ONERMANN,AN D (OLZWARTH !2  !NALYSISOFF THEABSORP TIONSPECTRUMOFF 0HOTOSYSTEM))REACTIONCENTERS4EMPERATURE DEPENDENCE PIGMENTASSIGNMENTAND INHOMOGENEOUS BROADEN ING"IOCHEMISTRY n +ONERMANN , 'ATZEN ' AND (OLZWARTH !2  0RIMARY + PROCESSESANDSTRUCTUREOFTHE0HOTOSYSTEM))REACTIONCENTER6 -ODELINGOFTHEmUORESCENCEKINETICSOFTHE$ $ #YT B COMPLEXAT+ * 0HYS#HEM"n + IEGER ,ISZKAY!AND +R D 2UTHERFOR F D !7  )NmUENCEOF HERBI R CIDEBINDINGONTHEREDOXPOTENTIALOFF THEQUINONEACCEPTORIN 0HOTOSYSTEM))2ELEVANCETOPHOTODAMAGEAND D PHYTOTOXICITY Y "IOCHEMISTRYn + IEGER ! 2UTHERFOR +R F D D !7 AND *OHNSON '.  /N THE DETERMINATIONOFTHEREDOXMIDPOINTPOTENTIALOFTHEPRIMARY QUINONE ACCEPTOR 1! IN 0HOTOSYSTEM )) "IOCHIM "IOPHYS Q !CTAn +RISHTALIK ,)  %NERGETICS OF MULTIELECTRON REACTIONS H !CTA 0HOTOSYNTHETIC OXYGEN EVOLUTION "IOCHIM "IOPHYS  n + HN/ 2ENGER4 -AY +à A 6 6OIGT* 0ULLERITS4AND3UN D DSTRÚM6  %XCITON VIBRATIONAL COUPLING IN PHOTOSYNTHETIC ANTENNA COMPLEXES THEORYMEETS EXPERIMENT4RENDS0HOTOCHEM0HO TOBIOLn + +àHN0 %CKERT( * %ICHLER( *AND2ENGER' !NALYSIS OFTHE0s REDUCTIONPATTERNANDITSTEMPERATUREDEPENDENCE INOXYGENEVOLVING03))CORECOMPLEXESFROMTHERMOPHILIC CYANOBACTERIA AND HIGHER PLANTS 0HYS H #HEM #HEM 0HYS H  n

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS +àHN0 0IEPER* +AMINSKAY A A/ %CKERT( * ,ECHNER2 3HUVALOV 6 AND2ENGER' 2EACTIONPATTERNOF0HOTOSYSTEM)) /XIDATIVEWATERCLEAVAGEANDPROTEINmEXIBILITY0HOTOSYNTH 2ESn +URRECK* 'ARBERS! 2EIFARTH& !NDRÏASSON,% 0ARAK&AND F BRANE 2ENGER' )SOLATIONANDD PROPERTIESOF03))MEM F GMENTSDEPLETEDD OF THENONHEMEIRONCENTER&%"3,ETT FRA  n +URRECK K * ,IU" .APIWOTZKI! 3ELLIN3 %CKERT(* %ICHLER(* AND D 2ENGER'A 3TOICHIOMETRY OFP F IGMENTSANDD RADICAL PAIRFORMATIONUNDERSATURATINGPULSEEXCITATIONIN$$#YT BPREPARATIONS"IOCHIM"IOPHYS!CTAn +URRECK* 'ARBERS! 0ARAK&AND2ENGER'B (IGHLYPURI lED$$#YTB PREPARATIONSFROMSPINACHDONOTCONTAIN THENONHEMEIRONCENTER&%"3,ETTn ,AIBLE0$ +IRMAIER# 5DAWATTE#3- (OFMAN3* (O F LTEN$ AND D (ANSON$+  1UINONEREDUCT D IONVIASECONDARY B BRANCH ELECTRONTRANSFER F INMUTANTBACTERIAL REACTIONCENTERS "IOCHEMISTRYn ,EIBL7 "RETON* $EPREZ*AND4RISSL(7 0HOTOELECTRONIC STUDYONTHEKINETICSOFTRAPPINGANDCHARGESTABILIZATIONIN ORIENTED03))MEMBRANES0HOTOSYNTH2ESn ,EUPOLD$ 4EUCHNER+ %HLERT* )RRGANG+$ 2ENGER'AND,OK STEIN (  4WO PHOTON EXCITED mUORESCENCE FR F OM HIGHER ELECTRONIC STATES OF CHLOROPHYLLS IN PHOTOSYNTHETIC ANTENNA COMPL M EXES! NEW AAPPROACHTO APP INVESTIGATE SPECTRAL HETEROGENEITY T @CHLOROPHYLL FORMS F "IOPHYS*n ,INCE-4AND6ERMAAS7 D  !SSOCIATIONOFF (IS INTHE $PROTEINOF0HOTOSYSTEM))WITHACHLOROPHYLLTHATAFFECTS EXCITATION %NERGY TRANSFER EFlCIENCY TO THE REACTION CENTER %UR*"IOCHEMn ,IU" VAN+AN0*-AND(OFF!* )NmUENCEOFTHE( SUBUNIT n AND D &E ONELECTRONTRANSPORTFROM) F TO1! IN &E FR F EE ANDOR F EE REACTION CENTERS FR F OM 2HODO D BACTERSPH S AEROIDE D S 2  ( FR &%"3,ETTn ,UBI U TZ7AND D ,ENDZIAN& %.$/23PECTROSCOPY)N Y !MESZ D (OFFF !* EDS "IOPHYSICAL4ECHNIQUESIN0HOTOSYNTHESIS *AND PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT 9 !BRESCH%# 0LATO-AND ,UBITZ7 7 )SAACSON2! /KAMURA-9 &EHER' %.$/2STUDIESOFTHEINTERMEDIATEELECTRON ACCEPTORRADICALANION)n s IN0HOTOSYSTEM))REACTIONCENTERS H  "IOCHIM "IOPHYS!CTAn -AC-ILLAN& ,ENDZIAN& 2ENGER'ANDD ,UUBITZ7 %02 AND D %.$/2 INVESTIGATION OF F THE PRIMARY ELECTRON ACCEPTOR RADICAL ANION 1!n INIRON DEPLETED0HOTOSYSTEM))MEMBRANE F GMENTS"IOCHEMISTRY n FRA -ÊNTELE 7  )NFRARED AND &OURIER TRANSFORMED INFRARED SPECTROSCOPY )N !MESZ * AND (OFF !* EDS "IOPHYSICAL 4 4ECHNIQUESIN0HOTOSYNTHESIS PPn+LUWER!CADEMIC 0UBLISHERS $ORDRECHT -ARCUS2!AND D 3UTIN. %LECTRONTRANSPORTINCHEMISTRY AND BIOLOGY" Y IOCHEM"IOPHYS!CTAn -ATYSIK K * 'AST!0 VAN'ORRKOM(*AND DE'ROOT(*-  D D NUCLEAR SPIN POLARIZATION IN REAC 0HOTOCHEMICALLY INDUCE TION CENTERS OFF 0HOTOSYSTEM )) OBSERVED BY # SOLID STATE .-2 REVEALS A STRONGLY ASYMMETRIC ELECTRONIC STRUCTURE OF THE 0s PRIMARYDONORCHLOROPHYLL0ROC.ATL!CAD3CI53! n -C-AHON"( -ULLER*$ 7RAIGHT#!AND.IENHAUS'5 F D PROTEIN DYNAMICS INTHEPHOTOSYNTHETIC %LECTRONTRANSFERAN REACTIONCENTER"IOPHYS*n

 -ERRY 3!0 .IXON0* "ARTER,-# 3CHILSTRA- 0ORTER' "ARB R ER * $URRANT*2AND+LUG$2 -ODULATIONOFQUANTUM YIELDOFPRIMARYRADICALPAIRFORMATIONIN0HOTOSYSTEM))BY SITE DIRECTEDMUTAGENESISAFFECTINGRADICALCATIONSANDANIONS "IOCHEMISTRYn -ICHEL (AND D $EISENHOFER* F  2ELEVANCEOFF THEPHOTOSYN THETICREACTIONCENTERFROM F PURPLE BACTERIATOTHESTRUCTUREOF 0HOTOSYSTEM))"IOCHEMISTRY n -ICHEL "EYERLE-% 0LATO- $EISENHOFER* F ICHEL( "IXONAND D *ORTNER*  5NIDIRECTIONALITYOFC F HARGESEPARATIONIN REACTIONCENTERSOFPHOTOSYNTHETICBACTERIA"IOCHIM"IOPHYS !CTAn -IMURO - 4OMO 4 .ISHIMURA9 9AMAZAKI ) AND 3ATOH +  )DENTIlCATIONOFAPHOTOCHEMICALLYINACTIVEPHEOPHYTIN MOLECULE INTHESPINACH $ $ #YT BCOMPLEX"IOCHIM "IOPHYS!CTAn -IMURO - !KIMOTO 3 'OTOH 4 9OKONO - !KIYAMA - A ASHI -AND9AMAZA D KI)  4SUCHIYA4 -IYASHITA( +OBAY )DENTIlCATION OF F THE PRIMARY ELECTRON DONOR IN 03)) OF F THE D D !CARYOCHLORISMARINA&%"3 #HL D DOMINATEDCYANOBACTERIUM ,ETTn -OÑNNE ,OCCOZ2 2OBERT"AND,UTZ- !RESONANCE 2AMAN CHARACTERIZATION OF THE PRIMARY ELECTRON ACCEPTOR IN 0HOTOSYSTEM))"IOCHEMISTRYn -OSER##AND$UTTON0# D  /UTLINEOFT F HEORY OFPROTE F IN ELECTRONTRANSFER)N"EN F DALL$3 ED 0ROTEIN%LECTRON4RANSFER F PP n")/33CIENTIlC0UBLI U SHERS /XFOR F D -àH& 2AUTTER*AND D ,U UBITZ7 4WODISTINCTCONFORMAT F IONS OFTHEPRIMARYELECTRONDONORINREACTIONCENTERSFROM 2HODO BACTERSPHAEROIDES E REVEALEDBY%.$/242)0,%3PECTROSCOPY "IOCHEMISTRYn -ULKIDJANIAN! 0HOTOSYSTEM))OFGREENPLANTS/NTHE POSSIBLEROLEOFF RETARDED PROTONICRELAXATION INWATEROXIDATION "IOCHIM "IOPHYS!CTAn -àLLER-' $ORRA$ (OLZWARTH !2 'ADON.AND$REWS' D  4IME DEPENDENT RADICAL PAIR RELAXATION IN CHROMATO PHORESOFANANTENNA F F FREEMUTANT F M 2HODO FRO D BACTERCAPSULATUS L  )N-ATHIS0ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE6OL  PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT -àLLER-' (UCKE- 2EUS-AND(OLZWARTH!2 0RIMARY PROCESSESANDSTRUCTUREOFTHE0HOTOSYSTEM))REACTIONCENTER )6 6 ,OWINTENSITYFEMTOSECON F D TRANSIENTABSORPTIONSPECTRAOF $ $ REACTION CENTERS *0HYS#HEMn .ABEDRYK K % !NDRIANAMBININTSOA 3 "ERGER ' ,EONHARD D - -ÊNTELE7AND"RETON* D  #HARACTERIZATIONOF BONDING INTERACTIONSOFT F HEINTERMEDIARYELECTRONACCEPTORINTHEREAC TIONCENTEROF0HOTOSYSTEM))BY&4)2SPECTROSCOPY"IOCHIM "IOPHYS!CTAn .ANBA/AND3ATOH+ )SOLATIONOFA0HOTOSYSTEM))REACTION N CENTERCONSISTINGOF$AND$POLYPEPTIDESANDCYTOCHROME B0ROC.ATL !CAD 3CI 53!n .ISHIGAKI! /HSHIMA3 .AKAY A AMA+ /KADA-AND D .AGASHIMA F LECULARORBI R TALCALCULATIONSTO IN 5 !PPLICATIONOFMO TERPRETTHECHLOROPHYLL SPECTRAL FORMS F INPEA0HOTOSYSTEM)) 0HOTOCHEM0HOTOBIOLn .OGUCHI4 4OMO4AND)NOUE9 &OURIERTRANSFORMINFRARED STUDYOFTHECATIONRADICALOF0INTHE0HOTOSYSTEM))REACTION CENTER%VIDENCEFORCHARGEDELOCALIZATIONONTHECHLOROPHYLL DIMER"IOCHEMISTRYn .OGUCHI4 +URRECK K * )NOUE9AND 2ENGER'  #OMPARATIVE &4)2ANALYSISOFF THEMICROENVIRONMENTOFF 1!nINCYANIDEAND

 HIGHP(TREATED D AND @IRONDEPLETED03))MEMBRANE FRA F GMENTS "IOCHEMISTRYn .ORRIS*2 3CHEER(AND+ATZ** -ODELSOFANTENNAAND REACTIONCENTERCHLOROPHYLLS!NN.9!CAD3CI .UIJS!- VAN'ORKOM(* 0LIJTER**AND$UYSENS,.- F HLOROPHYLL A IN 0RIMARY CHARGESEPARATIONANDD EXCITATIONOFC F INACH ASSTUDIED BY PICOSECOND 0HOTOSYSTEM))PARTICLESFROMSP ABSORB R ANCE DIFFERENCE F SPECTROSCOPYY "IOCHIM "IOPHYS!CTA  n /BA4AND D 4AMIAKI( 7HICHSIDEOFF THE / MACROCYCLE PLANEOFBACTERIO CHLOROPHYLLSISFAVOREDFORBINDINGTHElFTH LIGAND0HOTOSYNTH2ESn /GRODNIK! 6OLK- ,ETTERER2 &EICK2AND-ICHEL "EYERLE-%  $ETERMINATIONOFFREEENERGIESINREACTIONCENTERSOF2B SPHAEROIDES D  "IOCHIM "IOPHYS!CTAn 0AGE ## -OSER ## #HEN 8 ANDD $UTTON 0,  .ATURAL ENGINEERING PRINCIPLES OFF ELECTRON TUNNELING IN BIOLOGICAL OXIDATION REDUCT D ION.ATUREn 0ALAZZO' -ALLARDI! (OCHKOEPPLER! #ORDONE,AND D 6ENTUROLI ' %LECTRONTRANSFERKINETICSINPHOTOSYNTHETICREACTION CENTERSEMBEDDEDINTREHALOSEGLASSES4RAPPINGOFCONFORMA TIONALSUBSTATESATROOMTEMPERATURE"IOPHYS*n 0APAGEORGIOU'#AND'OVINDJEEEDS  #HLOROPHYLL A &LUO RESCENCE!3IGNATUREOFF 0HOTOSYNTHESIS3PRINGER "ERLIN 0ARK R ER#!AND*O D OYCE4!  $ELAY A ED mUORESCENCEANDD SOME PROPERTIES OFF THE CHLOROPHYLL TRIPLETS 0HOTOCHEM 0HOTOBIOL n 0ARSON77  4HERMODYNAMICSOFT F HEPRIMARYREACTIONSOF PHOTOSYNTHESIS0HOTOCHEM0HOTOBIOLn 0ARSON 77 AND 7ARSHEL  ! DENSITY MATRIX MODEL OF PHOTOSYNTHETICELECTRONTRANSFERWITHMICROSCOPICALLYESTIMATED VIBRATIONALRELAXATIONTIMES#HEM0HYSn 0ARSON77 #HU:4ANDD 7ARSHEL !  %LECTROSTATIC CONTROL OFF CHARGE SEPARATION IN BACTERIAL PHOTOSYNTHESIS "IOCHIM "IOPHYS!CTAn 6 +NOX00 0 +RASILNIKOV0- 2EDLIN 0ASCHENKO6: 'OROKHOV66 UBIN!" %NERGETICSANDD MECHANISMS ( 2ENGER'ANDD 2U OFHIGHEFlCIENCYOFCHARGESEPARATIONANDELECTRONSTRANSFER PROCESSESIN2HODOBACTERSPHAEROIDES REACTIONCENTERS"IO ELECTROCHEMISTRYn 0EARLSTEIN2- #HLOROPHYLLSINGLETEXCITON)N'OVINDJEE ED 0HOTOSYNTHESIS %NERGY#ONVERSIONBY0LANTSANDD "AC TERIA PP n!CADEMIC0RESS .EW9ORK R 0EARLSTEIN 2-  4HEORETICAL INTERPRETATION OF F ANTENNA SPECTRA)N3CHEER(ED #HLOROPHYLLS PPn#2# R 0RESS .EW9ORK 0ELOQUIN*- 7ILLIAMS*# ,IN8 !LDEN2' 4AGUCHI!+7 !LLEN *0AND7OODBURY.7 4IME DEPENDENTTHERMODYNAM ICSDURINGEARLYTRANSFERINREACTIONCENTERSFROM 2HODOBACTER SPHAEROIDES "IOCHEMISTRYn 0ETERMAN%*' 0ULLERITS4 VAN'RONDELLE2AND D 6AN!MERONGEN(  %LECTRON PHONONCOUPLINGANDD VIBRONIClNESTRUCTURE OF LIGHT HARVESTINGCOMPLEX))OF GREENPLANTS4EMPERATURE DEPENDENTABSORPTIONAND HIGH RESOLUTIONmUORESCENCESPEC TROSCOPY*0 Y HYS#HEM"n 0ETERSON !RSKÚLD3 -ASTERS6- 0RINCE"* * 3MITH0* * 0ACE2* AND +RAUSZ %  /PTICAL 3PECTRA OF 3YNECHOCYSTIS AND 3PINACH0HOTOSYSTEM))0REPARATIONSAT+)DENTIlCATION OFTHE$ 0HEOPHYTIN%NERGIESAND3TARK3HIFTS*!M#HEM 3OC D #HRISTOFFERSEN F 2% 0ETKE *$ -AGGIORA '- 3HIPMAN , AND

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI  3TEREOELECTRONICPROPERTIESOFPHOTOSYNTHETICANDRE LATEDSYSTEMS 6 !BINITIO CONlGURATIONINTERACTIONCALCULATION ONTHEGROUNDANDLOWEREXCITEDSINGLETANDTRIPLETSTATESOF ETHYLCHLOROPHYLLIDE A ANDETHYLPHEOPHORBIDE A0HOTOCHEM 0HOTOBIOL  n 0IEPER * 6OIGT * 2ENGER ' AND D 3MALL '*  !NALYSIS OF PHONONSTRUCTURE IN LINE NARROWEDOPT D ICAL SPECTRA#HEM0HYS ,ETT n 0IEPER* )RRGANG + $ 2ENGER'AND D ,ECHNER2% $ENSITY OFF VIBRATIONAL STATESOFF SOLUBILI U ZED,(#))COMP D LEXESOF GREEN PLANTSSTUDIEDBYINELASTICNEUTRONSCATTERING*0HYS#HEM" n 0ROKHORENKO6) AND (OLZWARTH!2  0RIMARY PROCESSES ANDSTRUCTUREOFTHE0HOTOSYSTEM))REACTIONCENTER!PHOTON ECHOSTUDY*0 Y HYS#HEM"n 2APPAPORT & 'UERGOVA +URAS - .IXON 0* $INER "! AND ,AVERGNE*  +INETICS AND PATHWAY A S OF CHARGE RECOMBINATION IN 0HOTOSYSTEM )) "IOCHEMISTRY  n  2ASZEWSKI' 3AENGER7AND2ENGER4 4HEORYOFOPTICAL SPECTRA OF 0HOTOSYSTEM )) REACTION CENTERS ,OCATION OF THE TRIPLET STATE AND THE IDENTITY OF THE PRIMARY ELECTRON DONOR "IOPHYS*n 2AYMOND*AN D D "LANKENSHIP2%  4HEEVOLUTIONARYDEVELOP MENTOFF THEPROTEINCOMPLEMENTOFF 0HOTOSYSTEM"IOCHIM "IOPHYS!CTAn 2EED$7AN D D LAY D# A TON2+ )SOLATIONOFF AREACTIONCENTER F FRACT ION FROM F 2HODOPSEUDOMONAS SP SPHAEROIDES "IOCHEM "IOPHYS2ES#OMMUNn 2EIFARTH&AND2ENGER' )NDIRECTEVIDENCEFORSTRUCTURAL CHANGESCOUPLEDWITH1"nFORMATIONIN0HOTOSYSTEM))&%"3 ,ETT n 2EMY!AND D 'ERWERT+ #OUPLINGOF LIGHT INDUCE D D ELECTRON F KEINPHOTOSYNTHESIS.ATURE3TRUCT"IOL TRANSFERTOPROTONUPTA  n 2EINMAN 3 AND D -ATHIS 0  )NmUENCE OFF TEMPERATURE ON F IONS"IOCHIM"IOPHYS 0HOTOSYSTEM))ELECTRONTRANSFERREACT !CTAn 2ENGER' -ECHANISTICASPECTSOFPHOTOSYNTHETICWATER CLEAVAGE0HOTOSYNTHETICAn 2ENGER' %NERGYTRANSFERANDTRAPPINGIN0HOTOSYSTEM 4 ICS IN 0HOTOSYNTHESIS 4HE0HOTOSYS )))N"ARB R ER* ED 4OP TEMS3TRUCTURE &UNCTIONAND D -OLECULAR"IOLOGY PPn Y  %LSEVIER !MSTERDAM 2ENGER' -OLECULARMECHANISMOFWATEROX F IDATION)N 3INGHAL'3 2ENGER' 'OVINDJEE )RRGANG +$AND D 3OPORY 3+EDS #ONCEPTSIN 0HOTOBIOLOGY0HOTOSYNTHESISAND0HO TOMORPHOGENESIS PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT AND.AROSA0UBLISHING#O $ELHI 2ENGER' 0HOTOSYNTHETICWATEROXIDATIONTOMOLECULAR OXYGEN!PPARATUS AND D MECHANISM "IOCHIM "IOPHYS!CTA H  n 2ENGER'  !PPARATUSAND D MECHANISMOFP F HOTOSYNTHETIC OXYGEN EVOLUTION ! PERSONAL PERSPECTIVE 0HOTOSYNTH 2ES  n 2ENGER '  #OUPLING OF ELECTRON AND PROTON TRANSFER IN OXIDATIVE WATER CLEAVAGE IN PHOTOSYNTHESIS "IOCHIM "IO PHYS!CTA 2ENGER4AND-ARCUS2!A /NTHERELATIONOFPROTEINDY NAMICSAND D EXCITONRELAXATIONINPIGMENT PROTEINCOMPLEXES F HESPECTRAL DENSITYAND D ATHEORY FORT F HECAL !NESTIMATIONOFT

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS CULATIONOFF OPTICAL SPECTRA*#HEM0HYSn 2ENGER4AND-ARCUS2!B 0HOTOPHYSICALPROPERTIESOF 03 REACTIONCENTERSANDADISCREPANCYINEXCITONRELAXATION TIMES *0HYS#HEM"n 2ENGER4AND-AY6 -ULTIPLEEXCITONEFFECTSINMOLECULAR AGGREGATES!PPLICATIONTOAPHOTOSYNTHETICANTENNACOMPLEX 0HYS2EV,ETTn 2ENGER'AND7O D LFF# F H  4HEEXISTENCEOFA F HIGHH PHOTOCHEMI CALTURNOVERRATEATTHEREACTIONCENTERSOFS F YSTEM))IN4RIS WASHED D CHLOROPLASTS"IOCHIM"IOPHYS!CTAn 2ENGER' 'LEITER(- (AAG%AND2EIFARTH& 0HOTO SYSTEM)) THERMODYNAMICSANDKINETICSOFELECTRONTRANSPORT FROM1!nTO1" 1"n ANDDELETERIOUSEFFECTSOFCOPPER)) : F RSCH C n .ATURFO 2ENGER' %CKERT(* "ERGMANN! "ERNARDING* ,IU" .API WOTZKI ! 2EIFART F H&ANDD %ICHLER*(  &LUORESCENCEAND SPECTROSCOPICSTUDIESONEXCITONTRAPPINGANDD ELECTRONTRANSFER F IN0HOTOSYSTEM))OF HIGHERPLANTS!USTR*0LANT0HYSIOL n 2IGBY3%* .UGENT*(!AND/-ALLEY0* %.$/2AND SPECIALTRIPLERESONANCESTUDIESOFCHLOROPHYLLCATIONRADICALS IN0HOTOSYSTEM)) "IOCHEMISTRYn 2ILEY+ *ANKOWIAK 2 2ÊTSEP- 3MALL'*AND:AZUBOVICH6  %VIDENCEFOR F HIGHLY L DISPERSIVEPR I IMARYCHARGESEPARATION KINETICSAND GROSS HETEROGENEITY INTHEISOLATED03))REACT D ION CENTEROF GREENPLANTS*0HYS#HEMn 2INY N U, -ARIN%7 4AKAHASHI % -AROTI 0AND7RA D IGHT#!  -ODU D LATIONOFF THE FREEENER F GY OFF THEPRIMARY QUINONEACCEP TOR 1! IN REACTION CENTERS FROM 2HODOBACTER SPHAEROIDES #ONTRIBUTIONSFROMTHEPROTEINANDPROTEIN LIPIDCARDIOLIPIN INTERACTIONS"IOCHIM"IOPHYS!CTAn 2OBERT "  2ESONANCE 2AMAN STUDIES IN PHOTOSYNTHE SISˆCHLOROPHY H LLANDD CAROTENOIDD MOLECULES)N!MESZ*AND (OFFF !* EDS "IOPHYSICAL4ECHNIQUESIN 0HOTOSYNTHESIS PP n +LUWER!CADEMIC0UBLI U SHERS $ORDRECHT 2OBERT" ,UTZ-ANDD 4IEDE$-  3ELECTIVE PHOTOCHEMICAL D IONOFE F ITHEROFF THETWO BACTERIOPHEOPHYTINSINREACTION REDUCT CENTERSOF 2PSSPHAEROIDES 2 &%"3 ,ETT n 2OELOFS4! AND (OLZWARTH!2  )N SEARCH OF A PUTATIVE LONG LIVEDRELAXEDRADICALPAIRSTATEIN CLOSED0HOTOSYSTEM)) +INETICMODELINGOFPICOSECONDmUORESCENCEDATA"IOPHYS*  n 2OELOFS4! F ,EE #( AND (OLZWARTH !2  'LOBAL TARGET ANALYSISOFF PICOSECONDD CHLOROPHYLL mUORESCENCEKINETICS FROM F PEACHLOROPLASTS"IOPHYS*n F HISON23 2UFmE36 7ANG * *OHNSTON(' 'USTAFSON4, (UTC -INAGAWA * #ROFTS! AND 3AYRE 24  0HOTOSYSTEM )) PERIPHERALACCESSORYCHLOROPHYLLMUTANTSIN #HLAMYDOMONAS R  "IOCHEMICAL CHARACTERIZATION AND SENSITIVITY TO RDTII R REINHA PHOTO INHIBITION0LANT0HYSIOLn 2UTHERFOR F D!7AN D D &ALLER0 0HOTOSYSTEM))%VOLUTIONARY PERSPECTIVES0HIL4RANS23OC,ONDD "n F D!7AN D D &ALLER0 4HESTABLETYROXYLRADICAL 2UTHERFOR IN 0HOTOSYSTEM )) 7HY $ "IOCHIM "IOPHYS !CTA  n 3CHATZ'( "ROCK(AND(OLZWARTH!2 0ICOSECONDKINET ICSOFmUORESCENCEANDABSORBANCECHANGESIN0HOTOSYSTEM)) PARTICLESEXCITEDBYLOWPHOTONDENSITY0ROC.ATL!CAD3CI 53! n 3CHATZ '( "ROCK K ( AND (OLZWARTH !2  +INETIC AND ENERGETICMODEL FORT F HEPRIMARY PROCESSESIN 0HOTOSYSTEM))

 "IOPHY H S*n 3CHEER( ED  #HLOROPHYLLS#2#0RESS "OCA2ATON 3CHILSTRA-* 2APPAPORT& .UGENT*(! "ARNETT#*AND+LUG$2 H TRANSFER AFFECTSTHE3 STATE DEPENDENT  0ROTONHYDROGEN MICROSECOND PHASES OF 0 REDUCTIONDURING WATER SPLITTING "IOCHEMISTRY n 3CHMIDT5AND D 2EINEKER0  4RIPLETEXCITONSINMOLECULAR PAIRS-OL 0HYSn D 2ENGER' 2ATEOF 3CHÚDEL2 )RRGANG+$ 6OIGT*AND CAROTENOID TRIPLET FFORMATION IN SOLUBILIZED LIGHT HARVESTING COMPLEX)),(#)) FROM SPINACH"IOPHYS*n 3CHOLES'$ *ORDANIDES8*AND&LEMING'2 !DAPTING THE&ÚRSTER THEORYOFENERGY TRANSFERFORMODELINGDYNAMICS IN AGGREGATED MOLECULAR ASSEMBLIES * 0HYS #HEM "  n 3CHWEITZER2( -ELKOZERNOV!. "LANKENSHIP2%AND D "RUDVIG '7 4IME RESOLVED mUORESCENCEMEASUREMENTSOFF 0HO TOSYSTEM))4HEEFFECTO F F QUENCHING BY OXIDIZED D CHLOROPHYLL :*0HYS#HEM"n 3EIBERT-AND7ASIELEWSKI- 4HEISOLATED0HOTOSYSTEM)) REACTIONCENTER&IRSTATTEMPTSTODIRECTLYMEASURETHEKINETICS OFPRIMARYCHARGESEPARATION0HOTOSYNTH2ESn 3ENGE-/ 2ENNER-7 +ALISCH77AND&AJER* *#HEM 3OC $ALTON4RANS D HRÚ H DER70 4HELOWMOLECULARMASSSUB U UNITS 3HI , 8AND3C OFF THEPHOTOSYNTHETICSUPRACOMPLEX PHOTOSYSTEM))"IOCHIM "IOPHYS!CTAn 3HKUROPATOV!9 +HATYPOV2! 6OLSHCHUKOVA43 3HKUROPATOVA 6! /WENS4'AND3HUVALOV6! 3PECTRALANDPHOTO CHEMICALPROPERTIESOFBOROHYDRIDE TREATED$ $ CYTOCHROME B COMPLEXOF0HOTOSYSTEM))&%"3,ETTn 3MALL$7 -ATYUSHOV$6AND6OTH'! 4HETHEORYOF ELECTRONTRANSFERREACT F IONS7HATMAY BEMISSING*!M#HEM 3OCn 7 RZYNSKI4 3TYRING 3 3MITH 0* 0ETERSON 3 -ASTERS6- 7YD D  -AGNETO OPTICAL MEASUREMENTS +RAUSZ%AND0ACE2* OFF THEPIGMENTSIN FU F LLY ACTIVE0HOTOSYSTEM))CORECOMPLEXES FROMPLANTS"IOCHEMISTRYn 3PIEDEL$ *ONES-2AND2OBERT" 4UNINGOFTHEREDOX POTENTIALOFTHEPRIMARYELECTRONDONORINREACTIONCENTRESOF PURPLEBACTERIA%FFECTSOFAMINOACIDPOLARITYANDPOSITION &%"3,ETTn 3TEFFEN2 +E F LLY !! (UYER0* $ÚRMANN0AND D 2ENGER'  T EREACTIONPATTERNOFF 0HOTOSYSTEM)) INLEAVES )NVESTIGATIONSONTH F FROM !RABIDO DOPSISTH T ALIANA WILDD TYPEPLANTSAND D MUTANTSWITH GENETICALLY MODIlED LIPIDD CONTENT))'ALACTOLIPID DElCIENCY Y "IOCHEMISTRYn 3TEWART$( .IXON0* $INER"!AND"RUDVIG'7 !S SIGNMENTOFTHE1Y ABSORBANCEBANDSOF0HOTOSYSTEM))CHRO MOPHORESBYLOW TEMPERATUREOPTICALSPECTROSCOPYOFWILD TYPE AND D MUTANTREACTIONCENTERS"IOCHEMISTRYn 3TIEHL ((  5NTERSUCHUNGENPERIODISCHANGEREGTER!BSORP TIONSÊNDERUNGENIM 2EAKTIONSZENTRUM)) DER0HOTOSYNTHESE 0H$ 4HESIS 4ECHNISCHE5NIVERSITÊT "ERLIN 3TIEHL (( AND D 7ITT (4  1UANTITATIVE TREATMENT OF F THE FUNCTION OF PLASTOQUINONE IN PHOTOSYNTHESIS : .ATURFORSCH Bn 3TOWELL -(" 0HILLIPS 4- 2EES $# 3OLTIS 3- !BRESCH % AND&EHER' ,IGHT INDUCEDSTRUCTURALCHANGESINPHO TOSYNTHETIC REACTION CENTER )MPLICATIONS FFOR MECHANISM OF ELECTRON PROTONTRANSFER3C F IENCEn

 3TRASSER2*AND3 D IRONVAL # )NDUCT D IONOFF 0HOTOSYSTEM)) ACTIVITYINmASHEDLEAVES&%"3,ETTn 3UMI (  4HEORY ON RATES OF EXCITATION ENERGY TRANSFER BETWEENMOLECULARAGGREGATESTHROUGHDISTRIBUTEDTRANSITION DIPOLES WITH APPLICATION TO THE ANTENNA SYSTEM IN BACTERIAL PHOTOSYNTHESIS*0HYS#HEMn 4AKAHASHI9 (ANSSON½ -ATHIS0AND3ATOH+ 0RIMARY RADICAL PAIR IN THE 0HOTOSYSTEM )) REACTION CENTER "IOCHIM "IOPHYS!CTAn 4ANG$ *ANKOWIAK2 3EIBERT-AND3MALL'* %FFECTSOF DETERGENTONTHEEXCITEDSTATESTRUCTUREANDRELAXATIONDYNAMICS OFTHE0HOTOSYSTEM))REACTIONCENTER!HIGHRESOLUTIONHOLE BURNINGSTUDY0HOTOSYNTH2ESn 4ELFER! 7HATIS` CAROTENEDOINGINTHE0HOTOSYSTEM)) REACTIONCENTRE0HIL4ANS23OC,ONDD "n 4 4ETEN KIN 6, 'ULYAEV "! 3EIBERT - ANDD 2UUBIN !"  3PECTRAL PROPERTIES OFF STABILIZEDD $$CYTOCHROME B  0HOTOSYSTEM )) REACTION CENTER COMPLEX &%"3 ,ETT  n 4IEDE $- AND $UTTON 0,  /RIENTATION OF THE PRIMARY Q QUINONEOFBACTERIALPHOTOSYNTHETICREACTIONCENTERSCONTAINED IN CHROMATOPHORE AND RECONSTITUTED MEMBRANES "IOCHIM "IOPHYS!CTAn 4 IAK 4RET K 3 -IDDLETON # #HERNYAKK 6 AND -UKAMEL 3  %XCITON (AMILTONIAN FOR THE BACTERIOCHLOROPHYLL SYSTEM IN THE,( ANTENNA COMPLEXOF PURPLE BACTERIA*0HYS#HEM"  n 4 ISSL(7AND 4R D ,AVERGNE* &LUORESCENCEINDUCT D ION FROM F 0HOTOSYSTEM))!NALYTICALEQUATIONSFORTHEYIELDSOFPHOTO CHEMISTRYANDmUORESCENCEDERIVEDFROMANALYSISOFAMODEL INCLUDINGEXCITON RADICALPAIREQUILIBRIUMANDRESTRICTEDENERGY TRANSFERBETWEENPHOTOSYNTHETICUNITS!USTRAL*0LANT0HYSIOL  n 6 HA & *OSEPHS $- $URRANT *2 4ELFER! 6AC F +LUG $2 0ORTER D "ARRBER* 0HOTOCHEMISTRY ANDD SPECTROSCOPY OF 'AND A lVE CHLOROPHYLLREACTIONCENTEROFF 0HOTOSYSTEM))ISOLATED BY USINGA#UAFlNITY COLUMN0ROC.ATL !CADD 3CI 53! n 6 6ACHA& $URCHAN-AND3IFFEL0 %XCITONICINTERACTIONSIN THEREACTIONCENTREOF0HOTOSYSTEM))STUDIEDBYUSINGCIRCULAR DICHROISM"IOCHIM"IOPHYS!CTAn VAN!MERONGEN( $EKKER*0  ,IGHT (ARVESTINGIN 0HOTO SYSTEM)))N'REEN"2 0ARSON77 EDS ,IGHT (ARVESTING !NTENNAS IN0HOTOSYNTHESIS PPn+LUWER!CADEMIC 0UBLI U SHERS $ORDRECHT VAN 'ORK R OM (*  )DENTIlCATION OFF THE REDUCE D DD PRIMARY ELECTRONACCEPTOROF0HOTOSYSTEM))ASABOUNDSEMIQUINONE ANION"IOCHIM"IOPHYS!CTAn VAN -IEGHEM &*% AND 2UTHERFORD !7  #OMPARATIVE SPECTROSCOPYOF PHOTOSYSTEM ))AND PURPLE BACTERIAL REACTION CENTRES "IOCHEM 3OC4RANSn VAN2ENSEN**3 8U#ANDD 'OVINDJEE  2OLEOF BICARB R ONATE INTHE0HOTOSYSTEM)) THEWATER PLASTOQUINONEOXIDO REDUCTASE D OFF PLANTPHOTOSYNTHESIS0HYSIOL 0LANTARUMn 6 ILEV3 "ERGMANN! 2EDLIN( %ICHLER( *AND2ENGER' 6AS  /NTHEROLEOFEXCHANGEABLEHYDROGENBONDSFORTHE KINETICSOF0 1!nFORMATIONAND0 0HEO< q RECOMBINATION IN0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn 6ASILEV 3 3CHRÚTTER4 "ERGMANN! )RRGANG +$ %ICHLER (* 6 AND D 2ENGER '  #RYOPROTECTANT INDUCE D DD QUENCHING OF CHLOROPHYLLAmUORESCENCE FROM,(#)) F INVITRO4IMERESOLVED

(FSOPU
3FOHFS
BOE
"MGSFE
3
)PM[XBSUI mUORESCENCEAND D STEADY STATESPECTROSCOPICSTUDIES0HOTOSYN THETICAn 6ASILEV3 /RTH0 :OUNI! /WENS4'AND$INER" %X CITED STATEDYNAMICSIN0HOTOSYSTEM))INSIGHTSFROMTHE8 RAY CRYSTALSTRUCTURE0ROC.ATL!CAD3CI53!n D "RUCE$  3TRUCTURE 6ASILEV3 ,EE# ) "RUDVIG'7AND BASED KINETIC MODELING OF EXCITED STATE TRANSFER AND TRAPPING IN HISTIDINE TAGGED D 0HOTOSYSTEM)) CORE COMPLEXES FROM F 3YN ECHOCY CYSTIS"IOCHEMISTRYn 6ASILEV3 "RUDVIG '7AND D "RUCE$ 4HE8 RAY A STRUCTURE OF0HOTOSYSTEM))REVEALSANOVELELECTRONTRANSPORTPATHWAY BETWEEN 0 CYTOCHROME B AND THE ENERGY QUENCHING CATION #HL:&%"3,ETTERSn 6ASILEV3 3HEN* 2 +AMIYA.AND"RUCE$ 4HEORI ENTATIONSOFF COREANTENNACHLOROPHYLLS IN 0HOTOSYSTEM))ARE OPTIMIZED D TOMAXIMIZETHEQUANTUMYIELDD OFP F HOTOSYNTHESIS &%"3,ETTn D 6ERMAAS 7  %NERGY AND 6AVILIN $ 8U ( ,IN 3 AND F IN0HOTOSYSTEM))OFF A#HLOROPHYLL B #ON %LECTRON4RANSFER TAINING 3YNECHOCYSTIS SP 0##  -UTANT "IOCHEMISTRY n 6OS-( 2ISCHEL# *ONES-2AND-ARTIN*, %LECTROCHRO MIC DETECTION OF A COHERENT COMPONENTINTHE FORMATIONOFTHE F CHARGE PAIR 0(,nINBACTERIAL REACTION CENTERS"IOCHEMISTRY  n 7 KEHAM-# 'OODWIN-' -C+IBBIN#AND 7A D *ONES-2 0HOTO ACCUMULATIONOFT F HE01"n RADICALPAIRSTATEINPURPLE BACTERIAL REACTIONCENTRESTHAT LACKT K HE1! UBI U QUINONE&%"3 ,ETTn 7 7ALZ$ .ONEQUILIBRIUMTHERMODYNAMICSAPPLIEDTOENERGY CONVERSIONINBIOLOGICALSYSTEMS)N'RÊBER0AND-ILAZZO' EDS "IOENERGETICS PPn "IRKHÊUSER6ERLAG "ASEL 7ANG* 'OSZTOLA$ 2UFmE36 (EMANN# 3EIBERT- 7ASIELEWSKI -2 (ILLE2 'USTAFSON4,AN F D AYRE24  &UNCTIONAL D3A ASYMMETRY Y OF 0HOTOSYSTEM))$AND$PER D IPHERAL CHLOROPHYLL M DOMONASREINHARD R TII0ROC.ATL!CAD 3CI MUTANTSOF #HLAMYD 53!n 7 7ARNCKE+AND$UTTON03 )NmUENCEOF1 ! SITEREDOXCOFAC TORSTRUCTUREONEQUILIBRIUMBINDING INSITUELECTROCHEMISTRY A ANDELECTRON TRANSFERPERFORMANCEINTHEPHOTOSYNTHETICREACTION CENTERPROTEIN"IOCHEMISTRYn 7 HEL ! #HU:4AND 7ARS D 0ARSON77  $ISPERSEDPO D LARON SIMULATIONS OFF ELECTRON TRANSFER F IN PHOTOSYNTHETIC REACTION CENTERS3CIENCEn 7 IELEWSKI-2 *OHNSON$' 3EIBERT-AND 7AS D 'OVINDJEE F HEPRIMARYCHARGESEPARATIONRATEIN ISOLATED $ETERMINATIONOFT 0HOTOSYSTEM))REACTIONCENTERSWITHFSTIMERESOLUTION 0ROC.ATL!CAD3CI53!n 7ATANABE 4 AND +OBAYASHI -  %LECTROCHEMISTRY OF 7 CHLOROPHYLLS )N 3CHEER ( ED #HLOROPHYLLS PP n #2# 0RESS "OCA2ATON 7ILLIAMS *# (AFFA!,- F -C#ULLEY *, 7OODBURY .7 AND !LLEN *0  %LECTROSTATIC INTERACTIONS BETWEEN CHARGED AMINOACIDD RESIDUESAN D D THE BACTERIOCHLOROPHYLL DIMERINRE ACTIONCENTERS FROM F 2HODO D BACTERSPHAEROIDES D "IOCHEMISTRY n 7 7OODBURY.7AND0ARSON77 .ANOSECONDmUORESCENCE D FROMISOLATEDPHOTOSYNTHETICREACTIONCENTERSOF2HODOPSEUDO MONASSP S HAEROIDES "IOCHIM"IOPHYS!CTAn D $UTTON 7OODBURY.7 0ARSON77 'UNNER-2 0RINCE2#AND 0,  2ADICAL PAIRENERGETICSAND DECAY A MECHANISMS IN

$IBQUFS

1SJNBSZ &MFDUSPO
5SBOTGFS REACTION CENTERS CONTAINING ANTHRAQUINONES NAPHTOQUINONES ORBENZOQUINONESINPLACEOFUBIQUINONE"IOCHIM"IOPHYS !CTAn 8IONG, 3EIBERT- 'USEV!6 7ASIELEWSKI-2 (EMANN# (ILLE #2AND3AYRE24 3UBSTITUTIONOFACHLOROPHYLLINTOTHE INACTIVE BRANCHPHEOPHYTIN BINDING SITEIMPAIRSCHARGESEPARA TIONIN 0HOTOSYSTEM))*0HYS#HEMn D 3CHU H LTEN+ #OUPLINGOFF PROTEINMOTIONTOELECTRON 8U$AND TRANSFER F INAPHOTOSYNTHETICREACTIONCENTER)NVESTIGATINGTHE LOWTEMPERATURE BEHAVIOURINTHEFRAMEWOR F RK OFT F HESPIN BOSON MODEL#HEM0HYSn 8U ( :HANG 2 " -A 3 ( 1U : 7 :HANG 8 + AND :HANG 1 9 4HEORETICALSTUDIESONTHEMECHANISMOFPRIMARY ELECTRONTRANSFERINTHEPHOTOSYNTHETICREACTIONCENTEROF 2HO D BACTERSPH DO S AEROIDE D S0HOTOSYNTH 2ESn 8U1 "ACIOU, 3EBBAN0AND'UNNER-2 D  %XPLORING n THEENERGY LANDSCAPEFOR1 F ! TO1" ELECTRONTRANSFERIN BACTERIAL PHOTOSYNTHETICREACTIONCENTERSEFFECTO F F UBSTRATEPOSITIONAND FSU F IONAL GATING STEP "IOCHEMISTRY TAIL LENGTH ON THE CONFORMAT n 9 9AKOVLEV!' 3HKUROPATOV!9AND3HUVALOV6! .UCLEAR WAVEPACKETMOTIONBETWEEN0 AND0"!nPOTENTIALSURFACES WITHASUBSEQUENTELECTRONTRANSFERTO Q (! IN BACTERIAL REACTION CENTERSAT+%LECTRONTRANSFERPAT F HWAY"IOCHEMISTRY n

 9RUELA) 4ORRADO% 2ONCEL-AND D 0ICOREL2 ,IGHT INDUCE D D ABSORPTIONSPECTRAOFTHE$ $ CYTOCHROME BCOMPLEX OF0HOTOSYSTEM))%FFECTOFMETHYLVIOLOGENCONCENTRATION 0HOTOSYNTH2ESn :ECH3' +URRECK* %CKERT(* 2ENGER' ,UBITZ7AND"ITTL2  0ULSED D %02MEASUREMENTOF THE DISTANCE BETWEEN0 AND 1!n IN0HOTOSYSTEM))&%"3,ETTn :HANG# "OUSSAC!AND D 2UTHERFOR F D !7  ,OW TEMPERA TUREELECTRONTRANSFER F IN0HOTOSYSTEM))!TYROSYL RADICALAND SEMIQUINONECHARGEPAIR"IOCHEMISTRY n :HELEVA $ (ANKAMER " AND "ARBER *  (ETEROGENEITY ANDPIGMENTCOMPOSITIONOFISOLATED0HOTOSYSTEM))REACTION CENTERS"IOCHEMISTRYn :INTH7AND+AISER7 4IME RESOLVEDSPECTROSCOPYOFTHE PRIMARYELECTRONTRANSFER F INREACTIONCENTERSOFF 2HODOBACTER SPHAEROIDESAND 2HODO DOPSEUDOMONASVIRI D DIS )N $EISENHOFER *AND D .ORRIS *2EDS 4HE0HOTOSYNTHETIC2EACTION#ENTER 6O 6L )) PP n !CADEMIC0RESS 3AN$IEGO :OUNI! 7I 7 TT(4 +ERN* &ROMME0 +RAU”. 3AENGER7AND D /RTTH 0 #RYSTALSTRUCTUREOF0HOTOSYSTEM))FROM 3YNECHOCOC CUSELONGATUSAT¯ RESOLUTION.ATUREn :UCCHELLI ' *ENNINGS 2# 'ARLASCHI &- #INQUE ' "ASSI 2 AND#REMONESI/ 4HE CALCULATED D IN VITRO AND D IN VIVO CHLOROPHY H LL A ABSORPTION BANDSHAPE"IOPHYS*n H 

$IBQUFS
 5IF
*SPO2VJOPOF
"DDFQUPS
$PNQMFY 7BTJMJ
1FUSPVMFBT 7 *OTUJUVUF
PG
.BUFSJBMT
4DJFODF
/$43
A%FNPLSJUPT


"HIJB
1BSBTLFWJ
"UUJLJT
(SFFDF

"OUPOZ
3
$SPGUT %FQBSUNFOU
PG
#JPDIFNJTUSZ

3PHFS
"EBNT
-BC

4
.BUIFXT
"WFOVF
BOE $FOUFS
GPS #JPQIZTJDT
BOE
$PNQVUBUJPOBM
#JPMP Z HZ
6OJWFSTJU Z UZ PG
*MMJOPJT
BU
6SCBOB$IBNQBJJHO 6SCBOB
*-

64"

4VNNBSZ   *

*OUSPEVDUJPO   **

1SPCJOH
UIF
*SPO2VJOPOF
$PNQMFY
5ISPVHI
UIF
*SPO
4JUF  "
(FOFSBM   #
-JHBOE
&YDIBOHF
3FBDUJPOT
BOE
&GGFDUT
PO
UIF
2"2# &MFDUSPO
5SBOTGFS
3BUF   /JUSJD 0YJEF   
$ZBOJEF    $BSCPYZMBUF
"OJPOT  
&GGFDUT
PG
UIF
*SPO
-JHBUJPO
PO
UIF
2"2# &MFDUSPO
5SBOTGFS
3BUF   $
3FEPY
1SPQFSUJFT
PG
UIF
*SPO  %
4QJO
4UBUFT
PG
UIF
*SPO
BOE
UIF 4FNJRVJOPOF*SPO
.BHOFUJD
*OUFSBDUJPOT   &
0O
UIF
3PMF
PG
UIF
*SPO   ***

0SHBOJ[BUJPO
PG
UIF
2VJOPOF
#JOEJOH
4JUFT
5IF
5XP&MFDUSPO
(BUF  "
4USVDUVSBM
.PEFMT
PG
UIF
2VJOPOF
4JUFT   #
.FDIBOJTN
PG
UIF
5XP&MFDUSPO
(BUF   
,JOFUJD
.PEFMT
PG
UIF
5XP&MFDUSPO (BUF   
5IF Q)
%FQFOEFODF
PG
UIF
"QQBSFOU
&RVJMJCSJVN
$POTUBOU
,BQQ   
&TUJNBUJPO
PG
UIF
3BUF
$POTUBOUT
GPS
UIF
3FBDUJPOT
*ODMVEJOH
UIF
1SPUPOBUFE
4QFDJFT BOE 1SPUPO6QUBLF
BU
UIF
2#TJUF   
5IF
4FDPOE
0SEFS
3BUF
$POTUBOU
GPS
#JOEJOH
PG
1MBTUPRVJOPOF   
5IF
1BUIXBZ
GPS
&MFDUSPO
5SBOTGFS
JO
4VTDFQUJCMF
BOE
3FTJTUBOU
#JPUZQFT   
3PMF
PG
%4FS
JO
UIF
.FDIBOJTN
PG
UIF 2#4JUF   $
#JOEJOH PG
*OIJCJUPST
BOE
&YPHFOPVT 2VJOPOFT BU
UIF
2#4JUF   %
4USVDUVSBM
BOE
.FDIBOJTUJD
*OGPSNBUJPO
GSPN
.VUBHFOFTJT
4UVEJFT  
)FSCJDJEF
3FTJTUBOU 4USBJOT
PG $IMBNZEPNPOBT Z BOE
$ZBOPCBDUFSJB  
4QFDJàD
.VUBHFOFTJT
PG
)FSCJDJEF
3FTJTUBODF
4JUFT
JO 4ZOFDIPD Z Z ZTUJT   
$PNCJOBUPSJBM
.VUBHFOFTJT
PG
B
$POTFSWFE
4QBO
PG
UIF EF -PPQ   
4QFDJàD
.VUBHFOFTJT
PG
%)JT
4IPXT
BO
*NQPSUBOU
3PMF
JO
) 1SPDFTTJOH   
&YDJTJPO
PG
3FTJEVFT
GSPN
UIF
1&45
4FRVFODF
PG
UIF EF-PPQ   
.VUBHFOFTJT
UP
&YQMPSF
UIF
#JDBSCPOBUF
&GGFDU   &
&GGFDUT
PG
0DDVQBUJPO
PG
UIF
2#TJUF PO
UIF &N GPS 2" BOE
PO
UIF
&RVJMJCSJVN
$POTUBOUT JO
UIF
5XP&MFDUSPO
(BUF 

"OTHAUTHORSWELCOMECORRESPONDENCE EMAIL  VPETR IMSDEMOKR K ITOSGR  A CROFTS F LIFEU F IUCEDU

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT

'
3PMF
PG
UIF
2#4JUF
JO
#BDL3FBDUJPOT
UP
UIF
44UBUFT   
#BDL
3FBDUJPO
GSPN 2#m UP
UIF 44UBUF   
#BDL
3FBDUJPO
GSPN 2) UP
UIF
44UBUF‡"
1PTTJCMF
3PMF
GPS
$ZUPDISPNF C   *7 $PODMVTJPOT  "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 4HE mUXOFF REDUC D INGEQUIVALENTSOUTOFF 0HOTOSYSTEM))03)) OCCURSTHROUGHTHETWO ELECTRONGATE FUNCT F ION CATALYZEDBYTHEIRON QUINONECOMPLEXONTHEACCEPTORSIDE4HEMECHANISMOFTHETWO ELECTRONGATEHASBEEN D MORECOMPLETELY IN BACTERIALREACTIONCENTERS WH W EREANUNDERSTANDINGOF FUNCT F ION HAS BENElTED FROM F STUDIED ASTRUCTURAL CONTEXT(OWEVER THETWO ELECTRONGATEWAS DISCOVERED INGREENPLANTS ANDD A LARGEBODYOFF WORRK HADSUGGESTEDTHATTHEMECHANISMANDMAINSTRUCTURALFEATURESARESIMILARINTHETWOSYSTEMS ANDTHISISNOW CONlRMED BYSTRUCTURES)N03))ANUMB M EROFF ADDITIONAL PROPERTIESAREFOUN F D W D WHICHRESULTFROMT F HEREDOXACTIVITY F HEACCEPTORCOMPLEX AND FROMT F HE LABILITYOF ITSLIGANDS0ENDINGSTRUCTURES FOR03)) F OFF THENON HEME IRONOFT ATAHIGHERRESOLUTION MUCHOFTHEDISCUSSIONONTHEMOLECULARARCHITECTUREHADBORROWEDTHESTRUCTURALCONTEX R T F FROMT HEBACTERIAL HOMOLOGUE/NETHEME INTHISCHAPTERISTHE JUSTIlCATION FORT F HIS BORROWING THATCOMES FROM F THEAPPLICATIONOFSPECTROSCOP F ICAPPROACHESTOTHE03))ACCEPTORCOMPLEX4HISHASBEENESPECIALLYSUCCESSFU F L INSTUDIESOFTHE1! SITESEMIQUINONE THEMAGNETICINTERACTIONBETWEENTHESEMIQUINONEFORMEDATTHESITE AND D THE IRON AND D THE INTERACTIONOFF EXTERNAL LIGANDSWITH THE IRON!SECONDD THEME REmECTINGTHEPOORSTABILITY OFF THESEMIQU Q INONEOFF THE1" SITE INISOLATED03))PREPARAT D IONS ISTHEUSEOF INDIRECTAPPROACHES INCLUDING KINETICSTUDIESANDSTRUCTURALMODELING TOUNDERSTANDTHESTRUCTURE FUNCTIONINTERFACE4HECRYSTALLOGRAPHIC F HESEALTERNATIVEAPPROACHES STRUCTURESNOWAVAILABLEPROVIDEA GRATIFYING VALIDATIONOFT *
*OUSPEVDUJPO 4HE mUX OF REDUCING EQUIVALENTS OUT OF 0HOTOSYS TEM))03)) OCCURSTHROUGHTHETWO ELECTRONGATE F FUNCT ION CATALYZED BY THE IRON QUINONE COMPLEX ONTHEACCEPTORSIDE4HECOMPLEXCONSISTSOFTWO PLASTOQUINONEMOLECULES 1! AND 1" SEPARATED BY D THEPROTEINMILIEUTHAT ANON HEME IRON)) ION AND HOUSESTHESEREDOXCENTERS4HETWO QUINONESO Q PERATE AS SEQUENTIAL ELECTRON ACCEPTORS 1! BEING A ONE F R AND 1" A TWO ELECTRON ACCEPTOR %LECTRON TRANSFE RATESBETWEENTHETWO QUINONESAREOFTHEORDEROFA Q F FEWTENT HSOFAM F ILLISECOND D BUTTHESEVARY DEPEND INGONTHETREATMENT3PECULATIONABOUTTHESPATIAL ORGANIZATIONOFTHEIRON QUINONECOMPLEXHADBEEN !BBREVIATIONS R !TRAZINEn CHLORO N ETHY H L .|  METHY H LETHY H L    TRIAZINE   DIAMINE"1nBENZOQU Q INONE"2#nBACTER IAL REACTION CENTER #HL n CHLOROPHYLL #YT n CYTOCHROME $ U UNITS OF F 03)) $#"1 n   $I $n REACTION CENTER CORE SUB CHLORO P

P P "1$#-5n  DICHLOROPHENYL   DIMETHYLUREA )3nISOMERSHIFT .2nNEUTRALRED O PHENANTHROLINEn  PHENANTHROLINE P "1n  BENZOQUINONE0$"nPROTEINDATA BANK0 P "1n0HENYL P

"101nPLASTOQUINONE03))n0HOTO Q

SYSTEM )) 1! 1" n PRIMARY ANDD SECONDARY 01 ACCEPTORS OF 03)) 13!2 n 1UANTITATIVE 3TRUCTURE !CTIVITY 2ELATIONSHIP 51nUBIQ U QUINONE

DERIVEDD MAINLY FROMSPECTROSCOP F ICSTUDIESANDPRO D

TEIN SEQUENCECOM Q MPARISONSWITH THTTHEBACTERIAL REACTION O SEUDOMONASVIRIDIS AND CENTER"2# FROM 2HODOP 2HODO D BACTERSPHAEROIDE D S NOWVALIDATED BYCRYSTAL LOGRAPHICSTRUCTURES SEE &IGFORANOVERV F IEW )N THE PRESENT CHAPTER WE AVOID REPEATING TOPICS THAT WEREEXTENSIVELYCOVERED INEARLIERREVIEWS#ROFTS F ANDD 7RAIGHT 2ICH ANDD -OSS $INERET AL $INERAND"ABCOCK  %MPHASISIS D F THEIRON QUINONE RATHERGIVENTOSELECTEDASPECTSO COMPLEX THEPROGRESSTHATHAS BEENMADEINRECENT YEARS ANDTOPICSVIEWSWHICHWERENOTADEQUATELY COVERED INTHEEARLIERREVIEWS **
1SPCJOH UIF
*SPO2VJOPOF
$PNQMFY 5ISPVHI
UIF
*SPO
4JUF "
(FOFSBM
4HEIRONLOCATEDBETWEENTHETWOQUINONESHASSTRONG SPECTRAL ANDD STRUCTURAL SIMILARITIES TO THE GENERAL CLASS NOT OFTEN F RECOGNIZEDD AS SUCH OFF NON HEME MONONUCLEAR IRONCENTERSFOUNDINOXYGEN ACTIVATING F IONS INCLUDING ENZYMESWITH DIVERSECATALYTIC FUNCT

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT OXIDATIONS MONO AND DI OXYGENATIONS AND HY H DRA TIONS-ICHELAND$EISENHOFER !NDERSONET AL $INERETAL ,IPSCOMBAND/RV D ILLE D /KAMURA   1UEETAL &EHERAND #HARACTERISTICOFTHISTYPEOFIRONCENTERISTHEmEX IBILITY OFF THE IRON COORDINATION 4HE COORDINATION NUMBERVARIESBETWEENAND D WITHTHEMOSTCOM MONLIGANDSBEINGNITROGENSHISTIDINE IMIDAZOLE AND D OXYGENS CARB R OXYLATE PHENOLATE WATER  4HE D LATESTOAGREAT VARIATION INTHELIGAND DONORSETMODU EXTENTTHEREDOX POTENTIALOFTHEIRONBETWEENEXTREME LIMITS!DIFFERENTNUM F BEROF LABILELIGANDSCANBE F FOUN D INEACH CASEAND D THIS IS LINKED D TOTHESPECIlC CATALYTIC FUNCTION OF THE IRON #HARACTERISTIC IS THE F LL MOLECULESLIKENITRICOXIDE REVERSIBLE BINDING OFSMA D CYANIDE ,IPSCOMBAND D /RVILLE /RVILLE AND AND,IPSCOMB  (EGGAND1UE COMPAR INGTHESTRUCTURESOFF ANUMBEROF DIFFERENTENZ F YMES D THAT THECOMMON CATALYZINGDIVERSEREACTIONSNOTED COORDINATIONFEATUREINTHISTYPEOFIRONCENTERSISA R OXYLATEFAC F IALTRIAD4HEOCCUPATIONOF  (IS  CARB THETHREEREMAININGCOORDINATIONSITESBYADDITIONAL ENDOGENOUS PROTEIN RESIDUES ANDOR EXOGENOUS LI GANDS EG SUB U STRATE MOLECULES TUNES TO A GREAT EXTENTTHEPROPERTIESOFT F HEMETALCENTER(EGGAND 1UE  1UE  4HE IRON OF THE "2# HAS PERH R APSTHEMOSTINERTCOORDINATIONWITH(ISPLUS D GLUTAMATE BIDENTATELIGAND $EISENHOFERE F T A lXED AL !LLENETAL  ANDAHIGH%M "EIJER AND D 2UTHERFOR F D $ D INERAND D 0ETROULEAS B  "ASED D ONEXTENSIVESPECTROSCOPICAND D SEQUENCE Q HO MOLOGIES2UTHERFORD -ICHELAND$EISENHOFER REVIEWED IN$INERETAL  THE IRONOF03)) F ISALSOASSUMED D TO BECOORDINATED BY FOUR F HISTIDINES TWOFROMEACHOFTHETWO PROTEINSUBUNITS$AND $ #OMPARED D TOTTHE"2# TH T EREAREIMPORTANTDIFFERENCES F INTHE lFT F HANDD SIXTHCOORDINATIONPOSITIONS!TLEAST ONE OF THESE POSITIONS IS OCCUPIED BY BICARBONATE IN 03)) $INER AND D 0ETROULEAS   !CCORDING TO &4)2 STUDIES BICARB R ONATE BINDS AS A BIDENTATE LIGAND IN THE REDUCED STATE OF THE IRON &E AND D STATE(IENERWADEL AND MONODENTATE INTHEOXIDIZED "ERTHOMIEU  4HEIRONOFF 03))HASAT LEASTTWO LABILELIGANDSANDISREDOXACTIVE ASDETAILEDBELOW 4HESE PROPERTIES DIFFERENT F IATE IT FROM F ITS BACTERIAL COUNTERPART AND D SUGGESTAMORERICHPHYS H IOLOGICAL F FUNCTION4HELABILITYOFTHEBICARBONATELIGANDPRO B ABLY EXPLAINSCERTAIN HETEROGENEITIESASSOCIATED D WITH THEIRON QU Q INONECOMPLEX

 #
-JHBOE
&YDIBOHF
3FBDUJPOT
BOE
&GG & GFDUT
PO G UF UIF
2"2# &MFDUSPO
5SBOTGFS
3BU )N03)) UNLIKETHE"2#WH W ERENO LABILITYOF LIGANDS HASBEENREPORTED ANUMBEROFMOLECULESCANBIND REVERSIBLY ATTHENON HEMEIRONSITEOFTEN F INCOMPE TITION WITH BICARB R ONATE RESULTING IN MOST CASES IN DECELERATION OFTHE ELECTRONTRANSFE F R 
/JUSJD 0YJEF !DIRECTDEMONSTRATIONOFEXO F GENOUS LIGAND BINDING TOTHE IRON HASBEENPROVIDED BYTREATMENTWITHNITRIC OXIDE WHICHGIVESRISETOAPRONOUNCED%02SIGNAL AT G CHARACTERISTICOFANIRON NITROSYLCOMPLEX G WITH 3 0ETROULEASAND$INER  4HIS IS F LD DECREASE OFTHE ACCOMPANIEDBYAN ATLEASTTENFO F BETWEEN 1! AND 1" FROMT F HE ELECTRONTRANSFERRATE SECONDD SATURATINGmASHEXCITATIONONWARD $INERAND 0ETROULEAS  4HE EFFECTS OF ./ ARE REVERSED BY THEADDITIONOFAPPROX F IMATELY M- BICARB R ON ATE$INERANDD 0ETROULEAS  4HISSUGGESTSTHAT BICARBONATEISALABILELIGANDTOTHEIRON CONlRMING F EARLIERPREDICTIONS-ICHEL ANDD $EISENHOFER  VAN2ENSENETAL  /RIENTATIONSTUDIESOFF THE G GSIGNALIMPLYTHAT./ ANDBYEXTRAPOLATIONONE OF F THE OXYGENS OF BICARB R ONATE OCCUPY A POSITION HOMOLOGOUSTOONEOFT F HEGLUTAMATEOXYGENSOFT F HE PURPLEBACTERIA$ELIGIANNAKISETAL  &ORMATE W CH ALSOCOMPETESWITH BICARB R ONATESEE ANANIONWHI BELOW BINDSSIMULTANEOUSLYWITH ./ ASITDOESNOT DECREASE THE &E ./SIGNALBUTITCHANGESSOMEWHAT ITS ROMBICITY $INER ANDD 0ETROULEAS   $IRECT EVIDENCE FFOR THE SIMULTANEOUS BINDING OFF ./ AND ANOTHERANIONTOTHEIRON HASBEENPROVIDEDBYTHE F D SUPERRHYPERlNESTRUCTURE OBSERVATIONOFPRONOUNCE ONTHE GSIGNALFROMTHEmUORIDENUCLEUS UNDER G THESIMULTANEOUSPRESENCEOF./AND&n3ANAKISET AL  ./AND &n HAVEBEENSUGGESTEDTOBIND CISTOEACHOTHERINA HEXA COORDINATEARRANGEMENT 3ANAKISETAL  )TISREASONABLETOASSUMETHAT ./AND FORMATE F BIND INASIMILARFAS F HION%XPERI MENTS WITH ORIENTEDD MEMBRANES * (ANLEY ANDD 6 0ETROULEAS UNPUBLISHED SHOW THATTHE ORIENTATION OF G

THE GSIGNALPRINCIPALAXESDOESNOTCHANGEEX CEPTFORAN F INTERCONVERSIONOFT F HEXANDYAX D IS INTHE PRESENCEOFmUORIDE4HISANDTHEFACTTHATTHEADDED ANIONS DONOTALTERSIGNIlCANTLYTHESPIN(AMILTONIAN PARAMETERS OF F THE &E ./ 3   CONlGURATION STRONGLYSUGGESTSTHATTHE IRON ./COMPLEXINTHE F GENOUSANIONSISALSOHEXA COORDINATE A SENCEOFEXO AB

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT

 )TISPOSSIBLETHATATTHE INITIALSTAGESOF BICARB R ONATE DISPLACEMENT BICARBONATEBINDSSIMULTANEOUSLYWITH ./ASAMONODENTATELIGAND&ULL DISPLACEMENTOF BICARB R ONATE ANDREP D LACEMENT FOREXAMP F LE BYWATER ISPROBABLYFACILITATEDBYTHEREDUCTIONOF1!n ASTHE EXPERIMENT DESCRIBED BELOW SUGGESTS4HIS WOULD OFFERA F POSSIBLEEXPLANATIONOFT F HEOBSERVATIONMEN TIONEDABOVE THATTHE./ INDUCEDDECELERATIONOFTHE ELECTRONTRANSFER F DEVELOPSFU F LLY FROMT F HESECOND mASH AND D ONWARD $INERAND D 0ETROULEAS  )TCOULD ALSOEXPLAINTHESMALLMODIlCATIONSMALLINCREASEIN F LLOWINGTHE ROMBICITY OFF THE&E ./%02SIGNAL FO lRST ILLUMINATIONDARK R ADAPTATIONCYCLEINASERIESOF CYCLESEFFECTINGSUCCESSIVEELECTRONTRANSFERSAMONG THEQUINONES 0ETROULEASAND D $INER   2ECENTLYANEWMODEOFTRANS F IENT BINDINGOF. F / TO THE IRONHASBEENREPORTED4HEAFlNITYFORTHIS D IONOF 1! MODEOF BINDING ISENHANCED BYTHEREDUCT 'OUSSIAS ET AL  4HE NEW TRANSIENT STATE IS TRAPP A EDATn O#ANDISCHARACTERIZEDBY%02SIGNALS WITH GVALUESOFINTHEPRESENCEOF./ALONEOR G AND D  INTHESIMULTANEOUSPRESENCEOF./ F AND#.n!N IMPORTANTIMPLICATIONOFTHERESULTSIS

ATRANSINmUENCEOF1! REDUCT D IONONTHE BICARB R ONATE LIGATIONTOTHEIRON4HISISTRANSMITTEDPRESUMABLY VIA HYDROGEN BONDING ( BONDING OF 1! WITH AN IMIDAZOLELIGANDD TOTHE IRON)NTHEABSENCEOFF #.n ./ HAS BEEN CONCLUDED TO BIND IN A  COORDINATE GEOMETRY UN Y DERTHESECONDITIONS 0ROLONGED INCUB U ATIONWITH./APPEARSTORESULT INADINITROSYL IRONSPECIES'OUSSIASETAL   2EDUCT D ION BY FREE./EXP F LAINSPROBABLY THEAPPEAR ANCEOFF WEAK %02SIGNALSIN DARK R ADAPTEDD MATERIAL VERYSIMILARTOASYNTHETIC&E./ IMIDAZOLE SPE CIES BUT FU F LL EVOLUTIONOFT F HESIGNAL ISEFFECTE F D BY ILLUMINATION0RESUMABLY1!n REDUCESTHEDINITROSYL COMPLEXUNDERTHESECONDITIONS 4HE PROPOSED IRON COORDINATION IN THE PRESENCE F ./ ANDD THE VARIOUS ANIONS IS SUMMARIZED IN OF 3CHEME  $ZBOJE $ F #YANIDE BINDSSTEPWISE +OULOUGLIOTISETAL  3ANAKIS ET AL   7I 7 TH AN APPROXIMATE +D OF  —- ITELIMINATESTHE GSIGNALINDUCED G

3 HEME 3UGGESTED 3C D MODESOFF ./BINDINGTOTHE IRON

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT BY ./ !T HIGHER CONCENTRATIONS +D   M- #.nCAUSESASHIFTOFTHE1!n &E SIGNALTO G G AND D MODIlES THE SPLIT PHEOPHYTIN SIGNAL 3ANAKIS AND D 0ETROULEAS  AND D WITH ANAPPROXIMATE +D OFM-ITCONVERTSTHEIRONTOLOWSPIN3 3 3ANAKISETAL  4HECYANIDEEXPERIMENTS IMPLY THATUPTOTHREESITESAT ORNEAR THENON HEMEIRON AREACCESSIBLETOEXOGENOUSLIGANDS4HEDATADONOT PROVIDEDIRECTEVIDENCETHATALLCYANIDES BIND D AS IRON LIGANDS SINCEBINDING INTHEVICINITYOFF THE IRONCOULD EXPLAINSOMEOFTHEEFFECTS)TISEXPECTED HOWEVER THATCONVERSIONOFF THE IRONIONTOITS LOWSPIN FORM F WOULDREQU D IRETHE BINDINGOFF MORETHANONE#.n LI GAND4HISISCORROBORATEDBY&4)2STUDIES WHICH REVEAL ATLEASTTWO#.n BANDSINTHE1!n  1! SPECTRA .OGUCHIETAL A 4HEEFFECTSOFCYANIDECAN BEREVERSEDBYPROLONGEDSTORAGEINACYANIDEFREE MEDIUMATP(3ANAKISETAL   
$BSCPYZ YZMBUF
"OJPOT !PARTFROM./AN F D CYANIDE ANUMB M EROFF CARRBOXYLATE ANIONSAPP A EARTOBINDATTHENON HEMEIRONINAPP A ARENT T BICARB R ONATE&ORMATEINPARTICULAR COMPETITIONWITH D ACETATEHAVE BEENSTUDIED D EXTENSIVELY INTHEPAST AND ASCOMPETITIVEINHIBITORSOFBICARBONATE2OBINSONET AL 3TEMLERAND-URP D HY %ATON 2 Y 2YEAND 'OVINDJEE  4HEEXTENSIONOFF THESTUDIESTOA SERIESOFCARBOXYLATEANIONSEG GLYCOLATE GLYOXYL ATE OXALATEAND LACTATE $ELIGIANNAKISETAL  0ETROULEASETAL  HASYIELDED IMPORTANTNEW INSIGHTSONTOTHEMODEOFACTIONOFTHECARBOXYLATE LIGANDSTOTHE IRON4HEANIONSCOMPETEWITH./FOR F BINDINGTOTHEIRON$ELIGIANNAKISETAL  IMPLY INGTHATTHEYBINDASBIDENTATELIGANDS&ORMATE THE R OXYLATEANIONSAPPEARSTOBETHE SMALLESTOFF THECARB ONLYEXCEPTION BINDINGSIMULTANEOUSLYWITH ./SEE 3ECTION))" $IFFERENCE&E&E &4)2SPECTROS COPYWITH # AND # LABELED LACTATE INDICATESTHAT THISANIONBINDSTHROUGH ONECARB R OXYLATEOXYGENAND THE HYDROXYLGROUP INBOTH OXIDATION STATESOFTHE IRON"ERTH T OMIEUAND D (IENERWADEL  'LYCOLATE ANANIONTHAT HASASIMILAREFFECTW F ITH LACTATEONTHE &E %02SPECTRA$ELIGIANNAKISETAL  ANDON SIGNIlCANTLY LOWERINGTHEMIDPO D INTPOTENTIAL OFF THE IRONDISCUSSED BELOW ISINFERRE F D TO BIND INASIMILAR F FASHION"ERTHOMIEUAND(IENERWADEL   
&GGFDUT
PG
UIF
*SPO
-JHBUJPO
PO
UIF
2"2# &MFDUSPO
5SBOTGFS
3BUF G /FF ALL TH T EMOLECULESTH T ATBIND D ATTTHE IRONSITE BICARB R ON

 ATEAPPEARSTO BEUNIQUE WITHTHEPOSSIBLEEXCEPTION OFCYANIDE WHOSEEFFECTSONTHEELECTRONTRANSFERRATE F LLYUNDERSTOOD +OU D LOUGLIOTISETAL  ARENOT FU INSUPPORTINGANUNDIMINISHEDD ELECTRONmOWINTHE IRON QUINONECOM Q PLEX2EPLACEMENTWITHFORMATEIS WELL KNOWNTO K INDUCEAPRONOUNCE D D SLOWINGOFT F HE F HREE TO FOUR F F LD FO D SLOWINGON ELECTRONTRANSFERRATEAT THElRSTmASH ANDA *TENFOLDSLOWING ONSUBSEQUENT mASHES2OBINSONETAL 3NEL ANDVAN2ENSEN D %ATON 2YEANDD 'OVINDJEE  ./ HASA SIMILAREFFECT$INERAND0ETROULEAS  !MONG THEVARIOUSCARB R OXYLATEANIONSEXAMINED D GLYCOLATE HASASIMILARPRONOUNCEDD EFFECTAN F D NOTABLYITEXERTS F TS AT A CONCENTRATION AT LEAST  FO F LDD LOWER ITS EFFEC THANTHATOF FORMATEW F ITH A +M OFF nM-ATP( 0ETROULEASETAL  'LYOXYLATEANDD OXALATE INDUCE LESS PRONOUNCED EFFECTS 0ETROULEAS ET AL R ONATEPLAY A S  )T HAS BEENSUGGESTEDD THATBICARB D H HY AROLEINTHEPROTONATIONOFF 1" 3TEMLERAND-URP VAN2ENSENETAL $INERETAL  4HE PARALLELEFFECTSO F F THEVARIOUSANIONSONTHEMIDPO D INT POTENTIAL AND ITSP(DEPENDENCE OFT F HE IRON SEE BELOW THE 1!n &E %02 SIGNALS SEE BELOW AND THE1!1" ELECTRONTRANSFERRATE COU F LD BEEXPLAINED IFWEASSUMET F HAT BINDINGOFT F HEANIONSTOTHEIRON MODIlESTHE P+ +A OFANEARBYGROUPS INVOLVEDINTHE PROTONATIONOF1 F " .UGENTETAL $ELIGIANNAKIS ETAL 0ETROULEASETAL  )NSIGHTSAREOF F F FEREDBY&4)2STUDIES WHICHINDICATEAHYDROGEN BONDD NETWORRK FFROM THE NON HEME IRON TOWARDD THE 1" POCKET INVOLVING BICARB R ONATE ANDD $ (IS "ERTHOMIEUAND(IENERWADEL  )NDEPENDENT SUPPORTOFF THESESUGGESTIONSCOMESFROMT F HECOUN TEREFFECTT F HAT1! REDUCT D IONHASONTHE BICARB R ONATE LIGATIONDETECTEDBYTHEUSEOF./ TRANSMITTEDVIA THE ( BONDING OFF $ (IS  TO 1!n 'OUSSIAS ET AL SEE3CHEME 4HESYMMETRICSHAPEAND +A OFITS BINDINGOFTHEBICARBONATEMOLECULE THEP+ PROTONATABLE GROUPSTOGETHERWITH ITSSIZECOULD BE F INTHESUPP U ORTOF ITS PROPOSEDD ROLE CRITICAL FACTORS 7HENBICARBONATEISEXCHANGEDBYOTHERMOLECULES EG ./ AN ALTERNATIVE SLOW PROTON PATHWAY A MAY A F FUNCT ION!S HOWEVER ACCORDING TO SCHEME  BI CARBONATEMAYSTILLREMAINBOUNDASAMONODENTATE LIGANDD UNTIL AFTERT F HEREDUCT D IONOF1 F ! BY AlRSTmASH INAmASHSERIES THEPROTONATION LIMITEDD SLOWINGOF THEELECTRONTRANSFERAPPEARSAFTERTHESECONDmASH )T ISPOSSIBLETHATCERTAINANIONSWH W OSEEFFECTONT F HE ELECTRONTRANSFERRATE F ISMODERATEEG GLYOXYLATE CAN SUBSTITUTE IN PART FOR BICARBONATE OR AFFECT THE P+ +A VALUESOFF PROTONATABLE GROUPSALONG THEPROTON TRANSLOCATIONPATHWAY

 $
3FEPY
1SPQFSUJFT
PG
UIF
*SPO 4HE INTERESTING REDOX PROPERTIES OF F THE IRON HAVE BEEN REVIEWEDD EXTENSIVELY IN THE PAST $INER AND 0ETROULEAS B "RIEmY THE&E&E COUP U LEHAS BEEN IDENTIlED D ASTHE 1 ELECTRONACCEPTOR)KEGAMI AND D +ATOH  WITH AMIDPO D INTPOTENTIALOFF ABOUT M6ATP(THATVARIESBYAPPROXIMATELYnM6 PERP(UNITBETWEENP(AND D  "OWESETAL A0ETROULEASAND D $INER  4HISPOTENTIAL ISRATHERHIGHFORPARTICIPATIONOFTHEIRONINELECTRON TRANSFERREACT F IONS/NCETHE IRON ISOXIDIZED D HOWEVER 1!n REDUCESITRAPIDLYTš —SATP( REVIEWED IN$INERAND0ETROULEAS B /NTHEOTHERHANDA NUMBEROFF EXOGENOUSQUINONES EG P "1 0 P

P "1 D $#"1 ACTINGTHROUGH THE1" SITECANOXIDIZE AND THE IRON ONCE REDUCED BY 1!n TO THE SEMIQUINONE F FORM :IMMERMANNAND D 2UTTHERFOR F D  D 0ETROULEAS D $INER A B  4HE CARB R OXYLATE ANIONS THAT AND COMPETE WITH BICARBONATE FOR BINDING AT THE IRON MODIFY SIGNIlCANTLY THEREDOXPROPERTIESOFF THE IRON $ELIGIANNAKIS ET AL   'LYCOLATE AND LACTATE LOWERTHEMIDP D OINTPOTENTIALTOM6ANDMAKEIT P( INDEPENDENT WHIL W EOXALATE MALATEAND FORMATE F INDUCEM D IDPO D INTPOTENTIALSHIGHERTHANM6 INTHE P(RANGETO'LYOXYLATEANDPYRUVATEHAVE INTERMEDIATEEFFECTS4 F HEEFFECTO F F HESEANIONS HAS FT BEINGATTRIBUTED D TOTHESHIFTO F F THEP+ +A OFAN F IONIZABLE GROUPTHATISRESPONSIBLEFORTHEP(DEPENDENCEOFTHE MIDPO D INTPOTENTIALOFF THEIRON-OLECULESTHAT BIND D AT THE 1" SITE $#-5 O PHENANTHROLINE ATRAZINE 0 P "1 APPEARTORAISETOAVARIABLEEXTENTTHEMIDPOINT POTENTIALOFT F HEIRON/XIDATIONOFF THE IRON ONTHE D LOWERSTHEAFlNITYFOR F BINDINGATTHE 1" OTHERHAND SITE7RAIGHT   %
4QJO
4 4UBUFT
PG
UIF
*SPO
BOE
UIF 4FNJRVJOPOF*SPO
.BHOFUJD
*OUFSBDUJPOT 4HE IRONCANATTAINAVARIETYOFF SPINSTATES FO F LLOWING A NUMBER OF REVERSIBLETREATMENTS 3   OBTAINED D INTTHE&E STATE4HE%02SPECTRA ARE CHARACTERIZED BY RESONANCES AT G G    0ETROULEASAND$INER  ASSIGNEDTOASYS TEMWITHPARAMETERS $ n+ %$ %  $INER AND D 0ETROULEAS B!ASAETAL  "INDING OFEXOGENOUSMOLECULESATTHE1" SITE REDUCES THE %$  VALUERESULTING INSOMECASES INAXIALSPECTRA )TOH ET AL  $INER AND D 0ETROULEAS A B  /RIENTATIONSTUDIESINDICATETHATTHE GYAXISRUNSALONG THE HOMOLOGOUS DIRECTION OF F THE BACTERIAL TWOFO F LD

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT SYMMETRYAXIS $ELIGIANNAKISETAL  4HE GZ AXIS IS INFERRED TO LIE ALONG THE . &E . DIRECTION DElNED BY THENITROGENLIGANDSFROMT F HEHISTIDINES F HETWOQUINONES NOTPARTICIPATING INTHEBINDINGOFT 0HOTOREDUCIBLE HIGH SPIN IRON SIGNALS INITIALLY AT TRIBUTEDD TO HEME IRONHAVE BEENNOWASSIGNEDD TOTHE NON HEMEIRONOFT F HEACCEPTORSIDE .UGENT   #ARBOXYLATEMOLECULESBOUNDONTHEIRONEXERTSMALL EFFECTSONT F HE $ PARAMETERBUTSOMEOFF THEANIONS INDUCERESONANCESAT D G CORRESPONDINGTO $   + AND %$  $ELIGIANNAKISETAL   .O EVIDENCE FFOR THE TRAPPING OFF THE INTERMEDIATE 1!n &E HASBEEN PRESENTED)TISPOSSIBLE HOWEVER THATTHELIGHT INDUCEDDECREASEOFTHE&E SIGNALAT +AND LOWERTEMPERATURES $INERANDD 0ETROULEAS D HEALTERATION B.UGENT  MAYBE DUETOT OFTHE &E SIGNALBYTHEMAGNETICINTERACTIONWITH 1!nRATHERTHANTHEREDUCTIONOFTHE&E WHI W CH MAY A BELIMITED BYTHEUPTAKEOFF APROTON 3  NATURALSTATE&E INTHEMAJORITYOFCENTERS .O%02SIGNALSHAVE BEENREPORTED FORT F HISINTEGER SPINSTATE BUT-OSSBAUERSPECTROSCOPY HASPROVIDED VALUABLEINFORMATIONREVIEWEDIN$EBRUNNER   5SEFU F L INSIGHTS HAVE BEENPROVIDED BY COMPARISON WITH THASERIESOFSYNT F THETICMODELCOMPOUNDS "OINNARD ETAL 'ARGEETAL 2AKOTONANDRASANAET AL -ARTINEZ,ORENTEETAL  4HE-OSS BAUERSPECTRAIN""9PREPARATIONSAREHETEROGENOUS CONTAININGATLEASTTWOCOMPONENTSASSIGNEDTOTHE F F  NON HEMEIRONWITH ACOMMONISOMERSHIFTO MMSANDD QUADRUPO D LESPLITTINGSMMSCOMP ANDCOMP 0ETROULEASETAL  #OM PONENT  APPEARS TO BE THE DOMINANT ONE IN MOST PREPARATIONS 0ETROULEASETAL 0ICORELETAL  'ARBERS ET AL   )N PREPARATIONS FROM #HLAMYD M DOMONAS REINHARD R TII A SINGLE COMPONENT WITH ASIMILARISOMERSHIFT F BUTANAVERAGEQUADRUPOLE SPLITTINGWASOBSERVED0ETROULEASAND$INER   4HE ISOMER SHIFT F IN ALL CASES INDICATES OCTAHEDRAL COORDINATION WITH OXYGENS OR NITROGEN LIGANDS AS WASORIGINALLYSUGGESTEDFORTHEHOMOLOGOUSIRONIN 2SPHAEROIDES D 2  $EBRUNNERETAL "OSO F LLOF ETAL  4REATMENTWITH FORMATECONVERTSA THEABSORPTIONAREATOACOMPONENTTYPESPECTRUM $INER ANDD 0ETROULEAS B 3EMIN ET AL  0ETROULEASETAL  )NTHEREDUCE D DSTATE T D HEPRI MARYQUINONE "OINNARDETAL 3š INTERACTS 3 MAGNETICALLYWITH THENON HEMEIRON 3 4HIS RESULTS INASEVEREBROADENINGOFF THE%02SPECTRUMOF THESEMIQUINONEANDTHEAPPEARANCEOFHETEROGENOUS F FEATURES INTHEGnREGIONOFT F HE%02SPECTRA

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT .UGENTETAL 2UTHERFOR F D AND :IMMERMANN .UGENTETAL  WITHPRONOUNCEDANISOT ROPY 2UTHERFOR F D  D .OTABL A EISTHEP( DEPENDENT EQU Q ILIBRIUM BETWEEN THE @ AND D THE @ FORM F OFTHE1!n &E SIGNAL2UTHERFORDAND:IMMERMANN D THE DRAMATIC ENHANCEMENT BY FORMATE F  AND OFF THE G FORMOFTHE 1!n&E SIGNAL 6ERMAAS AND2UTHERFORD  !CORRELATIONAPPEARSTOEXIST BETWEENCOMPONENT INTHE-OSSBAUERSPECTRA THE G FORMANDTHENON OXIDIZABLECONlGURATION OFTHEIRON0ETROULEASETAL $ELIGIANNAKISET AL  4HESMALLERQUADRUPOLESPLITTING VALUEOF COMPONENT0ETROULEASETAL  ASWELLASOF THE BACTERIAL &E $EBRUNNERETAL "OSOETAL D TOARADIAL EXPANSIONOFT F HE  HASBEENASSIGNED D ELECTRONSOFT F HEIRON ANEFFECTT F HATWOULD D STABILIZE D THE &E OXIDATIONSTATE ASOBSERVEDEXPERIMENTALLY F F FORMATEANUM BEROFCAR F RBOXYLATEANIONS !PARTFROM F BICARB R ONATEAPPEARTOSUPPORTEITHER SUB U STITUTING FOR THE@ORTHE@CONlGURATION$ELIGIANNAKIS ET AL   4HE P( DEPENDENT HETEROGENEITY IN THEPRESENCEOF BICARB R ONATEALONEAND D THECHANGES INDUCEDBYTHEVARIOUSCARBOXYLATEANIONSHAVE BEEN ATTRIBUTED D TOTHESHIFTO F F THEP+ +A OFF ACRITICAL IONIZABLE GROUP $ELIGIANNAKISETAL  POSSIBLY(IS$ "ERTHOMIEUAND(IENERWADEL  #YANIDE D IRELYNEW ATMODERATECONCENTRATIONS INDUCESANENT 1!n &E %02SIGNALWITH PRONOUNCED INTENSITYATG +OULOUGLIOTISETAL  4HESHIFTOFTHESIG NAL CLOSETO GMUSTBEDUEPRIMARILYTOCHANGES G INTHEZERO lELDSP D LITTINGOFT F HE IRON))  %02SIGNALSINTHEGTOREGIONHAVEBEEN ALSO REPORTED FFOR THE 1"n&E STATE (EATHCOTE AND 2 HERFOR 2UT F D (U D UBBARD D ETAL  !PROMINENT %02SIGNALAT G HASBEENDETECTEDFROMTHE STATE1!n&E1"n(ALLAHANETAL   3  O  BTAINED BYT B THE BINDINGOF OF ./TOTTHEIRON 4HE%02SPECTRAARECHARACTERIZEDBYAPROMINENT DERIVATIVEAT G  INDICATIVEOFAXIALSYMMETRYWITH ASMALLRH R OMBICITY0ETROULEASAND D $INER  4HE RESPECTIVESPIN(AMILTONIAN PARAMETERSARE $   ( CMn AND %$  AND D THESECHANGESLIGHTLY F LLOWING THE SIMULTANEOUS BINDING OF FORMATE FO F $INERAND0ETROULEAS  ORmUORIDE3ANAKISET AL  )NTHEPRESENCEOF mUORIDETHEPARAMETERS BECOME$CMn AND D %$  AS DETERMINED BYTHESIMULATIONOFTHE%02SPECTRAATTWODIFFERENT MICROWAVE FREQUENC F IES 8AND1 D BAND 3ANAKISET AL  3TUDIESOFF ORIENTED D SPINACHMEMBRANES TREATEDWITH./SHOWTHAT GX LIES ONTHE MEMBRANE PLANEWHIL W E GY ISORIENTED D ATO AND GZATO WITH

 RESPECTTOTHEMEMBRANEPLANE$ELIGIANNAKISETAL  !SIMILARAXESORIENTATIONISOBSERVEDINTHE SIMULTANEOUSPRESENCEOFF &n(ANLEYAND0ETROULEAS UNPUBLI U SHED 4HESIMULTANEOUS BINDINGOFF ./AND mUORIDE TO THE IRON INDUCES PROMINENT HYPERlNE SPLITTING OFF THE GG   RESONANCE FROM THE mUORIDE NUCLEUS) ) š &OLLOWINGREDUCTIONOF1! THESYSTEM OFTHETWOHALF INTEGERINTERACTINGSPINSšFORTHE F HE IRON ./COMPLEX DOES SEMIQUINONEANDD FORT NOT HAVEDETECTABLERESONANCESAT8 BAND FRE F QUE Q N CIESBUTITHASRESONANCESAT1BAND)NTHESIMUL TANEOUS PRESENCE OF mUORIDE HOWEVER PROMINENT RESONANCES WITH COMPONENTS IN PERPENDICULAR AND PARALLEL MODE %02 AT 8 AND 1 BAND FREQUENCIES Q ARE OBSERVED 4HE SYSTEM OF F THE TWO HALF F INTEGER INTERACTING SPINS HAS BEEN SUCCESSFU F LLY SIMULATED WITHANANTIFERROMAGNETIC COUPLINGCONSTANTOF CMn INTHEPRESENCE ANDD CMn INTHEABSENCEOF &n3ANAKISETAL  .OISOTOPICEFFECT./ &E VS ./ &E COULDBEDETECTEDINTHE#7%02AND %3%%-SPECTRA INDICATINGTH T ATTH T ESPINDENSITY TY ONTTHE F ISVERYSMALL $ELIGIANNAKIS NITROGENNUCLEUSOF./ ETAL  4HECOUPLINGTOTHREEORFOURIMIDAZOLE NITROGENSCOULD BE DETECTED BY THEAPPLICATIONOFT F HE (YSCORESPECTROSCOPY YIELDINGTH T E lRSTSPECTROSCOPIC EVIDENCE SUPPORTING HISTIDINE LIGATION TO THE IRON $ELIGIANNAKISETAL   3 š OBTAINEDD UNDERVARIA D BLECONDITIONS 4HESE CONDITIONSAREI CHEMICALOXIDATIONOFTHEIRONIN THE PRESENCE OF HIGH CONCENTRATIONS OF F CYANIDE  ORCYANIDEIONSBOUNDD ONTHE IRON 3ANAKISAND 0ETROULEAS  4HE%02SPECTRAARECHARACTERIZED INTHISCASEBYWEAKK RESONANCESAT GX AND GY  F D D#. INTHEPRES II BINDINGOF./OR./AN ENCEOFTHEREDUCED1! ATn O#'OUSSIASETAL  .EW 1!n&E %02SIGNALSAPPEARINTHISCASEAT  PRESENCEOF./A F LONE OR  INTHE SIMULTANEOUSPRESENCEOF./AND#.n AND UNLIKE ALL F HECOMPLEXWITH THEEXCEPTION OTHERSIGNALSFROMT F HEFREESEM F IQUINONESIGNAL THESIGNALSSATURATE OFT ATVERYLOWMICROWAVEPOWER%LECTRON3PIN%CHO %NVELOPE-ODU D LATION %3%%- EXPERIMENTSSHOW F PROTEIN .NUCLEICOUPL U EDD TO THEEXISTENCEOFTWO ELECTRONSPIN4HESETWONITROGENSHAVEBEENDETECTED F HESEMIQUINONE CONSISTENTLY INTHEENVIRONMENTOFT 1!n INANUMBEROF03))PREPARAT F IONS4HESIGNALSCAN BESIMULATEDWITHTHEASSUMPTIONOFA 3 šSTATE OF F THE IRON ./#. COMPLEX INTERACTING WITH THE F IC * VALUES SEMIQUINONERADICAL WITH ANIFERROMAGNET INTHERANGETCMn 'OUSSIASETAL   III BINDINGOFTWO./MO F LECULESONTHE IRON/NE

 ELECTRONREDUCT D IONOFF THIS DINITROSYLSPECIESYIELDSA G HALFINTEGERCONlGURATIONCHARACTERIZEDBY GVALUES OFF    'OUSSIASETAL   3  DIAMAGNETICSTATEOF OF THEIRON4HISSTATEIS OBTAINEDINTHEPRESENCEOFHIGHCONCENTRATIONSOF CYANIDEORCYANIDE IONSBOUND D ONTHE IRON AND ISCHARACTERIZED BY-OSSBAUERSPECTRAWITH ISOMER SHIFTMMSANDQUADRUPOLESPLITTING A SETAL  4HECONVERSIONOFT F THEIRON MM M S3ANAKI TOADIAMAGNETIC FORM F HASALLOWED D THEOBSERVATION ANDSTUDYOFTHEUNPERTURBEDSEMIQUINONERADICALAT G SEEBELOW -OSSBAUEREXPERIMENTSINA 03n MUTANTFROM #REINHARDTII # "URDAETAL  INDICATEDTHATTHENON HEMEIRONINTHESE PREPARATIONS OCCURSNATURALLY INA LOWSPINFORM F 4HEFREESEMIQUINONE 1 1!n RADICAL !PARTFROMTHE REVERSIBLE CONVE N RSION OFTHE IRONTO 3 BYCYANIDE 3ANAKIS ET AL  1!n CAN BE DECOUPLED FROM THEIRONBYTREATMENTSEXTENDINGANORIGINAL METHOD OF +LIMOV ET AL  INCLUDING TRYPSINIZATION -ACMILLANETAL  ORREPLACEMENTOFF THE IRON WITHTHEDIAMAGNETIC:N !STASHKINETAL  THEBINDINGOF:N HAS NOT BEEN DEMONSTRATED UNDER THESECONDITIONS ORBY TREATMENTOFF CORECOMPLEXES F FROM 3YNECHOCYSTIS WITH HIGH CONCENTRATIONS OF PHOSPHATEONTHEHYDROXYAPATITECOLUMN4ANGETAL  ORBY AHIGHP(TREATMENT $ELIGIANNAKISET AL  0ROBABLYALLTHESETREATMENTS EXCEPT FOR F THECYANIDETREATMENT RESULTINREMOVALOFTHEIRON +URRECK K ETAL $ELIGIANNAKISETAL  4HE DECOUPL U ED 1!n ISCHARACTERIZEDAT8 BAND%02BYA  ' WIDEDERIVATIVEAT G3ANAKISETAL G  WITHRESOLVED D ANISOTROPYAT1 BAND D %02AND ANUNUSUALLY LARGEGXX COMPONENT INDICATINGWEAKER HYDROGENBONDSCOMPAREDWITHPLASTOSEMIQUINONE INSOLUTIONAND D POSSIBLE / INTERACTIONWITH AROMATIC AMINOACID D RESIDUES D -AC-ILLANETAL A 4HE APPLICATIONOF ( %.$/2SPECTROSCOPY-AC-IL LAN ET AL B 2IGBY ET AL  :HENG AND $ISMUKES  HAS PROVIDED D USEFU F L INFORMAT F ION ABOUTTHEORIENTATIONOFTHEHYDROGENBONDSANDHAS INDICATED D ANASYMMETRY INTHE( BONDING%3%%D LINGOFT F HESEMIQUINONE SPECTROSCOPY DETECTEDCOUP ELECTRONSPINTOPROTEINNITROGENS!STASHKINETAL $ELIGIANNAKISETAL  -AC-ILLAN ET AL A 4ANG ET AL  !STASHKIN ET AL $ELIGIANNAKISETAL  4HESESTUDIESHAVE BEEN EXTENDEDD TO INTACT PREPARATIONS IN WHI W CH THE IRONIS IN ITS HIGH SPINFORM F 0ELOQUINETAL   !PARTFROMVARIATIONSATTRIBUTEDTOTHEEFFECTSOFTHE DIFFERENT F TREATMENTS AND THE P( THE EXPERIMENTS

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT AGREE GENERALLY ON THE PRESENCE OFF TWO INTERACT ING NITROGEN NUCLEI 4HESE HAVE BEEN ASSIGNED TO $ !LAANDD $ (IS !STASHKINETAL  $ELIGIANNAKISETAL  BUTTHIS ISCONTESTED BY 0ELOQUINETAL BASEDONSITEDIRECTEDMUTA GENESISANDD EARLIER(IS LABELINGSTUDIES 4ANGETAL  )T ISGENERALLYAGREEDHOWEVERTHAT( BONDING BETWEEN$ (ISAND1!n EXISTSALTHOUGHITSSPEC TROSCOPICSIGNATUREISNOTAGREEDD UPON ASSUGGESTED BY &4)2 STUDIES .OGUCHI A B TOO AND BY ANALOGYWITHTHEARRANGEMENTINTHEPHOTOSYNTHETIC BACTERIA 3POY O ALOV ET AL   4HIS ( BONDING D IT FFOR THE MAGNETIC SERVES PRESUMABLY AS A CONDU INTERACTIONBETWEEN 1!n ANDTHEIRON &
0O
UIF
3PMF
PG
UIF
*SPO 4HE IRONIS LOCATEDD MIDWA D AY BETWEENTH T ETWOQUINONES BUT ITS DIRECT PARTICIPATION IN THE ELECTRON TRANSFE F R REACTIONSHASNOTBEENDEMONSTRATED!CTUALLY Y THE USE INTHE IRON QUINONE COMPLEX OFF ATYPE OF IRON THAT ISNOTGENERALLYEMPLOYED IN ELECTRON TRANSFE F R REACTIONS BUTRATHERINCATALYTICREACTIONS SUGGESTS THATTHE IRONCOULD D SERVEALTERNATEROLES)TCANNOT BEA MERECOINCIDENCETHAT03)) APHOTOCHEMICALCENTER THAT PERFORMSONEOFTHEMOSTCRUCIALANDATTHESAME TIME POTENTIALLY HARMFU F L THROUGH ITS BYPRODUCTS D REACTIONS THE SPLITTING OFF WATER WOULD D EMPLOY A REACTIVEANDREDOXACTIVECONFORMATIONOFTHISTYPE F HERATHEREXTENSIVELISTOFPROPOSE F D ROLES OF IRON/FT OFT F HEIRONREVIEWED IN$INERANDD 0ETROULEAS B $INER ET AL  WE WILL CONCENTRATE ON A FEW LIKELY FUNCT F IONS !MINIMALL ROLE 4HESIMILARORGANIZATIONAND FUNC F TIONOFTHEACCEPTORSIDEOFTHEREACTIONCENTERSOFTHE BACTERIAAND03)) D DESPITETHEGROSSDIFFERENCES F INTHE DONORSIDEORGANIZATIONAND FUNCT F ION SUGGESTSTH T ATTH T E IRONPLAYSANESSENTIALROLECOMMONTOBOTHSYSTEMS )T ISREASONABLYASSUMEDD THATTHEIRONISAN IMPORTANT STRUCTURAL ELEMENTAROUNDD WHICHH THEREACTIONCENTERIS ORGANIZED$INERETAL  &URTHERMORE THEROLE F HE IRONINSTABILIZING THESEMIQUINONEFORMO F F 1! OFT HAS BEENDEMONSTRATED $UTTONETAL   2EGULATION OF THE ELECTRON TRANSFER RATE ON THE D 4HELABILITYOFT F HEIRONLIGANDS INNON ACCEPTORSIDE HEME NON IRON SULFUR F PROTEINS IS ASSOCIATEDD WITH SPECIlC CATALYTIC REACTIONS )N 03)) THIS PROPERTY ISASSOCIATEDD WITHATLEASTONE FUNCT F ION THEUNUSUAL REGULATIONOFT F HEELECTRONTRANSFERRATE F K KNOWNAST HE BICARBONATE EFFECT #HAPTER  VAN 2ENSEN AND +LIMOV 4HEPRESENCEOF BICARB R ONATEASA LIGANDD TO

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT THE IRON IS ESSENTIAL FFOR THE UNDIMINISHED D ELECTRON mOW BETWEEN THE TWO QUINONES4HE PHYSIOLOGICAL F H ACONTROL MECHANISM HAS BEENSTRESSED NEED FORSUC D CAN BE OUTLINED EARLIER $INER ET AL  AND BRIEmYASFOLLOWS)NCASESOFLOW#/(#/nSTROMAL CONCENTRATIONS BICARB R ONATE WOULD DISSOCIATE FROM F THEIRONRESULTINGINA DIMINISHED D ELECTRONTRANSFE F R RATEBY03))ANDACCORDINGLYLOWER/ PRODUCTION D INGTHEWASTEFU F L REACTIONOFF 2U"0WITH EXCESS REDUC / PHOTORESPIRATION !NATTRACTIVEEXTENSIONOFTHIS IDEAHASCONSIDEREDGLYCOLATE APRODUCTOFPHOTORES PIRATIONAND D ANEFlCIENTCOMPETITOROF BICARB R ONATE F DBACK K MECHANISMUNDER ASBEINGINVOLVED INA FEE THESECONDITIONS0ETROULEASETAL  )THASBEEN NOTED HOWEVER THATTHE BINDINGOF GLYCOLATEISPOOR H IOLOGICAL HIGH P(OFT F HESTROMALSIDE4HE ATTHEPHYS POSSIBILITYHOWEVER THATTHERELATIVEAFlNITYOFBICAR BONATEAND GLYCOLATEFORT F HE IRONSITECHANGES DUR D ING ILLUMINATION REMAINSTO BETESTED/F INTEREST INTHIS RESPECTARETHERECENTEXPERIMENTSWITH./IMPLYING THATTHE BINDING AFlNITYOF BICARB R ONATE ISSIGNIlCANTLY REDUCE D D INTHE1!n STATE'OUSSIASETAL   0ROTECTIVEFUNCTIONS 4HEFUNCTIONOFTHEIRON QU Q I NONECOMPLEX INAPOTENTIALLY/ RICH ENVIRONMENT RENDERS IT SUSCEPTIBLE TO DAMAGE BY THE REACTION WITHACTIVEOXYGENSPECIES3UCHSPECIESCOULDBE PRODUCE D D ATTHEDONORSIDEORVIA INTERACTIONOF / WITHTHEREDUCE D DQU D INONES #LELAND D AND'RACE  D .UGENT  4HEIRONHASACCORDINGLYBEENPRO POSED D TOHAVEAPROTECTIVEROLE ACTING ASANOXIDASE D 0ETROULEAS B ORASA ORACATALASE$INERAND WEAKSUPEROXIDEDISMUTASE.UGENT  &ROMA DIFFERENTPERSPECT F IVE THE BINDING OFF SUPEROXIDETOA F METALCENTER MOST LIKELYTHENON HEME IRON TOFORM APEROXIDEINTERMEDIATEHASBEENPROPOSEDRECENTLY A IN/(% PRODUCT D IONDUR D ING ASANIMPORTANTPATHWAY PHOTODAMAGE 0OSPISILETAL  )T ISNOTABLETHAT THEIRONHASALLTHE PROPERTIESTHATCOULDSUPP U ORTA CATALYTIC ROLE )T HAS LABILE LIGANDS IT IS REDOX AC TIVE HAS DIRECTACCESSTOTHEELECTRONTRANSFERC F HAIN 1!n REDUCES&E WITHA Tš —SATP( REVIEWED IN $INER AND D 0ETROULEAS B AND IS PROPERLY LOCATED BETWEENTHETWOQU Q INONES )NSUMMARY WHILETHEIRONINBOTH"2#AND03)) ISANESSENTIAL STRUCTURAL ELEMENTOFF THEELECTRON TRANS PORTCHAIN IN03))ITALSOHASACATALYTICROLE)T IS NOTABLETHATTHELOCATIONOFTHEIRONWOULDCOUP U LEA F IONTOPHOTOCHEMICAL ELECTRONTRANSFER F CATALYTIC FUNCT 4HIS ISARARECOMBINATIONNOTFOUN F D INOTHERPROTEINS EMPLOYINGTHISTYPEOFIRON

 *** 0SHBOJ[BUJPO
PG
UIF 2VJOPOF
#JOEJOH 4JUFT
5IF
5XP&MFDUSPO (BUF "
4USVDUVSBM
.PEFMT
PG
UIF
2VJOPOF 4JUFT .EITHEROFF THEEARLIERCRYSTALLOGRAPHICSTRUCTURESOF 03))AVAILABLEATTHETIMEOFF WRITING :OUNI ETAL 0$")$FE +AMIYAAND3HEN 0$" )$ IZL WAS AT A RESOLUTION THAT ALLOWED DETAILED DISCUSSION ANDD NEITHERSHOWEDD A 1" SITEOCCUP U ANT #HAPTERS 7ITTAND 3HENAND+AMIYA 4HIS LIKELY REmECTED BOTH A WEAKER OCCUPANCY OFF THE 1" SITE THAN OF F THE 1! SITE ANDD ALSO SOME LOSS OF STRUCTURALINTEGRITYONDETERGENTEXTRACTION SOTHAT THE ISOLATEDD PROTEIN HADD AN EVEN LOWER OCCUPANCY THAN IN SITU "ASEDD ON THE HOMOLOGY IN SEQUENCE BETWEEN $ $ AND , - SUBUNIT PAIRS AND THE COMMONPHYSICOCHEMICAL AND BIOCHEMICAL PROPER TIESOFF THEACCEPTORSIDE SEVERAL GROUPS HADD MODELED STRUCTURESFORTHE03))SITE USINGTHEREACTIONCENTERS F FROM 2PSVIRIDIS AND 2BSPHAEROIDE D S ASATEMPLATE #ROFTSETA F L "OWYERETAL 2UFmEET AL 8IONGETAL A  &ORTHE1! SITE MUCH SPECTROSCOPIC EVIDENCE IS AVAILABLE TO SUPPORT THE VALIDITY OF F THESE MODELS AS DISCUSSEDD ABOVE BUT NO DETAILED SPECTROSCOPIC INFORMATION OF A SIMILAR F HE1" SITE!SACONSEQUENCE QUALITY ISAVAILABLE FORT DISCUSSIONOFT F HESTRUCTURE FUNCT F ION INTERFACE F HASHAD TODEPENDONINDIRECTMETHODSFORINFORMATIONABOUT THEMOLECULARARCHITECTURE4HE"2# BASEDD MODELS SERVEDD ASAVALUABLEAID FORUN F DERSTANDINGTHEBIND ING OF PLASTOQUINONE AND INHIBITORS TO THE 1" SITE OF F 03)) AND ALTHOUGH THE EARLIER 03)) STRUCTURES D SHOWA WEREATARELATIVELYLOWRESOLUTION THEY DID CONFORMATIONAROUNDTHE PUTATIVE1" POCKETSIMILAR F HEMODELS3INCESUB U MISSION TOTHATEXPECTED FROMT OFT F HISREVIEW AMORECOMPLETESTRUCTUREHASBECOME AVAILABLE&ERREIRAETAL 0$")$SL#HAPTER  "ARB R ERANDD )WATA THATCONTAINSBOTH1! AND 1" QUINONES &IG AND INWHI W CHH SIDECHAINCOORDINATES HAVEBEENMODELED4HESTRUCTURESHOWSACONlGURA TIONOFT F HEACCEPTORSIDESIMILARINESSENTIALSTOTHAT F L  &IG MODELED INTHELATES #ROFTSETA  ALTHOUGHROTAMERSCHOSENFORSIDECHAINSINTHE EARLIERMODEL WEREINSOMECASESINAPPROPRIATE4HE CONlGURATIONCONSISTSOFT F HECARB R OXYL TERMINAL END OFTHE $ HELIX FOLLOWEDBYALOOPTHE@0%34 LIKE LOOP WITH NO HOMOLOGY INTHE BACTERIALSTRUCTURE W CH ISCON CONNECTINGTOATRANSVERSE DE HELIX WHI NECTEDBYASHARPTURNANDANEXTENDEDLOOPTOTHE F AMINO TERMINALENDD OF % HELIX4HESESPANS FRAME



7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT

&IG 0HOTOSYSTEM))VIEWEDFROMTHESIDETOSHOWTHETOPOLOGYOFTHEACCEPTORSIDE#OORDINATESWERETAKENFROMTHESTRUCTUREBY & &ERREIRAETAL 0$")$SL BYABSTRACTIONOFAMONOMEROFTHEMAINFUNCTIONALSUBUNITSANDCHROMOPHORES4HE$SUBUNIT WITHTHE@0%34 LIKELOOP AND$ARESHOWNBYRIBBONS 0ROSTHETICGROUPSARESHOWNASSTICKMODELS COMPRISINGTHE# ATOMSOFF 1! 1" AND BICARB R ONATEAND HEMEB/THERSTRUCTURAL FEATURES F DISCUSSED INTHETEXTARE INDICATED3EE#OLOR0LATE &IG

THE 1" BINDINGPOCKET4HEHELICALENDSOF$ AND % WEREEXPECTED D TOCONTAINTHECONSERVED D &E LIGANDS $ (ISAND D $ (IS NOWIDENTIlED D ASSUCH INTHE&ERREIRAETAL STRUCTURE )N "2#S THE ( BONDING OFF THE QUINONE AT THE 1" SITE AND D THEMECHANISTICIMPLICATIONS ARESTILL SOMEWHAT CONTROVERSIAL )NTERPRETATION OF EARLIER F D BY PARTIAL OCCUPANCY Y STRUCTURESWAS LIKELY CONFUSE AND BYCONTRIBUTIONSTOTHEELECTRONDENSITIES FROM F WATER ANDFROM QUINONEINTWODIFFERENT Q POSITIONS ,ANCASTERANDD -ICHEL 3TOWELLETAL  )N S AEROIDES D STRUCTURES MORERECENTSTUDIESWITH 2BSPH OF THE DARK ADAPTED REACTION CENTER SHOW MOST OF THEQUINONEDISPLACEDABOUT¯OUTOFTHEPOCKET 3TOWELLETAL &RITZSCHETAL +UGLSTAT TERETAL  /NILLUMINATION THEQUINONEROTATES D MOVESSOTHATTHE 1" POCKET IS FU F LLY OCCUPIED D AND LIKELYBYAREDUCE D D FORM F 3TOWELLETAL  4HE F FUNCTIONALSIGNIlCANCEOFTHEQUINONEDISPLACEMEN T SEEN IN THESE CRYSTALLOGRAPHIC STRUCTURES HAS BEEN CHALLENGED BY "RETON AND D COLLEAGUES ON THE BASIS IF &4)2 SPECTRA THAT FAVOR A PROXIMAL POSITION FOR THEACTIVEQUINONE SIMILARTOTHATOBSERVED FORT F HE K L SEMIQUINONE"RETONETAL .ABEDRYKETA  3TUDIESOFTHETEMPERATUREDEPENDENCEOFELEC TRONTRANSFER F FROM F 1!n TO1" INSPINACH CHLOROPLASTS D WITHMEASUREMENTSOFPROTE F INmEXIBILITY COMBINED THROUGH -OSSBAUER SPECTROSCOPY OF &E ENRICHED MATERIAL HAVE SHOWN AN INHIBITION THAT CORRELATES

WITH DECREASED mEXIBILITY 'ARBERS ET AL  2EIFART F HANDD 2ENGER  4HISWASINTERPRETEDD AS F LREORIENTATIONOF SHOWINGASIMILARNEED FORSTRUCTURA THEHEADGROUPOFPLASTOQUINONEINTHE 1" POCKETOF 03))(OWEVER INVIEWOFT F HE&4)2EVIDENCE THIS INTERPRETATIONSHOULD BEREGARDEDD ASTENTATIVE)NTHE "2# THEBONDINGOFTHEREDUCEDFORMAFTERILLUMINA TIONWASSIMILARTOTHATORIGINALLY SUGGESTED INTHE 2PSVIRIDIS STRUCTURESPRCANDD PRC$EISENHOFE F R ETAL ,ANCASTERAND-ICHEL  INCLUDING ( BONDSWITHTHE, SUB U UNITTO(IS AN&E LIGAND BACKBONE .(GROUPS FROM) F LEAND' D LY AND TO /(OF3ER4HELATTERWASALSO( BONDEDTO U QUINONEISDISPLACED BY Y    !SN!SP 7HENUBI TRIAZINE PRC THE HERBI R CIDE BINDSTHROUGHSIMILAR ( BONDSTOTHETWOBACKBONE .(GROUPSANDTO /( OF3ERAN F D ALSOTO /(OFF 4YR ,ANCASTERAND -ICHEL  4HE( BONDINGDISTANCESSUGGESTTHAT THESERINEBONDISNOSTRONGERWITHTRIAZINE¯ THANWITHTHE51 ¯  )N"2#S EXTENSIVEKINETIC MUTATIONALANDD COM PUTATIONAL STUDIES HAVE PROVIDED A COMPREHENSIVE PICTURE OFF THE MECHANISM OFF ELECTRON ANDD PROTON TRANSFER F REVIEWED BY/KAMURAETAL 7RAIGHT  BUTINTHESTRUCTURALCONTEXTOFTHEPROTEOLIPID DETERGENTMICELLE)N03)) KINETICCHARACTERIZATION OF F MUTANT STRAINS MODIlED IN THE $ SUB U UNIT AND COMPARISONOFTHEPATTERNSOFINHIBITORSENSITIVITY PROVIDEDD SOMEVALIDATIONOFT F HESTRUCTURAL MODELS AS

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT



&IG  4HE1" SITESTRUCTURECOMPAREDTOTHEMODEL4HESTRUCTUREISFROM0$")$SL&ERRIERAETAL  VIEWEDLOOKINGDOWN & 9 INTOTHE1" SITEFROMTHE. PHASE4HEMODELISTHATFROM#ROFTSETAL WITHRElNEMENTBY((2OBINSON #9ERKESAND!2 #ROFTSUNPUBLISHED VIEWEDFROMASIMILARPERSPECTIVE2ESIDUESREFERREDTOINTHETEXTAREHIGHLIGHTED

WILL BE DISCUSSEDATGREATER D LENGTH BELOW)NANEARLY MODELOFTHE03)) 1" POCKETBASEDONTHE 2PSVIRI DIS STRUCTUREASATEMPLATE&IG WEASSUMED D THAT PLASTOQU Q INONEAND D ATRAZINE BOUND D TOTHERESIDUESO D F T THE$ SUBUNITSHOWNBYALIGNMENTTOBEHOMOLOGOUS TO, SUBUNITRESIDUES EG (IS(IS  !SN 'LY L AND D POSSIBLY 3ER 3ER 3 BACTERIAL ! RESIDUESINITALICS THROUGH( BONDSINTHE 1" SITE! HYBRIDUS D SEQUENCE WITH THADDITIONAL GROUPSOF INTEREST (IS !SP !SN  0HE 0 0HE  #ROFTS ETAL  4HECHOICEOF .(BACKBONE( BONDS WASSOMEWH W ATARBI R TRARY S Y INCE ALTHOUGH THEPUTATIVE 3 IS WELL CONSERVED D SERINE LIGAND 3ER  3ER $HASANEXTRARESIDUEBETWEEN3ERAND(IS THEOTHER&E LIGAND COMPARED D TOTHE, SUB U UNIT4HE

lGURESHOWSTHEPUTATIVE LIGANDINGRESIDUES SEVERA D L OTHERS MODIlEDD IN STRAINS WITHHERB R ICIDE RESISTANCE SEEBELOW ANDD (IS SUGGESTEDD TOBETHERESIDUE D F BINDINGAPROTONTOSTABILIZE1"n INTHE RESPONSIBLE FOR SITE#ROFTSETAL "OWYERETAL  -ORE F HAT RECENTMODELSHADD SUGGESTEDD ADDITIONAL FEATUREST ARE ABSENT FFROM THE BACTERIAL TEMPLATE INCLUDING POTENTIALSITESFORBICARBONATEBINDING2UFmEETAL 8IONGETAL A 4HE BI DENTATE LIGANDING OFT F HE&E BY BICARB R ONATESUGGESTED INTHE LATTERMODEL ISWELLSUPPORTEDBY%02AND&4)2STUDIES4HESE F FEATURESARENOWA LLCONlRMED INTHE&ERREIRAETAL  STRUCTURE

 #
.FDIBOJTN
PG
UIF
5XP&MFDUSPO
(BUF U 
,JOFUJD
.PEFMT
PG
UIF
5XP&MFDUSPO
(BUF !LTHOUGHTHE 1" SITEHASBEENRECALCITRANTTOEXPLORA TIONBY MOLECULARSPECTROSCOPY AMA Y AJORADVANTAGEOF STUDIESOFF THETWO ELECTRONGATE INOXYGENICSYSTEMS HASBEENTHEABILITYTOFOLLOWTHEKINETICSOFELECTRON TRANSFER F ININTACTMEMBRANEPREPARATIONSTHROUGH USE OFF THECHANGE INmUORESCENCEYIELD!STHECLOSED REACTIONCENTERREOPENS THEHIGHmUORESCENCEYIELD ASSOCIATED D WITH REDUCT D ION OF 1!n EVOLVES TO A LOW mUORESCENCESTATEWITH T KINETICSREmECTINGTH T EELECTRON TRANSFERTOTHE1" SITEOCCUPANT*OLIOT "OWES D #ROFTS  F "ECAUSETHEREACTIONCENTERCOM AND MUNICATESWITHTHEPLASTOQU Q INONEPOOL INTHENATIVE CONlGURATION THERATESMEASUREDDEPENDNOTONLYON THERATECONSTANTSFORE F LECTRONTRANSFER F BUTALSOONTHE OCCUP U ANTOFF THE1" SITE QUINONEORSEMIQUINONE ANDONTHEKINETICSOFEXCHANGEOF1AND1( WITH THE POOL 2ECOGNITION OFF THE CONTRIBUTION OF F THESE EXCHANGEPROCESSESTOTHE KINETICSMADEITPOSSIBLETO OBTAINDETAILEDKINETICPARAMETERSDESCRIBINGBOTHTHE F D THEINTERACTIONWITH THEQUINONE ELECTRONTRANSFERAN POOL 2OBINSONAND D #ROFTS  F  4HE COMMONLY ACCEPTED MODEL IS THE TWO ELEC TRONGATEPROPOSED BY "OUGES "OCQUET AND 6ELTHYUS H   FFOR 03)) AND BY 7RAIGHT  FOR"2#S INWHICHQUINONEANDQUINOLFORMS F LY EXCHANGEATTHE 1" SITE AND FREE D THESEMIQUINONE

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT FORMGENERATEDAFTERONEmASHISNON EXCHANGEABLE (OWEVER WE SHOULD NOTE THAT THERE ARE DISSENTING F VIEWS +ASHINO ET AL   +INETIC MODELS FOR THEREACTIONSOFT F HETWO ELECTRONGATE IN"2#SAND CHLOROPLASTSHAVEBEENEXTENSIVELYREVIEWED#ROFTS D IGHT  ANDD 7RAIGHT 3HINKAREVAND7RA /KAMURAETAL 7RAIGHT   )NANEARLYMODELFORGREENPLANTS BASEDONEXPERI MENTSWITHPEATHYLAKOID D MEMBRANES 2OBINSONAND #ROFTS F  RECOGNIZEDEXP D LICITLYT L THERELATIVE I LYWEA L AK BINDINGOFPLASTOQUINONE REQUIREDTOACCOUNTFORTHE BIPHASICNATUREOFT F HEELECTRONTRANSFER F FO F LLOWINGA SINGLE mASH FROMT F HEDARK R ADAPTEDD STATE4HESLOWER PHASEWASATTRIBUTEDTOTHESECOND ORDERPROCESSOF D IONOFF QUINONEFROMT F HEPOOL 3CHEME ! REDUCT SIMILARMODEL HASRECENTLYBEENUSEDD TOMEASURETHE DEUTERIUMISOTOPEEFFECTONTHERATESOFTHESEPARATE F DE7IJ 7 NANDD VAN'ORRKOM  )N ELECTRONTRANSFERS THEEARLIERMODEL THE P( DEPENDENCEOFF THEAPPARENT EQUILIBRIUMCONSTANTFORSHARINGANELECTRONBETWEEN PRIMARYY AND SECONDARYQU Y INONES +APP WASACCOUNTED F BYPOSTULATINGASINGLERESIDUEC FOR D LOSETOTHE1" SITEWHOSEP+WASCHANGEDWHEN1" WAS REDUCEDD TO F FORMT HESEMIQUINONEANION 2OBINSONANDD #ROFTS F F L  7I 7 TH MODIlCATION  #ROFTSETA F RTHE KINETICS AND THIS MODEL ALSO ACCOUNTED WELL FO THERMODYNAMICSOF INHIBITORBINDING ATTHE1" SITE 4AOKAAND#RO D F FTS 4AO KA  OFF ELECTRON TRANSFER IN SUSCEPTIBLE AND RESISTANT !MARANTHUS HYBRIDUS D BIOTYPES "OWESETAL 4AOKA 

3 HEME 4HE KINETICMODEL DISCUSSED5NDERPHYSIOLOGICAL CONDITIONS MOSTCENTERSSTART INTHEDARK 3C R GRAY A BOX ANDPRO D GRESSTHROUGH A PATH4HEREMAINDERSTARTINTHELIGHT GRAY A BOX AND D REEJOINTHEMID GRAY A PATHWAY A AFTERT F HESECOND ORDERBINDING OF01 F THEMID GRAY FROMTHEPOOL3EETEXTFORDETAILS

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT 4AOKAAND D #ROFTS  F AND IN HERBI R CIDERESISTANT MUTANTS OF #REINHARDTI # I #ROFTSETAL  3INCE THISMORECOMPLETEMODEL HASBEEN DESCRIBED D ONLY BRIEmY 4AOKAAND D #ROFTS  F WEWILLSUMMARIZE ITSMAINPOINTSHERE 4HE BIPHASIC KINETICS IN THE  MS RANGE WERE D TOREmECT TWO DISTINCTPOPULATIONSOF03)) F ASSUMED 2EACTIONCENTERSAFTERLONGDARKADAPTATIONHAVEEITHER D ATTHE 1" SITE1!1" OR ASECONDARY QUINONE BOUND AVACANT1" SITE1!VACANT ASSHOWNIN3CHEME !NACTINICmASHPRODUCESEITHER1!nVACANT OR1!n 1" "OTH 1!nVACANTAND 1!n 1" ARECLOSEDCENTERS W D ITH THE SAME HIGH mUORESCENCE YIELD!T TIME  —S AFTERTHEmASH WHENREACTIONSONTHEDONORSIDEHAVE REACHEDEQU D ILIBRIUM THE mUORESCENCE YIELD D REmECTS THEELECTRONSREMAININGON1!n)NCENTERSINITIALLYIN THE1!1" STATE THEELECTRONISTRANSFERREDTO1" IN A lRST ORDERPROCESS GENERATING 1!1"nWITHLOWmUO RESCENCE WITH AHALFF TIMEOFAPPROX F IMATELYTO —S3INCETHEELECTRONON1! CAN BETRANSFE F RRED D TO 1" ONLYY WHENAPLASTOQUINONEISPRESENTATTH T E 1" SITE ELECTRONTRANSFER F INCENTERS INITIALLYWITHAVACANTSITE MUSTlRSTINVOLVEA BINDINGOFQUINONEINASECOND ORDERPROCESS AND D THENTRANSFERO F F THEELECTRON WITH A SLOWER KINETICS REmECTING THE CONVOLUTION OFF THE BINDINGANDELECTRONTRANSFERREACTIONS"OTHPHASES AREWELL lTTED BY EXPONENTIAL DECAY A CURVES3INCETHE F HAN HALFF THOSE MORERAPID PHASEACCOUNTED FORMORET CENTERSWHICHTRANSFERANELECTRONWITHINMS AND THEQUINONEPOOLWASOXIDIZED D UNDEROURCONDITIONS THEMINORFRACT F IONOF INITIALLYVACANTCENTERSWOULD BECOMEOCCUP U IEDFROMAPOOLOFPLASTOQUINONEIN Q GREATERTHAN lVE FO F LD D EXCESSOVERTHEREACTIONCEN TER2EACTION FROMT F HIS POOLWOULD D THEREFORE F F LLOW FO PSEUDO lRST ORDERKINETICS 2ATECONSTANTSFORE F LECTRONTRANSFERAN F D BINDING F HEKINETICCURVES REACTIONSCAN BECALCULATED FROMT F FORTHETWOSE PARATEELECTRONTRANSFERSOBSERVEDAFTER ONEORTWOACTINIC mASHESGIVENTOPREPARATIONSSTART ING INADElNED INITIALSTATEUSUALLYWITHALL CENTERS HAVING AN OXIDIZED 1" FOLLOWING PROLONGED DARK F HE DECAY A KINETICSAFTERT F HE ADAPTATION !NALYSISOFT lRST mASHCANBEGREATLYSIMPLIlED BYMAKINGUSEOF THEFOLLOWINGEXPLICITASSUMPTIONSDERIVEDFROMTHE MODELOUTLINEDD ABOVE I THERATIOOFT F HEAMPLITUDEOF THEFASTCOM F PONENTTOTHATOFT F HESLOWCOMPONENTIN THEDECAYKINETICSFOLLOWINGANACTINICmASHISEQUAL TOTHERATIOOFF THE FRACT F IONOFF REACTIONCENTERS INITIALLY INTHE 1!1" STATEOVERTHATINTHE1!VACANTSTATE II UNDER OUR EXPERIMENTAL CONDITIONS THE BINDING OF APLASTOQUINONETOTHEVACANTSITE ISAPSEUDO lRST

 F A ORDER REACTION III THE DISSOCIATION CONSTANT OF PLASTOQUINONE FROM ITS BINDING SITE FOLLOWING AN ACTINICmASH FROM F 1!n 1" ISTHESAMEASTHAT INTHE DARK R FROM1 F 1  ! " 3OLUTIONOFTHESTANDARDRATEEQUATIONSGIVES%QS  AND  FORT F HEOBSERVEDD RATECONSTANT R AND R4HE F HARINGANELECTRON APPARENTEQUILIBRIUMCONSTANTFORS BETWEENQUINONES Q 1! AND1" IS +APP DElNEDIN%Q  4HEDISSOCIATIONCONSTANT + OFF PLASTOQUINONE F FROMT HE1" SITE ISTHERATIOOFTHEON ANDOFF RATE CONSTANTSK!6 K6! %Q  +% ISTHEEQUILIBRIUM Q CONSTANT FFOR THE ELECTRON TRANSFER F REACTION WITH F FORWAR D AND REVERSERATECONSTANTSK!" ANDK"!4HE EQUILIBRIUM CONSTANTS ARE RELATED THROUGH %QS  F FURT F HERDISCUSSION  AND  SEEBELOW FOR R  R  K6!  6   +  K" "![  +APP + ] RR  K6!K"!  +   +AAPP . DS DSS 

¨ª4$4% ·¹ YDF ·¹  ¨ª4$ 4% ·¹

¨ª4$ N$% .( N%$   ¥ $ ´   ¦§   % µ¶ 

. 







$ N$9 ;4$ YDF =   % N9$ ; 4$ 4% =





)NTHESEEQUATIONS S Q PECIESINSQUAREBRACKETSRE Q PRE SENTFRACT F IONSOFF THEREACTIONCENTERSINAPARTICULAR STATEASINDICATED INTHEBRACKET 3TATES 1!VAC AND 1!nVAC REPRESENTCENTERSWITHAVACANT1" SITE WITH THE1! OXIDIZEDD AND REDUCE D DRESPECT D IVELY6AR Y IABLES R AND R ARETHEMEASUREDD RATECONSTANTSFORT F HESLOW ANDFASTPHASESOFELECTRONTRANSFERDERIVEDDIRECTLY F FROMT HEBIPHASIC DECAY A KINETICSOF 1!nBYlTTINGTHE KINETICCURVESWITH TWOEXPONENTIAL COMPONENTSAND ARESIDUAL4HE 1!n KINETICSAREOBTAINEDFROMTHEmUO RESCENCECURVESAFTERT F HE LATTERHAVEBEENCORRECTED F HENON LINEARRELATION BETWEENmUORESCENCEYIELD FORT ANDCONCENTRATIONOF1!n6ARIABLES 6 ! ANDD " ARETHE F F THE AMPLITUDESOFF THESLOWAND FASTCOMPONENTSO BIPHASICDECAY KINETICS AGAINOBTAINED DIRECTLY FROM F KINETICANALYSISOFTHEDATA F 5SINGTHEABOVEEQUATIONS THE FOURRATE CONSTANTS K!6 K K6! KK!" AND K"! DElNED IN4ABLE CAN BEDETER

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT



4ABLE 0HYSICO CHEMICAL CONSTANTS FORT F HEREACTIONSOFT F HETWO ELECTRONGATEFO F LLOWING ANACTINICmASH #ONSTANT

2EACTION

7ILDD TYPE

3'

!P+VALUES P+

.OTES 

1!1" ( ' 1!1"  ( 

P+

1!VAC ( ' 1!VAC(

P+

1!1"n ( ' 1!1"n  (















"%QUILIBRIUM+ + ORRATEK K CONSTANTS T +// P+

1!n 1" ' 1!n 1POOL

 ›

› 

+%

1!n 1" ' 1!1"n

 › 

 › 

K6!

1!n 1POOL ´ 1!n 1"





 

K|6!

1!n (  1POOL ´ 1!n 1"(





 

n !

n !

 

K!6

1 1" ´ 1 1POOL





 

K|!6

1!n 1"( ´ 1!n  ( 1POOL



 

 

K!"

1!n 1" ´ 1!1"n

 

 

 

n !



n "



K|!"

1 1"( ´ 1!1 (

 

 

 

K"!

1!1"n ´ 1!n 1"



 

 

K|"!

1!1"n( ´ 1!n 1"(





 

.OTES  )TISASSUMEDD THATVALUES FORP+ F D THEFRACT F IONOFVACANTREACT F IONCENTERSINTHEDARK R DETERMINED  AND P+ AND BY +O ARETHESAMEAFTER F DARK RK ADAPTATIONASTHOSEFO F LLOWING ANACTINIC mASH  !LTHOUGH THESEREACTIONS INVOLVETHE Q INONEPOOL CONSTANTSSHOWNARE BASEDD ONTHE PSEUDO lRST ORDERPROCESS WITH;1POOL=ASSUMED QU D TOBE 4HETRUE VALUECANBEOBTAINEDFROM+|O  +O ;01=POOLWE HAVEPREVIOUSLY ESTIMATED D ;01=POOL M-UNDEROXIDIZINGCONDI TIONS#ROFTSETAL   2ATECONSTANTSKIJ AREFORREACTIONATP(ABOVEP+2ATECONSTANTSK|IJ AREFORREACTIONAT P(BELOWP+ 2ATECONSTANTS KIJ AREFORREACTIONATP(ABOVEP+2ATECONSTANTSK|IJ AREFORREACTIONATP(BELOW P+ 4HEDIFFERENCEBETWEENRATECONSTANTSKANDK|DOESNOTREmECTACHANGEINTHERATECONSTANTFORTHEELECTRON TRANSFER PROCESS SINCE +% IS INDEPENDENT OF P( BUT A CHANGE IN CONCENTRATION OF SUBSTRATE FOR THE BACKREACTION ;1!1"n= ASSOCIATEDD WITH P+

MINED FROM F F FOURMEASURE D PARAMETERSTHE FASTAN F D F IONOFVACANT F SLOWAPPARENTRATECONSTANTSTHEFRACT OR 1" BOUND REACTION CENTERS BOTH DETERMINED F FROMT HECORRECTED DECAY A KINETICS AND D THEAPPAR ENTEQU Q ILIBRIUMCONSTANT +APP4HISLASTVARIABLE IS THEONLYTERMNOTOBTAINEDDIRECTLYFROMTHEKINETIC EXPERIMENTS)N mUORESCENCEEXPERIMENTS ITCAN BE F HERATIOOF BACK REACTIONSFROM F 1!n OR MEASURED FROMT n F FROM1 F RMER CAN BE MEASUREDD FROMTHE !1"  4HE FO DECAY A OF 1!n MEASURED D THROUGH mUORESCENCEYIELD D F LLOWING ILLUMINATION BY A OR SPECTROPHOTOMETRY FO SATURATINGmASHINTHEPRESENCEOF$#-5THELATTER F FROM LOSSOFF THEBINARY PATTERNWH W ENTHE mUORESCENCE YIELD DECAYWASMONITORED D AT^ +S FO F LLOWINGA SERIESOFmASHES2OBINSONAND#ROFTS   ORTHROUGH THE 3 A 3 DEACTIVATIONONTHE DONORSIDE Q ILIBRIUM CONSTANT CAN ALSO BE DETERMINED 4HE EQU THROUGHTHERMOLUMINESCENCEFROMTHETEMPERATURES F FOR1AN D "COMPONENTS2UTHERFOR F D ETAL  

F F IMPORTANCEIN lTTING THEORETICAL !NOTHERFEATUREO CURVESTOTHEEXPERIMENTAL RESULTSWASTHEFRACT F IONOF CENTERSINWHICHTHEDECAYOFmUORESCENCEOCCURSIN THESECONDSTIMERANGE ATTRIBUTEDD TONON FUNCT F IONAL CENTERS 4HIS FRACT F ION WAS OFTEN F HIGHER IN MUTANT STRAINSTHANINTHENATIVESTRAIN BUTWEDONOTHAVE AREADYEXPLANATIONFORT F HISINTHE FRAMEWOR F RKOFOUR F F MODEL)NHOMOGENEITIESINTHEPOPULATIONOF03)) THAT GIVE RISE TO DIFFERENCES IN KINETIC BEHAVIOR AS ASSAY A ED BY mUORESCENCEHAVE BEEN DISCUSSEDD EXTEN SIVELYELSEWH W ERE'OVINDJEE *OLIOTETAL  ,AVERGNEETAL  ANDAREOUTSIDETHESCOPEOF THISREVIEW W 
5IF Q)
%FQFOEFODF
PG
UIF
" G QQBSFOU
&RVJ MJCSJVN
$POTUBOU
, ,BBQQ -EASUREMENT OF +AAPP ASAFUNCTIONOFP(SHOWEDA DEPENDENCEOVERTH T ERANGEn INDICATING TH T ATTH T E

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT



1!1"n STATEWASSTABILIZEDBYUPTAKEOFA( 2OBIN SONAND#ROFTS  4HECURVECOULDBEWELLlTBY ASSUMING THATASINGLEGROUPUNDERWENTAP+CHANGE F FROM^ P+ TO^ P+ DUETOT D HE#OULOMBI M CEFFECT F EXPERIENCEDBYTHEGROUPONFORMATIONOFASEMIQUI NONEANIONONREDUCT D IONOF 1"3IMILARSTUDIESOFT F HE P(DEPENDENCEOFRATEAN F D EQU Q ILIBRIUMCONSTANTSIN A HERB R ICIDE RESISTANT STRAIN OF ! HYBRIDUS SHOWED THATTHEP( DEPENDENCECHANGED D ONMUTATIONOFF $ D F FTS #RO F T FTSE 3ERTOGLYCINE4AOKAAND#RO AL  0ARTOFTHECHANGECOULDBEATTRIBUTEDTO F IONOF ACHANGEIN + INTHEWILD BIOTYPE THEFRACT THEREACTIONCENTERSWITH AVACANT 1" SITEWASFOUND TOVARYWITHP( BUTTHISDEPENDENCEWASLOSTINTHE MUTANTSTRAIN4HEP( DEPENDENCEFOR F BINDINGCOULD BECHARACTERIZED BYTWOP+S GIVENIN%Q  . 

;4 YDF = ;4 4 = $

WRW

$



; ;

% WRW

=

–

=



S. 

S.

¥ ¦  § ¥ ¦  §

;4 ;

YDF =  ¨ª4$ YDF +  ·¹

$

=  ¨ª4 4 $

¨ª +  ·¹ ´ µ . G ¶ 

¨ª + ·¹ ´ µ .G ¶



%

 + ·¹



WHERE + IS THE VALUE OFF THE DISSOCIATION CONSTANT 1! MEASUREDATA PARTICULARVALUEOFP(4HETERMS;1 VAC=P++ AND;1!1"=P++ ARETHEFRACTIONALCONCENTRATIONS OFF VACANTAND 1"OCCUPI U ED D CENTERSATP(ABOVEP+ ASSUMEDTOBETHEHIGHERP+VALUE AND+D AND +D ARETHEACID DISSOCIATIONCONSTANTSOF 1!1"( AND D 1!VAC( RESPECTIVELY7I 7 THTHESIMPLIFY F ING ASSUMPTIONSGIVENABOVE %Q EXPRESSESTHEP( DEPENDENCEOF + AND HENCEOF !O"O %Q  4HE F FACTOR; 1!VAC=P++;1!1"=P++ GIVES + P+ THEDISSOCIA TION CONSTANT ABOVE P+4HE RESULTS SUMMARIZED IN 4ABLE  SHOW THAT IN THE PHYSIOLOGICAL RANGE PLASTOQUINONE BINDS MORE TIGHTLY AT THE 1" SITE IN THESUSCEPTIBLETHANINTHERESISTANTSTRAIN4HISRAISES INTERESTING MECHANISTIC QUESTIONS TO BE DISCUSSED F HERBELOW FURT 
&TUJNBUJPO
PG
UIF
3BUF
$POTUBOUT
GPS
UIF 3FBDUJPOT
*ODMVEJOH
UIF
1SPUPOBUFE 4QFDJFT 4 BOE
1SPUPO6Q 6 UBLF
BU
UIF
2#TJUF U 4HERATECONSTANTSFORREACT F IONS INVOLVINGBOTH PRO TONATEDANDUNPROTONATEDSPECIESCANBECALCULATED

FFROMTHREEP+SANDD THERATECONSTANTSOBTAINED FROM F KINETICEXPERIMENTS4HE PARAMETERSCANBEEXPRESSED INTERMSOFT F HESEP+S AND FO F LLOWINGTHESAMERATIO NALEASABOVE THEFO F LLOWINGEQUATIONSCANBEUSED INANALYSISOFTHEKINETICDATA

. DS DSS 



¥ ¦  .( § . ¥ ¦  §

¨ª +  ·¹ ´ µ . ¶ ¨ª +  ·¹ ´ µ . ¶



FROM WHI W CH

N$% N%$

¥ ¦  §  .( ¥ ¦  §

¨ª +  ·¹ ´ µ . ¶ ¨ª +  ·¹ ´ µ . ¶



0ARAMETERS FOR F + CAN BEOBTAINEDD THROUGH %Q   4ABLECOMPARESVALUESOBTAINEDD WITHSUSCEPTIBLE AND RESISTANT BIOTYPES 4HE VALUES OF THE ON RATE F INONE BINDING WERESIMILARFOR F BOTH CONSTANTSFORQU BIOTYPES INTHERANGEFROMTO F Sn AND INEACH CASEWEREP(INDEPENDENT)NTHERESISTANTBIOTYPE THERATECONSTANTSFORUN F BINDING OFQU F INONEWERETHE F HEPROTONATEDD AND DEPROTONATEDD SITES)N SAMEFROMT THESUSCEPTIBLEBIOTYPE THEOFF RATECONSTANTATP( VALUESABOVEP+ IE FROMT F HE DEPROTONATEDD SITE IS  TIMES SMALLER THAN IN THE RESISTANT STRAIN AND ABOUTTIMESSMALLERTHANTHATFROMTHE PROTONATED SITE!SACONSEQUENCE INTHEP(RANGEABOVEP+ THE LIKELYPHYS H IOLOGICAL RANGE THESUSCEPTIBLESTRAIN BINDSQUINONEABOUT TIMESMORESTRONGLYTHANTHE RESISTANTSTRAIN INTHESUSCEPTIBLESTRAIN AQUINONE CANREACH BOTH THE PROTONATEDD AND THEDEPROTONATED BINDINGSPECIESATTHESAMEFREQUENCY BUTITLEAVES THEPROTONATEDD SITETIMESMOREFREQUENT F LYTHANTHE DEPROTONATEDD SITE"ELOWP+ BOTH TH STRAINSSHOWAB A OUT THESAMEAFlNITYFORQUINONE $IRECT MEASUREMENT OF F THE KINETICS OFF THE TWO ELECTRONGATE FROMT F HEPROTONUPTAKEASSOCIATEDD WITH THE REDUCTION OF THE 1" SPECIES IN CHLOROPLASTS IS F BLEMSI DELAY A SDUE D COMPLICATED BYANUMBEROFPRO TOEXCHANGEWITH BUFFER F INGGROUPS INTHEMEMBRANE AND THE TORTUOUS PATHWAY THROUGH THE APPRESSED REGIONSOFF THEGRANASTACKSTOTHE BULKK PHASE 0OLLE ANDD *UNGE   AND II THE INDIRECT COUPLING

 THROUGH PROTONATION OFF PROTEIN SIDE CHAINS W WHICH LEADSTOUPTAKEOFAPROTONONEACHmASH ALTHOUGHTHE SEMIQUINONEISANIONIC &OWLER 3APHONAND #ROFTS (O F PEAND D -ORLAND (O D PEAND -ATTHEWS 2OBINSONAND#ROFTS #ROFTS ETAL  $ETAILED DISCUSSIONOFF THESE DIFlCULTIES IS BEYOND D THESCOPEOFT F HISREVIEW BUTTHE INTERESTED READERISREFERREDTOAPAPERBY(AUMANNAND*UNGE  W WHO MEASURED D RATES CORRESPONDING MORE F BYUSINGNEUTRAL CLOSELYTOTHEELECTRONTRANSFERRATES RED .2 AS AN INDICATOR )NTERESTINGLY THE PROTON F ( UPTAKEWASSLOWERONTHElRSTmASH  +S FOR U TAKECOMPARED UP D TO +SFORTHEELECTRONTRANSFER THANONTHESECONDmASH +S COMPAREDTO +SFORT F HEELECTRONTRANSFER F 4HISWAS INTERPRETED D AS SHOWINGTH T ATTRANSFERO F FT F THESECOND D ELECTRONDEPENDED ONPROTONATIONOFTHE1!n 1"n STATE ASSUGGESTEDIN BACTERIAL REACTIONCENTERS 7RAIGHT   
5IF
4FDPOE
0SEFS
3BUF
$POTUBOU
GPS
#JOE JOH
PG
1MBTUPRVJOPOF 4HETURN OVERTIMEOFTHETWO ELECTRONGATECANBE MEASURED FROMT F HEREDUCT D IONOFT F HEPLASTOQUINONE POOLONSTRONGILLUMINATION WHI W CHTAKESABOUT MS3TIEHLAND7ITT  7ITHABOUTQUINONE MOLECULES03)) INTHEPOOL THISREPRESENTSABOUT F HETRANSFERO F F FEAC H PAIROFF ELECTRONS INCLUD MSFORT INGALLBINDINGANDUNBINDINGREACTIONS!SIMILAR F D FROMT F HERATEOF GENERATIONOFT F HE VALUEWASFOUN SPECIESABLETOACCEPTA PROTON WITH HALFT F IMEOFF  MS(OPEAND-ATTHEWS  )NEXPERIMENTSIN WHICH THE REOPENING TIME WAS STUDIED BY VARYING THEDELAYTIME BETWEENTHESECOND D AND THIRD mASHES OFASERIES THE1" SITE WAS ABLETO EVOLVE FROMTHE 1!n 1"n STATE FORMEDIMMEDIATELYAFTERASECONDmASH F FROMT HE DARK R ADAPTEDSTATE TO D 1!1" STATE DETECTED BY THE CHARACTERISTIC RAPID KINETICS WITH HALF TIME OFF MS 2OBINSONAND#RO D F FTS  4HEOVERALL PROCESS INVOLVED ELECTRON TRANSFER F UNBINDING OF 1( AND BINDING OF 1 SO THE OBSERVED HALFTIME F HESEPARTIAL MUSTREmECTACONVOLUTIONOFF TIMESFORT PROCESSES EACH OFF WHICH WASLESS4HE PSEUDO lRST ORDERRATECONSTANTSFORBINDINGOFQUINONEDERIVED FROMTHEKINETICSAFTERTHElRSTmASHKK66! INVOLVED F IONOFCENTERS T F HOSEWITHAVACANT H BINDING ONLYAFRACT SITE7HENADJUSTEDTOCOMPENSATEFORTHISFRACTION THEAPPROXIMATEHALFT F IMES FOR F BINDING OFF QUINONEARE AND D MS FORT F HESUSCEPTIBLEAND D THERESISTANT BIOTYPE RESPECTIVELY INGOODAGREEMENTWITHEARLIER VALUES4HESECOND ORDERRATECONSTANT FORT F HEBIND

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT INGOFQUINONECANBEFOUNDBYUSINGTHESEVALUES ANDANESTIMATEDCONCENTRATIONFORPLASTOQUINONEIN Q THEMEMBRANE!SSUMING THESETO BEM- INTHE SUSCEPTIBLE ANDD  M- IN THE RESISTANT STRAINS THE SECONDORDERRATECONSTANTSREFERREDTOCONCENTRA TIONS INTHEMEMBRANE AREFOUN F D TOBE =   -nSn ANDD  =  -nSn RESPECTIVELY 4HE DISSOCIATION Q PECTTOMEMBRANE CONSTANTFORPLASTOQUINONEWITHRES 1POOL= ISTHEN = n CONCENTRATION +|  +;1 INTHESUSCEPTIBLEANDD  = n -INTHERESISTANT STRAINSEE4ABLE ANDLEGEND  
5IF
1BUIXBZ
GPS
&MFDUSPO
5SBOTGFS
JO
4VT DFQUJCMF
BOE
3FTJTUBOU
#JPUZ UZQFT )N THE PHYS H IOLOGICAL P( RANGE EG P( n THE DISTRIBUTIONOFSTATESISRATHERDIFFERENTINTHESUSCEP TIBLEANDD RESISTANTBIOTYPES SOTHATTHEPATHWAY A FOR F F HOWS DIFFERENTC F HARACTERISTICS)N ELECTRONTRANSFERS THESUSCEPTIBLESTRAIN MOSTCENTERS  HAVE THE 1" SITE OCCUPIED D AND ELECTRON TRANSFER F OCCURS FFROM 1!n 1" WITH THE FORWAR F D RATE CONSTANT OF  =  Sn ANDTERMINATESEITHERON 1!1"nWITH ORON 1!1"n( WITH  )NTHERESISTANTBIOTYPE F THE 1" SITES ARE INITIALLY ONLY ABOUT TWO THIRDS OF OCCUP U IEDINTHESECENTERS ELECTRONTRANSFEROCCURS F FROM 1!n 1" WITHTHERATECONSTANTOFF  SnINTHE F HROUGH REMAININGCENTERS ELECTRONTRANSFEROCCURST THESECOND ORDERROUTEFOLLOWINGBINDINGOFPLASTO QUINONE4HEREACTIONCOMESTOEQUILIBRIUMWITH A F IONOFCENTERSST F ILL INTHE1!n STATE SUB U STANTIAL FRACT OFTHOSECENTERSUNDERGOINGSUCCESSFULFORWARDELEC TRONTRANSFER T F HEELECTRONWASDISTRIBUTEDD WITH ł ON 1!1"n AND ̌ccō ON 1!1"n(  
3PMF
PG
%4FS
JO
UIF
.FDIBOJTN
PG
UIF 2#4JUF U 4HEMECHANISMDISCUSSEDD ABOVEWASDEVELOPED INTH T E F IMENTSWITHWILD TYPEAND HERBI R CIDE CONTEXTOFEXPER RESISTANTSTRAINS OFF ! !HYBRIDIS4HE RESISTANTSTRAIN FOL F LOWEDD THEGENERAL TREND FORATRAZ F INETOLERANTMUTANTS W ERE3ERISREPLACED BYGLYCINE OF HIGHERPLANTSWH (IRSCHBERGAND-C)NTOSH 'OLOUBINOFFETAL "ETTINI ETAL 4AOKAAND#RO D F FTS  # REINHARD R TII 3UNBY ET AL   )N CONTRAST IN # %RICKSONETAL  ANDCYANOBACTERIA 'OLDEN ! ANI ET AL A B THE ANDD (ASELKORN  !JL COMMONSUB U STITUTION IS BYALANINE-ORERECENTLY A RESISTANT MUTANT OF %UGLENA WAS DESCRIBED D THAT U STITUTIONOFT F HEHOMOLOGOUS3ERTO CARRIESASUB

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT THREONINE!IACH ETAL  4HISREPEATED LOSSOF 3ERISHIGHLYSIGNIlCANT INDICATINGTHATITMIGHT PLAY A ANIMPORTANTROLE IN HERBI R CIDEBINDING4REBST  AND D "OWYER ET AL  SUGGESTED D THAT 3ERCONTRIBUTESTOTHEBINDINGOFINHIBITORSOFTHE UREA ATRAZINEFAM F ILY TOTHE1"SITEBY( BONDINGOF ITSSIDECHAINHY H DROXYLGROUPTOTHE.(GROUPINTHE HERBICIDEMOLECULE INANALOGYWITHTHEOBSERVED( BONDINGOFT F HEHERBI R CIDETERB R UTRYNETO3ER INTHE , SUB U UNITOF BACTERIALREACTIONCENTERS -ICHEL ETAL  !NOTHERSTUDY4IETJENETAL  PROPOSED THAT3ERWOULD D STABILIZETHESTRUCTUREOFF THE 1" SITEBY( BONDINGTO(IS4HIS HISTIDINERESIDUE D COULDALSOBEINVOLVEDINBINDINGOFTHEPROTONTHAT STABILIZESTHESEMIQUINONE 1"n INTHEPOCKET#ROFTS ETAL   4HECHANGEOF3ERTOGLYCINEINTHERESISTANT STRAINWASASSOCIATED D WITHACHANGEINAFlNITY FOR F ATRAZINEOFF ABOUT FO F LD D BUTOFON F LYABOUT FFOLD FOR PLASTOQUINONE4HE RESISTIVITY FOR ATRAZINE F RGY OFF K* REPRESENTSACHANGE INBINDING FREE ENER D ( BOND D MOLn IN THE RANGE EXPECTED FFOR A BURIED BUTFORPLASTOQUINONE THE FOLDCHANGEINBINDING REPRESENTSONLY K*MOLn4HE DIFFERENT F IAL CHANGE INAFlNITYMAY BEUNDERSTOOD INTERMSOFT F HEMODEL DISCUSSED ABOVE IF ONE OR BOTH OFTHETWO BACK BONE ( BONDSARELIGANDSFORP F LASTOQUINONE BUTTHESERINE

/( FORMSON F L LYAWEA AK ( BOND#ONVERSELY T L THESERINE

/(FORMSASTRONG( BONDFORTHEHERBICIDE 0ERH R APSTHEMOST INTRIGUING ASPECTOFT F HESTUDIES D F  WASTHATTHEMECHANISTIC FTS OFF 4AOKAAND#RO PHENOTYPEOFTHERESISTANTSTRAINDIFFEREDFROMTHE SUSCEPTIBLESTRAINTHROUGH P+CHANGES3INCESERINE DOESNOTHAVEAP+INTHEPHYS H IOLOGICALRANGE THIS SUGGESTSTHATTHELOSSOF3ERINVOLVESALSOALOSSOF INTERACTIONWITHADISSOCIABLE GROUP)NOURSEQUENCE D THESAMEPOSITIONRELATIVE I ALIGNMENT (ISOCCUPIED TOACONSERVEDPHENYLALANINEIN$ IN 2PS VIRIDIS , SUB U UNIT AS, !SN!SP  INTHE"2#S D WASNATURALLYPOSITIONED INTHEMODEL TO FORMA F AND ( BONDEDWITH3ER4HESEINTERACTIONSMIGHTFA F CILITATEAPROTONRELAY A MECHANISM BYWHI W CH PROTONS COULD BETRANSFERRE F D TOTHE1" SITEONREDUCT D ION BYA SECONDELECTRON4HERESULTSCOULDTHENBEEXPLAINED BY A FRACT F IONALCONTRIBUTIONOFF THE3ER /(TOQUI NONE BINDING MODU D LATED BYASHARINGOFF THE( BOND WITH(IS4HESTRUCTURALSPECULATIONSUNDERLYING THISMECHANISTICMODEL HAVE BEENVALIDATED BYTHE &ERRERIAETAL  STRUCTURE#HAPTER "ARB R ER AND)WATA ANDTHESEAUTHORSALSOSUGGESTEDASIMILAR INVOLVEMENTOFF (ISININTERACTIONSWITH 3ER INSTABILIZATIONOFT F HE31$ETAILSOFT F HE FUNCT F IONAL

 RELATIONOFF THESTRUCTURETOTHE P+S IDENTIlED INTHIS WORKREMAINTOBEDETERMINED(OWEVER RESULTSFROM # HARD R TII 30ADDEN MUTAGENESISOFF (ISIN #REIN F UBLISHED AN D D SEE BELOW SUPPORT ANDD !#ROFTS UNPU A MODEL OFTHIS SORT $
#JOEJOH
PG
*OIJCJUPST
BOE
&YPHFOPVT 2VJOPOFT
BU
UIF
2#4JUF U 4HE STUDIES SUMMARIZEDD ABOVE HAVE STRONGLY SUG GESTED THAT 1" IS RELATIVELY WEAKLY BOUND4HIS IS ALSOAPPARENTFROMT F HEEASEWITHWHI W CH ITCANBE DIS PLACED BYEXOGENOUSELECTRONACCEPTORSANDD VARIOUS INHIBITORS6ELTHUYS "OWESAND#ROFTS A 6ELTHUYS #ROFTSAN F D 7RAIGHT ,AVERGNE A4AOKAETAL "OWYERETAL  4HIS OCCURSINSEVERALSTEPS INVOLVINGELECTRONTRANSFER BACKK TO 1! FO F LLOWED BY DISSOCIATIONOFQU F INONE TO GENERATEANINTERMEDIATE1!nVACANTSTATETHATISTHE SUBSTRATEFORBINDING)NTHECASEOFINHIBITORBINDING D D REDUCT D IONINVOLVING THERESULTISAN INHIBITOR INDUCE THEREACTIONS )NHIBITOR n "

n !

n !

1!1 ' 1 1" ' 1 [VACANT] ' 1!n )NHIBITOR %XOGENOUSQUINONESCAN FO F LLOWASIMILARREACTION PATH WITHANADDITIONALSTEP OFE F LECTRONTRANSFER F FROM F 1!n TO GENERATE A FOREIGN SEMIQUINONE AT THE SITE ,AVERGNE B4AOKAETAL :IMMERMANN F D 0ETROU D LEASANDD $INER   ANDD 2UTHERFOR 4HEMECHANISMOFLOSSOFTHE1"n SPECIESISTHOUGHTTO INVOLVEDISPLACEMENTOFF THEOXIDIZED FORM F 1" PRES ENTINA FRACT F IONOFCENTERSASARESU F LTOFT F HERELATIVELY SMALLEQUILIBRIUMCONSTANTFAVORINGELECTRONTRANSFER F FROM 1!n 1" TO1!1"n)FF THEEXOGENOUSREAGENTISAN ELECTRONACCEPTORFOREXAMP F LE BENZOQUINONE THE SEMIQUINONEISGENERATED)FTHESEMIQUINONEHASA HIGH ENOUGHPOTENTIAL ITCANACCEPTANELECTRON FROM F THENON HEME&E TOGENERATETHEOXIDIZED FORM F 1EX n "

n !

n !

1!&E1 ' 1 &E1" ' 1 [VACANT] ' 1!&E1EXn ' 1!&E1EXn



! CONSEQUENCE OF THE REACTION PATHWAY LEADING TOINHIBITOR INDUCEDREDUCTIONOF1! IS THATTHE TITER OF INHIBITORISHIGHERFOR F BINDING TOTHE 1!1"nSTATE THANTOTHE1!1" STATE ,AVERGNE C4AOKAAND #ROFTS  !SECONDCONSEQUENCEISTHATINHIBITOR BINDING ISP(DEPENDENT

 4AOKAAND D #ROFTS F  INVESTIGATED KINETICASPECT OFINHIBITORBINDINGTOTHEOXIDIZEDAND ELECTRON D D STATESOFT F HE 1" SITE AND D MEASURESONAND OFF REDUCE F IONOF P(FORSEVERA F L DIFFERENT F RATECONSTANTSASAFUNCT INHIBITORS$#-5 ATRAZINE O PHENANTHROLINE AND TWOCYANOACRYLATE DERIVATIVES3AND (UPPATZ D AND D 0HILLIPS  WHI W CH DIFFER F BYSTEREOROTATION ABOUTONE# ATOM ASSUMMARIZEDBELOW I "INDINGOF INHIBITORSTOTHEOXIDIZED D STATEOF THE SECONDARY QUINONE SITE WAS ESSENTIALLY P( INDEPENDENT II "INDINGTOTHEONE ELECTRONREDUCEDSTATEWAS SLOWER AND D STRONGLYP(DEPENDENT III &OR ALL THE INHIBITORS STUDIED BOTH THE DIF F F FERENCE INRATE AND D THEP( DEPENDENCE COULD BE F IN TERMS OFF A SIMPLE HYPOT H HESIS ACCOUNTED FOR 4HEYREmECTTHELOWEREDCONCENTRATIONOFTHEOPEN W CH THE INHIBITORBINDS ASSOCIATED D WITH SITESATWHI F FORMAT IONOFF ASTABLESEMIQU Q INONEATTHESITE4HE CONCENTRATIONOFOPENSITESWASDESCRIBEDBYTHESET OFEQU F ILIBRIUMCONSTANTSDETERMININGTHE DISTRIBU TIONOFANE F LECTRON BETWEENPRIMARYAND D SECONDARY Q QUINONEACCE PTORSDISCUSSEDABOVE IV 7HEN THESE FFACTORS WERE ACCOUNTED FFOR THE SECOND ORDERRATECONSTANTSFORINHIBITORBINDING F D TO BECONSTANTWITHP( AND D CLOSETO WERE FOUN T OSE FOR TH F BINDINGTOOXIDIZEDCENTERS ASSUM D INGTH T AT THEREACTIONPARTNERWASTHEVACANTQUINONEBINDING SITEINEITHERSTATE AND D THATTHE BINDING CONSTANT F PLASTOQU FOR Q INONEFROMT F HEPOOLWASTHESAME FO F R OXIDIZED AND ONE ELECTRON REDUCED CENTERS V 3ECOND ORDER RATE CONSTANTS FOR F BINDING AND UNBINDINGOFEACHOFTHEINHIBITORSWITHRESPECT TO CONCENTRATION IN THE MEMBRANE PHASE WERE DETERMINED BYTAKINGACCOUNTOFT F HEPARTITIONCO EFlCIENTSBETWEENAQUEOUSANDMEMBRANE Q PHASE $#-5AND D THETWOCYANOACRYLATESSHOWED D SIMILAR VALUESFOR F BINDINGRATECONSTANTn =  -nSn Q D CLOSETOTHEVALUEESTIMATEDFORPLASTOQUINONEBIN ING ^ -nSn SEEABOVE BUT O PHENANTHROLINE WASSLOWERABOUT =  -nSn $#-5AND 3AREEFFECTIVEINHIBITORSBECAUSEOFTHEIRVERY F SLOWUNBINDING Sn BUT2ISANINEFFEC TIVEINHIBITORBECAUSEOF ITSRAPID D UNBINDING n Sn 4HEBULKY O PHENANTHROLINEISA POORINHIBI TORBOTH BECAUSEOF ITSSLOWERRATEOF BINDING AND RELATIVELYHIGH RATEOFF UNBINDING Sn 

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT 3EVERALOFF THESECONCLUSIONSAREOFMOREGENERA F L IN TERESTTOWIDERSTUDIESOFBINDINGFROMTHELIPIDPHASE SINCEITISLIKELY THATSIMILARSITESINOTHERQUINONE PROCESSINGENZYMESWILL SHOWSIMILARCHARACTERISTICS )NHIBITOR BINDING FOLLOWS A PATTERN FAMILIAR FROM F LUBL U EENZYMES EXCEPTTHATTHERELEVANT STUDIESOFSO CONCENTRATIONTERM ISTHATFORT F HEMEMBRANEPHASE 4HECONCLUSIONSAREALSOOFINTERESTWITHRESPECTTO STUDIESOF INHIBITORBINDINGUSINGTHECONVENTIONAL 1UANTITATIVE3TRUCTURE!CTIVITY2ELATIONSHIP13!2 APPROACHFORACOMPREHENSIVEVIEW SEE(UPPATZ ANDD ARTICLESIN9OSHIDAETAL  SINCETHEY INDICATE CLEARLY THE PARAMETERS THAT UNDERLIE SOME OFTHElTTINGVARIABLESUSEDTOQUANTIFYDIFFERENTIAL ASPECTSOF INHIBITORBINDING %
4USVDUVSBM
BOE
.FDIBOJTUJD
*OGPSNBUJPO GSPN
.VUBHFOFTJT
4UVEJFT 
)FSCJDJEF
3FTJTUBOU
4USBJOT
PG
$IMBNZEP NPOBT
BOE
$ZBOPCBDUFSJ $ JB (ERBI R CIDERESISTANCEWASORIGINALLY FOUN F D IN HIGHER D  FOR F HISTORICAL REVIEW PLANTS SEE -ORELAND $RABERETAL /ETTMEIER   ANDTHESE CONTINUETOPROVIDEINTERESTINGNEWSTRAINS!LFONSO F RKON HERBI R CIDE ETAL  HOWEVER MOSTRECENTWORK RESISTANCEHASBEENINALGAEORCYANOBACTERIA WHICH AREMOREAMENABL A ETOGENETICMANIPULATION%RICKSON ETAL   ANDD 'ALLOWAYETAL STUDIED # GENER MUTATIONS INTHE $ SUBUNITIN #REINHARDTII ATED BYSELECTION FOR F HERBI R CIDERESISTANCE ANDD THESE HAVEALSO BEEN INVESTIGATED INGREATERDETAIL INOTHER LABS"AROLI 'OVINDJEEETAL $RABERET AL #ROFTSETA F L /ETTMEIERETAL  T EN ET AL   4HESE STUDIES HAVE COVERED 4IETJ DETAILEDD MEASUREMENTS OF INHIBITOR RESISTANCE FO F RA WIDE RANGE OF INHIBITORS KINETIC ANALYSIS ANDD USE OF F 13!2 AND COMPUTATIONAL MODELING TO PREDICT F OMTHE DIFFE FR F RENTIAL INHIBITOR SENSITIVITIES STRUCTURAL F FEATURESASSOC IATEDD WITH DIFFERENT F IAL BINDING )NADETAILED KINETICSTUDY FO F LLOWINGTHEPROTOCOLS OUTLINEDABOVE "AROLIANDCOLLEAGUES"AROLI  #ROFTSETA F L  WEREABLETODECONVOLUTETHECON TRIBUTIONSOFF RATECONSTANTSAND EQU Q ILIBRIUMCONSTANTS F FORPLASTOQUINONEBINDINGANDELECTRONTRANSFERTOTHE OVERALL PROCESS4WOMUTATIONS IN$ 3ER!LAAND !LA6AL LEDD TOAMARRKEDD SLOWINGOF KINETICS FOR F Q Q QUINONE REDUCTIONOFPLASTOQUINONETOTHEBOUNDSEMI )N3ER!LA THESECONDD ELECTRONTRANSFERWASA F LSO SLOWER BUT WAS NORMAL IN !LA6AL )N MUTANT

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT 'LY!SP THEELECTRONTRANSFER F KINETICSWERENOR MALAFTERTHElRSTmASH BUTSLOWEDAFTERTHESECOND )NMUTANTS,EU0HE 6AL)LE AND D 0HE4Y 4 R THEELECTRONTRANSFER F KINETICSAFTER F BOTH mASHESWERE SIMILARTOTHOSEINWILDTYPE!MONGTHEAMINOACID SUB U STITUTIONS IN $ COVERED BY THESE STUDIES TWO CLEARGROUPSCOULD BEESTABLISHED I -UTATIONS THAT CONFER F HERBI R CIDE RESISTANCE BUT HAD D NOMARRKED D EFFECTONT F HEELECTRONTRANSFE F R BETWEEN THE PLASTOQUINONE Q ACCEPTORS OF 03)) 4 R 4HESE MUTATIONS ARE 6AL)LE 0HE4Y ,EU0HEAND D 'LY!SP 4ABLE )NTHECASE OF 'LY!SP THE NORMAL KINETIC PARAMETERS D WERE IN CONTRAST TO A SLOWED D ELECTRON OBSERVED TRANSFERREPORTE F D FROMEAR F LIERWORK R %RICKSONETAL 'OVINDJEEETAL  (OWEVER THEEARLIER F D USINGAVERAGING WITH EXPERIMENTSWEREPERFORME ASHORT DARK R PERIOD BETWEENACTINIC mASHES7HEN THEEXPERIMENTWASPERFORMEDUNDERUNCOUP U LED CONDITIONSAND D WITHA LONGERDARK R INTERVAL ELECTRON TRANSFERONT F HE lRSTmASHWASSIMILARTOWILDTYPE D BUTTHAT AFTER A SECOND D mASH WAS INHIBITED)T SEEMS LIKELYTHATTHESLOWED KINETICSPREVIOUSLY REPORTED D A DOMINANT CONTRIBUTION FFROM A MIXED REmECTED POPULATIONOFSTATES WITHTHEINHIBITED ELECTRON REDUCE D D STATEDOMINATING ANDPOSS D IBLY SOMESLOW O ING DUETORE D D IONOFT DUCT F HEPLASTOQUINONEPOOL4HE SLOWEDELECTRONTRANSFERONTHESECONDmASHMAY HAVEREmECTED D AN INTERFERENCEO F F HEASPARTATESIDE FT CHAINWITH PROTONPROCESSINGASSOCIATED D WITHTHE SECOND ELECTRONTRANSFE F R II -UTATIONSTHATCONFERHERBICIDERESISTANCEAND F BETWEEN CONSIDERABLYMODIFYTHEELECTRONTRANSFER THE PLASTOQU Q INONE ACCEPTORS AS WITH 3ER!LA AND!LA6AL)NGENERAL THE3ER!LAMUTATION F HECHARACTERISTICSOF SHOWED INEXAGGERATED FORMT T E3ER'LYMUTAT TH L IONINHIGH H PLANTS/NLYAS L LIGHT CHANGE IN THE APPARENT EQUILIBRIUM CONSTANT FOR ELECTRONTRANSFERWASO F BSERVED4HESLOWELECTRON F INTHISMUTANTWAS DUETOA D HIGH DISSOCIA TRANSFER TIONCONSTANTFORPLASTOQUINONERATHERTHANACHANGE IN +APP4HISSUPPORTSTHESUGGESTIONTHAT3ER BESIDESBEINGINVOLVED INTHE BINDINGOF INHIBITORS ALSOPLAYSANIMPORTANTROLEINPLASTOQUINONEBIND ING4HEHIGH VALUEOFF + COMES FROMAN F INCREASED F IONOFCENTERS FRACT F INTH T ESTATE1!VACANTINTH T EDARK R  !LSO BOTHTHEON ANDOFF RATECONSTANTSFORBINDING AND DISSOCIATIONOFF PLASTOQUINONEARE INCREASED IN

 THISMUTANT)NCENTERSINTHE1!1" STATE HOWEVER THEELECTRONTRANSFERREACTIONPROCEEDSTIMES MORERAPIDLYTHANINWILDD TYPE!SSUMINGSTRUC TURAL HOMOLOGYWITHTHEBACTERIALREACTIONCENTER THELOSSOFTHEn/(ON3ERMIGHTBEEXPECTED TORESULTINAWEAKERBINDING ORBINDINGTOANAL TERNATIVESITE ATTHEENDD OFT F HE POCKETDISTAL FROM F THE &E INTHESE MUTANTSTRAINS AS DISCUSSED ABOVE 3INCEREACTIONCENTERSARESTRONGLY DEPENDENTON DISTANCE THEMORERAPID A D RATEFORT F HE BOUND QU Q INONE MIGHTREmECTASHORTERBONDINGDISTANCETOTHE. W CH PROVIDES A ( BONDD TO THE OFF $ (IS WHI OTHERQU Q INONE ˆ #/ATTHEPROXIMALENDD OF THE BINDINGPOCKETFORELECTRONTRANSFERREACTIONS)N F HEMUTATIONSMAY A @PUSHTHEPLASTOQUINONE EFFECT T MOLECULES CLOSER TOGETHER ALLOWING FFOR A FASTER F ELECTRONTRANSFE F R 4HESTRUCTURAL BASISFOR F DIFFERENT F IAL INHIBITORRESIS TANCEHASBEENEXPLOREDINDETAILED13!2STUDIES USINGTHEMUTANTSTRAINSTESTEDD AGAINSTAWIDERANGE F SU UBSTITUTEDD TRIAZINES ANDD TRIAZINONES 4IETJ T EN ET OF AL  AND NITRO  ALHYLPHENOLS$RABERETAL  #OMPLEMENTARY STUDIESATSOMEOFF THESESITES F IONS &ORSTER HAVEEXPLOREDD AWIDERRANGEOFMUTAT ETAL ,ARDANSETAL *OHANNINGMEIERET F DISCUSSIONOF AL  THATEXTENDD THEDATABASEFOR F IONRELATIONSHIPS STRUCTURE FUNCT  4QFDJGJD
.VUBHFOFTJT
PG
)FSCJDJEF
3FTJT 4  U UBODF
4JUFT
JO 4ZOFDIPDZTUJT 4 &OLLOWING EARLIERWORK RKONSPONTANEOUSMUTANTSSE LECTED FOR F HERBI R CIDERESISTANCE2OBINSONETAL  A SIMILAR SET OF MUTANTS SHOWING VARIOUS DEGREES R CIDERESISTANCEHAVEALSOBEENGENERATED IN OF HERBI 3 3YNEC HOCYSTIS SP0## BOTHASSINGLEMUTANTS ANDWITHCOMBINATIONSOFCHANGESATTHEINDIVIDUAL SITES!STIERETAL  %TIENNEETAL   4HESE SHOWED INTERESTING VARIATIONS IN SELECTIVITY F FORDIFFERENTHERBICIDESCOM PAREDTOTHERESULTSFOR #HLAMYD M DOMONAS AND HIGHERPLANTS 
$PNCJOBUPSJBM
.VUBHFOFTJT
PG
B
$POTFSWFE 4 4QBO
PG
UIF EF -PPQ 6ERMAASANDCOLLEAGUES6ERMAASETAL +LESS ETAL +LESSANDD 6ERMAAS  USEDD COMBI NATORIALMUTAGENESISTOEXPLORETHEROLESUB U STANTIAL SPANS BETWEEN HELICES $ AND % OF $ ANDD $ )N Y SPANS FFROM THE LOOP THE +LESS ET AL  STUDY

 STARTINGATTHEEND D OF HELIX $ OF$WERERE F PLACED BYRESIDUESFROMTHEEQUIVALENTSPANIN$)NTER ESTINGLY Y THE MUTANT STRAINS RETAINED FUNCT F ION WITH RELATIVELYMINORCHANGES BUTTHESE INCLUDECHANGES INHERBICIDEBINDINGORSTABILITYOFTHESEMIQUINONES F GLOWCURVES INDICAT AT BOTHSITES ASMEASURED FROM INGPERH R APSTHATALTHOUGHTHESPANCONTRIBUTES LITTLE TOEITHERCATALYTICSITE THEREAREPLEOTROPHICEFFECTS OVERA LARGERVOLUME4HIS ISINLINEWITHTHESTRUCTURAL DATA)NTHE+LESSAND D 6ERMAAS  STUDY THEROLE OFFOURCONSERVEDRESIDUES4YR 0HE 'LY !RG INTHEPUTATIVE DE HELIXOFT F HE$SUB U UNIT OFF THE 3YNECHOCYSTIS SP0##WASEXPLORED MUTANTSWITHFUNCTIONAL 03)) WERE ISOLATED ALL OF WHICHSHOWED DIFFERENTCO F DONCOMBINATIONSATPOSI TIONSTO.ONEOFT F HECONSERVED D RESIDUESWAS D F FOUNDTOBEMANDATORYFOR03))FUNCTION(OWEVER OFT F HE FUNCT F IONAL MUTANTSCONTAINED D 4YROR0HEAT POSITIONWHIL W EATTHEOTHERTHREEPOSITIONSMANY DIFFE F RENTAMINO ACID COMB M INATIONS COULD BE FU F NCTION ALLY ACCOMMODATED -OST OFF THE 03)) PROPERTIES WERESIMILARINTHEMUTANTSCOMPARED D TOWILDTYPE D .OTICEABLE MODIlCATIONSINTHE MUTANTS CONCERNED THE SEMIQUINONE EQUILIBRIUM ON ELECTRON TRANSFER F BETWEEN1! AND 1" AND D THEAFlNITYOFF 03)) INHIBI TORS4HEAUTHORSCONCLUDEDTHAT EVENTHOUGHMANY DIFFERENTCOM F BINATIONSOFAM F INOACIDS INPOSITIONS F HEPRIMARY FUNCT F ION COMPLEX TOMAYSATISFYT REQUIREMENTS Q NEED TO BE COMBINED FOR OPTIMIZED PERFORMANCE-OST F F FUNCT IONAL SEQUENCESMAINTAINED ANAMPHIPATHICARRANGEMENT ASSUMINGA HELICAL CON FFORMATION)NTHEIRALIGNMENT 4YRWASPROPOSEDTO BE FUNCT F IONALLY ANALOGOUSTO0HEOFF THE,SUB U UNIT INPURPLE BACTERIA AND D THEYSUGGESTED D THAT4YR F FACEDTHE1 " BINDINGPOCKETTOALLOWACONTRIBUTION TOBINDINGOF 1"4HEALTERNATIVECANDIDATE FORT F HE RESIDUE D FUNCT F IONALLYEQUIVALENTTO0HE IS0HE ASSUGGESTEDINTHE#ROFTSETAL MODEL AND THISISTHECONlGURATIONSHOWN INTHE&ERREIRAETAL  STRUCTURE #HAPTER "ARB R ERAND D )WATA 4HE ROTATIONOFTHEHELICALAXISBY^O BRINGSTHEPOLAR F FACEO F HE HELIXTOTHEAQUEOUSINTERFACE POS FT F ITIONING (ISFOR F INTERACTIONWITH 3ER  4QFDJGJD
.VUBHFOFTJT
PG
%)JT
4 4IPXT BO *NQPSUBOU
3PMF JO J ) 1SPDFTT 1 J JOH $INER ET AL  BRIEmY REPORTED D A MUTATION OF (ISINTHE DE HELIXOFT F HE1" SITE(IS,EU OF #REINHARDTI # I STUDIEDINCOLLABORATIONWITH0ETER .IXON WHI W CH SHOWED D AN INHIBITED D ELECTRONTRANSFER F

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT TO1"30ADDENAND!#RO D F UNPUBLI FTS U SHED HAVE CONSTRUCTED A MORE EXTENSIVE SERIES OF MUTANTS AT THISSITE(IS!SP ,Y , S !SNANDD 'LN INORDERTO Q INONEAND TESTTHEROLE INSTABILIZATIONOFF THESEMIQU PROTONDELIVERY/NLYTHE(IS!SPMUTANTWASABLE TOGROWPHOTOSYNTHETICALLY#E Y LLSEVOLVEDD / ATRATES nTHOSEOBSERVED INCELLSWITHWILD TYPE$ PROTEIN ANDSHOWEDRAPIDELECTRONTRANSFERTOTHE1" SITEFOR F BOTHODDD AND EVEN mASHES(OWEVER THERATE ONTHE lRST mASH WASSLOWERTHANTHATONTHESECOND REVERSINGTHEPATTERNOFTHEBINARYOSCILLATIONINTHE mUORESCENCE YIELD D MEASURED AT^ +SAFTEREAC F HOF ASERIESOF mASHES"OTH(IS!SNANDD (IS'LN SHOWEDSOMERAPIDELECTRONTRANSFERFROM 1!n TO1" F LLOWING THElRSTACTINIC mASHAFTER FO F DARK R ADAPTATION BUTWITHH AREDUCE D D YIELD"OTHH WERESEVERELYINHIBITED ONTHESECONDmASH(IS,YSWASSEVERELYINHIBITED EVENAFTERT F THElRSTmASH4HERMOLUMINESCENCESTUDIES AFTER F ILLUMINATIONWITH SATURATINGmASH IMMEDIATELY BEFOREFREEZINGSHOWEDGLOW CURVESFORALLSTRAINSIN W CHTHEPEAKWASS WHI K HIFTE F D TO HIGHERTEMPERATURES &ROM THIS WE CONCLUDE THAN EVEN IN THE STRONGLY INHIBITED (IS,YS STRAIN SOME ELECTRON TRANSFER TO1" OCCURRED F 4HE PATTERNOF DEPENDENCEOFF ELECTRONTRANSFERRATE ONmASHNUMBEROBSERVEDAT P(WITH(IS!SP WAS SIMILAR TO THAT SEEN IN WILD TYPE AT P( ABOVE THEP+AT FORT F HE1"n STATE4HISSUGGESTSTHATIN (IS!SP AP+SHIFTSIMILARTOTHATINWILD TYPE MIGHT BE OCCURRING BUT STARTING WITH A P+ IN THE DARK R LOWERTHANTHATINWILD TYPE ASMIGHTBEEX PECTEDFORTHEMOREACIDICASPARTATESIDECHAIN4HE RAPID D ELECTRONTRANSFERA F F FTERT HElRST mASH OBSERVED WITH STRAINS(IS'LNANDD (IS!SNSHOWSTHAT REDUCTIONOF1"WASNOTDEPENDENTONDELIVERYOFA PROTONTOTHESITETHROUGH THISPATHWAY AY4HESTRONGLY INHIBITEDD RATEAFTERT F HESECOND mASH CANBEREADILY INTERPRETED AS SHOWING THAT TRANSFER OF THE SECOND F BINDING OFT F HE lRSTPROTON ELECTRON HASTOWAIT FOR 7RAIGHT  DEPENDENTONA DISSOCIABLEGROUPAT THISSITE4HEBEHAVIOROFTHESEMUTANTSTRAINSSUPP U ORTS F LEFOR( F ISINPROTONDELIVERY THESUGGESTIONOFARO TOTHE1" SITE7EARECURRENTLYINVESTIGATINGTHEP( Q R DEPENDENCEOFRATESANDEQUILIBRIUMCONSTANTSFO F FROM F 1!n TO1" INTHESESTRAINS THEELECTRONTRANSFER F HERTESTTHIS HYPOT H HESIS TOFURT 
&YDJTJPO
PG
3FTJEVFT
GSPN
UIF
1&45
4F RVFODF
PGG UIF EF-PPQ 4HESPANLEADING FROMT F HETOPOF HELIX $ TOTHE DE HE

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT LIXCONTAINSASEQUENCECALLED D THE@0%34 LIKEREGION 'REENBERGETAL  WHICHHASBEENSUGGESTED F LYSIS INTHETURNOVEROFF $ ASALIKELYSITE FORPROTEO D WITH PHOTOPROTECTION.IXONATAL  ASSOCIATED DELETEDASUBSTANTIALSPANRESIDUES  INTHIS D SHOWED D THATTHERESULTANTSTRAINWASABLE REGION AND TOGROWPHOTOSYNTHETICALLY WITH LIGHT SATURATED D RATES SIMILARTOWILDTYPE2EMARKABLY ELECTRONTRANSFER D DESPITEEXCISION ONTHEACCEPTORSIDEWASSTILLRAPID U STANTIAL SECTION OF F THE DE LOOP AND THE OFF THIS SUB LIKELYSTRUCTURALCONSEQUENCES.OTSURPRISINGLY THE PROPERTIESOFT F HESITEWEREMODIlED AN D D MEASURE MENTSUSINGTHERMOLUMINESCENCEAND mUORESCENCE YIELDKINETICCHANGESSHOWEDTHAT1"n WASDESTABILIZED RELATIVETO1!n-EASUREMENTSOFF $TURNOVERSHOWED ANENHANCED D TURNOVERINTHE DELETIONSTRAININDICATING THATTHE 0%34SEQUENCEWASNOTRE Q Q QUIRED3IMILARDE

LETIONSTUDIES HAVEBEENREPORTED BYY -ULOETAL  W CH EXCISIONOFSPAN RESU F LTED IN  INWHI ANAUTOTROPHICSTRAINWITHAFUNCTIONALBUTMODIlED 1" SITE AND D WITHASIMILARSUSCEPTIBILITY TOPHOTO INHIBITIONTOWILDTYPE%XC D ISIONOFSPANS  F OR GAVEHETEROTROPHICSTRAINSWITHMODIlED 1" POCKETS SEVERELY INHIBITED D ELECTRONTRANSFER AN F D IMPAIREDRECOVERY D F FROMP HOTOINHIBITION4HEAUTHORS CONCLUDEDTHATNOSPECIlCSEQUENCEINTHE Q DE LOOP WASESSENTIAL FOR$PO F LYPEPTIDEDEGRADATION4HE &ERREIRAETAL  STRUCTURESHOWSTHATTHELOOP BETWEENANDISWELLREMOVEDFROMTHE 1" SITE CONSISTENTWITHTHELACKO K FE F FFECTONTWO E F LECTRON F IONSHOWN BYTHESERESULTS GATE FUNCT  .VUBHFOFTJT
UP
J & MPSF
UI &YQ U F
#JDBS J SCPOBUF &GGFDU & 'OVINDJEEANDD COLLEAGUES HAVEUSED D SPECIlCMUTA GENESISIN 3YNECHOCYSTIS AND #HLAMYDOMONAS D TO TEST THEHYPOTHESISTHATARGININERESIDUESINTHENEIGHBOR HOOD D OFF THE QUINONE BINDING SITES MIGHT SERVE AS LIGANDSTO BICARB R ONATE-UTATIONOF$ !RGTO F GLYCINELEDTOSUBSTANTIALCHANGESINSTABILITYOFTHE F LD D CHANGE INRATEOFF ELECTRONTRANSFE F R COMPLEX  FO n F FROM1 F HERMOLUMINESCENCEPEAKS ! TO1" LOSSOFT BUTNOTTOANYCHANGEINFORMATEBICARBONATEBINDING F HE 1!n&E%02SIGNAL (UTCHISON ASJUDGED FROMT ETAL 8IONGETAL  -UTATIONAT!RG 8IONGETAL B TOGLUTAMATE!RG'LU OR F IONWITH METHIONINE !RG-ET WEREABLETO FUNCT NEAR NORMAL KINETICS OFF ELECTRON TRANSFER F BETWEEN THE1! AND1"SITES BUTWITHASOMEWHATREDUCED F D REACTION )N EQUILIBRIUM CONSTANT FFOR THE FORWAR

 TERESTINGLY BOTH MUTATIONS LED TO GREATLY REDUCED SENSITIVITYTOINHIBITIONBYINCUBATIONWITHFORMATE R ONATEINNATIVESTRAINS3EV W CH DISPLACED BICARB WHI ERAL OFF SINGLE AND DOUBL U E MUTANTS IN THE DE HELIX 3 0## ANDTHELOOP AROUND3ERIN 3YNECHOCYSTIS SHOWEDD SIMILARDIFFERENT F IALEFFECTSON F F FORMATE SENSITIVITY 6ERNOTTEETAL OR 3INCETHE BICARBONATE EFFECT IS NORMALLY STUDIED THROUGH THE F FORMATEE FFECT F "LUB U AUGHANDD 'OVINDJEE VAN 2ENSENETAL VAN2ENSEN  CONCLUSIONS ABOUTTHEROLEOFANYOFTHESERESIDUESINBINDINGOF BICARB R ONATEMUSTREMAINTENTATIVE &
&GGFDUT
PG
0DDVQBUJPO
PG
UIF
2#TJUF
PO
UIF &N GPS
2" BOE
PO
UIF
&RVJMJCSJVN $POTUBOUT
JO UIF
5XP&MFDUSPO (BUF U -ANY N REPORTSHAVE BEENPUBLI U SHEDD SHOWINGCHANGES IN%M OFT F HE 1!1!n COUPLEONADDITIONOFINHIBITORS THAT BIND TO THE 1" SITE BOTH IN "2#S AND 03)) R ERSHADD ASSUMED THATANY N EFFECTS F !LTHOUGH MOSTWORK R HASSUGGESTED WITH $#-5WERESMALL RECENTWORK THATTHISMIGHTNOTBETHECASE+RIEGER ,ISZKAYAND 2 HERFOR 2UT F D  D &OREXAMPLE THE %M FOR F 1!1!nIS SHIFTE F D FROMnTOnM6 F BY$#-5 ANDD TOn M6INTHEPRESENCEOFBROMOXYNIL!NINTERESTING DISCUSSIONOFF THESEEFFECTS F INTHELIGHTOFT F HESENSI TIVITYTOPHOTO OXIDATIVEDAMAGEHASRECENTLYBEEN PUBLISHED 2UTHERFORD AND +RIEGER ,ISZKAY  &UFEZANETA F L  )TSHOULD BENOTEDD THAT VALUESFO F R +AAPP CALCULATEDD USINGTHEKINETICAPPROACH ABOVEDEPENDONTHEASSUMPTIONTHATTHE%M VALUE F HE 1! COUPLEISTHESAMEWH W ETHERTHE1" SITEIS OFT OCCUPIED BY$#-5ORBYTHE1"1"un COUPLETHE POTENTIALOF1! INTHEABSENCEOFINHIBITORASALWAYS MEASUREDD WITH 1( INTHESITE!CHANGE IN %M FORT F HE 1!1!n COUPLECHANGESTHEPROBABILITYFORPOPULATION OFDIFFERENTINTERMEDIATESTATES ANDSHOULDTHEREFORE AFFECTT F HEBACKK REACTIONRATEMEASURED)TISNOTCLEAR F HISWOULD BECOMPAREDD TOTHAT HOWLARGEANEFFECTT ARISINGFROMOCCUPANCYBY 1" BUTTHE +AAPP VALUES MEASURED FROMT F HERATIOOF BACKK REACTIONRATESWILL LIKELY HAVETOBECORRECTED)NADDITION ESTIMATIONOF THE %MFORTHE1"1"u n COUPLEFROMTHEEQUILIBRIUM CONSTANTDEPENDS DIRECTLY ONTHEVALUEASSUMED FO F R %M 1!1!n )NARECENTSTUDY 2APPAPORTETAL  MEASUREDTHEBACKREACTIONRATESFROM 1!n TO0 IN STRAINS INWHI W CH THE FREE ENER F GY GAPBETWEEN1! AND H INACCEPTOR0HEO OFF THE THEINTERMEDIATEPHEOPHYT PHOTOCHEMICALREACTIONWASVARIED4HEIRANALYSISHAS CALLED INTOQUESTIONTHECOMMONLYASSUMEDD VALUES

 FFOR MIDPOINT POTENTIALS OF F THE 1!1!n 0HEO0HEOn AND D 00 COUP U LES4HESE RECENT DEVELOPMENTS WILL F HERREVISION ANDPER D RHAPSSOME OPENTHISAREATO FURT REINTERPRETATIONOFEAR F LIERWORK R !LTHOUGH 1! ACTS AS A SINGLE ELECTRON ACCEPTOR U E REDUCT D ION UNDER PHYSIOLOGICAL CONDITIONS DOUBL ISPOSSIBLEUNDEREXTREMEREDUC D INGCONDITIONSSEE VAN-IEGHEMETAL  FORAREINTERPRETATIONOF THEOBSERVATIONSOFF %VANSETAL  $INERAND "ABCOCK  PRESENTACRITICAL DISCUSSIONOFT F HE RELATEDD LITERATURE '
3PMF
PG
UIF
2#4JUF
JO
#BDL3FBDUJPOT
UP
UIF ' 44UBUFT 
#BDL
3FBDUJPO
GSPN
2#m
UP
UIF
44UBUF U 4HE BACKREACT K IONAFTERAS F INGLEmASH FROMT F HEDARK R ADAPTED D STATE31"n BACKREACTION CANBEASSAYEDBY mUORESCENCEMEASUREMENTS ASDISCUSSEDATLENGTHIN THECONTEXTOFT F HE KINETICMODELABOVE4HEREACTION HASALSOBEENSTUDIED D EXTENSIVELYTHROUGHRELAXATION OF THE 3 STATE AND THROUGH DELAYED mUORESCENCE THERMOLUMINESCENCE AND ELECTROLUMINESCENCE "OWESAND#RO D F FTS VAN'OR RKOM 6ASS AND 'OVINDJEE  DE 7IJN ET AL   4HE D OVERALL REACTIONISCLOSETOSTOICHIOMETRICIN YIELD BUTENERGYLEVELSSTRONGLYAFFECTT F HEPATH ESPECIALLY THE FRACTION OF CENTERS DECAYING THROUGH PATHWAYS POPULATINGTHEEXCITED D STATE3INCETHERATEOBSERVED THROUGH mUORESCENCEYIELDEXPER D IMENTSAND 3 STATE Q DEACTIVATIONARESIMILAR ANDSINCETHEEQUILIBRIUM CONSTANT CALCULATED D AS ABOVE AND FFROM THERMOLU MINESCENCE ARESIMILAR ABACKREACT K IONVIA1!n AND PHEOPHYTINISGENERALLYASSUMED GENERATINGASMALL F IONOF INTERMEDIATESTATESWITHSUFlCIENTENERGY FRACT TOPOPULATETHESINGLETLEVEL TOGIVE DELAYED mUORES CENCEORTHERMOLUMINESCENCE#ROFTSETAL  "OWESAND D #ROFTS  F B2UTHERFOR F D ETAL  $EMETERETAL *OHNSONETAL +RIEGER ETAL   
#BDL
3FBDUJPO
GSPN
2) UP
UIF 44UBUF‡" 1PTTJCMF
3PMF
GPS
$ 1 $ZUPDISPNF
C 4HERELAXATIONOFT F HE 3 STATEGENERATED BYTWO mASH EXCITATIONFROMTHEDARK ADAPTEDSTATE ASMEASURED BY / YIELD S D HOWSKINETICSSIMILARTOTHATOFF THE3 RELAXATION$INER  (OWEVER INTHISCASE THE SOURCE OF ELECTRONS IS NOT OBVIOUS 4HE ACCEPTOR SIDE COMPLEX EVOLVES TO THE INITIAL STATE 1!1" OR

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT 1!VACANT WITHIN  MS AFTER F TWO mASHES LEAVING THE TWO REDUCING EQUIVALENTS AS 1( IN THE POOL 2OBINSONANDD #ROFTS  F 4HE %M OFT F HEPOOL IS F HE ^M6 SOTHEPROBABILITYOFF RE POPULATIONOFT REDUCEDACCEPTORCOMPLEXWOULDBELOWFROMTHE VALUESABOVE ATNEUTRALP( + +EEQQ FORTHEREACTION1!1"  1( ' 1!n1"n 1( IS^n -OREOVER $INER  FOUNDTHATTHERATEOFTHEBACKREACTIONAFTER TWOmASHES 1"n3 A 1"n 3 WASONLY WEAKLY DEPEN DENTONTHEDEGREEOFRE F DUCT D IONOFF THEQUINONEPOOL SUGGESTINGTHATTHISISNOTTHEPATHWAY!NALTERNATIVE F LECTRONSWOULD BEFROMT F HEENDOGENOUS SOURCEOFE D CAROTENOIDS ANCILLARY#HLS #YT B THAT REDUCTANTS ARE KNOWNTO DONATETO 0 WHENTHE-N COMPLEX IS DISRUPTED3INCETHEEQUILIBRIUMCONSTANTS BETWEEN 0ANDD THERELEVANT3 STATETRANSITIONSARERELATIVELY SMALL THISREDUCTANTPOOLWOULDALSOBEAVAILABLETO D THE3 AND 3 STATES(OWEVER NOACCUMULATION REDUCET F HEOXIDIZED FORMSO F F THESEREDUCTANTS D ISOBSERVED OFT WHENTHE3 STATESYSTEMISINTACT$INER  )F THEY ARE INVOLVED APAT D HWAY A FORT F HEIRREDUCT D IONFROM F F BE POSTULATED4HE ANADDITIONAL POOL MUSTTHEREFORE OBVIOUS SOURCE IS THE QUINONE Q POOL AND INDEED A $#-5 SENSITIVEPATHWAY A FORRE F D IONOF# DUCT F YT B HAS BEEN DEMONSTRATED "USER ET AL  ANDD A PLASTOQUINOLOXIDASEACTIVITYINVOLVING#YT B HAS BEENSUGGESTED +RUKAN K D3TRZA D LKA "ONDARAVA ETAL  &ROMTHEINHIBITORSENSITIVITY THISMUST INVO N LVETHE 1" SITE4HESTRUCTURESCURRENTLYAVAILABLE ALLOWCALCULATIONOFF POSSIBLERATECONSTANTSINPUTATIVE F B IS^ STEPS4HESESHOWTHATTHEHEMEOF#YT ¯FROMTHEEXPECTEDPOSITIONOF1"7ITHADRIVING F FORCEO FFM6 AND AVALUE FO F R h OFF 6 ARATE CONSTANTOFF ^Sn ASSUMINGOCCUPANCY CAN BECALCULATEDUSINGTHE-OSERETAL EQUATIONS F F DEAC 4HISISIN LINEWITHMEASUREDD VALUES FORRATESO TIVATION ANDAP D LAUSIBLEOCCUPANCY4HEMOSTLIKELY ACCEPTORWOULDBETHEANCILLARY#HLOF$ LIGANDED BY (IS SINCEMUTATIONOFF THE LIGAND LEADSTOLOSS OF# F HLPHOTOOXIDATION#HL : 2UFmEETAL   4HE EDGE TO EDGE DISTANCE BETWEEN HEME B AND THIS#HLIS^¯WITHADRIVINGFORCEOF^M6 F h THISSTEP WOULD D NOT ANDD ANYREASONABLEVALUEFOR BERATE LIMITING)NTHISSIMPLESCENARIO ELECTRONSFOR D IONOF BOTHTHE3 STATESCOULD FO F LLOWTHESAME REDUCT PATHWAYTHROUGH #YT B SINCETHE1"nWOULDBEA BETTERSUBSTRATETHAN1( FORREDUCTIONOF1! !NALTERNATIVEAND INSOMEWAY A SMOREATTRACTIVE F MECHANISM USINGASIMILARREACTIONPATHWAY IS FOR THE 1" SITE TO CATALYZE AN @OXIDANT INDUCED REDUC TIONREACTION

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT



1(  #YT B  1! ' 1  #YT B n  1!n(  4HEREACTIONWOULD PROCEED D VIAANINTERMEDIATE 1!1"n STATE IN WHICH THE SEMIQUINONE WOULD BE SUFlCIENTLY REDUC D ING TO FFORCE THE SECOND D ELECTRON ONTO 1! THEOVERALL EQUILIBRIUMCONSTANTWOULD BE A SFORTHETWO ^ THUSPROVIDINGPARALLELPATHWAY F WHICH WOULD D RETURN VIA #YT B ELECTRONS ONE OF AND D #HL : AND D THEOTHERVIA1!n ANDTHENORMAL3 DEACTIVATIONPATHWAY 3  #HL: J 3  #HL:



#HL: #YT B n J #HL:  #YT B 1( #YT B  1! ' #YT B n1!1"n( 1!1"n ' 1!n 1" ' 1!n1 3 1!n J 3  1! 3 1( J 3 1( $ISCRIMINATION BETWEEN THESE TWO POSSIBILITIES MIGHTNOTBEEASY SINCEINBOTHCASES DEACTIVATION Y F FROM F OFF THE3 STATEWOULD INVOLVEELECTRONTRANSFER PLASTOQUINOLTO Q 3 VIA#YT B(OWEVER ANINVOLVE MENTOFBOTH#YT B ANDTHEANCILLARY$#HL #HL: COULD BEREADILY TESTED BYOBSERVATIONOFT F HE3 DECAY A KINETICSINMUTANTSTRAINSWITHTHELIGANDSTOTHESE TWO GROUPSMODIlED-ORAISETAL 2UFmEET AL   !ROLEFORINTERACTIONBETWEENTHE1" SITEAND#YT B IN THE PROTEIN PROCESSING CONSEQUENT ON PHO TODAMAGE ISSUGGESTED BY ANINTERESTINGSTUDYOFF A COVALENT BONDD FORMED BETWEEN BAND$THROUGH (IS,UPINKOVAETAL   $EACTIVATION OFF THE HIGHER 3 STATES BY THE BACK REACTIONPLAYSANOBVIOUSROLEINREMOVALOFPOTEN TIALLY HARMFULOXIDANTS,ESSOBVIOUSISTHEFACTTHAT THIS ALSO REMOVES POTENTIALLY HARMFU F L REDUCTANTS D n 1!n HASAREDUCINGPOTENTIALINTHERANGETOGEN ERATE SUPEROXIDE BY REDUCTION OF / AND EVEN THE 1"1"n COUPLEREACTSWITH/ ATALOWRATE ASSEEN INTHE SLOWED D KINETICS FO F R OXIDATION UNDER ANAEROBIC CONDITIONSWHENTHEBACKREACTIONISPREVENTEDBY HYDROXYLAMINE((2OBINSONAND D !2#ROFTS F UNPUBLISHED  /HAD AND COLLEAGUES +EREN ET AL  HAVEEXAMINEDTHEINACTIVATIONOF03))UNDER

mASH ILLUMINATIONATAREPETITIONRATE INTHERANGEOF THELIFETIMEOFTHE1!nAND1"nSTATESBACKREACTING WITH 3 OR3)NACTIVATION OR$ DEGRADATION SHOWED A DEPENDENCEONTHE INTERVAL BETWEEN mASHES WITH HALF MAXIMALEFFECTSAT^SCOMPAREDTOTš ^ S FFOR THE BACK K REACTION FROM F 1!n IN THE PRESENCE OF $#-5 ANDD ^S COMPAREDD TOTHE BACKREACT K ION TIMEFROM1"nWITHTš ^S 4HEINACTIVATIONWAS MUCH LESS UNDER ANAEROBIC CONDITIONS SHOWING A Q IREMENTFOR/ F REQU 4HEAUTHORSINTERPRETEDTHEDATA ASSHOWINGAMECHANISMINWHICHTHEBACKREACTION GENERATESDAMAGING OXYGENRADICALSTH T ROUGH INVOLVE MENTOFF THE#HL TRIPLET(OWEVER ITSEEMSPOSSIBLE THATADIRECTREDUCTIONOF/ BY THEREDUCEDACCEPTOR QUINONESMIGHTPROVIDEANALTERNATIVEMECHANISM W ERE 4HESETOPICSAREDISCUSSED INGREATERDETAILELSEWH INTHIS VOLUME *7 $PODMVTJPOT 4HESTRUCTURAL INFORMAT F ION FROMSPECTROSCOPY SPE F

CIlCMUTAGENESIS KINETICSTUDIESANDMODELINGHAS BEEN REMARK R ABLY SUCCESSFU F L IN PREDICTING SPECIlC F LATINGTOTHEMECHANISMOFF THE STRUCTURAL FEATURESRE TWO ELECTRONGATE NOWREVEALEDBYCRYSTALLOGRAPHIC STUDIES4HIS SUCCESS MAY A BE VIEWEDD AS VALIDATING THESEAPPROACHES BUTALSO PROVIDESSOMECONlDENCE INTHEKINETICMODELS ANDTHECONCLUSIONSDRAWNFROM THEM7I 7 TH MORE DETAILEDSTRUCTURES T D HESCOPEFO F R F FUTUREWOR RK INWHI W CHH THEMOLECULARARCHITECTUREOFT F HE TWO ELECTRONGATEISEXPLOREDTHROUGHACOMBINATION F HESEAPPROACHESHOLDSTHEPROMISEOFF A DETAILED OFT UNDERSTANDINGOFT F HEREACTIONSONTHEACCEPTORSIDE OF03 )) "DLOPXMFEHNFOUT 60 ACKNOWLEDGES INSIGHTFUL DISCUSSIONS WITH $RS .)OANNIDISAND93ANAKIS2ESEARCHSUPP U ORTFOR THE LAB OFF 60 WAS PROVIDEDD THROUGH THE %UROPEAN #OMMUNITY PROGRAMS %2"&-28#4 AND %2"&-28#4!2#ACKNOWLEDGESEARLIER SUPPORT FFROM $EPARTMENT OF %NERGY ANDD 5NITED F ICULTURE THAT COVEREDD THE 3TATES $EPARTMENT OF!GR RESEARCH WORK DISCUSSED IN THE SECOND PART THE MAJ A OR CONTRIBUTIONS OFF $R (OWIE 2OBINSON AND 3HINICHI 4AOKATO DEVELOPMENTOFF THE KINETICMODELS DISCUSSED ANDMORERECENTUSEFULDISCUSSIONSWITH $R6LADD 3HINKAREV V

 3FGFSFODFT !ASA2 !NDREASSON, % 3TYRING3AND6ANNGARD4 4HE NATUREOFTHE&E))) %02SIGNALFROMTHEACCEPTOR SIDEIRONIN 0HOTOSYSTEM))&%"3,ETTn !IACH! /HMANN% "ODNER5AND*O D HANNINGMEIER5  ! HERBICIDE R RESISTANT %UGL U ENA MUTANTCARRYINGA3ERTO4HRSUB U STITUTIONOFF POSITIONINTHE$PROTEINOFF 0HOTOSYSTEM)) :.ATURFORSC F H Cn !JLANI' +IRILOVSKY$ 0ICAUD-AND!STIER#A -OLECULAR ! ANALYSISOF PSB! MUTATIONSRESPONSIBLEFORVARIOUSHERBICIDE RESISTANCEPHENOTYPESIN3YNECHOCYSTIS 0LANT-OL"IOL  n !JLANI' -EYER) 6EMOTTE#ANDD !STIER#B -UTATION IN PHENOL TYPEHERBI R CIDERESISTANCEMAPSWITHINTHE PSB! B GENE IN 3Y 3 NECHOCY CYSTIS &%"3 ,ETTn F YO** 'ADDOUR+ %TIENNE!, +IRILOVSKY $AND !LFONSO- 0UE D D NEWMUTATIONOFF $SERINE  IN 0ICOREL 2 )NDUCE SOYBEANPHOTOSYNTHETICCELLCULTURESPRODUCEDATRAZINERESIS TANCE INCREASEDSTABILITYOF31"n AND31"nSTATES ANDINCREASED SENSITIVITYTOLIGHTSTRESS0LANT0HYSIOLn !LLEN*0 &EHER' 9EATES4/ +OMIYA(AND2EES$# 3TRUCTUREOFT F HEREACTIONCENTERFROM F 2HODO D BACTERSPH S AEROIDES D F QUINONESANDD &E INTERACTIONS0ROC 2 0ROTEIN COFACTOR .ATL!CAD 3CI 53!n & "AKER(- .ORRIS'% 2ICE$7ANDD "AKER%. !NDERSON"&  3TRUCTURE OF HUMAN LACTOFERR F IN #RYSTALLOGRAPHIC STRUCTUREANALYSISANDRElNEMENTAT¯RESOLUTION* -OL "IOLn !STASHKIN!9 +AWAMORI! +ODERA9 +UROIWA3AND!KABORI +  !N ELECTRON SPIN ECHO ENVELOPE MODULATION STUDY OFF THE PRIMARY ACCEPTOR QUINONE IN :N SUBSTITUTED PLANT 0HOTOSYSTEM))*#HEM0HYSn !STASHKIN!6 (ARA( +UROIWA3 +AWAMORI!ANDD !KABORI +  !COMPARATIVEELECTRONSPINECHOENVELOPEMODU D LATION STUDY Y OF THEPRIMARYE Y LECTRONACCEPTORQUINONE IN:N SUB U STITUTED ANDCYANIDE TREATEDPREPARATIONSOF0HOTOSYSTEM))*#HEM 0HYSn !STIER# -EYER) 6ERNOTTE#AND%TIENNE! , 0HOTOSYS TEM))ELECTRONTRANSFERINHIGHLYHERBICIDERESISTANTMUTANTSOF 3YNECHOCYSTIS &%"3 ,ETTn !STIER# 0EREWOSKA) 0ICAUD- + D IRILOVSKY$ANDD 6ERNOTTE#  3TRUCTURAL ANALYSISOFT F HE1" POCKETOFT F HE$SUB U UNIT OFF 0HOTOSYSTEM )) IN 3YNECHOCY CYSTIS 0## ANDD  : F H Cn .ATURFORSC "AROLI) 4HETWO ELECTRONGATEOF0HOTOSYSTEM))INWILD TYPE AND HERBICIDE RESISTANT MUTANTS OF # REINHARDTII -3C 4HESIS 5NIVERSITYOF)LLINOIS "EIJER#AND2UTHERFORD!7 4HEIRON QUINONEACCEPTOR COMPLEX IN 2HODOS D SPIRILLUMRUBRUM CHROMATOPHORESSTUDIED BY %02"IOCHIM "IOPHYS!CTAn "ERTHOMIEU#ANDD (IENERWADEL2 )RONCOORDINATIONIN R ONATEANDD THE1" 0HOTOSYSTEM)))NTERACTIONBETWEEN BICARB POCKET STUDIED BY &OURIER TRANSFORM F INFRARE F DD SPECTROSCOPY Y "IOCHEMISTRY n "ETTINI0 -C.ALLY3 3EVIGNAC- $ARMENCY( 'ASQUEZ*AND $RON- !TRAZINERESISTANCEIN #HENOPODIUMALBUM ,OWANDHIGHLEVELSOFRESISTANCETOTHEHERBICIDEARERELATED TOTHESAMECHLOROPLAST03"!GENEMUTATION0LANT0HYS H IOL  n

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT "LUB U AUGH $ AND D 'OVINDJEE 4HE MOLECULAR MECHANISM OFTHEBICARBONATEEFFECTATTHEPLASTOQUINONEREDUCTASESITE OFPHOTOSYNTHESIS0HOTOSYNTH2ESn "OINNARD $ #ASSOUX 0 0ETROULEAS 6 3AVARIAULT *- AND 4U CHAGUES *0  )RON)) COMPLEXES OF  | BIIMIDAZOLE AND  | BIBENZIMIDAZOLE AS MODELS OF THE PHOTOSYNTHETIC F ITES3YNTHESIS MOLECULARAND MONONUCLEARNON HEMEFERROUSS CRYSTAL STRUCTURE AND D -OSSBAUERAND D MAGNETICSTUDIES)NORG #HEMn "ONDARAVA. $E0ASCALIS, !L "ABILI3 'OUSSIAS# 'OLECKI *2 "EYER0 "OCK2AND+RIEGER ,ISZKAY! %VIDENCE THAT#YT B MEDIATESTHEOXIDATIONOFREDUCEDPLASTOQUINONE INTHEDARK*"IOL#HEMn "OSO" $EBRUNNER0 /KAMURA-9AND&EHER' -OSS BAUERSPECTROSCOPY STUDIESOFF PHOTOSYNTHETICREACTIONCENTERS F OM 2HODO FR DOPSEUDOMONASS D SPHAEROIDES D 2 "IOCHIM "IOPHYS !CTA  n "OUGES "OCQUET "  %LECTRON TRANSFER F BETWEEN THE TWO PHOTOSYSTEMSINSPINACH CHLOROPLASTS"IOCHEIM"IOPHYS!CTA n "OWES*-AND#ROFTS!2 "INARYOSCILLATIONSINTHERATEOF REOXIDATIONOFTHEPRIMARYACCEPTOROF0HOTOSYSTEM))"IOCHIM "IOPHYS!CTAn "OWES*-AND D #ROFTS!2 F A %FFECTO F F $"-)"ONTHESEC ONDARY ELECTRONACCEPTOR"OFF 0HOTOSYSTEM))!RCH "IOCHEM "IOPHYSn "OWES*-AND D #ROFTS!2 F B 4HEROLEOFF P(AND MEMBRANE POTENTIAL INTHEREACTIONSOFF 0HOTOSYSTEM))ASMEASURED BY EFFECTSONDELAYEDmUORESCENCE"IOCHIM"IOPHYS!CTA n "OWES*- #ROFTS!2AND)TOH3A !HIGHPOTENTIALACCEPTOR FORPHOTOSYSTERN))"IOCHIM"IOPHYS!CTAn "OWES*- #ROFTS!2AN F D )TOH 3B %FFECTSO F F P(ONREAC TIONSONTHE DONORSIDEOFF 0HOTOSYSTEM))"IOCHIM "IOPHYS !CTA  n "OWES*- #ROFTS!2AN F D D!RNTZEN#*  2EDOXREACTIONS ONTHEREDUC D INGSIDEOFF 0HOTOSYSTEM)) INCHLOROPLASTSWITH ALTEREDHERBICIDEBINDINGPROPERTIES!RCH"IOCHEM"IOPHYS n "OWYER* (ILTON- 7HITELEGGE* *EWESS0 #AMILLERI0 #ROFTS !2 AND 2OBINSON ((  -OLECULAR MODELLING STUDIES ONTHE BINDINGOFF PHENYLUREAINHIBITORSTOTHE$PROTEINOF F HCn 0HOTOSYSTEM)):.ATURFORSC "OWYER*2 #AMILLERI0AND6ERMAAS7&* D  0HOTOSYSTEM)) AND ITS INTERACTIONWITH HERBI R CIDES)N "AKER.2AND D 0ERCIVAL -0 EDS 4OPICS IN 0HOTOSYNTHESIS 6OL  (ERBI R CIDES PP n%LSEVIER3CIENCE0UBLISHERS"6 6 !MSTERDAM "RETON * * "OULLAIS # -IOSKOWSKI # 3EBBAN 0 0 "ACIOU , AND .ABEDRYK% 6IBRATIONALSPECTROSCOPYFAVORSAUNIQUE 1" BINDINGSITEATTHEPROXIMALPOSITIONINWILD TYPEREACTION CENTERS AND INTHE 0RO L A 4YRMUTANT FROM F 2HODOBACTER SPHAEROIDES D  "IOCHEMISTRY n "URDA + +RUK K * "ORGSTADT 2 3TANEK K * 3TRZALKA + 3CHMID '(AND+RUSE/ D  -OSSBAUERSTUDIESOFF THENON HEME IRON AND D CYTOCHROME B IN A #HLAMYD M DOMONAS REINHARD R TII 03)n MUTANTANDTHEIRINTERACTIONSWITH _ TOCOPHEROLQUINONE &%"3,ETTn "USER#! $INER"!AND"RUDVIG'7 0HOTOOXIDATION OF CYTOCHROME B  IN OXYGEN EVOLVING 0HOTOSYSTEM )) "IOCHEMISTRYn #LELAND D 2% AND D 'RACE 3#  6OLTAMMETRIC DETECTION OF

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT SUPEROXIDE PRODUCT D ION BY 0HOTOSYSTEM )) &%"3 ,ETT  n #ROFTS!2AND7RAIGHT#! 4HEELECTROCHEMICALDOMAIN OFPHOTOSYNTHESIS"IOCHIM"IOPHYS!CTAn #ROFTS!2 7RAIGHT#!AND&LEISCHMAN$% %NERGYCON SERVATIONINTHEPHOTOCHEMICALREACTIONSOFF PHOTOSYNTHESISAND ITSRELATIONTODELAY A ED mUORESCENCE&%"3,ETTn F 2OBINSON (( ANDD 3NOZZI -  2EACTIONS OF #ROFTS!2 Q INOLSATCATALYTICSITESADIFFUS QU F IONAL ROLE IN( TRANSFER)N F 3YBESMA#ED !DVANCES IN0HOTOSYNTHESIS2ESEARCH 6OL) PPn-ARTINUS.IJHOFF$R7*UNK 4HE(AGUE #ROFTS!2 2OBINSON(( !NDREWS+ 6AN$OREN3AND"ERRY%  #ATALYTICSITESFORREDUCTIONANDOXIDATIONOFQUINONES )N0APA3 #HANCE"AND%RNSTER,EDS #YTOCHROME3YSTEMS -OLECULAR "IOLOGY ANDD "IOENERGETICS PP n 0LENUM R 0UBL U #ORP .EW9ORK #ROFTS!2 F "AROLI ) +RAMER $ ANDD 4AOKA 3  +INETICS OFF ELECTRON TRANSFER F BETWEEN 1! AND 1" IN WILD TYPE AND HERBI R CIDE RESISTANT MUTANTS OF # # REINHARD R TII : .ATURFORSC F H Cn DE 7IJN 2 AND VAN 'ORKOM (*  +INETICS OF ELECTRON TRANSFERFROM 1! TO1" IN0HOTOSYSTEM))"IOCHEMISTRY n DE7IJ 7 N2 3CHRAMA4ANDVAN'OR D RKOM(* 3ECONDARY STABILIZATIONREACTIONSAND PROTON COUPL U EDD ELECTRONTRANSPORT IN 0HOTOSYSTEM )) INVESTIGATED BY ELECTROLUMINESCENCE AND mUORESCENCESPECTROSCOPY" Y IOCHEMISTRYn Y D $EBRUNNER 0'  -OSSBAUERSPECTROSCOPY)N!MESZ*AN (OFF * EDS "IOPHYSICAL 4ECHNIQUES IN 0HOTOSYNTHESIS PP n+LUWER!CADEMIC0UBLISHERS $ORDRECHT $EBRUNNER0' 3CHULZ#% &EHER' AND/KAMURA-9 -OSSBAUER STUDY OF REACTION CENTERS FROM 2 SP S HAEROIDES H "IOPHYS*A F D -ICHEL (  4HEPHOTOSYNTHETICREACTION $EISENHOFER*AN CENTERFROMT F HE PURPLE BACTERIUM 2HODO DOPSEUDOMONASVIRI D DIS 3CIENCEn F IKI+ (UB U ER2ANDD -ICHEL( $EISENHOFER* %PP/ 3TRUCTUREOFTHEPROTEINSUBUNITSINTHEPHOTOSYNTHETICREACTION CENTREOF2HODOPSEUDOMONASVIRIDIS AT ¯ RESOLUTION.ATURE n $EISENHOFER* %PP/ 3INNING)AND-ICHEL( #RYSTALLO GRAPHICRElNEMENTAT ANGSTROMRESOLUTIONANDD RElNEDD MODEL OFF THEPHOTOSYNTHETICREACTIONCENTREFROM F 2HODO DOPSEUDOMONAS D VIRIDIS * -OL"IOLn $ELIGIANNAKIS9 4SEKOS. 0ETROULEAS6ANDD $INER"!  /RIENTATIONDEPENDENCEOFF THE&E ./ANDD &E %02SIGNAL ASSOCIATEDWITHTHENON HEMEIRONOF0HOTOSYSTEM))"IOCHIM "IOPHYS!CTAn $ELIGIANNAKIS9 0ETROULEAS6AND$INER"! "INDINGOF CARBOXYLATEANIONSATTHENON HEME&E)) OF03)))%FFECTSON D !&E %02SPECTRAANDD THEREDOXPROPERTIES THE1!n &E AND1 OFF THEIRON"IOCHIM "IOPHYS!CTAn F D!7 D  %3%%$ELIGIANNAKIS9 "OUSSAC!ANDD 2UTHERFOR STUDY OFF THE PLASTOQUINONE ANION RADICAL 1!% n IN . AND L . LABELED D 0HOTOSYSTEM )) TREATEDD WITH #.n "IOCHEMISTRY n $ELIGIANNAKIS9 *EGERSCHOLD#AND2UTHERFORD!7 %02 AND %3%%- STUDY OF THE PLASTOQUINONE ANION RADICAL 1!n IN0HOTOSYSTEM))TREATEDATHIGHP(#HEM0HYS,ETT n $ELIGIANNAKIS9 )OANNIDIS.ANDD 0ETROULEAS 6  L$ AND

 $ %3%%-STUDY OFF THE;&E ./=3 COMPLEXOFF 03)))N 'ARAB'ED 0HOTOSYNTHESIS-ECHANISMSAND%FFECTS 6OL)) PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT $ELIGIANNAKIS9 (ANLEY *AND2UTHERFORD!7 L$ AND$ %3%%-STUDYOFTHESEMIQUINONERADICAL 1!n OF0HOTOSYSTEM )) * !M #HEM3OCn $EMETER3 'OUSSIAS#H "EMAT' +OVACS,AND D 0ETROULEAS6  0ARTICIPATIONOFF THE G  AND G %02FORMS OFF THESEMIQU Q INONE IRONCOMPLEX 1!n &E OFF 0HOTOSYSTEM)) INTHE GENERATIONOFT F HE1AND#T D HERMOLUMINESCENCE BANDS RESPECTIVELY&%"3,ETTn $INER"! $EPENDENCEOFTHEDEACTIVATIONREACTIONSOF 0HOTOSYSTEM))ONTHEREDOXSTATEOFTHEPLASTOQUINONEPOOL! Q PTOR VARIEDUNDERANAEROBICCONDITIONS%QUILIBRIAONTHEACCE SIDEOFF 0HOTOSYSTEM))"IOCHIM "IOPHYS!CTAn $INER"! !PPLICATIONOFF SPECTROSCOPICTECHNIQUESTOTHE STUDY OFF 0HOTOSYSTEM))MUTATIONSENGINEERED IN 3YNECHOCY CYSTIS AND #HLAMYD M DOMONAS-ETHOD D %NZYMOLn $INER"!AND D "AABCOCK'4 K  3TRUCTURE DYNAM Y ICS AND D ENERRGY CONVERSIONEFlCIENCYIN0HOTOSYSTEM)))N/RT$2AND9OCUM #&EDS /XYGENIC0HOTOSYNTHESIS4HE,IGHT2EACTIONS PP n+LUWER!CADEMIC0UBLISHERS $ORDRECHT $INER"!AND0ETROULEAS6A ,IGHT INDUCEDOXIDATIONOFTHE ACCEPTOR SIDE&EL) OFF 0HOTOSYSTEM)) BYEXOGENOUSQUINONES ACTINGTHROUGHTHE1" BINDINGSITE))"LOCKAGE BY INHIBITORS AND D THEIREFFECTSONT F HE&E))) %02SPECTRA"IOCHIM"IOPHYS !CTAn $INER"!AND D 0ETROULEAS6 B 1 THENON HEMEIRONOF THE 0HOTOSYSTEM )) IRON QUINONE COMPLEX ! SPECTROSCOPIC PROBEOFQUINONEANDINHIBITORBINDING TOTHEREACTIONCENTER "IOCHIM"IOPHYS!CTAn $INER"!AND0ETROULEAS6 &ORMATIONBY./OFNITROSYL ADDUCTSO D F REDOXCOMPONENTSOFF THE0HOTOSYSTEM))REACTION CENTER))%VIDENCETHAT(#/n#/ BINDSTOTHEACCEPTOR SIDE NON HEMEIRON"IOCHIM"IOPHYS!CTAn $INER"! 0ETROULEAS6AND D 7ENDOLOSKI**  4HEIRON QUI NONE ELECTRON ACCEPTOR COMPLEX OFF 0HOTOSYSTEM )) 0HYSIOL 0LANTn $RABER7 4IETJEN+ +LUTH*&AND4REBST! (ERBICIDES IN PHOTOSYNTHESIS RESEARCH !NGEW #HEM )NT %D %NGL  n $RABER7 (ILP5 ,IKUSA( 3C K HINDLER-AND D 4REBST! )NHIBITION OF F PHOTOSYNTHESIS BY  NITRO  ALKYL PHENOLS 3TRUCTURE ACTIVITY STUDIES IN WILD TYPE AND lVE MUTANTS OF #HLAMYD M DOMONAS REINHARD R TII THYLAKOIDS : .ATURFORSC F H C n $UTTON0, 0RINCE2#AND4IEDE$- 4HEREACTIONCENTEROF PHOTOSYNTHETICBACTERIA0HOTOCHEM0HOTOBIOLn %ATON 2YE ** AND 'OVINDJEE  %LECTRON TRANSFER THROUGH THEQUINONEACCEPTORCOMPLEXOFPHOTOSYSTERN))AFTERONEOR R ONATE DEPLETEDSP D INACHTHY H LAKOID TWOACTINICmASHESIN BICARB MEMBRANES"IOCHIM "IOPHYS!CTAn %RICKSON*- 2AHIRE- "ENNOUN0 $ELEPELAIRE0 $INER"AND R CIDERESISTANCEIN #HL # AMYD M DOMONAS 2OCHAIX*$  (ERBI R TII RESULTS FFROM A MUTATION IN THE CHLOROPLAST GENE REINHARD FORTHEK$APROTEINOF03))0ROC.ATL!CAD3CI53! n %RICKSON*- 0lSTER+ 2AHIRE- 4OGASAKI2 -ETS,AND2OCHAIX * $ -OLECULARANDBIOPHYSICALANALYSISOFHERBICIDE D REINHARD R TII 3TRUCTURE RESISTANT MUTANTS OF #HLAMYDOMONAS F FUNCT ION RELATIONSHIP OFF THE PHOTOSYSTERN )) $L POLYPEPTIDE

 0LANT#ELL Ln %TIENNE ! , $UCRUET * - !JLANI ' AND 6ERNOTTE #  #OMPARATIVESTUDIESONELECTRONTRANSFERIN0HOTOSYSTEM))OF HERBICIDE RESISTANTMUTANTSFROMDIFFERENTORGANISMS"IOCHIM "IOPHYS!CTAn %VANS-#7 !TKINSON9%ANDD &ORD 2#  2EDOXCHARAC TERIZATIONOFT F HEPHOTOSYSTERN))ELECTRONACCEPTORS%VIDENCE F FORTWOE LECTRONCARRIERS BETWEENPHEOPHYTINAND1" D IOCHIM "IOPHYS!CTAn D KAMURA-9 4HEPRIMARYANDD SECONDARY &EHER'AND/ ACCEPTORSINBACTERIALPHOTOSYNTHESIS!PPL-AGN2ESON n &ERREIRA+. )VERSON4- -AGHLAOUI+ "ARBER*AND)WATA3  !RCHITECTUREOFTHE0HOTOSYNTHETIC/XYGEN %VOLVING #ENTER 3CIENCE  F DANS! "OY O NTON*% 'ILLHAM.7 &ORSTER" (EIFETZ0" ,AR (ERBI R CIDERESISTANCEAND GROWTH OFF $!LA MUTANTSIN #HLAMYD M DOMONAS:.ATURFORSC F HCn &OWLER #&  0ROTON TRANSLOCATION IN CHLOROPLASTS AND ITS RELATIONSHIPTOELECTRONTRANSPORTBETWEENTHEPHOTOSYSTEMS "IOCHIM"IOPHYS!CTAn &RITZSCH' +AMPMANN, +APAUN'AND-ICHEL( 7ATER CLUSTERS IN THE REACTION CENTRE OF 2HODOBACTER SP S HAEROIDES 0HOTOSYNTH 2ESn &UFEZAN# 2UT F HERFOR F DD!7AND +RIEGER ,ISZKAY A !  3INGLET OXYGENPRODUCT D IONINHERBI R CIDE TREATEDD 0HOTOSYSTEM))&%"3 ,ETTn A 2AND-ETS,* D  !TRAZINE BROMACILAND DIURON 'ALLOWAY # S!SINGLENON MENDELIANGENETIC RESISTANCEIN #HLAMYDOMONA LOCUSCONTROLSTHESTRUCTUREOFTHETHYLAKOIDBINDINGSITE0LANT 0HYSIOLn 'ARBERS! 2EIFARTH& +URRECK* 2ENGER'AND0ARAK& F R #ORRELATION BETWEEN PROTEIN mEXIBILITY ANDD ELECTRON TRANSFE F FROM 1!n TO1" IN03))MEMBRANE FRA F GMENTSFROMSP F INACH "IOCHEMISTRYn 'ARGE 0 #HIKATE 2 0ADHYE 3 3AVARIAULT *- DE ,OTH 0 AND 4 HAGUES* 0 )RON)) COMPLEXESOFORT 4UC F HO FUNCT F IONAL IZEDPARA NAPHTOQUINONES#RYSTALANDMOLECULARSTRUCTURE OFBISAQUO BISLAWSONE IRON)) ANDINTERMOLECULARMAGNETIC EXCHANGE INTERACTIONS IN BIS AMINOLAWSONE IRON))  )NORG #HEMn D (ASELKORN2 -UTATIONTO HERBI R CIDERESIS 'OLDEN33AND TANCEMAPSWITHINTHE PSB! GENEOF !NACY CYSTISNIDU D LANS 2 3CIENCEn 'OUSSIAS #( $ELIGIANNAKIS 9 3ANAKIS 9 )OANNIDIS . AND U TLECOORDINATIONCHANGESIN 0ETROULEAS6 0ROBING SUB THEIRON QUINONECOMPLEXOF0HOTOSYSTEM))DURINGELECTRON TRANSFER BYTHEUSEOF./"IOCHEMISTRYn 'OVINDJEE  0HOTOSYSTEM )) HETEROGENEITY4HE ACCEPTOR SIDE0HOTOSYNTH2ES  D  #HLORO 'OVINDJEE %GGENBERG0 0lSTER+AND3TASSER2* PHYLL A mUORESCENCEDECAY A IN HERBI R CIDE RESISTANT$MUTANTSOF #HLAMYD M DOMONASREINHARD R TII ANDD THE FORMATE EFFECT "IOCHIM "IOPHYS!CTAn 'REENBERG "- 'ABA 6 -ATTOO !+ ANDD %DELMAN -  )DENTIlCATIONOFAPRIMARYINVIVODEGRADATIONPRODUCTOFTHE RAPIDLY TURNING OVER K$PROTEINOF0HOTOSYSTEM))%-"/ *n (ALLAHAN "* 2UFmE 36 "OWDEN 3* AND .UGENT *(!  )DENTIlCATIONANDD CHARACTERISATIONOFF %02SIGNALSINVOLVING 1" SEMIQUINONE IN PLANT 0HOTOSYSTEM )) "IOCHIM "IOPHYS

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT !CTAn (AUMANN - AND *UNGE7  4HE RATES OF PROTON UPTAKE ANDELECTRONTRANSFERATTHEREDUCINGSIDEOF0HOTOSYSTEM))IN THYLAKOIDS&%"3,ETTn (EATHCOTE0AND2UTHERFORD!7 !N%02SIGNALARISING F FROM 1"n&E IN #HROMATIUM VINOSUM STRAIN $ )N "IGGINS * ED 0ROGRESSIN 0HOTOSYNTHESIS2ESEARCH 6OL) PPn -ARTINUS.IJHOFF0U F UBLISHER $ORDRECHT (EGG %,AND1UE,*R D  4HE (IS  CARB R OXYLATEFAC F IAL TRIAD!NEMERGING STRUCTURALMOTIF INMONONUCLEARNON HEME IRON)) ENZYMES%UR*"IOCHEMn (IENERWADEL2AND"ERTHOMIEU# "ICARBONATEBINDINGTO THENON HEMEIRONOF0HOTOSYSTEM))INVESTIGATEDBY&OURIER TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY AND # LABELED BICARB R ONATE"IOCHEMISTRY n (IRSCHBERG *AND D -C)NTOSH ,  -OLECULARBASISOF HERBI R CIDE T USHYBRIDUS D 3CIENCEn RESISTANCE IN !MARANTH (OPE!"AND D -ATTHEWS$" &URTHERSTUDIESONPROTON F INGLE TURNOVERmASHES TRANSLOCATIONS INCHLOROPLASTSAFTERS 0ROTONUPTAKE!UST*0LANT0HYSIOLn (OPE!AND-ORLAND! 0ROTON4RANSLOCATIONINISOLATED SPINACHCHLOROPLASTSAFTERSINGLE TURNOVERACTINICmASHES!UST *0LANT0HYSIOLn (UBB U ARD D *!- #ORRIE!2 .UGENT*(!AND%VANS-#7 D  0ROPERTIESOFT F HE0HOTOSYSTEM))ELECTRONACCEPTORCOMPLEXOF 0HORMIDIUM LAMINOSUM"IOCHIM"IOPHYS!CTAn (UPPATZ *,  1UANTIFYING THE INHIBITOR TARGET SITE IN TERACTIONS OFF 0HOTOSYSTEM )) HERBI R CIDES 7EED D 3CIENCE    (UPPATZ*,AND0HILLIPS*. #YANOACRYLATEINHIBITORSOF THE(ILLREACTION)))3TEREOCHEMICALANDELECTRONICASPECTSOF INHIBITORBINDING:.ATURFORSCHCn (UTCHISON23 8IONG* 3AYRE24AND'OV D INDJEE  #ON STRUCTIONAND D CHARACTERIZATIONOFA0 F HOTOSYSTEM))$MUTANT ARGI R NINE  GLYCINE OF #HLAM L MYDDOMONASREINHARD RDTII "IOCHIM "IOPHYS!CTAn )KEGAMI AND D +ATOH3  3TUDIESONCHLOROPHYLL mUORESCENCE INCHLOROPLASTS))%FFECTOFFERRICYANIDEONTHEINDUCTIONOF mUORESCENCE IN THE PRESENCE OF    DICHLOROPHENYL   DIMETHYLUREA0LANT#ELL0HYSIOLn )TOH3 4ANG83AND3ATOH+ )NTERACTIONOFTHEHIGHSPIN &EATOMINTHEPHOTOSYSTERN))REACTIONCENTERWITH THEQUINONES 1! AND 1" INPURIlED D OXYGEN EVOLVING 03))REACTIONCENTER COMPLEXAND IN03))PARTICLES&%"3,ETTn F LDD 5 /RAWSKI ' *OHANNINGMEIER5 3OPP' "RAUNER- !LTENFE R CIDERESISTANCEAND D SUPERSENSITIVITY /ETTMEIER7 (ERBI IN!LA OR!LA MUTANTSOFTHE$PROTEININ#HLAMYDO D MONASREINHARDTII0ESTICIDE"IOCHEM0HYSIOLn *OHNSON'. "OUSSAC!AND2UTHERFORD!7 4HEORIGIN OF n  # THERMOLUMINESCENCE BANDS IN 0HOTOSYSTEM )) H  "IOCHIM "IOPHYS!CTAn *OLIOT0 %FFECTO F F LOWTEMPERATUREnTOn O# ONTHE F THE 0HOTOSYSTEM )) PRIMARY ELECTRON TRANSFER F REOXIDATION OF INTHEPRESENCEAND D ABSENCEOF F   DICHLOROPHENYL N   DI METHYLUREA"IOCHIM"IOPHYS!CTAn *OLIOT0 ,AVERGNE*AND"EAL$ 0LASTOQUINONECOMPART MENTATIONINCHLOROPLASTS)%VIDENCEFORDOMAINSWITHDIFFERENT RATESOFPHOTO REDUCTION"IOCHIM"IOPHYS!CTAn +AMINSKAYA /0 $RACHEV,! +ONSTANTINOV!! 3EMENOV!9AND 3KU K LACHEV60  %LECTROGENICREDUCT D IONOFF THESECONDARY Q INONEACCEPTORINCHROMATO QU H PHORESOF 2HODOS D SPIRILLUMRU L BRUM

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT 2APID KINETICMEASUREMENTS&%"3,ETTn +AMIYA.AND3HEN*2 #RYSTALSTRUCTUREOFOXYGEN EVOLV ING0HOTOSYSTEM))FROM4HERMOSYNECHOCOCCUSVULCANUS AT  ¯ RESOLUTION0ROC.ATL!CAD3CI53!n +ASHINO9 9AMASHITA - /KAMOTO9 +OIKE ( AND 3ATOH +  -ECHANISMS OF F ELECTRON mOW THROUGH THE 1" SITE IN F F HEPRESENCEOFMEM FT F BRANESTRUC 0HOTOSYSTEM))%FFECTSO TUREONTHEREDOXREACTIONSATTHE1" SITE0LANT#ELL 0HYSIOL n +EREN. "ERG! 6ANKAN0*- ,EVANON(ANDD /HADD )  -ECHANISMOF0HOTOSYSTEM))PHOTOINACTIVATIONAND$PROTEIN DEGRADATIONATLOWLIGHT ˆ THEROLEOFBACKELECTRONmOW0ROC .ATL!CAD3CI53!n +LESS(AND6ERMAAS7&* -ANYCOMBINATIONSOFAMINO ACIDSEQUENCES D INACONSERVEDD REGIONOFF THE$PROTEINSATISFY 0HOTOSYSTEM)) FUNCT F ION*-OL "IOL n D DELMAN-AND6ERMAAS7&* D +LESS(/REN 3HAMIR- /HAD) %  0ROTEINMODIlCATIONS INTH T E$PROTEINOFF 0HOTOSYSTEM)) AFFECTPROPERT F IESOFF THE1" HERBI R CIDE BINDINGENVIRONMENT: .ATURFORSCHCn +LIMOV66 $OLAN % 3HAW %2 AND +E "  )NTERACTION BETWEENTHEINTERMEDIARYELECTRONACCEPTORPHEOPHYTIN ANDA POSSIBLEPLASTOQUINONE IRONCOMPLEXIN0HOTOSYSTEM))REACTION CENTERS0ROC.ATL!CAD 3CI 53!n +OULOUGLIOTIS $ +OSTOPOULOS 4 0ETROULEAS 6 ANDD $INER "!  %VIDENCE FOR#. F BINDINGATTHE03))NON HEME&E n  F HE%02SIGNAL FOR1 F ANDD ON 1!1" ELECTRON %FFECTSONT ! &E TRANSFER" F IOCHIM"IOPHYS!CTAn +RIEGER! 2UTHERFORD!7AND*EGERSCHOLD# 4HERMOLUMI NESCENCEMEASUREMENTSONCHLORIDE DEPLETEDANDCALCIUM DE PLETED0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn +RIEGER ,ISZKAY!AND2UTHERFORD!7 )NmUENCEOFHERBI CIDE BINDINGONTHEREDOXPOTENTIALOFT F HEQUINONEACCEPTORIN D HYTOTOXICITY Y 0HOTOSYSTEM))2ELEVANCETOPHOTODAMAGEANDP "IOCHEMISTRY  K *AND3TRZA D LKA+ 2EDOXCHANGESOFC F YTOCHROME B +RUK INTHEPRESENCEOFF PLASTOQUINONES*"IOL#HEM  +UGLSTATTER! %RMLER5 -ICHEL( "ACIOU,AND&RITZSCH' 8 RAY STRUCTURE ANALYSES OF PHOTOSYNTHETIC REACTION CENTER VARIANTS FROM 2HODOBACTER SPHAEROIDES 3TRUCTURAL CHANGES INDUCEDBYPOINTMUTATIONSATPOSITION,MODULATEELECTRON AND D PROTONTRANSFER" F IOCHEMISTRYn K * 'ARRBERS ! 2EIFART F H & !NDREASSON , % 0ARAKK & +URRECK F BRANE 2ENGER' )SOLATIONANDD PROPERTIESOF03))MEM F GMENTSDEPLETEDD OF THENON HEME IRONCENTER&%"3,ETTERS FRA n ,ANCASTER #2$ AND -ICHEL (  4HE COUPLING OF LIGHT INDUCEDELECTRONTRANSFERANDPROTONUPTAKEASDERIVEDFROM CRYSTALSTRUCTURESOFREACTIONCENTRESFROM 2HODOPSEUDOMONAS VIRIDISMODIlEDATTHEBINDINGSITEOFTHESECONDARYQUINONE 1" 3TRUCTUREn ,ANCASTER#2$ANDD -ICHEL (  2ElNEDD STRUCTURESOFREA F C TION CENTRES FR F OM 2HODO DOPSEUDOMONAS D VIRIDIS IN COMPLEXES R CIDEATRAZINEANDD TWOCHIRAL ATRAZINEDERIVATIVES WITHTHE HERBI ALSO LEAD D TOANEWMODELOFT F HEBOUNDD CAROTENOID*-OL"IOL n ,ARDANS ! 'ILLHAM .7 "OYNTON *%  3ITE DIRECTED MUTATIONSATRESIDUEOFTHE0HOTOSYSTEM))$PROTEINOF # #HLAMYDOMONAS THATRESULTINANONPHOTOSYNTHETICPHENOTYPE AND IMPAIR$SYNTHESISANDD ACCUMULATION*"IOL#HEM n

 ,AVERGNE*A )NTERACTIONOFF EXOGENOUSBENZOQUINONEWITH 0HOTOSYSTEM))INCHLOROPLASTS4HESEMIQUINONEFORMACTSAS ADICHLOROPHENYLDIMETHYLUREA INSENSITIVESECONDARYACCEPTOR "IOCHIM"IOPHYS!CTAn ,AVERGNE*B -ODEOFACTIONOF   DICHLOROPHENYL   DIMETHYLUREA%VIDENCETHATTHEINHIBITORCOMPETESWITH PLASTOQUINONE FOR F BINDING TOACOMMONSITEONTHEACCEPTORSIDE OFF 0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn ,AVERGNE*C 4WOTYPESOFPR F IMARYACCEPTORS INCHLOROPLAST 0HOTOSYSTEM)))$IFFERENTRECOM F BINATIONPROPERTIES0HOTO BIOCHEM0HOTOBIOPHYSn ,AVERGNE * "OUCHAUD *0 AND *OLIOT 0  0LASTOQUINONE COMPARTMENTATION IN CHLOROPLASTS )) 4HEORETICAL ASPECTS "IOCHIM"IOPHYS!CTAn ,IPSCOMB *$ AND D /RVILLE!-  -ECHANISTIC ASPECTS OF DIHYDROX D YBENZOATEDIOXYGENASES)N3IGEL(AND3 D IGEL! EDS -ETAL)ONS IN"IOLOGICAL 3YSTEMS 6OL PPn-ARCEL $EKKER)NC .EW9ORK R ,UPINKOVA, -ETZ*' $INER"! 6ASS)AND+OMENDA*  (ISTIDINERESIDUEOFTHE0HOTOSYSTEM))$POLYPEPTIDE ISINVOLVEDINALIGHT INDUCEDCROSS LINKINGOFTHEPOLYPEPTIDE WITHTHEALPHASUBUNITOFCYTOCHROMEB 3TUDYOFASITE DIRECTED D MUTANTOF 3YNECHOCYSTIS 3 0##"IOCHIM"IOPHYS !CTA  n -AC-ILLAN& 'LEITER( 2ENGER' ,UBI U TZ7  %02 2 %. $/2STUDIESOFF PLASTOQUINONEANIONRADICAL IN0HOTOSYSTEM n F KY -ED #UR )) 1! AND INORGANICSOLVENTS)N"ALTSCHEFFS RENT2ESEARCH IN0HOTOSYNTHESIS 6OL ) PPn+LUWER !CADEMIC0UBLISHERS $ORDRECHT -AC-ILLAN& & +URRECK) !DIR. ,ENDZIAN& & +ASS( 2EIFARTH& & 2ENGER'AND,UBITZ7A %02 %.$/2AND%3%%n% INVESTIGATION OF THE ELECTRON ACCEPTOR RADICAL ANION 1! IN PHOTO SYSTEM )) 03)) REACTION CENTRES )N -ATHIS 0 ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL ) PPn +LUWER!CADEMIC0UBLI U SHERS $ORDRECHT -AC-ILLAN& ,ENDZIAN& 2ENGER'AND D ,U UBITZ7 B %02 AND D %.$/2 INVESTIGATION OF F THE PRIMARY ELECTRON ACCEPTOR RADICALANION 1!% n INIRON DEPLETED0HOTOSYSTEM))MEMBRANE FRAGMENTS"IOCHEMISTRYn ,ORENTE-! $AHAN& 3ANAKIS9 0ETROULEAS6 "OUSSEKSOU! AND4UCHAGUES*0 .EWFERROUSCOMPLEXESBASEDONTHE  | BIIMIDAZOLELIGAND3TRUCTURAL -OSSBAUERAND-AGNETIC PROPERTIES)NORG#HEMn -ICHEL (AND D $EISENHOFER* F  2ELEVANCEOFF THEPHOTOSYN THETICREACTIONCENTERFROM F PURPLE BACTERIATOTHESTRUCTUREOF 0HOTOSYSTEM))"IOCHEMISTRY n -ICHEL ( %PP  AND $EISENHOFER *  0IGMENT PROTEIN INTERACTIONSINTHEPHOTOSYNTHETICREACTIONCENTREFROM 2HO DOPSEUDOMONASVIRIDIS %-"/*n -ORAIS& +UHN+ 3TEWART$( "ARBER* "RUDVIG'7AND.IXON 0* 0HOTOSYNTHETICWATEROXIDATIONINCYTOCHROME B MUTANTSCONTAINING A DISRUPTED HEME BINDING POCKET*"IOL #HEMn -ORELAND D $%  2ESEARCH ONBIOCHEMISTRY OF HERBI R CIDES!N HISTORICALOVERVIEW:.ATURFORSC F HCn -OSER## 0AGE## &ARID2AND$UTTON0, "IOLOGICAL ELECTRONTRANSFER*"IOENERG"IOMEMBn -ULO0 4YYSTJARVI4 4YYSTJARVI% 'OVINDJEE -AENPAA0AND !RO%- -UTAGENESISOFTHED ELOOPOF0HOTOSYSTEM)) F IONAND D ASSEMBLYOFF 0HOTO REACTIONCENTREPROTEIN $ ˆ FUNCT SYSTEM))0LANT-OL "IOL n

 -ULO 0 ,AAKSO 3 -AENPAA 0 ANDD !RO %-  3TEPWISE PHOTOINHIBITIONOF0HOTOSYSTEM))nSTUDIESWITH 3YNECHOCYSTIS 3 SPECIES0##MUTANTSWITHAMODIlEDDELOOPOFTHEREAC TIONCENTERPOLYPEPTIDE$0LANT0HYSIOLn .ABEDRYK% "RETON* 3EBBAN0AND"ACIOU, 1UINONE1" BINDINGSITEANDPROTE D INSTRUCTURAL CHANGES INPHOTOSYNTHETIC REACTIONCENTERMUTANTSAT0RO ,REVEALED BYVIBRATIONAL SPECTROSCOPY" Y IOCHEMISTRY n .IXON0* * +OMENDA* * "ARB R ER* * $EAKK : 6ASS)ANDD $INER"!  $ELETION OF F THE 0%34 LIKE REGION OFF 0HOTOSYSTEM )) MODIlES THE 1" BINDING POCKET BUT DOES NOT PREVENT RAPID TURNOVEROF$*"IOL#HEMn .OGUCHI4 +URRECK* )NOUE9AND2ENGER'A #OMPARATIVE &4)2ANALYSISOFTHEMICROENVIRONMENTOF1!n s INCYANIDE D HIGH P( TREATEDD AND IRON DEPLETEDD 0HOTOSYSTEM )) TREATED F GMENTS"IOCHEMISTRYn MEMBRANE FRA .OGUCHI4 )NOUE9 ANDD 4ANG 8 3 B (YDROGEN BONDING INTERACTION BETWEENTHEPRIMARYQUINONEACCEPTOR1! ANDD A HISTIDINESIDECHAININ0HOTOSYSTEM))ASREVEALED BY &OURIER TRANSFORMINFRAREDSPECTROSCOPY"IOCHEMISTRYn .UGENT*(! 0HOTOREDUCIBLEHIGHSPINIRONELECTRONPARA MAGNETICRESONANCESIGNALSINDARK ADAPTED0HOTOSYSTEM)) !RETHEYOXIDISEDNON HAEMIRONFORMEDFROMINTERACTIONOF OXYGENWITH 03))ELECTRONACCEPTORS"IOCHIM "IOPHYS!CTA H  n .UGENT)(! $INER"!AND%VANS-#7 D  $IRECT DETEC TIONOFF THEELECTRONACCEPTOROFF 0HOTOSYSTEM&%"3,ETT  n .UGENT *(! #ORRIE !2 $EMETRIOU # %VANS -#7 AND ,OCKETT#* "ICARBONATEBINDINGANDTHEPROPERTIESOF 0HOTOSYSTEM))ELECTRONACCEPTORS&%"3,ETTn .UGENT*(! $OETSCHMAN$#AND-ACLACHLAN$* #HARAC TERIZATIONOFT F HEMULTIPLE%02LINESHAP A ESOF IRON SEMIQU Q INONES INPHOTOSYSTEM"IOCHEMISTRYn R CIDESOFF 0HOTOSYSTEM)))N"ARB R ER*ED /ETTMEIER7  (ERBI 4 ICSIN0HOTOSYNTHESIS6OL4HE0HOTOSYSTEMS3TRUCTURE 4OP D -OLECULAR"IOLOGY PPn% Y LSEVIER3CIENCE &UNCTIONAND 0UBLISHERS"6 6 !MSTERDAM /ETTMEIER7 (ERBICIDERESISTANCEANDSUPERSENSITIVITYIN 0HOTOSYSTEM))#ELL-OL,IFE3CIn /ETTMEIER 7 -ASSON + +LOOS 2 AND 2EIL %  /N THE ORIENTATION OFF 0HOTOSYSTEM )) INHIBITORS IN THE 1" BINDING Q INONES : .ATURFORSC F H NICHE !CRIDONES XANTHONES AND QU C n /KAMURA-9 0ADDOCKK -, 'RAIGE-3ANDD &EHER'  0RO TONAND D ELECTRONTRANSFER F INBACTERIALREACTIONCENTERS"IOCHIM "IOPHYS!CTAn /RVILLE!-AND,IPSCOMB*$ #YANIDEANDNITRICOXIDE BINDINGTOREDUCEDPROTOCATECHUATE  DIOXYGENASE)NSIGHT INTOTHEBASISFORORDER DEPENDENTLIGANDBINDINGBYINTRADIOL CATECHOLICDIOXYGENASES"IOCHEMISTRYn 0ELOQUIN*- 4ANG8 3 $INER"!ANDD "RITT2$ !NELEC TRON SPIN ECHO ENVELOPE MODU D LATION %3%%- STUDY OFF THE 1! BINDINGPOCKETOF03))REACT F IONCENTERSFROMSP F INACH AND CYSTIS"IOCHEMISTRY n 3 NECHOCY 3Y 0ETROULEAS 6 AND $INER "!  )NVESTIGATION OF THE IRON COMPONENTS IN 0HOTOSYSTEM )) BY -OSSBAUER SPECTROSCOPY &%"3,ETTn 0ETROULEAS6AND$INER"! )DENTIlCATIONOF1 AHIGH POTENTIAL ELECTRONACCEPTOROFF 0HOTOSYSTEM)) WITHTHE IRONOF THE QUINONE IRON ACCEPTOR COMPLEX "IOCHIM "IOPHYS!CTA

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT n 0ETROULEAS6AND$INER"! ,IGHT INDUCEDOXIDATIONOFTHE ACCEPTOR SIDE&E)) OF0HOTOSYSTEM))BYEXOGENOUSQUINONES ACTINGTHROUGHTHE1" BINDINGSITE)1UINONES KINETICSAND P( DEPENDENCE"IOCHIM"IOPHYS!CTAn 0ETROULEAS6AND D $INER"!  &ORMATIONBY./OFF NITROSYL ADDUCTSO D F REDOXCOMPONENTSOFF THE0HOTOSYSTEM))REACTION CENTER)./BINDSTOTH T EACCEPTOR SIDENON HEME IRON"IOCHIM "IOPHYS!CTAn 0ETROULEAS6 3ANAKIS9 $ELIGIANNAKIS9 $INER"! 4HE NON HEME &E)) OF 03))  "INDING OF NEW CARBOXYLATE ANIONS  3TUDYOFTWO-OSSBAUERCOMPONENTS)N-URATA .ED 2ESEARCHIN0HOTOSYNTHESIS 6OL)) n+LUWER !CADEMIC0UBLISHERS $ORDRECHT 0ETROULEAS6 $ELIGIANNAKIS9 AND D $INER"!  "INDING OF CARB R OXYLATEANIONSATTHENON HEME&E)) OF03))))#OMPET F I TIONWITH BICARB R ONATEAND D EFFECTSONT F HE 1!1" ELECTRONTRANSFER RATE"IOCHIM"IOPHYS!CTAn 0ICOREL 2 7ILLI 7 AMSON$, 9RUELA)AND D 3EIBERT- 4HE STATEOFIRONINTHEOXYGEN EVOLVINGCORECOMPLEXOFTHECYA NOBACTERIUM0HORMIDIUMLAMINOSUM -OSSBAUERSPECTROSCOPY "IOCHIM"IOPHYS!CTAn 0OLLE!AND*UNGE7 4HESLOWRISEOFTHEmASH LIGHT INDUCED ALKALIZATIONBY0HOTOSYSTEM))OFF THESUSPENDINGMEDIUMOF D TOTHYLAKOIDD STACKING"IOCHIM THYLAKOIDSISREVERSIBLY RELATED "IOPHYS!CTAˆ 0OSPISIL 0 !RATO! +RIEGER ,ISZKAY A !AND D 2UTHERFOR F D D!7 (YDROX D YL 2ADICAL 'ENERATION BY0HOTOSYSTEM))"IOCHEMISTRY n 1UE,*R /NEMOTIF ˆ MANYDIFFERENTREACTIONS.ATURE 3TRUCT"IOLn 1UE , *R AND 2AYMOND9.(  $IOXYGEN ACTIVATION BY ENZYMESWITH MONONUCLEARNON HEME IRONACTIVESITES#HEM 2EV n 2AKOTONANDRASANA ! "OINNARD D $ 3AVARIAULT *- 4UCHAGUES D 6ERDAGUER- )RON)) *0 0ETROULEAS6 #ARTIER#AND COMPLEXESWITHPOLYDENTATE3CHIFF BASE LIGANDSASMODELSOF THEPHOTOSYNTHETICMONONUCLEARNON HEMEFERROUSSITES3YN THESIS CHARACTERIZATION MOLECULARCRYSTALSTRUCTURE %8!&3 AND8!.%3STUDIES -OSSBAUERSPECTROSCOPYANDMAGNETIC PROPERTIES)NORG#HIM!CTAn 2APPAPORT & 'UERGOVA +URAS - .IXON 0* $INER "! AND ,AVERGNE* +INETICSANDPAT D HWAY A SOFC F HARGERECOMBINA TIONIN0HOTOSYSTEM))"IOCHEMISTRYn 2EIFART F H&AND D 2ENGER'  )NDIRECTEVIDENCE FORSTRUCTURA F L CHANGESCOUPLED D WITH 1"% n FORMATIONIN0HOTOSYSTEM))&%"3 ,ETTn 2IGBY3%* (EATHCOTE0 %VANS-#7AND.UGENT*(! %.$/2ANDSPECIAL42)0,%RESONANCESPECTROSCOPYOF1!% nOF PHOTOSYSTEM"IOCHEMISTRY  2OBINSON((AND D #ROFTS!2 F  +INETICSOFT F HEOXIDATION REDUCT D ION REACTIONS OF F THE 0HOTOSYSTEM )) QUINONE ACCEPTOR COMPLEX AND D THEPATHWAY A FOR F DEACTIVATION&%"3,ETT n 2OBINSON((AND#RO D FFTS!2 +INETICSOFF PROTONUPTAKEAND THEOXIDATION REDUCTIONREACTIONSOFTHEQUINONEACCEPTORCOM PLEXOF0HOTOSYSTEM)) FROMPEACHLOROPLASTS)N3YBESMA# ED !DVANCESIN0HOTOSYNTHESIS2ESEARCH 6OL PPn -ARTINUS.IJHOFF$R7*UNK0UBLISHERS 4HE(AGUE 2OBINSON (( AND D #ROFTS!2 F  +INETICS OFF THE CHANGES INOXIDATION REDUCT D IONSTATESOFF THEACCEPTORSAND DONORSOF

$IBQUFS

2VJOPOF
#JOEJOH 4JUFT 0HOTOSYSTEM))INPEATHYLAKOIDSMEASURED BY mASH mUORES CENCE)N"IGGINS*ED 0ROGRESSIN0HOTOSYNTHESIS2ESEARCH 6 6OL PPn-ARTINUS.IJHOF FF$R7*UNK0UBLISHERS 4HE(AGUE 2OBINSON (( %ATON 2YE ** VAN 2ENSEN **3 AND 'OVINDJEE  4HEEFFECTSO F F BICARB R ONATE DEPLETIONAND FORMATE F IN CUBATION ON THE KINETICS OF OXIDATION REDUCTION REACTIONS OF F H THE0HOTOSYSTEM))QUINONEACCEPTORCOMPLEX:.ATURFORSC C n 2OBINSON (( 'OLDEN 3 "RUSSLAN * ANDD (ASELKORN 2  &UNCTIONINGOF0HOTOSYSTEM))INMUTANTSTRAINSOFTHECYANO BACTERIUM !NACYSTISNIDULANS 2)N"IGGINS*ED 0ROGRESSIN 0HOTOSYNTHESIS2ESEARCH 6OL)6 PPn-ATINUS.IJHOFF 0UBLISHERS $ORDRECHT 2UFmE36 $ONNELLY $ "LUNDELL4,ANDD .UGENT*(!  ! THREE DIMENSIONAL MODELOFF THE0HOTOSYSTEM))REACTIONCENTRE OF 0ISUMSATIVUM0HOTOSYNTH2ESn 2 FmE36 7ANG * *OHNSTON(' 'USTAFSON4, (UTC 2U F HISON23 F D AYRE24  0HOTOSYSTEM)) D3A -INAGAWA* #ROFTS!2AN PERIPHERALACCESSORYCHLOROPHYLLMUTANTSIN #HLAMYDOMONAS REINHARDTII "IOCHEMICAL CHARACTERIZATION AND SENSITIVITY TO PHOTO INHIBITION0LANT0HYSIOLn 2 2UTHERFORD!7 /RIENTATIONOF%02SIGNALSARISINGFROM COMPONENTS IN 0HOTOSYSTEM))MEMBRANES"IOCHIM "IOPHYS H !CTA  n F D!7 D  (OW CLOSE IS THE ANALOGY BETWEEN THE 2UTHERFOR F 03)) AND THAT OF PURPLE BACTERIA 4HE REACTION CENTRE OF ELECTRON ACCEPTOR SIDE )N "IGGINS * ED 0ROGRESS IN 0HO TOSYNTHESIS 2ESEARCH 6OL  PP n -ARTINUS .IJHOFF 0UBLISHERS $ORDRECHT 2UTHERFORD !7 AND +RIEGER ,ISZKAY !  (ERBICIDE IN D DUCEDOXIDATIVESTRESSIN0HOTOSYSTEM))4RENDS"IOL3CI n 2UTHERFOR F D!7AN D D +RIEGER ,ISZKAY A !  3INGLETOXYGEN PRODUCT D ION IN HERBI R CIDE TREATEDD 0HOTOSYSTEM )) &%"3 ,ETT  n  2UTHERFOR F D!7AN D D :IMMERMANN* ,  !NEW%02SIGNAL ATTRIBUTEDTOTHEPRIMARYPLASTOQUINONEACCEPTORIN0HOTOSYSTEM ))"IOCHIM"IOPHYS!CTAn 2UTHERFORD!7 #ROFTS!2 AND )NOUE9  4HERMOLUMI NESCENCE AS A PROBE OF 0HOTOSYSTEM )) PHOTOCHEMISTRY THE ORIGINOFF THE mASH INDUCE D D GLOWPEAKS"IOCHIM "IOPHYS!CTA H  n 3ANAKIS9AND D 0ETROULEAS6 #YANIDE BINDING ATTHENON HEME IRON OFF 03)) AND EFFECTS F ON CERTAIN %02 SIGNALS )N -ATHIS0ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL) PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT 3ANAKIS9 0ETROULEAS6 AND $INER "!  #YANIDE BIND ING AT THE NON HEME &E OF THE IRON QUINONE COMPLEX OF 0HOTOSYSTEM))!THIGHCONCENTRATIONSCYANIDECONVERTSTHE &E FROM F HIGH 33  TOLOW3 SPIN"IOCHEMISTRY 3 n 3ANAKIS9 0ETASIS$ 0ETROULEAS6ANDD (ENDRICH - 3I MULTANEOUSBINDINGOF mUORIDEANDD ./TOTHENONHEMEIRONOF F K HANGE KEXC 0HOTOSYSTEM))1UANTITATIVE%02EVIDENCE FORAWEA INTERACTIONBETWEENTHESEMIQUINONE1!nANDTHEIRON NITROSYL COMPLEX*!M#HEM3OCn 3APHON3AND#ROFTS!2 4HE (ERATIOINCHLOROPLASTS IS  0OSSIBLE ERRORSINITS DETERMINATION :.ATURFO F RSCH C n 3EMIN"+ ,OVIAGINA%2 !LEKSANDROV!9 +AUROV9.ANDD .O

 VAKOVA!!  %FFECTO F F FORMATEON-OSS F BAUERPARAMETERS OFTHENON HEMEIRONOF03))PARTICLESOFCYANOBACTERIA&%"3 ,ETTn 3HINKAREV6AND7RAIGHT#! %LECTRONANDPROTONTRANSFER Q PLEXOFREACTIONCENTERSOFPHOTO INTHEACCEPTORQUINONECOM SYNTH T ETICBACTERIA)N$EISENHOFER* F ED 4HE0HOTOSYNTH T ETIC2E ACTION#ENTER 6OL PP n!CADEMIC0RESS .EW9ORK R 3NEL*&(ANDVAN2ENSEN**3 D  2EEVALUATIONOFT F HEROLEOF BICARB R ONATEAND FORMATE F INTH T EREGULATIONOFF PHOTOSYNTH T ETICELEC TRONmOW IN BROKENCHLOROPLASTS0LANT0HYSIOLn 3POYALOV!0 (ULSEBOSCH2* 3HOCHAT3 'AST0 (OFF!* %VIDENCETHAT!LA-ISHYDROGEN BONDEDTOTHEREDUCED PRIMARYACCEPTORQUINONE1!% n INREACTIONCENTERSOF2BSPHAER R OIDES#HEM0HYS,ETTn 3TEMLER!AND D -URPHY * $ETERMINATIONOFF THEBINDING CONSTANTOFF (#/nTOTHE0HOTOSYSTEM))COMPLEXINMAIZE CHLOROPLASTS%FFECTSO F F INHIBITORSAND LIGHT0HOTOCHEM0HO TOBIOLn D -URPHY*"  "ICARB R ONATE REVERSIBLEAND IR 3TEMLER!AND REVERSIBLEINHIBITIONOF0HOTOSYSTEM))BYMONOVALENTANIONS 0LANT0HYSIOLn 3TIEHL (( AND 7ITT (4  1UANTITATIVE TREATMENT OF THE FUNCTION OF PLASTOQUINONE IN PHOTOSYNTHESIS : .ATURFORSCH C n 3TOWELL -(" -C0HILLIPS4- 2EES$# 3OLTIS3- !BRESCH % AND D &EHER'  ,IGHT INDUCE D D STRUCTURAL CHANGESINPHO TOSYNTHETICREACTIONCENTER ˆ IMPLICATIONSFORMEC F HANISMOF ELECTRON PROTONTRANSFER3C F IENCEn 3UNDBY# #HOW73AND!NDERSON*- %FFECTSON0HOTO SYSTEM)FUNCTION PHOTOINHIBITION ANDPLANTPERFORMANCEOF THESPONTANEOUSMUTATIONOFSERINEINTHE0HOTOSYSTEM) A US REACTIONCENTER$ PROTEININTRIAZINE RESISTANT "RASSICANAP ,0LANT0HYS H IOLn 4ANG8 3 0ELOQUIN*- ,ORIGAN'! "RITT2$AND D $INER"!  4HE BINDING ENVIRONMENTOFF THEREDUCE D DPR D IMARYQU Y INONE ELECTRONACCEPTOR1! OFF 03)))N-ATHIS0ED 0HOTOSYNTHESIS &ROM,IGHTTO"IOSPHERE 6OL) PPn+LUWER!CADEMIC 0UBLISHERS $ORDRECHT 4AOKA3 +INETICSOFELECTRONTRANSFERANDBINDINGOFIN HIBITORSINTHETWO ELECTRONGATEOFCHLOROPLASTS0H$4HESIS 5NIVERSITYOF)LLINOISAT5RBANA #HAMPAIGN D F FTS!2  #OMPETITIONOFF )NHIBITORSWITH 4AOKA3AND#RO THESECONDARYQUINONEIN DARK R ADAPTED D THYLAKOID D MEMBRANES )N"IGGINS*ED 0ROGRESS IN0HOTOSYNTHESIS2ESEARCH 6OL PPn -ARTINUS.IJHOFF $R7*UNK K 0UUBLISHERS 4HE (AGUE 4AOKA3AND#ROFTS!2 4WO ELECTRONGATEINTRIAZINERE SISTANTANDSUSCEPTIBLE !MARANTHUSHYBRIDUS )N"ALTSCHEFFSKY - ED #URRENT2ESEARCHIN0HOTOSYNTHESIS 6OL) PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT D #ROFTS!2 F  +INETICS OF F THE 4AOKA 3 2OBINSON (( AND REACTIONSOFF THETWO ELECTRON GATEOFF 0HOTOSYSTEM))3TUDIES ONTHECOMPETITION BETWEEN PLASTOQU Q INONEAND INHIBITORS)N F INDJEE -URATA. 2ENGER'AND3ATO D H )NOUE9 #ROFTS!2 'OV + EDS 4HE/XYGEN%VOLVING 3YSTEMOFF 0HOTOSYNTHESIS PP n!CADEMIC0RESS 4OKYO 4IETJEN+' $RABER7 'OOSSENS* *ANSEN*2 +LUTH*& 3CHINDLER - 7ROBLOWSKY( * (ILP5AND4REBST! "INDINGOF TRIAZINESINTHE1" BINDINGNICHEOF0HOTOSYSTEM )):.ATUR FORSCH C n R CIDE 4REBST!  4HETOPOLOGY OFF THEPLASTOQUINONEAND HERBI

 BINDINGPEPTIDESOFF 0HOTOSYSTEM)) INTHETHYLAKOIDD MEMBRANE :.ATURFORSCHCn VAN 'ORKOM (*  %LECTROLUMINESCENCE 0HOTOSYNTH 2ES n VAN-IEGHEM& .ITSCHKE7 -ATHIS0AND2UTHERFORD!7 4HE INmUENCEOFF THEQU Q INONE IRONELECTRONACCEPTORCOMPL M EXON THEREACTIONCENTREPHOTOCHEMISTRYOFF 0HOTOSYSTEM))"IOCHIM "IOPHYS!CTAn R ONATEATTHEACCEPTORSIDE VAN2ENSEN**3  2OLEOF BICARB OFF 0HOTOSYSTEM))0HOTOSYNTH2ESn VAN2ENSEN**3 4ONK7*-ANDDE"RUIJN3- )NVOLVEMENT OF BICARBONATE IN THE PROTONATION OF THE SECONDARY QUINONE ELECTRONACCEPTOROF0HOTOSYSTEM))VIATHENON HAEMIRONOF Q PTORCOMPLEX&%"3,ETTn THE QUINONEIRONACCE R ONATE VAN2ENSEN**3 8U#(ANDD 'OVINDJEE  2OLEOF BICARB IN 0HOTOSYSTEM)) THEWATER PLASTOQUINONEOXIDOREDUCTASEO D F PLANTPHOTOSYNTHESIS0HYSIOL 0LANTn 6ASS ) AND 'OVINDJEE  4HERMOLUMINESCENCE FFROM THE 6 PHOTOSYNTHETICAPPARATUS0HOTOSYNTH2ESn 6ELTHUYS "2  #HARGE ACCUMULATION AND RECOMBINATION 6 INSYSTEMOFPHOTOSYNTHESIS0H$4HESIS 2IJKSUNIVERSITEIT TE,EIDEN 6ELTHUYS "2  %LECTRON DEPENDENT COMPETITION BETWEEN 6 PLASTOQUINONE AND INHIBITORS FOR F BINDING TO 0HOTOSYSTEM )) &%"3,ETTn 6 LTHUYS"2 4HE FUNCT 6E F IONOFP F LASTOQUINONEINELECTRON F )N 4RUMPOWER ", ED &UNCTION OF 1UINONES IN TRANSFER %NERGY #ONSERVING 3YSTEMS PP n!CADEMIC 0RESS .EW9ORK R 6 6ELTHUYS"2AND!MESZ* #HARGEACCUMULATIONATTHE REDUCINGSIDEOFSYSTEMOFPHOTOSYNTHESIS"IOCHM"IOPHYS !CTA  n 6 6ERMAAS7&*AN D 2UTHERFOR F D !7 %02MEASUREMENTSON F F BICARB R ONATEANDD TRIAZINERESISTANCEINTHEACCEPTOR THEEFFECTSO SIDEOFF 0HOTOSYSTEM))&%"3,ETTn 6 6ERMAAS7&* 2EN GER'AND!RNTZEN#* D  (ERBI R CIDEQUI NONE INTERACTIONS IN PHOTOSYSTEM  : .ATURFORSC F H C n 6ERNOTTE# "RIANTAIS*- !STIER# 'OVINDJEE $IFFERENTIAL 6 EFFECTSOFFORMATEINSINGLEANDDOUBLEMUTANTSOF$IN3YN ECHOCYSTIS 0##"IOCHIM"IOPHYS!CTAn

7 7BTJMJ
1FUSPVMFBT
BOE
"OUPO Z 3 $SPGUT T ETIC 7RAIGHT#!  %LECTRONACCEPTORSOF BACTERIAL PHOTOSYNTH REACTIONCENTERS"IOCHIM"IOPHYS!CTAn 7RAIGHT#! /XIDATION REDUCTIONPHYSICALCHEMISTRYOFTHE ACCEPTORQUINONECOMPLEXINBACTERIALPHOTOSYNTHETICREACTION CENTERS%VIDENCEFORANEWMODELOFHERBICIDEACTIVITY)SR* #HEMn 7RAIGHT#! -ODU D LATIONOF HERBI R CIDE BINDING BY THEREDOX STATE OF 1 AN ENDOGENOUS COMPONENT OFF 0HOTOSYSTEM )) "IOCHIM"IOPHYS!CTAn 7RAIGHT#!  0ROTONAND D ELECTRONTRANSFER F INTHEACCEPTOR Q QUINONECOMPLEXOFPHOTOSYNTHETICREACTIONCENTERSFROM 2HO D DOBACTERSPHAEROIDES &RONTIERSIN"IOSCIENCES n 8IONG* (UTCHISON23 3AYRE24 'OVINDJEE -ODIlCATION OFTHE0HOTOSYSTEM))ACCEPTORSIDEFUNCTIONINA$MUTANT ARGI R NINE  GLYCINE OF #HLAM L MYDDOMONASREINHARD RDTII "IOCHIM "IOPHYS!CTAn 8IONG* 3UB U RAMANIAM3AND D 'OVINDJEEA !KNOW K LEDGE BASED D  $ MODEL OFF THE 0HOTOSYSTEM )) REACTION CENTER OF #HLAMYD M DOMONAS REINHARD R TII 0HOTOSYNTHESIS 2ESEARCH  n 8IONG * -INAGAWA * #ROFTS ! 'OVINDJEE B ,OSS OF INHIBITION BY FORMATE IN NEWLY CONSTRUCTED 0HOTOSYSTEM )) $MUTANTS $ 2%AND$ 2- OF #HLAMYDOMONAS R  "IOCHIM "IOPHYS!CTAn RDTII H R REINHA 9 HIDA 3 /ETTMEIER 7 AND 9OS D "OGER 0  0ROCEEDINGS OF R SHOP ON #HLOROPLAST -ETABOLISM AND ITS )NTERNATIONAL 7ORK R CIDES 3AITAMA *APAN :.ATURFORSC F H )NHIBITIONBY (ERBI C n :HENG- AND$ISMUKES'# 4HE#ONFORMATIONOFTHE ISOPRENYLCHAINRELATIVETOTHESEMIQUINONEHEADINTHEPRIMARY ELECTRONACCEPTOR1! OFHIGHERPLANT03))PLASTOSEMIQUINONE DIFFERSFROMTHATINBACTERIALREACTIONCENTERSUBISEMIQUINONEOR MENASEMIQUINONE BYCA "IOCHEMISTRY n D 2UTHERFOR F D!7 D  0HOTOREDUCTANT D

:IMMERMANN * , AND INDUCED OXIDATIONOFF &E INTHEELECTRONACCEPTORCOMPLEXOF 0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn 7ITT ( 4 +ERN * &ROMME 0 +RAU” . 3AENGER 7 :OUNI! 7 AND/RTH0 #RYSTALSTRUCTUREOF0HOTOSYSTEM))FROM 3YNECHOCOCCUS ELONGATUS AT  ¯ RESOLUTION .ATURE  n

$IBQUFS
 5IF
3FEPY"DUJWF
5ZSPTJOFT
:
BOE
:% #SVDF
"
%JOFS $FOUSBM
3FTFBSDI
BOE
%FWFMPQNFOU
&YQ Y FSJNFOUBM
4UBUJPO &
*
EV
1POU
EF
/FNPVST

$P
8JMNJOHUPO
%&

64"

3
%BWJE #SJUU U %FQBSUNFOU
PG
$IFNJTUSZ SZ
6OJWFSTJUUZ PG
$BMJGPSOJB
BU
%BWJT
%BWJT
$"

64" 4VNNBSZ   * *OUSPEVDUJPO  ** $IFNJDBM
/BUVSF
PG 4JHOBM
**  "
&MFDUSPOJD
4USVDUVSF   # .O:;r
*OUFSBDUJPO   ***

1SPUPOBUJPO 4UBUFT
PG
UIF 0YJEJ[FE
BOE
3FEVDFE
'PSNT
PG
5ZSPTJOF  " 3FEVDFE 5ZSPT 5 JOF   # 0YJEJ[FE
5ZSPTJOF  *7 7 -PDBMJ[BUJPO
PG :; BOE :%   7 5IF 1SPUPO "DDFQUPS
BOE )ZESPHFO#POEJOH   7 " 4QFDUSPTDPQJD
5FDIOJRVFT   # :%   $ :;   7*

,JOFUJDT
PG :; 0YJEBUJPO
BOE
3FEVDUJPO   "
*OUBDU
.O
$MVTUFS   # .O%FQMFUFE   7**

,JOFUJDT
PG :% 0YJEBUJPO
BOE
3FEVDUJPO
BOE
$PNQBSJTPO   7*** .FDIBOJTNT GPS
:; 0YJEBUJPO
BOE 3FEVDUJPO   " 0YJEBUJPO  # 3FEVDUJPO   *9 $PODMVEJOH
3FNBSLT   "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 0HOTOSYSTEM))03)) CONTAINSTWOREDOX ACTIVETYROSINES 9$ ANDD 9: LOCATEDINHOMOLOGOUSLOCATIONSIN THEREACTIONCENTERPOLYPEPTIDES$AND D $ RESPECTIVELY4 Y HESEARETHEBESTCHARACTERIZEDD OFT F HEENZYMATIC F ION4HEREASONSFORT F HISEXTENSIVEKNOW K LEDGE INCLUDETHEABILITY REACTIONSTHATINVOLVETYROSYL RADICAL FORMAT IN03))TOMEASURETYROSINEOXIDATIONANDREDUCTIONWITHNANOSECONDTIMERESOLUTIONFROMLIQUIDHELIUMTO ROOMTEMPERATUREAND INPREPARATIONSTHATHAVE BEENMODIlED BY SITE DIRECTEDD MUTAGENESIS4HEABUNDANCE AND D PURITY OF F 03)) PREPARATIONS AND D THE RECENT ABILITY TO CRYSTALLIZE THEM ALSO ALLOWS THE APPLICATION OFF A WIDEVARIETYOFSPECTROSCOPICTECHNIQUESTOTHECHARACTERIZATION OFTHEREDOX ACTIVETYROSINES4HISCHAPTER DESCRIBESTHE DISCOVERYOFF THESEREDOXCOMPONENTS THE KINETICS ENERGETICSANDD MECHANISMOFT F HEIROXIDA TIONAND D REDUCT D IONAND DETAILED INFORMAT F IONONTHEIRSPECTROSCOPICCHARACTERISTICSWITHA PARTICULAREMPHASIS

!UTHORFORCORRESPON F DENCE EMAIL"RUCE!$INER USADUPONTCO D M

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



#SVDF " %JOFS BOE 3 %BWJE
#SJUU

ONMAGNETICRESONANCEMETHODS4HECLOSE INTERACTIONOFTYROS F INE9: WITHTHEOXYGEN EVOLVINGCOMPLEX HAS IMPLICATED9: DIRECTLYINTHEMECHANISMOFWATEROXIDATION4HEROLEOFTHISTYROSINEINTHISMECHANISMHAS BEENTHESOURCEOFF AGREATDEALOF DEBATEAND INTEREST INRECENTYEARS HEIGHTENED BY THEIMPROVING RESOLUTION OFF THE03))CRYSTALSTRUCTURE *
*OUSPEVDUJPO 4 ROSYL RADICALS HAVENOW BEEN DESCRIBED 4Y D AS INTER MEDIATESINNUMEROUSENZYMATICREACTIONS!PARTIAL LISTINCLUDESRIBONUCLEOTIDEREDUCTASE PROSTAGLANDIN SYNTHASE GALACTOSEOXIDASE CYTOCHROMEOXIDASEAS WELLAS0HOTOSYSTEM)) 03)) 3TUBB U EAND D VAN DER $ONK  4HELATTERHASTHEADVANTAGEOFBEING TRIGGERED BY LIGHT WHI W CHALLOWSTHE KINETICSOFF TYRO SINEOXIDATIONTOBEFO F LLOWED D ONTHENANOSECOND D TIME SCALEANDOVERAWIDERANGEOFTEMPERATURESFROM TO+)NADDITION THEABILITYTOREADILY OBSERVE IN03 ))REVERSIBLEOXIDATION REDUCT D IONREACTIONSOF TYROSINE WITH EXCELLENT SIGNALNOISE USING A WIDE VARIETY OFF SPECTROSCOPIC TECHNIQUES HAS PROVIDED DETAILED INFORMAT F ION ON THE MECHANISM AND D ENER GETICSOFTHISPROCESS4HISCHAPTERWILLPROVIDEAN F WHATWEHAVE LEARNED D ABOUTTHETYROSYL OVERVIEWOFW RADICALSOFF 03 )) #OMMONERETAL lRSTREPORTEDTHEOBSERVA TIONINTOBACCOCHLOROPLASTSOFF ALIGHT INDUCE D D ELECTRON PARAMAGNETICRESONANCE%02 SIGNALTHATCAMETO BE KNOWN AS 3IGNAL))4HEREWASLITTLEINDICATIONAT THETIMEOFF THECHEMICAL NATUREOFF THERADICAL THOUGH ITS GREATER LINEWIDTH n' AND ITS HYPER H lNE STRUCTUREDISTINGUISHEDITFROM3IGNAL) ACHLOROPHYLL CATIONRADICAL THATARISES FROMT F HEOXIDIZED D PRIMARY DONOR OFF 0HOTOSYSTEM ) 03) 0 7EAVER   )N EXTENSIVE STUDIES OF 3IGNAL )) "ABCOCK "LANKENSHIPAND3AUER D "AB A COCK K AND3AUER A D B A B C"LANKENSHIPETAL A B"ABCOCKK ET !BBREVIATIONS #%0n CONCERTED ELECTRONPROTON TRANSFER #7nCONTINUOUSWAVEASOPPOSEDTOPULSEDMODE$#-5n   DICHLOROPHENYL   DIMETHYLUREA%.$/2nELECTRONNU CLEARDOUBL U ERESONANCE%02nELECTRONPARAMAGNETICRESONANCE %3%%-n ELECTRON SPIN ECHO ENVELOPE MODU D LATION %3% %. $/2nPULSED D ELECTRONSPINENVELOPE%.$/2%404nELECTRON F FO F LLOWED BYPROTONTRANSFER&4)2n&OUR F IERTRANSFORM F TRANSFER INFRARE F D(!4nHYDROGENATOMTRANSFER/%#nOX F YGENEVOLV INGCOMPLEX0n03))PRIMARYELECTRONDONORCHLOROPHYLL COMPLEX03)n0HOTOSYSTEM)03))n0HOTOSYSTEM))04%4n PROTON TRANSFER FOLLOWED BY ELECTRON TRANSFER 3IGNAL))n %02 SIGNAL ATTRIBUTED TO 9$s 3IGNAL ))S AND9 9:s 3IGNAL ))F AND VF  3NnSTATESOFT F HEWATEROXIDIZINGCOMPLEXFOR F N   OR 74nWILDD TYPE9$ $ nREDOX ACTIVETYROSINEONTHE$PROTEIN $ 4Y 4 R 9: : nREDOX ACTIVETYROSINEONTHE$PROTEIN $ 4Y 4 R

AL  WEREABLETO DISTINGUISH THREEKINETICCOM PONENTS ALLOFWHICHSHOWEDSIMILAR%02SPECTRA 4HESEWERECALLEDD 3IGNAL ))S FAN F D VF FORS F LOW FAST F F IVELY ANDD WERESHOWNTOARISE ANDD VERYFAST RESPECT F FROM03))ASINDICATEDBYTHEIRINDUCTIONBYREDLIGHT ASOPPOSEDD TOTHEFAR RE F D SENSITIZEDD 03) INSPINACH F HE CHLOROPLASTSANDD THEIRSENSITIVITYTOTHESTATEOFT OXYGEN EVOLVINGCOMPLEX/%# ANDUNDERCERTAIN R CIDEINHIBITOROF03)) F CONDITIONSTO$#-5 AN HERBI F HE3 AND3 STATESOF 3IGNAL))SWASGENERATED FROMT THE/%#WITHARISETIMEOF^SATROOMTEMPERATURE ANDD WASSENSITIVETO/%#INACTIVATION"ABCOCKK AND 3AUER A  3IGNAL ))S HADD A LIFET F IME MEASURED ONTHEHOURSTIMESCALE3IGNAL))F OBSERVEDUPON INACTIVATIONOFF THE/%#EG BY4RISWASH "AB A COCK ANDD 3AUER B HADD ARISETIMEINITIALLYSHOWN TO BE  —S AND A LIFETIME OF CLOSE TO  MS RESULTING FROMC F HARGERECOMBINATIONWITHREDUCE D D ACCEPTOR 1!n INTHEPRESENCEOFF $#-54HE3IGNAL ))FLIFETIMEWASSHOWNTODEPENDUP U ONTHE PRESENCE ACCESSIBILITY ANDD REDUCT D ION POTENTIAL OF F EXOGENOUS DONORS"LANKENSHIPETAL B SHOWED BY%02 THATTHEPRECURSOR RESPONSIBLEFOR3IGNAL))F WASA DIRECTDONORTO0 WITH ARISETIME FOROX F IDATION CONSISTENTWITH THE o —SREPORTED BY'LÊSERET AL FOR0 REDUCTIONUSINGOPTICALMETHODS !DIRECTCOMPARISONOFF THERISEOFF 3IGNAL))FF AND THE  DECAYOF0 F ANDD THEIRRESPECTIVEP( DEPENDEN CIESCONlRMEDTHISCONCLUSION"OSKAETAL   7ITH BETTER TIME RESOLUTION A SIGNAL 3IGNAL ))VF WITH SIMILARLINESHAPEANDD AMPLITUDETO3IGNAL))F F COULDBEOBSERVEDUSING%02INTHEPRESENCEOFTHE /%#WITH ARISETIMEOFF —S"LANKENSHIPETAL B ANDD A DECAYTIMEOF )MSTHATDEPENDEDD ON THE3 STATEOFTHE/%#"ABCOCKETAL  4HE F IGNAL))FAN F D VF REDOXCOMPONENTS RESPONSIBLE FOR3 HADD SIMILAR%02(OGANSONANDD "ABCOCK  AND OPTICALDIFFERENCESPECTRA'ERKENETAL  AND WERETHEREFORE F LIKELY ONEANDD THESAMESPECIES THE F IMEOFW F WHICH DEPENDEDD ONTHEPRES RISETIMEAND LIFET ENCEOFTHE /%#4HEMARKEDINmUENCEOFTHE /%#ON THEPOWERSATURATIONCHARACTERISTICSOF3 F IGNAL ))F VF 7ARDENETAL 9OCUMANDD "AABCOCK  ALSO IMPLIEDTHATTHESPECIESRESPONSIBLEFORTHISSIGNALWAS LOCATED INCLOSEPROXIMITYTOTHE-NCLUSTER-UCH

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% MOREACCURATEESTIMATESOFF THEDISTANCE BETWEENTHE TWOHAVEBEENOBTAINEDMORERECENTLY0ELOQUINET AL ,AKSHMIETAL  BASED D ONTHEWEAK DIPOLAR COUPLING OBSERVED BETWEEN THE TWO UNDER CONDITIONSTHATGENERATETHE3 SPLITSIGNAL"OUSSAC ETAL A(ALLAHANETAL -AC,ACHLANAND .UGENT  SEE BELOW  4HEDONORSRESPONSIBLEFOR3IGNAL))SAND))FVF BECAME KNOWNAS K DONORS$AND D : RESPECTIVELY4 Y HE D THATTHEYAROSE SIMILARITY INTHEIR%02SPECTRA IMPLIED F FROMTHESAMECHEMICALS PECIES **
$IFNJDBM
/BUVSF
PG
4JHOBM
** 4HElRSTATTEMPTSATESTABLISHINGTHECHEMICALNATURE OFTHESEDONORSWEREDESCRIBEDINAPAPERBY+OHL D 7OOD  WH W OREPORTED D THATLIPID D EXTRACTION AND D RECONSTITUTIONOFC F HLOROPLASTFRAGMENTSW F ITH PER AND DEUTERATED PLASTOQUINONEMODIlEDTHE%02S Q PECTRUM F OF 3IGNAL))4HECONCLUSIONTHATAPLASTOQUINONE FREE RADICAL CONTRIBUTEDTO3 D IGNAL)) INmUENCED D THETHI T NKI N NG Q OFSUBSEQUENTWORKWHICHINCLUDED%02STUDIESOF MODEL QUINOIDD RADICALS+OHLETAL (ALESAND $AS'UPTA 'HANOTAKISETAL /-ALLEY AND "ABCOCK  THEORETICAL ARGUMENTS FOR A PARTICULAR LOCALIZATION OF F THE ELECTRON SPIN DENSITY D RADICALSTHATCOULDEXP D LAINTHEHYPER H lNE OFF QUINOID STRUCTURE OF THE %02 AND ELECTRON NUCLEAR DOUBLE RESONANCE %.$/2 SPECTRAOF3 F IGNAL))/-ALLEY ETAL  AND D OBSERVED D SIMILARITIESBETWEENTHE OPTICALSPECTRAFOR::ANDTHOSEKNOWNFORQUIN Q OID D RADICALS$EKKERETAL A B$INERAND D $E 6 TRY  7HILENEUTRAL ORCATIONICPLASTOQUINONE 6I RADICALSREMAINEDTHEFAVOREDASSIGNMENTBASEDON SPECTROSCOPIC DATA THERE REMAINED D SOME DOUB U T IN THATTHEPLASTOQU Q INONECONTENTOF ISOLATED D 03))CORE COMPLEXES^PERREACTIONCENTER$E6ITRYETAL  WASINSUFlCIENTTOACCOUNT FORT F HEPRESENCEOF 1! :AND D $4HEULTIMATEPROOFF THAT$WASINREALITY ATYROSINEWASPROVIDEDINAKEYPAPERBY"ARRYAND "ABCOCK  WH W OSHOWED D THAT LABELINGOF 3YN ECHOCYSTIS SP0##WITH PERDEUTERATEDTYROS D INE RESULTEDINTHELOSSOFTHEHYPERlNESTRUCTUREOF3IGNAL ))S!SNOLABELWASINCORPORATED INTOPLASTOQUINONE ITWASCLEARTHAT3IGNAL ))SWASATYROSINEFREERA F DI CAL4HESIMILARITYINTHE%02SPECTRAOFOXIDIZED$ AND D : IMPLIEDD THAT BOTH WERETYROSYLRADICALS WITH THEGISO VALUEOFF  INBOTH CASESCONSISTENTWITH THEFORMATIONOFTHENEUTRAL$s AND :s RATHERTHAN THECATIONRADICAL $s AND :s 4HETYROSINEASSIGN

 MENTWASFURTHERSTRENGTHENEDBYTHEOBSERVATIONSOF )KEUCHIAND)NOUE ANDOF4AKAHASHIAND3ATOH  OFA F LIGHT INDUCE D D IODINATIONOFF $ 4 4YR 4HE LATTER AUTHORS AS WELL AS $EBUS ET AL A AND6ERMAASETAL REMARKEDONTHEPRESENCE OF HOMOLOGOUSTYROSINESAT$ ANDD $ OF THE $ AND D $ POLYPEPTIDES RESPECTIVELY 4HESE WERE LOCATED ATTHE LUMENAL SIDE OFTHE THIRDD TRANS MEMBRANEHELIXOF BOTH POLYPEPTIDESINREGIONSOF CONSIDERABLE HOMOLOGY 3ITEDIRECTEDD REPLACEMENTOF $ 4YRWITHPHENYLALANINE$EBUSETAL A 6ERMAASETAL  RESULTED INTHE LOSSOF $s !N ANALOGOUSREPLACEMENTAT$ 4YR$EBUSETAL B-ETZETAL  RESULTEDINTHELOSSOF:s 4HEOPTICALABSORB R ANCECHANGESASSOCIATEDD WITH : D 6ITRY OXIDATION$EKKERETAL B$INERAND$E6 'ERKENETAL  WERESHOWNTOBEARASTRONG RESEMBLANCETOTHOSEASSOCIATEDD WITHTYROSINEOXIDA TIONINWATER"ENTAND(AYON  D !STRONGCASE HADD THUS BEEN ESTABLISHEDD IN FAVOR OFTHE CONCLUSION THAT BOTH $s AND :s WERETYROSYL RADICALS4HE LOSSOF THE%02HYPER H lNESTRUCTURE SEEBELOW OF :s IN 74 "OERNERAND"ARRY  ANDIN$ LESS4ANGETAL A 03))CORECOMPLEXESCONTAINING PERDEUTER ATEDD TYROSINESEALEDD THEASSIGNMENT4HEABILITYTO OBSERVE PHOTOAUTOTROPHIC GROWTH ALBEIT IMPAIRED INTHECASEOFT F HE$ 4Y 4 R0HE $ MUTATIONAND NONE FORT F HE$ 4YR0HE : MUTATION WH W ERE/ ACTIVITYWASCOMPLETELYLOST FAVOREDTHELOCATIONOF :ANDD NOT$ASTHE INTERMEDIATEREDOXCOMPONENT BETWEEN 0 ANDD THE /%# CONSISTENT WITH THE DEPENDENCE OF THE FORMATION AND DECAY KINETICS 7 TH THE OF :s ON THE OXIDATION STATE OFF THE /%#7I CLEARIDENTIlCATIONOF BOTH:ANDD $WITHTYROSINE THEYHAVECOMETOBECALLED9: ANDD 9$ RESPECTIVELY 9 BEINGTHELETTERSYMBOL FORT F YROSINE4HEEARLIER F LTERATION IN%023IGNAL ))UPON OBSERVATIONSOFANA RECONSTITUTIONWITHDEUTERATEDPLASTOQUINONE+OHL D REMAINUNEXPLAINED BUTMAY A HAVE ANDD 7OOD  BEENACONSEQUENCEOFF STRUCTURAL CHANGESASSOCIATED WITHTHERECONSTITUTIONPROCEDURETHATRESULTEDINTHE LOSSORBROADENINGOFT F HERADICAL HYPERlNESTRUCTURE !NOTHEREXAMPLEOFT F HELOSSOFT F HE HYPER H lNESTRUCTURE OF 9$ WASFOUNDBY"OUSSACAND%TIENNE IN THEIRREDOXTITRATIONOFF THISSPECIES "
&MFDUSPOJD
4USVDUVSF S %02SPECTROSCOPYHAS BEENEXTENSIVELYUSEDD TOCHAR ACTERIZETHEELECTRONICSTRUCTUREOFTYROSINERADICALS INCLUDING9$s AND9:s&IGURESHOWSTHE8 BAND%02



#SVDF " %JOFS BOE 3 %BWJE
#SJUU

&IIG  8 BAND & D CONTINUOUS WAVE #7 %02 SIGNAL OF 9$s 4 ) + INASPINACH 03)) ENRICHED ""9 PREPARATION "ERTHOLD ETAL  

SPECTRUM OF 9$s FROM SPINACH4HIS FAIRLY COMPLEX LINESHAPERESULTSFROMANINTERPLAYOFG ANISOTROPY WITHCOMPARABLEHYPERlNE INTERACTIONSWITH SEVERAL D PROTONS STRONGLYCOUPLED 6 6ARIOUSADVANCE D %02 METHODS HAVE BEEN USEFU F L F FORPRO BING FURT F HERSPECTROSCOPIC DETAILSOFF THETWO 03))TYROSINERADICALS AND FORCOMPAR F INGTHESETO TYROSINE RADICALS IN OTHER ENZYMES &OR EXAMPLE HIGH FREQUENC F YlELD D %02 i * '(Z PROVIDESA F L SPECTROSCOPICHANDLE FORT F HESETYROSINE VERYUSEFU RADICALS INTHATBYGOINGTOHIGHlELDTHE G ANISOTROPY IS GREATLY INCREASED A D LLOWINGRESOLUTIONOFT F HETHREE PRINCIPAL G MATRIXCOMPONENTS 'ULINETAL  5NETAL  &ARRARETAL $ORLETET AL (OFBAUERETAL #HAPTER "ITTLAND +AWAMORI 3PECIlCALLY AT HIGH FREQUENCY F lELD T D HE @POWDERPATTERNFEATURESASSOCIATEDWITHTHETHREE G VALUESARESPLITWELL BEYOND D THESPECTRAL BREADTH INTRODUCE D D BYTHE lELD INDEPENDENTHYPER H lNETERMS 4HISISSHOWNIN&IG4HE GY 5  AND GZZ5  VALUES FFOR TYROSINE RADICALS ARE RELATIVELY INSENSITIVETOENVIRONMENTAL EFFECTS F BUTTHE GX COM PONENT WHICHISALIGNEDALONGTHE# /BONDAXIS VARIESAPPRECIABLY 5 TO FOR F DIFFERENT F PROTEINENVIRONMENTS AND D THE PRECISEVALUEISOFTEN F TAKENASAMEASUREOFTHEDEGREEOFHYDROGENBONDING F 3ECTION6 "ECAUSETHEORIENTATIONOFT F HE G AXES IS

s &IG (IGHlELD'(Z %02SIGNALSAT+OF! 4YR OF % COLI RIBONUCLEOTIDEREDUCTASE GX " 9$s IN -N DEPLETED D 03))CORECOMPLEXESFROMT F HEMUTANT$ (IS'LN OF 3YNECHOCY CYSTIS GX  # 9:s MEASUREDIN-N DEPLETED 03))CORECOMPLEXES FROMT F HE 3YNECHOCY CYSTIS 9$ LESSMUTANT $ s D SPECTRUM FOR9 F 4 R0HE$OTTED LINEnCALCULATED 4Y : ASSUMINGA GX VALUEOFF  ADDITIONAL DETAILSPROVIDED IN5NETAL  GX $ 9$sIN-N DEPLETED03))CORECOMPLEXESFROM74 3YNECHOCYSTIS GX AND% 9$s INSPINACH03)) ENRICHED MEMBRANES GX &ROM5NETAL 

KKNOWNRELATIVETOTHETYROSINERINGMOLECULARAXES RECENT HIGH lELDD STUDIESOF 9$s USINGORIENTED03)) MEMBRANES $ORLET ET AL  OR 03)) SINGLE CRYSTALS(OFBAUERETAL  PROVIDEDETAILED IN

$IBQUFS

5Z 5 SPTJOFT
:; BOE :%

&IG  3PIN DENSITY DISTRIBUTION FOR9$s $ 4YR HYDROGEN & BONDED D TO $ (IS

FFORMATIONABOUTTHEORIENTATIONOFT F HEPHENOXYL RING OFTHE9$s RADICALRELATIVETOTHEMEMBRANENORMALAND THE03))CRYSTALLINEAXES4HEPHENOXYL RING ISTILTED F HEMEMBRANENORMAL ABOUT  FROMT ! PRECISE MEASUREMENT OF HYPERlNE COUPLINGS WITHMAGNETICNUCLEIATNATURALABUNDANCE( OR D D ( # / INTO THE 9$s SPECIlCALLY INTRODUCE s AND 9:TYROSINERADICALS PROVIDESADIRECTMEASURE OFF THEIR DISTRIBUTIONS OF F UNPAIRED D SPIN DENSITY Y ! SUMMARYOFF THESPINDENSITIES IS ILLUSTRATED IN&IG !COMPARISONWITH PARALLELEXPERIMENTALRESULTS ON OTHER PROTEIN TYROSINE RADICALS AS WELL AS ON TYROSINE RADICALS GENERATED BY 56 IRRADIATION IN FFROZEN SOLUTION (ULSEBOSCH ET AL  CAN BE MADE &OR EXAMPLE THE / HYPERlNE INTERACTION F HE%02SPECTRUMOF CAN BEDIRECTLYOBSERVED FROMT 9$s OF3YNECHOCYSTIS 03))INCORPORATING / LABELLED TYROSINE $OLEETAL  &ROMTHISMEASUREMENT THESPIN DENSITYONTHEOXYGENOFF THE9$s RADICALWAS DETERMINEDTOBE WHICHISCLOSETOTHATDETER MINED BYSIMILAR / LAB A ELLINGOFT F HE2 9 TYROSINE D (OGANSONETAL  OF RIBONUCLEOTIDEREDUCTASE ANDOFTHENEUTRALTYROSINERADICALINFROZENSOLUTION (ULSEBOSCHETAL  (ISTORICALLY MOSTO Y F HE FT

 DATALEADINGTOSPINDENSITYASSIGNMENTSWITHINTHE PHENOXYLRINGSOFTHETWOTYROSINESHAVECOMEFROM PROTON %.$/2 (OGANSON ANDD "ABCOCK  2IGBYETAL 'ILCHRISTETAL 4OMMOSET AL  ANDDEUTERONELECTRONSPINECHOENVELOPE D LATION%3%%- EXPERIMENTS 7ARNCKEETAL MODU 4OMMOSETAL  &OREXAMPLE &IGSHOWS PULSEDPROTON %.$/2FOR9$s FROMSPINACH ALONG WITH %.$/2SIMULATIONSFORT F HEDIFFERENTCOUP F LED PROTONSANDD THEIRSUM 'ILCHRISTETAL  'OOD HYPERlNEVALUESALSOCOMEFROMHIGHFREQUENCY%02 SIMULATIONS$ORLETETAL  PARTICULARLY FROM F THESINGLECRYSTAL DATA (OFBAUERETAL   4HE MOST SPECIlC ASSIGNMENTS OF MATCHING HYPERlNE FFEATURES TO SPECIlC HYDROGENS COME FFROM SPECIlC DEUTERON LABELING STUDIES 7ARNCKE ET AL  4OMMOSETAL  )TISSTRAIGHTFORWARDTORELATE THEHYDROGEN HYPERlNEVALUESTOTHESPINDENSITIES F HECARB R ONSTOWHI W CHTHEYAREBOUND -C#ONNELL OFT -C#ONNELLAND#HESNUT  !TLEASTINA BROADD SENSE THESPIN DENSITIESAROUNDD THEPHENOXYL RING ARE RELATIVELY INVARIANT OVER VARIOUS TYROSINE RADICALS INCLUDING9$sAND9:s FROMDIFFERENTORGAN ISMS!SSEEN IN&IGSANDD  LARGECOUPLINGSARE F HEANDD RINGHY H DROGENS SEE&IG OBSERVED FORT F FORTHENOMENCLATURE ANDONTHEORDEROFO F THESPIN DENSITY ISONEACHOFT F HEADJACENTCARB R ONS )N CONTRAST SMALL COUPLINGS ARE OBSERVEDD TO THE  ANDRINGHYDROGENS ANDTHESPINDENSITIESOFTHEIR R ONSAREONLYABOUTnEACH4HE# ADJACENTCARB H DROGEN CARB R ONDOESNOTHAVEA DIRECTLYBONDINGHY BUTITSLARGESPINDENSITY5n PROJECTSON R ON THETWOPROTONSOFF THEADJACENT ` METHYLENECARB INANANGULARDEPENDENT _COSe FAS F HION INSETOF &IG ANDTHUSAMEASUREMENTOFTHETWOHYPERlNE COUPLINGS FORT F HESETWOPROTONSCANDETERMINETHE W ERE e    e&OREXAMPLE FOR F ANGLES e AND e WH SPINACH9$s THE ` METHYLENEPROTON%.$/2&IG LEADSTOANASSIGNMENTOFT F HESEANGLESTO BEAPPROXI MATELY ANDD  4HESEANGLES WHI W CH DESIGNATE THERINGORIENTATION VARYAPPRECIABLYAMONGVARIOUS PROTEINTYROSINERADICALS INCLUDING 9$s AND9:s AND AREALSOSPECIES DEPENDENT"ECAUSEATLEASTONEOF THE TWO ` METHYLENEPROTONSISUSUALLYONEOFTHE STRONGESTCOUPLEDD TOTHEELECTRONSPINOFF THERADICAL D LATED BY THE #7 %02 LINESHAPESARESTRONGLYMODU THESEANGLESASWELL/FPARTICULARNOTEISARECENT %02STUDY FROMT F HE(OFF LABORATORY .IEUWENHUISET W CHEMPLOYEDD ENANTIOSELECTIVE DEUTERA AL  WHI TION OFTHE ` METHYLENEGROUPHYDROGENSOFTYROSINE INCORPORATED INTO 3Y 3 NECHOCY CYSTIS 03))&OR 3YNECHO



#SVDF " %JOFS BOE 3 %BWJE
#SJUU

&IIG 3IMULATIONOF PULSED PROTON%.$/2%3% %.$/2 FOR & F 9$sFROMSPINACH ALONGWITH%.$/2SIMULATIONSFORTHEDIFFERENT COUPLEDPROTONSAN D D THEIRSUM&ROM'ILCHRISTETAL  

CYSTIS 9$s THE PROCHIRAL(2 HYDROGENHASTHELARGEST HYPERlNECOUPLING SMALLESTDIHEDRALANGLE WHI W CH ISCONTRARYTOTHESTRONGER(3COUPLINGOBSERVEDFOR ATYROSINERADICALINFROZENSOLUTION4HISOBSERVA TIONWASTAKENASSTRONG EVIDENCEFORACONSTRA F INED F THE 9$s TYROSINE RADICAL CONSISTENT ENVIRONMENT OF WITHATIGHTDISTRIBUTIONOFTHEDIHEDRALANGLEFOR9$s D EARLIERFROMANA F LYSISOF DEUTERON%3%%REPORTED 7ARNCKEETAL  )NCONTRAST %3%%-OF ( LABELLED9s: OF-N DEPLETED3YNECHOCYSTIS 3 03))SHOWS APPRECIABLEDISORDERINTHE DIHEDRAL ANGLE4OMMOS ETAL 7ARNCKEETAL   # .O: :;r *OUFSBDUJUJPO !MAJORSOURCEOFKNOWLEDGEABOUTTHEINTERACTIONOF THE9: TYROSINEWITHTHECATALYTIC-NCLUSTERHAS BEEN

THE%02SPECTROSCOPYOFANINTERESTING RADICALSIGNAL OBSERVED IN INHIBITED D 03)) SAMPLES &OR EXAMPLE F ICLES BY.A#L%'4! CALCIUMDEPLETIONOF03))PART WASHING"OUSSACAND2UTHERFORD "OUSSACET AL A ORLOWP(CITRATETREATMENT /NO ANDD )NOUE   ELIMINATES OXYGEN EVOLV ING ACTIVITY )N SUCH #A DEPLETED 03)) PARTICLES ILLUMINATIONATATEMPERATUREOFF + LEADSTOTHE F FORMAT IONOFA F BROAD n'FU F LLWIDTH AT HALF MAXIMUM G   %02SIGNALWITHANUNUSUAL@SPLIT LINESHAPE/THERTREATMENTSTHAT BLOCK / EVOLUTION U ATION LEADD TOSIMILAR SUCH ASACETATEORmUORIDEINCUB SIGNALSUPONSUCHILLUMINATION ALTHOUGHTHESIGNAL WIDTHSVARY APPRECIABLY DEPENDING ONTHEDETAILSOF THETREATMENTANDD THERESULTINGEXTRINSICPOLYPEPTIDE COMPOSITION"AUMGARTENETAL !NDREASSON ANDD ,INDBERG "OUSSACETAL (ALLAHAN

$IBQUFS

5Z 5 SPTJOFT
:; BOE :%



&IG  #7 %02@ILLUMINATEDD MINUSANNEALED DIFFERENCES &I F PECTRUMOFF THESPLIT%02SIGNAL IN ACETATE TREATED D SPINACH03))MEMBRANES 4HESOLID LINESREPRESENTEXPERIMENTAL DATAANDD THE DOTTED LINESNUMERICALSIMULATIONS4HEEXPERIMENTAL AND D SIMULATION DETAILSARE PROVIDED IN0ELOQUINETAL 

ETAL -AC,ACHLANAND.UGENT  &IGURE SHOWSTHE#7%02SPECTRUMOFT F HESPLITSIGNAL F FORME D BYACETATEINCUB U ATION4HE mASH DEPENDENCE M LES OFTHESPLITSIGNALAMPLITUDEIN#A DEPLETEDSAMP DEMONSTRATES THAT IT ORIGINATES FFROM A DONOR SIDE F LLY EQUIVALENT TO THE 3 STATE CONlGURATION FORMA "OUSSACETAL A !LSO 56ABSORPTIONCHANGES ASSOCIATED D WITHTHE FORMAT F IONOFT F HESPLITSIGNALWERE INTERPRETED D AS FAVOR F INGANOXIDIZED HISTIDINEASTHE ORIGINFORTHISSIGNAL WITHTHEUNUSUALBREADTHAT TRIBUTED D TOAMAGNETICCOUPLINGWITH THE-NCLUSTER "OUSSAC ET AL A  4HE HISTIDINE ASSIGNMENT WASINITIALLYSUPPORTEDBY &OURIERTRANSFO F RM INFRARED &4)2 DIFFERENCE F SPECTRA CORRELATED D WITH RADICAL F FORMAT ION "ERTHOMIEU AND D "OUSSAC  BUT LATER RE EVALUATED IN SUPPORT OF9 9:s AS THE ORGANIC D WITH THE RADICAL THEAPPEARANCEOFF WHICH COINCIDED SPLITSIGNAL "ERTHOMIEUETAL  !NALTERNATE ORIGINFORTHESPLITSIGNALWASALSOPUTFORWARDBY (ALLAHANETAL  WH W OSE#7%02EXPERIMENTS WEREINTERPRETEDTOFAVOR9:s ASTHERADICALCOMPONENT OFTHEBROADSPECTRUM 'ILCHRISTETAL USEDPU D LSED%02SPECTROS D

COPYTOADDRESST D HEMOLECULARORIGINOFT F HESPLITSIGNAL IN #A DEPLETED03)))NTHIS%3% %.$/2STUDY THEPROTON%.$/2SPECTRUMOFT F HESPLITSIGNAL WAS SHOWNTOCLOSELYMATCH THESPECTRAOF BIOLOGICALTY ROSINE RADICALS SUCH AS9$s SEE&IG &IGURESHOWS THECOMPARISONOFF THE (%.$/2OFF THESPLITSIGNAL F FROM #A DEPLETEDSPINACH03))WITHTHE9 9:s SIGNAL TRAPPEDINA 9$ LESS$ 4YR0HE 3YNECHOCYSTIS 3 MUTANT"RITTETAL  4HESPECTRAL MATCH ISQUITE GOOD PROVIDINGSTRONGSUPPORTFORA9 9:s ASSIGNMENT F FORTHEORIGINOFTHESPLITSIGNAL4HEDETAILEDASSIG N MENTOFF THEDIFFERENTPROTONRESONANCESCAN F BEMADE

&IG %3% %.$/2SPECTRAINTHEPROTONRESONANCEREGIONSFOR ! 9:sIN-N DEPLETED03))PARTICLESOFTHE 3YNECHOCYSTIS9$ LESS MUTANT$ 4YR0HE" 3PLITSIGNALTRAPPEDIN#A DEPLETED SPINACH03)) ENRICHED D MEMBRANES&ROM"RITTETAL  

BYCOMPARISONWITHTHE9$s %.$/2SPECTRUMSHOWN EARLIER&IG WITH WEAKLY COUPLEDD PROTONSATTHE D  POSITIONS MORE STRONGLY COUPLEDD  AND   AND POSITIONPROTONS ANDASTRONGLYCOUPLED ` METHYLENE PROTON!S DESCRIBEDD EARLIER THE FREQUENC F IESOFT F HE ` METHY H LENEPROTONCOUPLINGSPROVIDEANACCURATE MEASUREMENTOFTHEPHENOXYRINGORIENTATIONRELA TIVETOTHE ` METHYLENE GROUP#(BONDD ANGLES AND F FROMT HEIRSIMILARFRE F QUENC Q IESWECAN INFERT F HATTHE CORRESPONDINGANGLESAREQUITESIMILARWITHIN 5   F R 3YNECHOCY FO CYSTIS 9:s AND9 9:s TRAPPED INTHE#A DE PLETEDD SPLITSIGNAL FORM F "RITTETAL  4ANGET ALB USEDTHE%3%%-PULSED%02EXPERIMENT ALONG WITH 3Y 3 NECHOCY CYSTIS 03))PARTICLESGROWNAL

 TERNATIVELYWITH DEUTERATED D ORNATURALABUNDANCE TYROSINETOSHOWTHATTHERATIOOFTHE %3%%-SPECTRA D VS OFF THESPLIT%02SIGNAL GENERATED IN DEUTERATED D SIMILARFEATURES F NATURAL ABUNDANCESAMPLESSHOWED TOTHOSEFOUNDFORTHE9 9$s TYROSINE ANDOFTHESORT PREVIOUSLYREPORTEDFOR9 9$s BY 7ARNCKEETAL  RKWASTAKENASVERY DIRECTEVIDENCETHATTHE 4HISWORK SPLITSIGNALARISESFROMATYROSINERADICAL PRESUMABLY 9:s 0ELOQUINETAL ALSOUSED HIGH FREQUENC F Y %3% %.$/2TOOBSERVETHE -NTRANSITIONSOFTHE SPLITSIGNAL ANIMPORTANTOBSERVATIONTOCONCLUSIVELY SHOWTHATTHE3 STATE-NCLUSTERISTHEOTHERSPIN PARTNERTOTHE9 9:s RADICAL 'IVENTHIS9:s 3 STATEASSIGNMENT ONECANSIMULATE THE%02$ORLETETAL ,AKSHMI ETAL  0ELOQUINETAL  AND %3% %.$/20ELOQUIN ET AL  SPECTRA TO EXTRACT MAGNETIC COUPLING PARAMETERS AND D TOMODEL ADISTANCE BETWEENTHE-N CLUSTER AND THE9 9:s RADICAL4HE ESTIMATED DISTANCE RANGEHASVARIEDINDIFFERENTPAPERSFROMDIFFERENT GROUPS&OREXAMPLE 'ILCHRISTETAL USED DA DIPOLARAPPROXIMATION FORT F HE INTERSPINCOUPLINGAND EXTRACTEDA¯DISTANCE-ORERECENTLY$ORLETETAL  AND D ,AKSHMIETAL PROVIDED D EXCELLENT SPLITSIGNAL%02SIMULATIONSWITH DOMINANTEXCHANGE INTERACTIONS0ELOQUINETAL USEDACOMMON F EXCHANGE AND DIPOLAR COUPLINGS TO SIMULATE SET OF BOTHTHEACETATE TREATED03))SP D LITSIGNAL %02AND  -N%.$/2SPECTRA4HE%02SIMULATIONDOTTED LINE ISSHOWNALONG WITH THEEXPERIMENTALTRACE IN &IG4HEDISTANCE BETWEENTHE#TYROSINECARB R ON AND D THE NEAREST-NOFTHE CLUSTER WAS ESTIMATED D TO BEBETWEENAND¯INTHISSTUDY4HERECENT Y ¯RESOLUTION8 RAYSTRUCTUREOF03))&ERREIRAET AL  SHOWSA¯DISTANCEBETWEENTHETYROSINE D THE#A OFT OXYGENAND F HE/%# SOMEWH W ATLONGER TOTHECLUSTER-NAND D CONSISTENTWITH THEPREVIOUS %02 AND%.$/2 DERIVEDDISTANCES$ORLETETAL ,AKSHMI ETAL 0ELOQUINETAL   %VIDENCEFORASHORT-NCLUSTER 9 9:s DISTANCE PRO VIDEDAMAJORLINEOFEVIDENCEFORMECHANISTICMODELS INVOLVING9 9:s DIRECTLY IN WATER OXIDATION THROUGH A D PROTONTRANSFER F 'ILCHRIST COUPLINGOFF ELECTRONAND ETAL (OGANSONETAL (OGANSONAND "ABCOCK   )T IS INTERESTING THAT A NUMBER OF TREATMENTS #L DEPLETION #A DEPLETIONAND D ACETATE T TREATMENT ALLBLOCKTHE3 A3 STATETRANSITION RESULT ING IN A TRAPPABLE 3 9 9:s CONlGURATION )T IS LIKELY THATTHEREISACOMMONORIGINOFT F HISINDUCE D D LESION INTHE+OKCYCLE6RETTOSETAL HAVEARGUED THAT DISRUPTION OF ( BONDING BETWEEN9 9:s AND D THE

#SVDF " %JOFS BOE 3 %BWJE
#SJUU -NCLUSTER POSSIBLYTHROUGHTHELOSSOFASUBSTRATE -N BOUNDWATERCOULDDISRUP U TTHIS3 STATETRANSITION )N THIS CONTEXT IT IS INTERESTING TO NOTE THE RECENT %3%%-RESULTSONACETATE TREATEDD 03))WHI W CH SHOWS THATACETATEBINDINGRESULTSINADEHYDRATIONOFTHE -NCLUSTER#LEMENSETAL   ***
1SPUPOBUJPO 4UBUFT
PG
UIF 0YJEJ[FE
BOE 3FEVDFE
'PSNT
PG
5Z 5 SPTJOF (AVINGESTABLISHEDD THATTHEREARETWOREDOXACTIVE TYROSINES IN 03)) THE QUESTION REMAINS AS TO THE NATUREOFTHEINITIALSTATEOFTHETYROSINETYROSINEOR F HEOXIDATIONPRODUCT D TYROSINATE ANDD THE lNAL STATEOFT NEUTRALORCATIONRADICAL  " 3FEVDFE 5ZSPT 5 J F JO #ANDEIASETAL COMPAREDTHEOPTICALDIFFERENCE 4 RINPULSERADIOLYSISEXPERIMENTSAT SPECTRAOFF 4YRs4Y P(ANDD TOTHOSEREPORTED INTHE LITERATURE FO F R 9:s 9: ANDD 9$s 9$ AND CONCLUDEDTH D AT BOTH OFTHESE TYROSINESWEREALWA L AYS INTHE TYROSINATE FORM F INSITU WITH ORWITHOUTTHEPRESENCEOFT F HE-NCLUSTER4HE REFERENCE DIFFERENCE SPECTRA REPORTED BY #ANDEIAS F HOWEVER MARK R EDLY FROMT F HOSE ETAL  DIFFER REPORTEDD USING 56 mASH PHOTOLYSIS BY "ENT AND (AYON ATP(PROTONATEDTYROSINE WHICH MUCH MORECLOSELYRESEMBLETHEBIOLOGICAL SPECTRA (AUMANNETAL HAVESUGGESTEDD THEPRESENCE OF A TYROSINATE IN -N INTACT 03)) AT PHYSIOLOGICAL P(BASEDONOPT D ICAL DIFFERENCESPECTRAAN F D ONTHE D ELECTRONTRANSFER F LIMITEDD RATE FORT F HEOXIDATION RAPID OF 9: BY 0 AT PHYSIOLOGICAL P( SEE BELOW  4HESESPECTRAREQUIRED D HOWEVER SUB U STANTIALCORREC TIONFORT F HEPRESENCEOF 1!n ANDD WERECOMPAREDD TO WHATWEREERRONEOUSLYATTRIBUTEDTOABSOLUTEINVITRO SPECTRA 2APPAPORTANDD ,AVERGNE  (IENERW ADEL ETAL  COMPAREDD THE9$s 9$ &4)2 DIFFERENCESPECTRUMTOTHATOFCRESOLsCRESOL.EGA TIVESIGNALSARISING FROM9 F D 03)) $ IN-N DEPLETED CORECOMPLEXESATANDD nCMn WERE ATTRIBUTED D TO THE i## AND TO THE i## RING MODESOFF THESIDECHAINOF 9$ RESPECTIVELY4 Y HESE MODESWOULD BEEXPECTEDD TOBELOCATEDD ATAND  CMn RESPECTIVELY IF9$ WEREINTHETYROSINATE FORM )N ADDITION A NEGATIVE SIGNAL AT  CM Mn WASATTRIBUTEDD TOTHE b#/( MODEOF 9$3IMILAR SIGNALSWEREOBSERVEDBY.OGUCHIETAL IN F HE-NCLUSTER4HESEOBSERVATIONS THEPRESENCEOFT

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% ARGUETHAT 9$ ISPROTONATED D AT P(INTHE PRESENCE ANDABSENCEOFTHE-NCLUSTER.EGATIVE&4)2SIG NALSATAND D CMn FORT F HE i## AND D TO i## RINGMODES RESPECTIVELY OFT F HESIDECHAIN OF 9: AND AT CMn FO F RTHE b#/( MODEINTHE 9:s 9: DIFFERENCESPECTRUMWERE F LIKEWISEATTRIBUTED TOTHEPROTONATED FORMO F F 9: AT P( IN -N DEPLETED 03))CORECOMPLEXES"ERTHOMIEUETAL  4HE OBSERVATIONOFSIMILARSIGNALSANDCM Mn F FORATYROS INECOUPLED D TOTHEOXIDATIONOFF THE/%# 33 IN03))CORECOMPLEXESCONTAININGANINTACT -NCLUSTER.OGUCHIETAL  MIGHT INDICATETHAT 9: ISINTHEPROTONATEDSTATEAT P(INTHISCASEAS WELL4HISASSIGNMENT HOWEVER ASSUMESTHATTHETYRO W SINETHATISSENSITIVETOTHEOXIDATIONOFF THE/%# IS9: 4HUS THE&4)2MEASUREMENTSINTHEABSENCEOFF THE -NCLUSTERARGUESTRONGLYTHATTHEREDUCEDTYROSINES 9$ AND9 D : AREBOTHPROTONATED D ATPHYSIOLOGICAL P(S F !SIMILARCASECAN BEMADE FOR9 $ INTHEPRESENCE OFTHE/%#(OWEVER THECASEFORPROTONATED9: IN THEPRESENCEOFF THE/%# ISWEAKERAND D NEEDSTO BE WHYTHEREAREDIFFE F R RE EXAMINED)TREMAINSUNCLEARWH ENCESBETWEENTHEMODELTYROSINESPECTRAOF#ANDEIAS D "ENTAND (AAYON  4HE LATTER ETAL AND U ORTTHECONCLUSIONARRIVEDD AT SPECTRA HOWEVER SUPP BY &4)2 7ESTPHAL ET AL  HAVE ARGUED THAT THE4Y 4 Rs4YRRn REDUCT D IONPOTENTIALOFF 6MEASURED INWATERIS LIKELYTO BELOWEREDTO6 D INSITUFOR F 9:s 9:n BECAUSEOFTHEELECTROSTATICPENALTYASSOCIATED WITHTHE FORMAT F IONOFT F HETYROSINATEINAMEDIUMOF LOWDIELECTRIC4HISPENALTYWOULDMAKE9 9:s A POOR OXIDANT WITHAREDUCTIONPOTENTIALTHATISINCONSISTENT WITHTHATOFF n6 NECESSARYTODRIVETHEOXIDA TION OFF (/ TO /4HIS THERMODYNAMIC ARGUMENT THEREFORELENDSFURTHERSUPP U ORTFORTHELIKELIHOODOF APROTONATED D STATEFOR F 9: AND 9$  # 0YJEJ[FE
5Z 5ZSPTJOF 4WOPOSSIBILITIESTHENEXISTFORT F HEPRODUCTO D F FTYROS INE OXIDATION THENEUTRALRADICALWHERETHEREISDEPROT ONATIONOFF THEPHENOLIC /(ACCOMPANYI N NGELECTRON TRANSFERORT F HE PROTONATED D RADICAL INTHEABSENCEOF DEPROTONATION4HE ZERO CROSSING POINT GISO VALUE RANGES FFROM  FFOR THE PROTONATED D RADICAL TO F HENEUTRALRADICAL $IXONAND D -URPHY H  FORT  "ARRYAND"ABCOCK POINTEDOUTTHATAS THEGISOVALUEOF9 9:s AND D 9$sISITISLIKELYTHAT9 9:s s AND9$ AREBOTHNEUTRALRADICALS&4)2MEASUREMENTS HAVE SHOWNTHATTHE 9$ AND D 9: PHENOLICPROTONSARE EXCHANGEABLE UPON REPLACEMENT OF (/ WITH $/

 (IENERWADELETAL "ERTHOMIEUETAL   4HE REPLACEMENT OF THE EXCHANGEABLE PROTON WITH A DEUTERON IS ALSO OBSERVABLE IN THE %.$/2 AND %3%%-SPECTRAOF9 9$s AND9:s 2ODRIGUEZETAL  %VELOETAL 4ANGETAL  A B$INERET AL 4OMMOSETAL  AND BY KINETICMEANS F SEEBELOW 4HE MAKINGUSEOF A KINETIC ISOTOPEEFFECT STRENGTHOFTHEDIPOLARHYPERlNECOUPLINGn -(Z ANDD THEVIRTUAL ABSENCEOFF AN ISOTROPIC HYPER lNECOMPONENTFORTHEEXCHANGEABLEPROTONIN9 9$s 2ODRIGUEZETAL %VELOETAL (OGANSON AND"ABCOCK 2IGBYETAL  AND9:s &ORCE ETAL 4OMMOSETAL 4ANGETAL A INDICATESTHESETOBEHYDROGENBONDEDNEUTRALTYROSYL RADICALS&URTHERMORE THESITE DIRECTEDREP D LACEMENT OF F THE PHENOLIC PROTON ACCEPTOR $ (IS SEE BELOW WITHAGLUTAMINE4ANGETAL  RESULTED INTHECOMPLETELOSSOFTHEHYPERlNECOUPLINGTO9 9$s D DUETOT HEEXCHANGEABLEPROTON4HESEOBSERVATIONS ALLSUPP U ORTTHEFORMATIONOFTHENEUTRALRADICALUP U ON OXIDATION OF BOTH 9: ANDD 9$ AN OBSERVATION THAT IS CONSISTENT WITH THE EXTREMELY ACIDIC P+A OFF n REPORTEDFORTHECATIONRADICAL$IXONAND-URPHY F YROSINEALSOARGUE  4HEREDOXPROPERTIESOFT F HE FORMAT FORT F IONOFT F HENEUTRALRADICAL4HEFORMA F

TIONOFTHECATIONRADICAL4YR/(nEn A 4YR/(s IS THERMODYNAMICALLY RATHER UNFAVORA F BLE (ARRIMAN 4OMMOSETAL  5SINGLITERATUREVALUES FORTHE P+AOFTHEOXIDIZEDn $IXONAND-URPHY  ANDD REDUCE D D  (ARRIMAN   4OMMOS ET AL  FFORMS OF TYROSINE ANDD THE MEASURED REDUCTIONPOTENTIALOF4YR/s4YR/n%P+ARED 6VS.(% (ARRIMAN  6 4OM MOSETAL  ONECANCALCULATE FROMT F HETHER MODYNAMICCYCLETHATTHEREDUCTIONPOTENTIALFORTHE REDOXCOUPLE4Y 4 R/(s4Y 4 R/(SHOULD BEn6 $INER  4HATTHEREDUCT D IONPOTENTIALOFF 0  IS ESTIMATEDD TO BE %|0 0 6+LIMOVAND+RAS NOVSKII  MEANSTHATTHEOXIDATIONOFF TYROSINE TOTHECATIONRADICAL BYTHEOXIDIZEDPR D IMARY DONOR WOULDBEHIGHLYUNFAVORABLEANDINCONSISTENTWITH MEASUREDD EQUILIBRIUMCONSTANT +ZP WH W ERE + ZP 9:s ( 09:0 SEEBELOW 4HETERM9:s ( ISMEANTTOINDICATETHATTHE PHENOLICPROTONRELEASED F IONOFF THENEUTRALRADICAL REMAINS BOUND UPONFORMAT TOTHEREACTIONCENTER BUTNOTTO9 9:s FORAT F LEASTTHE lRSTFEWHUNDREDMICROSECONDS 4HEABOVECALCULA TIONISBASEDD ONTHEAQUEOUSSOLUTIONPROPERTIESOF TYROSINE4HEOXIDATIONTOTHECATIONRADICAL IS LIKELY TO BE EVEN MORE UNFAVO F RABLE IN A MEDIUM OF LOW DIELECTRIC 4OMMOSETAL  

 4HEABOVEDISCUSSIONTHUS FAVORST F HELOSSOFT F HE PHENOLIC PROTONUP U ONOXIDATIONTOFORMTHENEUTRAL F BOTH 9: AND D 9$ 4Y 4 R/(nEn A 4Y 4 R/s  RADICAL FOR ( *7 -PDBMJ[BUJPO
PG :; BOE
:% 4WO GROUPS$EBUSETAL A6ERMAASETAL  CONSTRUCTED D SITE DIRECTED D MUTATIONS IN 3YNEC 3 HOCYSTIS AT$  REPLACINGTHETYROSINEWITHAPHENYLALANINE $ 4Y 4 R0HE 4HEMUTATIONRESULTED INTHECOM PLETE LOSSOFF THE %02SIGNAL ARISING FFROM 9$DESPITE T THEPRESENCEOFWILDTYPE74 LEVELSOF03))REACTION CENTERS7HILEABLETOGROWPHOTOAUTOTROPHICALLY T Y HE MUTANT DIDD SOATARATEnTIMESSLOWERTHAN744HE CONSTRUCTION OFTHE SITE DIRECTEDD MUTATION AT$  WAS FO F LLOWED D SHORTLYTHEREAFTER F BYTHECORRESPOND INGMUTATION $ 4YR0HE $EBUSETAL B -ETZETAL  4HISMUTATIONRESULTEDINTHELOSS OFTHE%02SIGNALARISINGFROM9 9:s )TALSOSLOWED D THE  REDUCT D IONOFF 0 TOTHEMILLISECOND D TIMERANGE IN 03))CORECOMPLEXESWHEREREDUCTIONOCCURREDVIA CHARGERECOMBINATIONWITH 1!n4HISOBSERVATIONIM PLIED D THE LOSSOFF RAPID D ELECTRON DONATIONTO0 A CHARACTERISTIC OF 9:4HISMUTANTWASUNABLETOGROW PHOTOAUTOTROPHICALLY CONS Y ISTENTWITHTHELOSSOFT F HE REDOXCOMPONENTLINKING0 TOTHE/%#3ATURA TION RECOVERY%02MEASUREMENTSPLACED9 9:s AND9 9$s EQUIDISTANTFROMT F HENON HEME IRONOFF THEREACTION CENTERATADISTANCEOF›¯+OULOUGLIOTISETAL   4HIS DISTANCE WAS CONSISTENT WITH PREDIC TIONS FORT F HE LOCATIONOFF $ 4 4YRAND D $ 4 4YR HOMOLOGOUSTO, !RGAND D - !RGOF 2HO D DOBACTERS SPHAEROIDES BASEDONAMAPPINGOF03)) 2UFmEETAL 3VENSSONETAL 8IONGET F HE AL  ONTOTHETHREE DIMENSIONALSTRUCTUREOFT BACTERIALREACTIONCENTERS!LLENETAL -ICHEL D $EISENHOFER % F L +ABBANIETAL  4HE AND LOCATIONOFT F HESETYROSINESHASNOW BEENCONlRMED BYTHE8 RAYCRYSTALLOGRAPHICSTRUCTUREOF03))CORE COMPLEXES FFROM THE THERMOPHILIC 3YNECHOCOCCUS ELONGATUS NOW 4H 4 ERMOSYNECHOCOCCUS ELONGATUS :OUNIETAL +AMIYAAND3HEN &ERREIRA ETAL #HAPTERSn  7
5IF
1SPUPO "DDFQUPS
BOE )ZESPHFO 7 #POEJOH !SPOINTED D OUTABOVE THEOXIDATIONOF BOTH9:AND9 D $

#SVDF " %JOFS BOE 3 %BWJE
#SJUU INTHE PRESENCEANDABSENCEOFTHE-NCLUSTEROCCURS WITHTHELIBERATIONOFAPROTON4HEREHASBEENAGROW INGAPPRECIATION INRECENTYEARSOFT F HEIMPORTANCEOF 9: DEPROTONATION UP U ONOXIDATION FORT F HEMECHANISM OF WATER OXIDATION9: OXIDATIONDEPROTONATIONALLOWS THISTYROSINETO BEA DIRECTPARTICIPANT INTHEMECHA NISMOFWATEROX F IDATION BOTH ASANELECTRONSINK FOR F THEOXIDATIONOFTHE-NCLUSTERANDASAPROTONSINK FFOR THE PROTONS OFF THE COORDINATEDD SU UBSTRATE WATER MOLECULES 'ILCHRISTETAL (OGANSONETAL  "RITT A 4OMMOS AND "ABCOCK  7 7ESTP HAL ET AL  #UKIER   3UPPORT FOR F THISVIEW HASCOME FROMANUM F BEROFSOURCES F I THE PROPOSALBY+RISHTALIK  FORTHECOUPLING F IDATIONTOTHEPROTONATIONOFA F BASEWH W OSE OFWATEROX P+AISREDOXSTATE DEPENDENT II THESOLUTIONP+A OF^FORTHEPHENOLIC /(OFTYROSINEANDOFnFOR THECATIONRADICALIII THEPHYSICAL PROXIMITYOF 9: TOTHE-NCLUSTERBASEDD ONSPINCOUPLING"OUSSAC ETAL A(ALLAHANETAL 'ILCHRISTETAL 4ANGETAL B$ORLETETAL ,AKSHMI ET AL  0ELOQUIN ET AL   IV SUGGESTED HYDROGEN BONDED CONTACT BETWEEN 9: ANDD THE -N CLUSTER BASEDD ON THE SENSITIVITY OF 9: VIBRATIONAL MODESTOTHE3 STATE.OGUCHIETAL  ANDD OF THESPINSTATEOFTHE-NCLUSTERTOTHEREDOXSTATEOF 9: 3ZALAI ETAL  AND V THESUGGESTEDD EXO THERMICITYOFF (s TRANSFERASSOC F IATEDD WITHTHEBREAKING OFTHE /n(BONDOF(/ORn/(n BOUNDTO-N AND THEFORMAT F IONOFF AN/ ( BOND INTYROSINE ,INDD ET AL "ORDWELL ANDD #HENG 'ARDNERAND -AYER 0ECORAROETAL  #ONSEQUENTLY HOWPROTON COUPLEDD ELECTRONTRANSFEROCCURS F IN03)) IS OFF CRITICAL IMPORTANCE FFOR THE UNDERSTANDING OF DONOR SIDE ELECTRON TRANSFER IN THIS PHOTOSYSTEM !SWEWILLSEEBELOW THEPROTON COUPLEDD ELECTRON TRANSFER F OCCURS MOST RAPIDLY BETWEEN A HY H DROGEN BONDEDPROTONDONORACCEPTORPAIR4HEEXPERIMENTAL OBSERVATIONSLEADING TOTH T E IDENTIlCATIONOFT F THEPROTON ACCEPTORTOTHEREDOXACTIVETYROSINESWILL BEEXPLORED INTHEPARAGRAPHSTHATFOLLOW W "
4QFDUSPTDPQJD
5FDIOJRVFT 4 4HREE INDEPENDENT SPECTROSCOPIC TECHNIQUES %. $/2ANDD %3%%- HIGH lELD D %02AND &4)2 HAVE INDICATED THAT THE TWO REDOX ACTIVE TYROSINES ARE HYDROGENBONDED INTHEIRRADICALSTATES MOST LIKELY BYTHEPROTONATED BASETHATSERVES IN ITS DEPROTON ATED FORM AS THE IMMEDIATE PROTON ACCEPTOR UUPON K L  4HE lRSTOFF THESE OXIDATION"ABCOCKETA

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% EXPERIMENTS #7 AND PULSED%.$/2AN D D %3%%- PROVIDE THE MOST DIRECT PROBE OF ( BONDING FOR TYROSINE RADICALS "RITT B  )N GENERAL THESE F HEPRESENCEOFF MAGNETIC EXPERIMENTSCANPROBE FORT NUCLEIINTHEVICINITYOFAPARAMAGNETICMOLECULE 3PECIlCALLY T Y HISCAN BEAPROTONORDEUTERON INTRO D D INTOTHEEXCHANGEABLE( BOND HY DUCE H DROGENSITE ANDHYPERlNECOUPLEDTOTHEUNPAIREDELECTRONSPIN DELOCALIZED D ONTOTHETYROSINERING SEE3ECTION))!  Q HAVEALL INDICATED D THE PRESENCEOFF A 4HESETECHNIQUES PROTONHYPERlNEINTERACTIONTHATISSENSITIVETO (( EXCHANGE2ODRIGUEZETAL %VELOETAL  4ANGETAL &ORCEETAL 4ANGETAL A $INERETAL   (IGHlELD%02CANRESOLVETHEGX COMPONENTOFF THETYROSYLRADICAL ANISOTROPIC G TEN SOR4HEVALUEOFF THISCOMPONENT ISQU Q ITESENSITIVE TOTHEORIENTATIONANDSTRENGTHOFAHYDROGENBOND

 TOTHEPHENOLICOXYGENOFT F HERADICAL AND HAS BEEN USEDD TO CHARACTERIZE HYDROGEN BONDING TO SEVERAL TYROSYLRADICALS5NETAL   &4)2CAN DETECT A i#/ VIBRATIONALMODE THEVALUEOFF WHICH ISA SENSITIVEMEASUREOFTHEHYDROGENBONDINGSTRENGTH F HETYROSYLRADICAL6ALUES TOTHEPHENOLICOXYGENOFT BETWEEN  AND  CM Mn HAVE BEEN OBSERVED FORTHE03))TYROSYLRADICALSINTHE74ANDINNON HYDRO D GEN BONDINGMUTANTS(IENERWADELETAL  "ERTHOMIEUETAL  !NIMPORTANTADVANTAGEOF Q THISTECHNIQUEISTHATITCANALSOBEUSEDTOCHARACTERIZE HYDROGENBONDING INTHEREDUCE D D STATEOFT F HETYROSINE EG b#/( BENDINGMODE SEEBELOW  7 7EHAVEHADGOODSUCCESSINOBSERVINGDEUTERONS EXCHANGED INTO THE TWO 03)) TYROSINE SITES USING PULSED(%.$/2&ORCEETAL   (%.$/2 AND %3%%- OFFERSTHEADVANTAGETHATTHEEXCHANGED

&IG  -IMS (PULSED%.$/2%3% %.$/2 FORSTARTINGFROMTHETOP 9$sIN03))ENRICHEDMEMBRANESFROMSPINACH 9$sIN-N & DEPLETED 3YNEC 3 HOCYSTIS 03))CORECOMPLEXES 9:sIN-N DEPLETED3YNEC 3 HOCYSTIS 03))CORECOMPLEXESFROMT F HE 9$ LESS$ 4YR0HE s CYSTIS 03)) CORE COMPLEXES FFROM THE $ (IS'LN MUTANT AND 9 IN % COLI RIBONUCLEOTIDE MUTANT 9$s IN -N DEPLETED 3YNECHOCY D DAPTED FROM&ORCEETA F L  REDUCTASE!

 HYDROGENS ARE OBSERVED DIRECTLY RATHER THAN AS A H SUBTRACTIONBETWEENTWOSPECTRAOBTAINEDBEFOREAND F HANGE4HETOPTWOTRACESOFF &IGSHOW AFTEREXC THE (%.$/2FOR9 9$s INSPINACH AND 3YNEC 3 HOCYSTIS 03))PREPARATIONS4HESE9 9$s ( BONDINGSIGNATURES F ISE ARERATHERSIMILAR4HE HIGHLY RESOLVED FEATURESAR F FROM DIPOLARHYPER H lNECOUPLINGAND FROMT F HEELECTRIC QUADRUPOLARCOUPLINGOFTHESPIN )   ( NUCLEUS &ROM THE THROUGH SPACE DIPOLAR COUPLING AND D THE SPINDENSITYONTHETYROSINEOXYGEN› MEASUREDFROM03))INCORPORATING / LABELEDTYRO SINE ONECANESTIMATEADISTANCEOF¯BETWEEN THEDEUTERONANDTHE9 9$s TYROSINEOXYGEN$OLEETAL  &IG  )T WAS POINTED D OUT BY $EBUS ET AL A THAT THEREEXISTINTHE HOMOLOGOUS$AND$PO D LYPEP TIDESA PAIROFCONSERVEDHISTIDINES $ (ISAND $ (IS THATARELIKELY LOCATED D NOT FAR F FROMT F HE REDOXACTIVETYROSINES4HISSUGGESTIONWASREINFORCE F D BYEFFORTSATWHATISLARGELYMOLECULARREPLACEMENT TYPEMODELING OFT F HE03))REACTIONCENTER2UFmEET AL 3VENSSONETAL 8IONGETAL   4HE ABOVE MENTIONED SPECTROSCOPIC TECHNIQUES Q AS WELLASMEASUREMENTSOFT F HERATESOFF ELECTRONTRANSFE F R TO0 HAVEIMPLICATED D THESEHISTIDINESASEITHER THE HYDROGEN BONDING PARTNERS OR AS LIKELY PROTON ACCEPTORSUPONTYROSINEOXIDATIONORBOTH #
: :% 4HE SPECTROSCOPIC EVIDENCE FOR F HY H DROGEN BONDING BETWEEN 9$ AND9 9$s AND$ (ISISQUITESTRONG D MUTATIONSAT$ (ISRESULT INSOME 3ITE DIRECTED R ATIONOFF THE8 BAND D %02SPECTRUM CASESINAPERTURB OF9 9$s 4ANGETAL 4OMMOSETAL  7HILE NOT DIRECTLY DUETOT D HE LOSSOFA F HYDROGEN BOND T D HESE F LTERATIONSINTHEORI CHANGESPROBABLYRESULT FROMA ENTATION OFTHE ` METHYLENEPROTONSRELATIVETOTHE PZ ORBI R TALOFF THE#CARB R ONOFF THEAROMATICRING AL TERINGTHEHYPER H lNECOUPLINGOFF THESEPROTONSTOTHE ELECTRONICSPIN4HEMOSTSTRUCTURALLYCONSERVATIVE LEASTPERTURB R ED D 8 BAND D %02SPECTRUM OFF THESESITE DIRECTED D CHANGES $ (IS'LN RESULTED INTHE LOSS OF A (( EXCHANGEABLEPROTONHYPERlNECOMPONENT AT-(Z 4ANG ETAL  #ONSISTENTWITH THIS OBSERVATION ISTHE LOSS INTHE ( %3% %.$/2OF 9$s INTHE03))CORECOMPLEX OFTHISMUTANT&IG SECOND D TRACEFROM F BOTTOM OFT F HEDEUTERONCOUPLING D TO THE HY H DROGEN BOND 4HIS SPECTRUM IS ASSIGNED IDENTICALTOTHATOFTHETYROSYLRADICALOF %COLI RIBO NUCLEOTIDEREDUCTASE D &IG BOTTOMTRACE WHI W CH IS

#SVDF " %JOFS BOE 3 %BWJE
#SJUU K KNOWNTO LACKK A HY H DROGENBOND "ENDERETAL  5NETAL  4HISSAMEMUTATIONRESULTEDINTHE DISPLACEMENTOFT F HEGX COMPONENTOFT F HEANISOTROPIC G TENSOROF9$s FROM F INTHE74TOIN THE$ (IS'LNMUTANT ONLYSLIGHTLYLOWERTHAN THAT OBSERVED IN THE NON HYDROGEN BONDEDD TYROSYL D  RADICAL OF %COLI RIBONUCLEOTIDEREDUCTASE &IG "ENDERETAL .ORDLUNDETAL  5NETAL   4HE POSITION OF F THE &4)2 DETECTED i#/ MODE OF A TYROSINE RADICAL IS UPSHIFTED UPON INCREASING STRENGTHOF HYDROGEN BONDING&4)2SPECTRA INDICATE THE DISPLACEMENT OFF THE i#/ MODE OF9 9$s FROM n INTHE74TOCM M INTHE$ (IS'LN MUTANT (IENERWADEL ETAL  4HELATTERISALSO VERY CLOSE TO THE i#/ SIGNAL OBSERVED AT  CMn FOR THE NON HYDROGEN BONDED TYROSYL RADICAL D BY RESONANCE OF % COLI RIBONUCLEOTIDE REDUCTASE 2AMAN SPECTROSCOPY "ACKES ET AL  !LL OF THESEOBSERVATIONSINDICATETHATTHEREPLACEMENTOF $ (ISBY'LNRESULTSINTHE LOSSOFF THE HYDROGEN BONDTOTHEPHENOLICOXYGENIN9$s "ASEDD ONTTHE i## AND i#/ STRETCHMODESANDTHE b#/( BENDING MODEDETECTED BY&4)2(IENERWADEL ETAL  ITAPPEARSTHAT9$ INTHEMUTANTREMAINSAHY H DROGEN BONDDONOR NOTTOHISTIDINEASINTHE74BUTTOTHE N OXYGENOFF THE GLUTAMINESIDECHAIN CARB R ONYL 4HE IDENTITYOFF THESPECIlC( BOND DONORS CAN BE INTERROGATED BY %.$/2 METHODS AS WELL &OR EXAMPLE &IGSHOWSASERIESOF . %3% %.$/2 EXPERIMENTSTARGETINGTHE9$s TYROSINEIN 3YNECHOCYSTIS I 03))PREPARATIONSINCORPORATINGDIFFERENT .LABEL INGS #AMPBELLETAL  4RACEA SHOWSTHE . %3% %.$/2FORAG F LOBALLY . LABELED74SAM D PLE !PAIROFFEATURESISSPLITSYMMETRICALLYABOUTTHE^ -(Z .,ARMORFREQUENC F Y DUETONON ZERO D HYPER Y lNE INTERACTIONSWITHONEORMORE . NUCLEI4RACE B SHOWSTHE%3% %.$/2FORASAMPLE . LABELLED AT ONLYTHE HISTIDINENITROGENSIDECHAINPOSITIONS)NTHIS CASEWEOBSERVEA PAIROFF SHARP ISOTROPICLINESSPLIT BY-(ZABOUTTHE .,ARMORFREQUENCY4HESE PEAKSARENOTOBSERVEDD WHENONLY THE HISTIDINE / NI TROGENIS LAB A ELED TRACEC INDICATINGTHATAHISTIDINE o NITROGENISTHEHYDROGENBONDDONOR&IG &IGURE  TRACE D SHOWSTHE .%3% %.$/2OFA F GLOBALLY  . LABELLEDD SAMPLE FFROM MUTANT $ (IS'LN )NTHISCASE THEISOTROPICHISTIDINEPEAKSAREGONE DIRECTLY DEMONSTRATINGTHAT$ (ISIS INDEEDD THE H DROGENBOND DONOR4HUSWE HAVEAVERYCLEARDE HY SCRIPTIONOFHYDROGENBONDINGFORTHE 3YNECHOCYSTI 3 S 9$s TYROSINE RADICAL WITH A STRONG HYDROGEN BOND

$IBQUFS

5Z 5 SPTJOFT
:; BOE :%



&IG -IMS.PULSED%.$/2%3% %.$/2 FOR-N DEPLETED03))CORECOMPLEXESFROM 3YNECHOCYSTIS & 3 PREPAREDFROMA 74GLOB ALLYLABELEDWITH ..A./ B AHISTIDINE TOLERANTSTRAINOF74LABELEDWITH . HISTIDINE / AND o NITROGENSLABELEDWITH . C AHISTIDINE TOLERANTSTRAINOF74LABELEDWITH . HISTIDINE/ NITROGENLABELEDWITH . D THE$ (IS'LNMUTANTGLOBALLYLABELED WITH . E THE DIFFERENCE F A n D  F THE$ 'LN'LUMUTANTGLOBALLYLABELED D WITH .&ROM#AMPBELL ETAL 

DONATEDBYTHE o NITROGENOFTHE$ (ISRESIDUE &IG  4HIS ASSIGNMENT HAS BEEN CONlRMED IN A U SHED D 8 RAYCRYSTAL STRUCTUREOF03)) F RECENTLYPUBLI &ERREIRAETAL   5SING . LABELLED D TYROSINE WE HAVEASSIGNED D THE BROAD D REMNANTFEATURES F INTHE$ (IS'LNMUTANT  . %.$/2SPECTRUMTOTHEPEPTIDENITROGENOFTHE 9$s TYROSINE +! #AMPBELL 2$ "RITT AND D "!

$INER PERSONAL COMMUNICATION  2EMARKABLY A SUB U TRACTIONOFT F THE$ (IS'LNMUTANT .%.$/2 F HECORRESPONDINGWILD D TYPESPECTRUM SPECTRUM FROMT GIVESADIFFERENCESPECTRUMTRACEE VIRTUALLYIN DISTINGUISHABLE FROMT F HEHISTIDINELABELEDSPECTRUM D TRACEB  7HILE (%.$/24ANGETAL  HIGHlELD %025NETAL  ANDD &4)2"ERTHOMIEUETAL

  ALL INDICATETHELOSSOFA F ((EXCHANGEABLE9$s HYDROGENBONDEDPROTONINTHE$ (IS'LNMUTANT 3YNECHOCY CYSTIS 03))CORECOMPLEXES SIMULATIONSOF THREE PULSETIMEDOMAIN%3%%-STUDIESAPP A EARMORE CONSISTENTWITHTWOEQUALSTRENGTHDEUTERONBONDSTO 9$s K(Z INTHEEXCHANGED D CENTERS $INERETAL  4HESEOBSERVATIONSWOULDD THEN IMPLYTHATTHE MUTATIONRESULTSINTHELOSSOFTWONEARLYEQUIVALENT HYDROGEN BONDSONLYONEOFW F WHICHCANCOME FROM F $ (IS7HATTHESOURCEOFF THESECOND HY H DROGEN BONDISANDWHYITWOULDBELOSTUPONMUTATIONAT $ AREUNCLEAR %3%%-STUDIES INSPINACH""9 MEMBRANES "ERTHOLDD ETAL  SIMILARTOTHOSE PERFORMEDIN 3YNECHOCYSTIS 3 INDICATEHOWEVERONLYA SINGLEDEUTERONBONDTO9 9$s $INERETAL   $
: :; 4HE CHARACTERIZATION OF F THE PROTON ACCEPTOR TO 9: HASBEENMORECOMPLICATEDTHANFOR9$"ECAUSE9 9:s ISRAPIDLY )MS REREDUCE D D BY 3 STATETURNOVER ITIS 3 NOTPOSSIBLETOTRAPTHE9:s RADICAL INUNINHIBITED INTACT 03)) PREPARATIONS(OWEVER WECANUSEPULSED%02 TOSTUDYTHE9:s ENVIRONMENTIN-N DEPLETED D PREPARA TIONS WHERETHE9:s RE REDUCT D IONISSLOWED4HECENTER TRACEOF&IGSHOWSTHE (%3% %.$/2FOR9 9:s IN SUCH ASAMPLE &ORCEETAL  /NEOBSERVESTHAT THEOVERALLWIDTHOFT F HE (%.$/2 FEATURE F ISABOUT THESAMEASFORTHEHYDROGEN BONDED9$s RADICALS BUT THERESOLUTIONOFS F HARP FEATURES F ISCOMPLETELYABSENT 4HE LARGE (%.$/2LINEWIDTH IS INSHARPCONTRAST TO THE CASE FO F R 9$ INMUTANT$ (IS'LNANDFOR s THETYROSYL9 RADICAL FROM F % COLI RIBONUCLEOTIDE D BOTTOM TWO TRACES OFF &IG  TYROSINE REDUCTASE RADICALSWHICH ASMENTIONEDABOVE ARENOTHYDROGEN BONDED7EINTERPRETTHE9 9:s %3% %.$/2LINEWIDTH ASSUPPORTFORHYDROGENBONDINGFORTHE9 9:s RADICAL BUTTHELACKOFRESOLVEDSPECTRALFEATURESINDICATES DISORDER IN THE HYPERlNEANDOR QUADRUPOLAR COU PLINGS WHI W CH MAY INDICATE APPRECIABLE STRUCTURAL DISORDERINTHE SITE (IGH lELD '(Z %02SHOWSTHE GX COMPONENT OFTHEANISOTROPICG TENSORFOR9:s TOBE BROADENED BUT CLOSEINVALUE TOTHATOF9$s  &IG  4HESE GX VALUESARE INCONTRASTTOWH W AT ISOBSERVED FOR9 9$s INTHE$ (IS'LNMUTANT AND %COLI RIBONUCLEOTIDEREDUCTASE NEITHEROF WHICHARE HYDROGEN BONDED #7%.$/24ANG ET AL A MEASUREMENTSALSOREmECTDISORDERINTHE HYDROGENBONDINGOF9 9:s 4HEDISORDERINTHEHYDROGEN s BONDINGOF9 9: ISLIKELYAREmECTIONOFT F HE%3%%-

#SVDF " %JOFS BOE 3 %BWJE
#SJUU DETECTED4OMMOSETAL 7ARNCKEETAL  CONFO F RMATIONAL DISORDER THAT THIS RADICAL SHOWS IN THEBONDD ANGLES BETWEENTHETYROSINERING PLANEAND THETYROSYL ` METHY H LENE# (BONDS IN -N DEPLETED 03))9 9:s WITHA i#/ MODEATCMn IS EVEN MORESTRONGLYHYDROGENBONDEDTHAN9 9$s WH W ERETHIS MODEISCMn "ERTHOMIEUETAL   4HREE PULSE%3%%-MEASUREMENTSOF9 9:s (( EXCHANGEDD -N DEPELETEDD 03))CORECOMPLEXESSHOW TWODIPOLARCOUPLED DEUTERONS$INERETAL   !GOODlTTOTHETIMEDOMAIN ((RATIOEDSPEC TRA COULD BE OBTAINEDD WITH EITHER TWO EQUIVALENTLY COUPLED DEUTERONSK(Z ORWITH TWODISSIMILAR DEUTERONSANDK(Z -OSTLIKELYTHEREISA DISTRIBUTIONOFF MULTIPLEDEUTERONSAS INTHECASEOF W CH WASSIMULATED BY SPINACH""9MEMBRANESWHI ONE STRONGLY COUPLED AND SEVERAL WEAKLY COUPLED DEUTERONS 4OMMOSETAL   !S POINTEDD OUT ABOVE THERE IS PRESENT IN THE $ POLYPEPTIDEAHISTIDINE$ (IS INALOCA TION HOMOLOGOUS TO THAT OFF $ (IS $ETAILED MEASUREMENTS OFF THE P( DEPENDENCE OFF THE RATE AND OF A DEUTERIUM ISOTOPE EFFECT ON9: OXIDATION IN-N DEPLETEDD 7403))CENTERS !HLBRINKETA K L  $INER ET AL  (AYS ET AL   INDICATEDTHATAT P( ) THE RATE OF 9: OXIDATIONBY 0 WAS LIMITED BYPROTON OR(s TRANSFER F BUTSEE BELOW 4HAT$ (ISACTSASAPROTONACCEPTORFO F R 9: SIMILARTOTHEROLEOF$ (ISINTHEOXIDATION OFF 9$ ISINDICATED BYAMARK R EDD SLOWING OFF THERATEOF OXIDATIONOF 9: BY0 UP U ONSITE DIRECTEDD REPLACE MENTOF$ (ISBYOTHERRESIDUES$INERETAL +RAMERETAL .IXONANDD $INER  2OFFEYETA F L A B#HUETAL $INERAND .IXON (AYSETAL  -AMEDOVET AL  4HESLOWING OFF THE KINETICS INTHESEMU TANTSCOULD BEINPARTREVERSED BYCHEMICAL RESCUE THEP(DEPENDENCEOFWHICHDEPENDSONTHE P+AOF THEADDEDD EXOGENOUSORGANIC BASESEG IMIDAZOLE ETHANOLAMINE (AYS ET AL    4HESE OBSERVATIONSIMPLYTHATHISTIDINEISTHEENDOGENOUS BASE INWILD D TYPE03))CENTERS Y &4)2SPECTRAOF9:s 9: "ERTHOMIEUETAL  AND9 9$s 9$ (IENERWADEL ETAL  SHOWFEATURESSIMILARTOTYROSINEINTHE PRESENCEOFF METHYL IMIDAZOLE i|A#/ ATn CM Mn AND b#/( ATnCM Mn (IENERWADEL ET AL  THATWEREATTRIBUTEDTO9: AND 9$ HYDROGEN BONDEDD TOAHISTIDINESIDECHAIN%NHANCEMENTOFA F FFEATURE AT n CMn ARISING FFROM THE iA## MODEOFTYROSINEWASSUGGESTEDTOREmECTHYDROGEN BONDINGOF9:s AND9$s BY HISTIDINE&URTHERMORE LOSS

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% OFFEATURESATCM Mn ANDCM Mn UP U ON9: AND 9$ OXIDATION RESPECTIVELY HAVE BEEN ATTRIBUTED TO # .STRETCHMODESOF HISTIDINE 7HILETH T EAB A OVEOBSERVATIONSIMPLYAC L LOSE INTERAC TION BETWEEN 9: AND$ (IS .%3% %.$/2 SPECTRAOF9 9:s IN-N DEPLETED D 03))CORECOMPLEXES SHOWNOEVIDENCEOF ISOTROPICALLYCOUPLED HISTIDINE NITROGEN#AMPBELL  ASOBSERVEDABOVEFOR9$ #AMPBELL ETAL  &IGURE FOCUSESONT F HE . %.$/2SPECTRAFOR9 9:s OF-N DEPLETED3YNECHOCY 3 YS TIS 03))PREPARATIONS#AMPBELL  4RACESA ANDB RESPECTIVELYSHOWTHEILLUMINATED9 9:s  9$s TRAPPED ANDTHEDARK9 9$s ONLY . %.$/2SPECTRA  F FORGLOBALLY . LABELLED -NDEPLETED 3YNECHOCY CYSTIS

 03))4RACEC SHOWSTHE DIFFERENCESPECTRUM W F WHICH SHOULDCORRESPONDTOTHE . %.$/2SPECTRUMOF 9:s 4HIS DIFFERENCESPECTRUM F ISVERY SIMILARTOTHE 9:s . %.$/2SPECTRUMOBTAIN INANILLUMINATED 9:s TRAPPED 9$ LESSMUTANT$ 4YR0HE SAMPLE TRACE D )N NEITHER9 9:s SPECTRUM DO WE OBSERVE THE SHARP ISOTROPICALLY COUPLED . PEAKS OF F THE SORT ASSIGNED TO THE HYDROGEN BONDED $ (IS PARTNEREDWITH9 9$s &IG TRACEE 4HEOBSERVED BROAD .%.$/2FEATURESAPPEARTOCLOSELYMATCH THETYROSINEPEPTIDENITROGENPEAKSINTHE9 9$s SPECTRA &IG TRACE D  . HISTIDINESPECIlCALLY LABELED WILD D TYPE03))PREPARATIONSREVEAL NO.%.$/2 OR%3%%-FEATURESDATANOTSHOWN PRODUCEDUPON

& &IG  .PULSED D %.$/2 %3% %.$/2 SPECTRAMEASURED IN -N DEPLETED D 03))CORECOMPLEXESFROMG F LOBALLY . LABELLED 3YN 3 ECHOCY CYSTISA SHOWS9:s  9$s TRAPPEDB ONLY9$s TRAPPEDC A nB WHICHCORRESPONDSTOTHE . %.$/2SPECTRUMOFF 9:s D 9:s IN COMPLEXESFROMT F HE GLOBALLY . LABELLED 9$ LESS$ 4Y 4 R0HE MUTANT!DAPTED FROM#AMP F BELL  

 ILLUMINATIONANDTRAPPINGOF9 9:s INCLUDINGTHESMALL -(Z SEPARATEDFEATURESSHOWNBYTHEARROWSIN &IG 4HUS WE HAVE BEEN UNABLE TO IDENTIFY ANY N HISTIDINEHYPERlNECOUPLINGSTHATWOULDSUPPORTA9:s $ (ISHYDROGEN BONDINGINTERACTIONCOMPARABLE TOTHE9$s $ (ISCASE)TISCERTAINLYPOSSIBLETHAT SUCH AN INTERACTION IS DISRUPTED BY THE -N DEPLE TION STEP NEEDED TO TRAP THE MAGNETICALLY ISOLATED 9:s RADICAL ALTHOUGHASNOTED D ABOVE EVEN FO F LLOWING -N DEPLETION THERE ARE DRAMATIC REDUCT D IONS IN THE 9:s OXIDATIONRATESIN$ (ISMUTANTSCOMPARED TO744HISOBSERVATION HASBEENTAKENASEVIDENCE THAT$ (IS ISTHEDIRECTPROTONACCEPTORINTHIS PROTON COUP U LED ELECTRON TRANSFER REACTION)NDEED INTHEMOSTHIGHLYRESOLVED D 8 RAAY CRYSTALSTRUCTURE OF03))&ERREIRAETAL  9 9:s ISREPORTEDTOBE DIRECTLY( BONDEDTO$ (IS!LTHOUGHTHEDATA DESCRIBED D ABOVEDONOTRELATEDIRECTLY TOTHE-N INTACT CASE THEY SUGGEST THE POSSIBILITY THAT $ (IS MIGHTNOTBEDIRECTLYHYDROGENBONDEDTO9 9:s IN -N DEPLETED03)) D 4HAT$ (ISISNECESSARY FORRAP F ID OXIDATION OF 9: AT ROOM TEMPERATURE EVEN IN THE -N DEPLETEDSAMPLES COULDIMPLYANINTERMEDIATE HYDROGEN BONDED D CHAIN BETWEEN 9: AND D $ (IS AGATEDINTERACTIONBETWEEN9 9:s AND D $ (IS ORA DISCONNECTBETWEENTHETWOATTHELOWTEMPERATURE OFF THE%3% %.$/2EXPERIMENTS(OWEVER ARECENT DENSITYFUNCT F IONAL THEORETICALANALYSIS -"RYNDA PERSONALCOMMUNICATION OFTHE4YRs (IS ( BONDED PAIRINDICATESTHATTHESTRENGTH OFF THEISOTROPIC . (IS COUPLING DEPENDSONTHERELATIVEORIENTATIONSOF THE4YRAND(ISRINGPLANES&ORCERTAINORIENTATIONS THISCOUPLING IS LOSTCOMPLETELY4 Y HUS WESHOULD D NOT RULEOUTTHEPOSSIBILITYTHAT9:s MAY INDEED BE DIRECTLY ( BONDEDBY$ (ISINTHE-N DEPLETEDCASE BUT THATTHEDIRECT( BONDING MAY A INSOMECASESBEMORE DIFlCULTTO DETECTBY .%3% %.$/2 7*
,JOFUJDT
PG :; 0YJEBUJPO
BOE
3FEVDUJPO "
*OUBDU
.O
$MVTUFS 4HE RATE OF OXIDATION OF9 F : BY0 IN 03 ))CORECOM PLEXESCONTAININGANINTACT-NCLUSTERISMULTIPHASIC WITH THMAAJORSUB U MICROSECOND D COMPONENTSTš OFF n D n NS FO F RTHE NSFORSTATES3 AND3 AND  AND STATES 3 AND3 D  RESPECTIVELY -AUZERALLAND D -ALLEY Y  "RETTEL ET AL  3CHLODDER ET AL  -EYERETAL +ARGEETAL (AUMANNET K ETAL 3CHILSTRAETAL   AL !HLBRINK

#SVDF " %JOFS BOE 3 %BWJE
#SJUU 4HESLOWING INSTATES 33 RELATIVETO33 HASBEEN ATTRIBUTEDTOTHEACCUMULATIONOFAPOSITIVECHARGE ONTHE-NCLUSTERANDD A LOWERING OFF THE9:0 W9 9:s ( 0EQUILIBRIUMCONSTANT + +ZZP "RETTELET AL  RESULTING FROM A LACK OF PROTON RELEASE ONTHE3A3 TRANSITIONOFF THE/%# &OWLER  F D ,AVERGNE 3APHONANDD #ROFTS 2APPAPORTAN ,AVERGNEAND*UNGE  ORTOASTRUCTURAL CHANGEASSOCIATEDD WITH THE FORMAT F IONOF 3 #HRISTEN AND2ENGER  4HEEQUILIBRIUMCONSTANTS+ +ZZP IN THESUBMICROSECONDTIMEDOMAINFOR3 AND3 FOR3 F D  "RETTELETAL  INCREASEUPON  AND3 RELAXATIONATLONGERTIMES"OUGES "OCQUET  3CHLODDERETAL 3HINKAREVAND7RAIGHT  BUTREMAINLOWERINTHE3AND 3 STATES4HUSTHERE ARECOMPONENTSOF 9: OXIDATIONINTHEMICROSECOND RANGEn—S THATOSCILLATEWITHPERIODFOUR THE MOSTMARK R EDD OFW F WHICHARE INTHE 3 ANDD 3 STATES AS F HEDEQUENCHING SHOWNBY INDIRECTMEASUREMENTSOFT OF THE mUORESCENCE YIELD IN THE MICROSECOND TIME RANGE #HRISTEN ET AL  $ELOSME ANDD *OLIOT F  REDU D C  AND BYDIRECTMEASUREMENTSOF0 TION3CHLODDERETAL 3CHILSTRAETAL  #HRISTENANDD 2ENGER  4HESELOWEQUILIBRIUM CONSTANTS RESULT IN KINETIC COMPONENTS ASSOCIATED WITH9:s REDUCTIONBYTHE-NCLUSTERSEEBELOW TOBE  D IONOF0 F 3CHLOD VISIBLE INTHE KINETICSOFF REDUCT DERETAL  01!nCHARGERECOMBINATIONANDTHE NON NEGLIGIBLEEQUILIBRIUMCONCENTRATIONSOF0 AND 1!n PARTICULARLY UPONEXCITATIONINTHE3 AND3 D  STATES ACCOUNTFORT F HEMISSESOBSERVED INTHEPROGRES SIONOFTHE+OK *OLIOTCYCLEOFTHE/%#3CHLODDER ETAL 3HINKAREVANDD 7RAIGHT   4HESUB U MICROSECONDD COM MPONENTSFORT F HEOXIDATION OF 9: INEACH3STATESHOWONLYAWEAKP(DEPENDENCE #HRISTENETAL  VERYSMALL DEUTERIUMISOTOPE F ) ANDD SMALL ACTIVATION ENERGIES ^ EFFECTS K*MOL %CKERT AND 2ENGER   #OMPONENTS INTHEn —STIMERANGEAREMOSTENHANCED BYTHE REPLACEMENTOFF (/ WITH$/ INTHE3 AND LESSSO INTHE3 STATE3CHILSTRAETAL  #HRISTENAND 2ENGER OBSERVEDD THEENHANCEMENTOFF A —S COMPONENTINTHE3 AND 3 STATES LESSSO INTHE3 AND 3 STATEUPON($EXCHANGE4HEYREPORTEDTHATSLOWER COMPONENTSANDD  —S AREMOREAFFECTE F D INTHE 3 AND 3 STATESTHANIN 3 AND 3!CONSEQUENCEO Q F THESLOWINGOF0 REDUCTIONBYSOLVENTREPLACE MENTISANENHANCEDD CHARGERECOMBINATIONBETWEEN 1!n ANDD 0 RESULTINGINANINCREASEINTHEMISS PARAMETER _ OFTHE+OK *OLIOTCYCLE#HRISTENAND 2ENGER  4HEMICROSECOND KINETICCOMPONENTS

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% THUSA H APPEARTOBECOUPL U ED D TOINTERNAL PROTONMOVEMENT ASPROTONRELEASETOTHEBULKPHASETAKESLONGER OC CURRING INn —S!HLBRINK K ETAL 3CHILSTRA ETAL #HRISTENAND D 2ENGER  )NACONTRARY OPINION HOWEVER 4OMMOSAND"ABCOCK HAVE D THAT@DOMINODEPROTONATIONCOULD D REACH SUGGESTED THE BULK K PHASE RAPIDLY ENOUGH TO ACCOUNT FFOR THE MICROSECONDPHASES2APPAPORTAND,AVERGNE HAVEPROVIDED D ADETAILEDCOMPAR D ISONOFF THESEMODELS AND D THEIRCORRESPONDINGENERGETICS4HEYPOINTOUT THESIGNIlCANTINmUENCEOFTHEELECTROSTATICINTERAC TION BETWEEN 9:/n AND(IS( ANDPOSS D IBLY OFF 0 ITSELFONT F HE FORMAT F IONOFF TYROSINATE INAPROTONlRST MECHANISM FO F R THE OXIDATION OF 9:4HEY CAUTION HOWEVER THATONE GENERALLY ASSUMESTHATTHE03)) CENTERSAREPARTICIPATINGINAHOMOGENEOUSPROCESS W WHERETHEREISNOINTRINSICHETEROGENEITYBETWEENTHE F HEMULTIPHASICKINETICRELAX CENTERSTOACCOUNTFORT ATION ANASSUMPTIONTHATMAYNOT BECORRECT 4HE RATE OF REDUCTION OF 9: BYTHEINTACT-NCLUSTER R ED DEPENDENCEON3STATE WITH LIFET F IMES SHOWSAMARK F F R OFF TO—SREPORTED FOR3  A 3 n—S FO 3 A 3 n—SFOR3A3 ANDnMSFOR3 A 3 2APPAPORTETAL $INERAND D "ABCOCK F D ET AL  AND REFERENCES F  2AZEGHIFAR THEREIN 4HESERATESSHOWTHEFOLLOWINGDEUTERIUM KINETIC ISOTOPE EFFECTS F AND ACTIVATION ENERGIES IN PARENTHESES n K*MOL FOR F 3 A 3 n K*MOL FOR3 A 3nK*MOL FOR 3 A 3 AND D   K*MOL FOR3 F  A 3 A 3 (AUMANNETAL +ARGEETAL 7ESTPHAL ETAL  /FTHE3 STATETRANSITIONS 3 A 3 SHOWS THEMOSTMARK R EDP( D DEPENDENCE 2APPAPORTETAL F F  BETWEEN (AUMANNETAL  FACTORO P(—S AND—S ANDISOTOPEEFFECTON BOTHTHERATEAND D THEACTIVATIONENERGY K*MOL IN $/ IMPLYINGTHATTHISSTEP ISTHEONEMOSTLIMITED BYPROTONTRANSFER #
.O%FQMFUFE 5PON LOSSOFT F HE-NCLUSTER THERATEOFF 9: OXIDA TION BY0 ISMARK R EDLYSLOWED D DESPITETHELARGE Q 9:0 INCREASEINTHE +ZP EQUILIBRIUMCONSTANTFOR  9:s ( 0 n FFOR THE P( RANGE n 9ERK R ESETAL "USERETAL  #ONJ N EAUD AND-ATHIS REPORTEDRATESRANGINGFROM—S ATP(TO—SATP(4HISRATEWASEXAMINED IN MOREDETAIL BYANUMBEROFWOR F RKERSUSING03))CORE COMPLEXESFROMPEA!HLBRINKETAL  ANDFROM 3YNECHOCY CYSTIS $INERETAL (AY A SETAL 

 ANDD SHOWNTO BEMULTIPHASICWITH THE DATAlTBYUP TOFOURCOMPONENTSRANGINGFROM oNSTO—S (AY A SETAL  4HEFASTESTCOMPONENTSREPORTE F D BY!HLBRINKETAL AND(AYSETAL o    —SANDNS RESPECTIVELY SHOWEDP( INDEPENDENT RATES THEAMPLITUDESOFF WHICH WEREP( DEPENDENT P+ASOFAN F D  RESPECTIVELY !SIMILARP+A WAS DETECTED INDIRECTLY BY -AMEDOV ET AL  THROUGH MEASUREMENTSOF mUORESCENCEINDUCT D IONIN -N DEPLETEDD THY H LAKOIDSOF #HL # AMYDOMONAS D $INER ETAL WITH MICROSECONDTIMERESOLUTION BUT USINGAMUTANTLACKING 9$ $ 4Y 4 R0HE AND lT TINGTHEDATAWITH TWORATECONSTANTSALLOWEDD TOVARY WITHP( PLUSANOFFSET FOUNDASLIGHTLYHIGHERP+A F HLBRINKETA K L  ANDD (AAYSETAL  OF! ASSUMEDD THATONEORMOREOFT F HECOMPONENTREACTION RATESWERE P( INDEPENDENTWITHTHECOMPONENTAM PLITUDESP( DEPENDENT!HLBRINK  ANDD $INERET RK OF#ON F NJEAUD AL  ASSUMEDD ASINTHEEARLIERWORK AND-ATHIS THATTHEMICROSECONDCOMPONENT RATESWEREP( DEPENDENT4HESEREmECTSLOWAND FAS F T PROTONEQUILIBRATION RESPECTIVELY WITHRESPECTTOTHE RATEOFELECTRONTRANSFERANDARENOTEASYTODISTIN GUISH FOR F DISCUSSION SEE2APPAPORTANDD ,AVERGNE F F  !LLTHREEGROUPSALSOEXAMINEDD THEEFFECTO ($EXCHANGEONTHERATEOF9: OXIDATION AND AS A F FUNCT IONOFF P()SOTOPEEFFECTSO F F BETWEENANDD  F DOMINATINGATP() WEREREPORTED FORCOMPONENTS "OTH!HLBRINKETAL AND$INERETAL F D THATTH FOUN T EDEUTERIUMISOTOPE DISAPPEAREDATP( D K(K$ ) WHIL W ETHE FASTESTNSCOMPONENTO F F (AYSETAL WHICHCONTRIBUTES *INTHIS F P(RANGE WASREPORTEDD TOSTILLSHOWANISOTOPEEFFECT K ET AL  ALSO REPORTED OF K(K$!HLBRINK ANACTIVATIONENERGYOFE6FORTHEMAJORSLOW KINETICCOMPONENTWITH AP( DEPENDENT Tš OFF  —S ATP(! FAST F KINETICCOMPONENTWITH ALARGELYP( INDEPENDENT Tš OF  —S THAT DOMINATES ABOVE P( F  E64WO POSSIBLE HAS AN ACTIVATION ENERGY OF F HE BEHAVIORINTHEABSENCEOF MODELS&IG FORT THE-NCLUSTER!HLBRINKETAL  ATTRIBUTETHE F HEPROTON P+ASMENTIONEDD ABOVETOEITHERTHATOFT ACCEPTOREG $ (IS IMIDAZOLIUMIMIDAZOLE ORTO 9: ITSELF4YR/(4YR/n )NBOTHMODELS 9 9:s IS THENEUTRALRADICAL)NTHElRSTCASE THEPROTONACCEP TOR (IS ABOVEITSP+AIS( BONDED BY9: ANDD ALLOWS RAPIDDEPROTONATIONOF9 9: COUP U LEDTOITSOXIDATION4HE PHENOLICPROTONTRANSFERTOT F HEPROTONACCEPTORISFAS F T AND 9: OXIDATIONISELECTRONTRANSFER F LIMITED"ELOW THEP+A 9: CANNOTDEPROTONATETO(IS( WHICHIS D FORC F INGTHEPROTONTRANSFERTOA F ALREADY PROTONATED

#SVDF " %JOFS BOE 3 %BWJE
#SJUU



& &IG 4WOPOSSIBLEMODELSFORTHEOXIDATIONOF9: BY0 INTHEABSENCEOFTHE-NCLUSTER3EETEXTFORDETAILS

LESS FAVORA F BLE LOWERP+AORMORE DISTANT ACCEPTOR "n ORTO(IS FO F LLOWINGTHEDEPROTONATIONOFF (IS( 4HERATEISSLOWER WITHAHIGHERACTIVATIONENERGYAND P( DEPENDENT POSSIBLY THROUGH ACHOICEOFF SEVERAL LESS FAVORA F BLEORSECONDARYPROTONACCEPTORS)NTHE -N INTACT CENTERS THE P+A OF THE PROTON ACCEPTOR WOULD BEWHIL W EINTHE-N DEPLETED D CENTERSTHE P+AWOULD BE)NTHESECONDCASE T D HE FORMAT F ION OFTHETYROSINATEABOVETHEP+ARESULTSINOXIDATION F AS NO PROTON TRANSFER F IS VIA PURE ELECTRON TRANSFER NECESSARY"ELOWTHEP+A THETYROSINEMUSTGIVEUP ITSPROTONTOTHEACCEPTOR RESULTINGINADEUTERIUM ISOTOPEEFFECTAN F D ANELEVATED D ACTIVATIONENERGY R Y)NTTHIS MODEL THEP+AOF 9:9:n WOULDBEIN-N INTACT CENTERSANDIN-N DEPLETEDCENTERS4HEMAJOR SLOW P( DEPENDENTCOMPONENTOBSERVED BELOWTHE P+AINTHE-N DEPLETED D CENTERSCOULD D ARISEFROMT F HE LOWERP+AVALUESOFTHESECONDARYPROTONACCEPTORS HYDROGEN BONDED D TO THE PRIMARY PROTON ACCEPTOR

/NEMAJ A ORDIFlCULTYWITH THISMODEL ISTHEP+A) F HE-N INTACTCENTERS4HISISUNUSUALLY LOW FO FORT F R ATYROSINE THEP+AOFWHICHINAQUEOUSSOLUTIONIS NORMALLY(OWEVER THEPRESENCEOFF NEARRBY-N D POTENTIALLY LOWER THE TYROSINE P+A OR #A COULD !NUMBEROFAUTHORSHAVEARGUEDTHAT9: ISINTHE TYROSINATESTATEIN-N INTACTCENTERS #ANDEIASETAL (AUMANNETAL   (AYSETAL HAVESHOWNTHATIN-N DEPLETED 03))CORECOMPLEXESOFT F THE$ (IS!LAMUTANT KI NETICCOMP M ONENTS FOR F 9: OXIDATIONTš—SCONSTITUTE  OFTHEOXIDATIONOF9: ATP( )(OWEVER AT P(ANEWRAPIDCOMPONENT D Tš—S APPEARS TH ANAAPPROXIMATEP+AOF W F WHICHH THESEAUTH T ORS WITH ATTRIBUTE TO THATOF 9:9 9:n 4HE ADDITION OFTHE BASES IMIDAZOLEANDD ETTHANOLAMINETOCORECOMPLEXESOFT F THIS D ID KINETICCOMPONENTS Tš MUTANT INTRODUCESRAP —S WITHP+ASOFAND RESPECTIVELY%ACHOF THESEP+ASCORRESPONDSTOTHEP+AOFT F HERESPECTIVE

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% BASE INAQUEOUSSOLUTION4HE INTERPRETATIONOFF (AYS ETAL FORTHESEOBSERVATIONSISTHATTHEP+A THAT GOVERNSTHEAPPEARANCEOFF THERAPIDCOMPONEN D T F 9: OXIDATION ISTHATOFT FOR F HEBASEWHI W CHACTSASTHE PROTON ACCEPTOR TO 9: $ (IS IN THE 74 AND THEEXOGENOUSBASESINTHE$ (IS!LAMUTANT CONSISTENTWITH -ODEL ABOVE !SMENTIONEDABOVE %3% %.$/2SPECTRAOF. LABELED D -NDEPLETED D 03))CORESSHOWED D EVIDENCE FO F R ANISOTROPICALLYCOUPLED HISTIDINENITROGEN INTHECASE OF9 9$s INDICATIVEOFADIRECT( BONDBETWEEN9 9$s AND $ (IS #AMPBELL ET AL   .O SUCH COU s PLINGISOBSERVED FOR9 F : IN-N DEPLETED03))CORES IMPLYINGTHAT 9: AND$ (ISARENOTDIRECTLY ( BONDED4HESEMAY A BE LINKED BY A HYDROGENBONDED CHAIN (AYSETAL  TOEXPLAINTHEDEPENDENCE OFTHE RATE OF 9: OXIDATION ON $ (IS!LTERNA TIVELY T Y HETWOMAY A BELINKED INTERMITTENTLY ATROOM TEMPERATURE GATED D MECHANISM $INERETAL  SUCHTHAT$ (IS FAC F ILITATES9: DEPROTONATIONBUT WHERETHESTATE FROZEN F INATTHE LOWTEMPERATUREOF THE%3% %.$/2MEASUREMENTSHOWSNOSUB U STANTIAL PRESENCEOFALINKAGEBETWEENTHETWO 7** ,JOFUJDT PG :% 0YJEBUJPO
BOE
3FEVDUJPO BOE
$PNQBSJTPO 'IVENTHAT9$ ISLOCATEDINAPOSITIONSYMMETRICWITH RESPECTTO9: INTHEREACTIONCENTERAND D THAT9$ FORMS F A WELL DElNED HY H DROGEN BOND D TO $ (IS IT IS SURPRISINGTHAT9$ DOESNOTSHOW UNDERPHYSIOLOGICAL F 4HE CONDITIONS ASRAPIDD ELECTRONTRANSFERTO0 IMPORTANCEOF 9$ TOTHE FUNCT F IONOFF 03)) IS HOWEVER DEMONSTRATEDBYITSROLEINTHEASSEMBLYOFTHE-N N EVETAL  AND D THE IMPAIRMENT CLUSTER!NANY OFF PHOTOSYNTHETICGROWTH 6ERMAASETAL  IN THE$ 4YR0HEMUTANT!GLOBALKINETICSTUDYOF ELECTRONTRANSFER F IN03)) "USERETAL  ATTRIBUTED F HEOXIDATIONOF 9$ BY0 A Tš OFnMSTOT BETWEENP(AND2ECENTLY THEOXIDATIONOF9$ WASRE EXAMINED INA9: LESSMUTANTOF 3YNECHOCY CYSTIS $ 4YR0HE &ALLERETAL  FOUN F D THATA FAST F COMPONENTOF9$ OXIDATIONTš NS APPEARED WITH INCREASINGP( SHOWING AP+AOF4 F HISP+A D 3TYRING AGREESWITHTHATPROPOSED BY6ASSAND F FORTHE P( DEPENDENCEOFTHERATEOFOXIDATIONOF9$ BY THE 3 STATE P+An  &ALLER ET AL  ALSOFOUN F D ASIMILARP+A  FORT F HEOXIDATIONOF 9$ ATCRYOGENICTEMPERATURESn+ 4HISABILITY TOOXIDIZE 9$ AT LOWTEMPERATURECONTRASTSWITHTHE

 OXIDATIONOF9: WHI W CH CANNOLONGEROCCURAT )  #IN-N DEPLETED03))+UEHNEAND"RUDVIG   F 9: TWOMODELS FORT F HEORIGINOFF THEP+A !SABOVE FOR D INVOLVINGEITHERTHE(IS(ISORTHE WEREPROPOSED 9$ 9$n ACID BASECOUPLES&ALLERETAL  )NTHE lRSTCASE THE9 9$s (IS COUPLEWOULDD CARRYAPOSITIVE CHARGEONTHE$ (ISUNLESSTHEDISTAL PROTONOF THEIMIDAZOLERINGWERETRANSFERREDTOASECONDARY PROTONACCEPTOR/-ALLEY  HASMADEPREDIC TIONS FROM F H BRID DENSITY FUNCT HY F IONAL CALCULATIONSOF PHENOLIC( BONDDISTANCESANDRELATIVESPINDENSITIES ONTHEPHENOLICOXYGEN THE#RINGCARB R ONANDD THE ( BONDED o IMIDAZOLE NITROGEN4HESE PARAMETERS DIFFE F R FO F R THE IMIDAZOLE ANDD THE IMIDAZOLIUM ( BONDEDD TOTHE4YR 4 s4HOSEMEASUREDFOR9$s &IG LIE BETWEENTHETWO BUTCLOSERTOTHENEUTRAL IMIDAZOLE IMPLYINGTHEPRESENCEOFASECONDARYPROTONACCEPTOR IN( BONDEDD CONTACTWITH THE / IMIDAZOLENITROGEN OFF $ (ISABLETOATLEASTPARTIALLY DISSIPATETHE CHARGEONTHEIMIDAZOLEBYPROTONTRANSFER$INER   !NUMBEROF QUEST Q IONSSTILLREMAINUNANSWERED 7HY IS THE OXIDATION OF9: SO MUCH FAS F TER IN THE -N INTACT VERSUS THE -N DEPLETEDD CENTERS DESPITE F F THEREACTIONFO F L THEINCREASE INTHEDRIVING FORCEO LOWINGLOSSOFTHE/%#7HY BELOWTHEP+A THERATE F IDATIONOF 9: ISSOMUCH FASTERT F HANTHATOFF 9$ OFOX DESPITETHE LARGERDRIVING FORCE F F HE LATTER7HY FORT H IS THEREDUCTIONPOTENTIALOF9 9$s 9$ SO MUC M H LESSTHANTH A AT OF9 9:s 9: IN-N INTACTANDD -N DEPLETEDD CENTERS 4HESLOWINGOFT F HEOXIDATIONOF 9: UP U ONDEPLE TIONOF-NCOULDBETHECONSEQUENCEOFTHELOSSOFA Q WELL STRUCTUREDHYDROGENBONDEDLINKBETWEEN9: AND $ (IS7ESTPHALETAL  4HELATEST 8 RAY CRYSTALSTRUCTUREOF03))FROM4HERMOSYNECHOCOCCUS ELONGATUS G &ERREIRAETAL  PLACES$ (IS CLOSETO9: INTHE-N INTACTCENTERS4HE INABILITYTO DETECTANISOTROPICALLYCOUPLED(ISo NITROGENINTHE %3% %.$/2SPECTRUMOF9 9:s IN-N DEPLETED03)) D H DRO D CORES #AMPBELL  ANDD THE DISORDERINTHE HY GENBONDINGTOTHEPHENOLOXYGEN&ORCEETAL  ANDINTHEORIENTATIONOFTHERINGOF9:s 4OMMOSETAL F WHICHCONTRAST 7ARNCKEETAL  BOTH OFW WITH9$ (OGANSONAND"ABCOCK  COULDEX PLAINTHESLOWING OF9 F : OXIDATIONBY AN IMPAIRMENTOF F BCOCKETA K L  HAVEPROPOSED PROTONTRANSFER"A THATTHE REASON FFORTHE SLOW OXIDATION OF 9$ RELATIVE TO9: MIGHT HAVETO DOWITH A LARGERREORGANIZATION ENERGYASSOCIATEDD WITHTHERETENTIONOFF THEPHENOLIC PROTONCLOSEBY9 9$s 7HILETHISPROPOSALWASINVOKED TOSUPPORTTHERELEASEOFF THEPHENOLICPROTONOFF 9:

#SVDF " %JOFS BOE 3 %BWJE
#SJUU

 TOTHE BULK K PHASE IT ISPOSSIBLETHATTHEGREATERPO LARIZABLITY OF THE ENVIRONMENT OF9: FAC F ILITATES THE D LUSCHARGE4HEABILITYTO STABILIZATIONOFF ARETAINEDP OBSERVESUCH A LARGESTIMULATION INRATEASSOCIATED WITHTHE OXIDATION OF 9$ ATALKALINEP(&ALLERETAL  MIGHTALSOREmECTANINCREASED HYDROPHILICITY POLARIZABILITY INTHEENVIRONMENTOFF THISTYROSINEAS THEP(INCREASES4HEREDUCTIONPOTENTIALOF9:s 9: AT D "ABCOCK  ISCLOSETOWH W ATONE ^6 $INERAND WOULDEXPECT D F FORTYROS INEINAQUEOUSSOLUTION7HAT REMAINSAPUZZLEISWHYTHEREDUCTIONPOTENTIALOF 9$s 9$ ISSO LOWn6 "OUSSACAND D %TIENNE D ING  0LACINGTHETYROSINE 6ASSAND3TYR IN A HYDROPHOBIC ENVIRONMENT WOULD BE EXPECTED TO EXERT THE OPPOSITE EFFECT F ON REDUCT D ION POTENTIAL 4OMMOSETAL   7***
.FDIBOJTNT
GPS
:; 0YJEBUJPO
BOE 3FEVDUJPO "
0YJEBUJPO 4HEREARETHREEPOSSIBLEMECHANISMS BY WHI W CH THE F TYROSINE MIGHT OCCUR TO PRODUCE D THE OXIDATION OF NEUTRAL RADICAL $INER ET AL  2APPAPORT AND ,AVERGNE 3J 3 ÚDINETAL  4HESEARE I ELECTRON TRANSFER F PRODUC D ING 4YR/(s FO F LLOWED BY PROTONTRANSFER%404 II PROTONTRANSFERPRODUCING 4 R/n FOLLOWEDBYELECTRONTRANSFER04%4 ANDIII 4Y CONCERTED D ELECTRONANDPROTONTRANS D F #%0 WITH FER BOTHREACTIONSOCCURRINGSIMULTANEOUSLY THROUGHA COMMON TRANSITION STATE4HESE REACTIONS HAVE RE CENTLY BEEN DISCUSSED INANINTERESTINGCOMPARISON BETWEENATYROSINE LINKEDPHOTOACTIVE2U)) BPY  COMPLEXAND03)) D 3J 3 ÚDINETAL 3J 3 ÚDINETAL  4HESEAUTHORSCONCLUDETHATTHEOXIDATIONOF 9: ISVIATHE #%0MECHANISMIN-N DEPLETED03)) ASWELL4HEIRARGUMENTSARETHAT BELOWTHEP+AOF n %404 WOULD D NOT SHOW THE OBSERVED D P( DEPENDENCEOFTHESLOWKINETIC PHASESANDTHAT04%4 WOULD D NOTSHOWAP(DEPENDENCEATALL BECAUSETHE F F INTHETYROSINE PROTONACCEPTOR FER RATEOFPROTONTRANS PAIREG $ (IS WOULDBEEXPECTEDTOBEP( INDEPENDENT4HEY CONCLUDE FURT F HERMORE THAT THE DEUTERIUMISOTOPEEFFECT ANE F LEVATED REORGANIZATION I ENERGYE6 ANDANELEVATEDP( DEPENDENTACTIVA TIONENERGYALL POINTTOA#%0MECHANISM FOR03)) F ASINTHECASEOFF THESYNTHETIC RUTHENIUMCOMPLEX EVENTHOUGH INTHELATTERCASE THEPHENOLICPROTONIS TRANSFERRE F D TOWATER4HEP( DEPENDENCEOFT F HERATE

ISRATIONALIZED BYITS INmUENCEON 6' nME66P( UNIT DUE TO THE P( DEPENDENCE OF THE REDUCTION POTENTIALOF9 9:s 9: (ARRIMAN 4OMMOSETAL D ION  $INERETAL  HAVEVARIEDD THEREDUCT POTENTIAL %| OF 00 BY INTRODUCING SITE DIRECTEDD MUTATIONSATPOSITION$ AND$  D REPLACINGTHE74HISTIDINEATTHESEPOSITIONSWITH OTHERRESIDUESIN-N DEPLETED03))CORECOMPLEXES !VARIATIONINREDUCT D IONPOTENTIAL OF^M6RE F LA TIVETO74 ATP( MODU D LATESTHERATEOFF OXIDATION OF 9: BY 0 BYAFACTOROF$INERETAL   FROM›M F SnATn M6TO ›MSn ATM6 BOTH POTENTIALSRELATIVETO74 › MSn &IGURESHOWSASIMILARDEPENDENCEOFTHE RATEOF 9: OXIDATION BY 0 ONTHECHANGE IN 6' THE DIFFERENCE F INREDUCT D IONPOTENTIAL BETWEENREDOX COUPLES9 9:s 9: ANDD 00 VARIEDBYTHESETWO INDEPENDENTMEANS3HOWNARETHESAMEP( DEPEN DENTPHASEMEASURED INTH T ETENSOFF MICROSECONDD RANGE BY$INERETAL ANDBY!HLBRINKETAL  4HEEFFECTO F F P(ONW WHAT!HLBRINKAN K D CO WORRKERS F HERATEOFF 9:OXIDATION OBSERVEASA@SLOWPHASEOFT ISEQUIVALENTTOTHE Q K KINETICPHASEOF(AYSETAL  4HEDEPENDENCEOFF THERATEON 6' OVERFOU F R P(UNITS INDICATESTHAT 6'  h CONSISTENTWITHTHE REORGANIZATIONENERGY h OFE6THAT3JÚDINETAL  DEDUCE D F FROMT HETEMPERATURE DEPENDENCEOF THERATE!SIMILARSLOPETOTHATOBSERVED BYVARYING THEP(ISOBSERVEDFORTHEDEPENDENCEOFTHERATEOF 0 REDUCT D IONON b6' BY VARYINGTHEREDUCT D ION  POTENTIAL OF0 F  0 WH W ERE b6'  n&b%|AND b%|  %|MUT n %|7447HILENOTANABSOLUTEPROOF THIS AGREEMENTISCONSISTENTWITH A#%0MECHANISMIN WHICHTHERATE DEPENDSONTHE DRIVINGFORCEO F F HE FT OVERALL REACTION 3JÚDIN ET AL  $INER ET AL   #
3FEVDUJPO %404 04%4 #%0AND HY H DROGEN ATOMTRANS D F (!4 FER MECHANISMS HAVE ALSO BEEN COMPARED 7ESTPHAL ETAL #UKIER  FORTHEREDUCTIONOF9 9:s BYTHE-NCLUSTERINTHE INTACTSYSTEM)NTHE(!4 MECHANISM AHYDROGENATOMISTRANSFERREDTO9:s FROM AWATERBOUNDD TOTHE-NCLUSTER4HESEAUTHORSHAVE F IONOFC F HARGED INTERMEDIATES ARGUEDD THATTHEFORMAT 9:n -N(/ AND9:s -N/(n RESULTSINANELECTRO STATICPENALTYARISING FROMT F HECREATIONOFF CHARGED 

.OTETHATIN4ABLEOFF REF F $INERETAL  ATYPOGRAPHICAL ERRORLEFTOUTTHEMINMSn FORTHERATECONSTANTK

$IBQUFS

5Z 5 SPTJOFT
:; BOE :%



&IIG  $EPENDENCEOFF THERATEOFF 9: OXIDATION BY 0 ONTHECHANGE INTHEDRIVING FORCE & F 6' OFF THEREACTION b6' ISTHECHANGEIN THEDIFFERENCEINREDUCTIONPOTENTIALBETWEENREDOXCOUPLES 9:s 9: AND00 BYCHANGING%|OF00THROUGHSITE DIRECTED MUTATIONORBYCHANGINGTHEP(WHICHMODIlESTHEREDUCTIONPOTENTIALOF F 9:s 9: b6' nME66P(UNIT 4HERATESSHOWNCORRESPOND TOTHESAMEP( DEPENDENTKINETICPHASEMEASUREDBY$INERETAL   N ANDBY!HLBRINKETAL S 

SPECIES INA LOW DIELECTRICENVIRONMENT)NTHE%404 F F FORMAT F IONOF 9:n-N(/ CASE THE FREEENERGYO  KCALMOL FOR 3 3 AND HIGHER FOR THE OTHER 3 STATE TRANSITIONS LEADS TO A PREDICTED D ACTIVATION W ATISMEASURED ENERGYCONSIDERABLYHIGHERTHANWH EXPERIMENTALLY ) KCALMOL +ARGE ET AL  7 7ESTP HAL ET AL   ,IKEWISE THE FORMAT F ION OF 9:s-N/(n FOR04%4W F ITHA 6P+AOF * FORT F HE PROTONATIONOF9:s PREDICTSA 6' OFKCALMOLAND ASMUCHAS KCALMOL INCLUDING THEELECTROSTATIC PENALTY4HESEESTIMATESAREALSOFARGREATERT F HANTHE MEASUREDACTIVATIONENERGIESFORTHE3 STATETRANSI TIONS#UKIER "ABCOCKAN K D COWORRKERS 7ESTPHALET AL  #UKIER  CONSIDERTHE(!4MODEL AN ADIABATICPROCESSWHERETHEELECTRONDONORANDACCEP TORARESTRONGLY COUPLED TOBETHEMOST LIKELY FOR F 9:s REDUCT D IONBYTHE-NCLUSTER3MALL &RANCK #ONDON FACTORS ASSOCIATED WITH THIS MODEL ARE CONSISTENT WITH THE SMALL !RRH R ENIUS PRE EXPONENTIAL FACTORS F THAT HAVE BEEN SEEN FFOR 3 STATE OXIDATION (YDRO GEN ATOMTRANSFERWOULDALSOBECONSISTENTWITHTHE F D MICROSECOND D RATES MODESTKINETIC ISOTOPEEFFECTSAN F FARS F LOWERTHANTHENSORFASTER F OFF ELECTRONTRANSFER RATESPREDICTEDFORNON ADIABATICELECTRONTUNNELING 0AGE ET AL  4OMMOS AND D "ABCOCK  

4HEMOSTRECENT8 RAY A STRUCTUREOFF 03)) &ERREIRA ETAL  SHOWS9: TO BE INCLOSEPROXIMITYTOA PUTATIVEWATERMOLECULE 8 COORDINATINGTHE#A OF F THE /%# 7ERE 8 TO BE THE SECONDD SU UBSTRATE WATERMOLECULEPOISED FORNUC F LEOPHILICATTACKONTO K AN OXYGEN INTERMEDIATE BOUND TO -N THEN IT IS F HE3 STATECYCLE9:s POSSIBLETHATINTHE lNAL STEPOFT PARTICIPATESINA(!4MECHANISMINVOLVING84HIS MECHANISM HOWEVER SEEMSLESSLIKELYINTHEEARLIER 3 STATETRANSITIONSWH W ERETHE lRSTSUB U STRATEWATER 8 IS FAR F FROM F 9:s 4HEMOBILITY DESCRIBEDD ABOVE FOR9 F : COULDBECONSISTENTWITHTHENEEDFORTHISTYROSINETO HYDROGENBONDD WITH $ (ISEARLY INTHE3 STATE CYCLEANDD WITH 8 LATE INTHECYCLE *9 $PODMVEJOH
3FNBSLT 0ROBABLY ONEOFT F HEMOSTANXIOUSLYAWAITEDD RESULTS INTHEPHOTOSYNTHESISCOMMUNITYISAHIGHRESOLUTION 8 RAYSTRUCTUREOFTHE03))REACTIONCENTERANDIN PARTICULARTHATOFF THE-NCLUSTER!SEVIDENCEPOINTS TO A MORE INTIMATE ROLE OFF 9: IN THE MECHANISM OF WATER OXIDATION THE POSITION ORIENTATION AND HYDROGENBONDINGPARTNERSOFF THISTYROSINEWILL BE

 CRITICAL TOOURUNDERSTANDINGOFT F HEPROCESSOFF WATER OXIDATION7HILECONSIDERABLEPROGRESSHASALREADY BEENMADE WEEXPECTTHATWITH FURT F HERRElNEMENTOF THE03))STRUCTURE THEREWILL BEAlRMERSTRUCTURAL BASISWITHINWHICHTHEKINETICANDTHERMODYNAMIC CHARACTERISTICS OF F TYROSINE OXIDATION AND D REDUCT D ION D TO BETTERUNDERSTAND D THECOUPLING CAN BE INTERPRETED OFELECTRONANDPROTONTRANSFERFORBOTHTYROSINES! HIGHRESOLUTIONSTRUCTURESHOULD D ALSOHELPTO IDENTIFY THEPROTONACCEPTORFOR9 F INTHEINTACTSYSTEM THE : PATHWAYSBYWHICHPROTONSAREEXPELLEDTOTHETHY LAKOID LUMEN FROMT F HE-NCLUSTERORFROM9 F : ORBOTH D THEREASONSFORT F HELARGEDIFFERENCE F INREDUCTION AND POTENTIALOF9: AND D 9$ "DLOPXMFEHNFOUT 4HEAUTHORSGRATEFULLYACKNOWLEDGETHESUPPORTOF THE5NITED3TATES$EPARTMENTOF!GRICULTURE.2) #'0AWARD D NO   TO"!$AND D OF THE .ATIONAL 3CIENCE &OUNDATION -#"  ANDTHE.)('- TO2$"4HEAUTHORSHAVE GREATLY BENElTED FROM F DISCUSSIONS OVER THE YEARS WITH$RS'ERALD D 4"ABCOCK #ATHERINE"ERTHOMIEU + +RISTY!#AMPBELL 2ICHARD*$EBUS 0ETER&ALLER ,EIFF (AMMARSTRÚM 2AINER (IENERWADEL -ELVIN /KAMURA -ARK RK 0ADDOCK K AND &ABRICE 2APPAPORT REGARDINGTHEINSANDOUTSOFCOUPLEDELECTRONAND PROTONTRANSFERO F F THEREDOXACTIVETYROSINESOF03)) F 4HIS PAPERISCONTRIBUTIONOFF THE#ENTRAL2E SEARCHAND$EVELOPMENT$EPARTMENT %)DU0ONT DE.EMOURS#O 3FGFSFODFT !HLBRINK K 2 (AUMANN - #HEREPANOV $ "OEGERSHAUSEN / D GE7  &UNCTIONOFT F YROSINE: -ULKIDJANIAN!AND*UN INWATEROXIDATIONBY0HOTOSYSTEM))%LECTROSTATICALPROMOTOR INSTEADOFHYDROGENABSTRACTOR"IOCHEMISTRY¥ !LLEN*0 &EHER' 9EATES4/ 2EES$# $EISENHOFER* -ICHEL( AND(UBER2 3TRUCTURALHOMOLOGYOFREACTIONCENTERS F OM 2HODO FR DOPSEUDOMONASS D SPHAEROIDES D AND 2HODO DOPSEUDOMONAS D VIRIDISASDETERMINED BY8 RAY A DIFFRACT F ION0ROC.ATL!CAD3C D I 5 3! ¥ !NANY N EV'- 3AKIYAN) $INER"!ANDD $ISMUKES'# ! FUNCT F IONAL ROLEFORT F YROSINE $ INASSEMBLYOFT F HEINORGANIC COREOFTHEWATEROXIDASECOMPLEXOF0HOTOSYSTEM))ANDTHE KINETICSOFWATEROXIDATION"IOCHEMISTRY¥ !NDREASSON,%AND,INDBERG+ 4HEINHIBITIONOFPHO TOSYNTHETIC OXYGEN EVOLUTION BY AMMONIA PROBED BY %02 H  "IOCHIM "IOPHYS!CTA¥

#SVDF " %JOFS BOE 3 %BWJE
#SJUU "ABCOCK'4AND3AUER+A %LECTRONPARAMAGNETICRESO NANCESIGNAL))INSPINACHCHLOROPLASTS )+INETICANALYSISFOR UNTREATEDCHLOROPLASTS"IOCHIM"IOPHYS!CTA¥ "ABCOCK'4AND3AUER+B %LECTRONPARAMAGNETICRESO NANCESIGNAL))INSPINACHCHLOROPLASTS))!LTERNATIVESPECTRAL F FORMSAN D INHIBITOREFFECTSON F KINETICSOFS F IGNAL )) IN mASHING LIGHT"IOCHIM "IOPHYS!CTA¥ "ABCOCK K '4AND 3AUER+A 2APIDCOMPONENTO D F ELECTRON PARAMAGNETICRESONANCESIGNAL ))#ANDIDATE FORT F HEPHYSIOLOGI CAL DONORTO0HOTOSYSTEM)) INSPINACHCHLOROPLASTS"IOCHIM "IOPHYS!CTA¥ "ABCOCK'4AND3AUER+B 2APID LIGHT INDUCEDTRANSIENT INELECTRONPARAMAGNETICRESONANCESIGNAL))ACTIVATEDUPON INHIBITIONOFPHOTOSYNTHETICOXYGENEVOLUTION"IOCHIM"IOPHYS !CTA  ¥ "ABCOCK K '4AND 3AUER+C 4WOELECTRONDONATIONSITESFOR F EXOGENOUSREDUCTANTS D INCHLOROPLAST0HOTOSYSTEM))"IOCHIM "IOPHYS!CTA¥ "ABCOCK K '4 "LANKENSHIP 2% AND D 3AUER +  2EACTION KINETICSFORPOSITIVECHARGEACCUMULATIONONTHEWATERSIDEOF CHLOROPLAST0HOTOSYSTEM))&%"3,ETT¥ "ABCOCK'4 "ARRY"! $EBUS2* (OGANSON#7 !TAMIAN- -C)NTOSH, 3ITHOLE)AND9OCUM#& 7ATEROXIDATION IN 0HOTOSYSTEM)) &ROM RADICAL CHEMISTRY TO MULTIELECTRON CHEMISTRY" Y IOCHEMISTRY¥ "ABCOCK K '4 %SPE- (OGANSON# ,YD , AKIS 3IMANTIRIS. -C #RACKEN* 3HI7 3TYRING3 4OMMOS#AND D 7ARNCKE+ 4 ROSYL RADICALS INENZYMECATALYSISSOMEPROPERTIESAND 4Y DA FOCUS ON PHOTOSYNTHETIC WATER OXIDATION!CTA #HEM 3CAND ¥ "ACKES' 3AHLIN- 3JOBERG"- ,OEHR4-AND3ANDERS ,OEHR *  2ESONANCE 2AMAN SPECTROSCOPY OF RIBONUCLEOTIDE D %VIDENCE FFOR A DEPROTONATED D TYROSYL RADICAL AND REDUCTASE PHOTOCHEMISTRY OFF THE BINUCLEAR IRON CENTER "IOCHEMISTRY  ¥ "ARRY "!AND D "ABCOCK K '4 4Y 4 ROSINERADICALSAREINVOLVED INTHEPHOTOSYNTHETICOXYGEN EVOLVINGSYSTEM0ROC.ATL !CAD 3CI53!¥ "AUMGARTEN- 0HILO*3AND$ISMUKES'# -ECHANISMOF PHOTOINHIBITIONOFPHOTOSYNTHETICWATEROXIDATIONBYCHLORIDE DEPLETION AND mUORIDE SUBSTITUTION /XIDATION OF A PROTEIN D IOCHEMISTRY¥ RESIDUE" "ENDER#* 3AHLIN- "AB A COCK'4 "ARR K Y "! #HANDRAS D HEKAR4+ 3ALOWE30 3TUBB U E* ,INDSTRÚM" 0ETERSSON, %HRENBERG! AND D 3JÚBERG" -  !N%.$/2STUDY OFT F HETYROSYL FREE F D F FROM %SCHERICHIACOLI*!M RADICAL INRIBONUCLEOTIDEREDUCTASE #HEM3OC¥ "ENT$6AND(AYON% %XCITEDSTATECHEMISTRYOFAROMATIC AMINOACIDSANDRELATEDPEPTIDES)4YROSINE*!M#HEM3OC  ¥ "ERTHOLDD $! "ABCOCK K '4 AND D 9OCUM #&  ! HIGHLY D OXYGEN EVOLVING 0HOTOSYSTEM)) PREPARATION FROM F RESOLVED SPINACH THYLAKOIDD MEMBRANES %02 AND D ELECTRON TRANSPORT PROPERTIES&%"3,ETT¥ "ERTHOMIEU#AND D "OUSSAC! (ISTIDINEOXIDATION INTHE 3TO3TRANSITIONPROBEDBY&4)2DIFFERENCESPECTROSCOPY INTHE#A DEPLETED0HOTOSYSTEM))#OMPARISONWITHHISTI DINERADICALSGENERATEDBY56IRRADIATION"IOCHEMISTRY ¥ "ERTHOMIEU# (IENERWADEL2 "OUSSAC! "RETON*AND D $INER F REDOX ACTIVE TYROSINE : "!  (YDROGEN BONDING OF

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% OFF 0HOTOSYSTEM)) PROBED BY &4)2 DIFFERENCE F SPECTROSCOPY Y "IOCHEMISTRY¥ "LANKENSHIP2% "ABCOCK'4AND3AUER+A +INETICSTUDY OF OXYGEN EVOLUTION PARAMETERS IN TRIS WASHED REACTIVATED CHLOROPLASTS"IOCHIM"IOPHYS!CTA¥ "LANKENSHIP2% "ABCOCKK '4 7ARDEN*4ANDD 3AUER+B /BSERVATIONOFF ANEW%02TRANSIENT INCHLOROPLASTSTHATMAY A REmECTTH T EELECTRONDONORTO0HOTOSYSTEM))ATROOMTEMPERATURE &%"3,ETT¥ "OERNER2*AND D "ARRY "! )SOTOPICLABELING AND%02SPEC D

TROSCOPYSHOWTHATATYROSINERESIDUEISTHETERMINALELECTRON DONOR : INMANGANESE DEPLETED0HOTOSYSTEM))PREPARATIONS *"IOL#HEM¥ "ORDWELL &' AND #HENG *  3UBSTITUENT EFFECTS ON THE STABILITIESOFP F HENOXYL RADICALSANDD THEACIDITIESOFP F HENOXYL RADICAL CATIONS* !M #HEM 3OC¥ "OSKA- 3AUER+ "UTTNER7ANDD "ABCOCKK '4 3IMILARITY OFF %02SIGNAL ))FF RISEANDD 0  DECAY A KINETICSIN4RIS WASHED CHLOROPLAST0HOTOSYSTEM))PREPARATIONSASAFUNCT F IONOFP( F "IOCHIM"IOPHYS!CTA¥ "OUGES "OCQUET" +INETICMODELSFORTHEELECTRONDONORS OF0HOTOSYSTEM))OFPHOTOSYNTHESIS"IOCHIM"IOPHYS!CTA  ¥ "OUSSAC!AND D %TIENNE!,  -IDPO D INTPOTENTIAL OFS F IGNAL )) SLOW IN 4RIS WASHEDD 0HOTOSYSTEM)) PARTICLES "IOCHIM "IOPHYS!CTA¥ D 2UTHERFOR F D!7 D  .ATUREOFF THE INHIBITION "OUSSAC!AND OFF THE OXYGEN EVOLVING ENZYME OFF 0HOTOSYSTEM)) INDUCE D D BYSODIUMCHLORIDEWASHINGANDREVERSEDBYTHEADDITIONOF CALCIUM ORSTRONTIUM "IOCHEMISTRY¥ "OUSSAC! :IMMERMANN *, AND 2UTHERFORD!7  %02 SIGNALS FROM MODIlED CHARGE ACCUMULATION STATES OF THE OXYGEN EVOLVINGENZYMEINCALCIUM DElCIENT0HOTOSYSTEM)) "IOCHEMISTRY¥ F D !7 AND ,AVERGNE * "OUSSAC! :IMMERMANN *, 2UTHERFOR A (ISTIDINEOXIDATION INTHEOXYGEN EVOLVING PHOTOSYS TEM ))ENZYME.ATURE¥ "OUSSAC! :IMMERMANN*,AND2UTHERFORD!7B &AC TORSINmUENCINGTHEFORMATIONOFMODIlED3%02SIGNALAND THE3%02SIGNALINCALCIUM DEPLETED0HOTOSYSTEM))&%"3 ,ETT ¥ F 0 AND 2UTHERFOR F DD !7  )NHIBITION OF "OUSSAC ! 3ÏTIF F FORMAT F IONOFT F HE3 STATEIN TYROSINE:PHOTOOXIDATIONAFTER CALCIUM DEPLETEDD AND CHLORIDE DEPLETEDD PHOTOSYSTEM ))"IO CHEMISTRY ¥ D "RETTEL + 3CHLODDER%ANDD 7ITT(4 .ANOSECONDD REDUC TIONKINETICSOFPHOTOOXIDIZEDCHLOROPHYLL A))0  INSINGLE mASHESASAPROBEFORTHEELECTRONPATHWAY PROTON RELEASEAND CHARGEACCUMULATIONINTHEOXYGEN EVOLVINGCOMPLEX"IOCHIM "IOPHYS!CTA¥ "RITT 2$ A /XYGEN EVOLUTION )N /RT $2 ANDD 9OCUM #& EDS /XYGENIC0HOTOSYNTHESIS4HE,IGHT2EACTIONS PP ¥+LUWER!CADEMIC0UBLI U SHERS $ORDRECHT "RITT2$B %LECTRONSPINECHOMETHODS INPHOTOSYNTHESIS RESEARCH)N(OFF!*AND!MESZ* EDS "IOPHY H SICAL4ECHNIQUES IN0HOTOSYNTHESIS PP¥+LUWER!CADEMIC0UBLISHERS $ORDRECHT "RITT 2$ 2ANDALL $7 "ALL *! 'ILCHRIST -, *R &ORCE $! 3TURGEON"% ,ORIGAN'! 4ANG8 3 $INER"! +LEIN-0 D !RMSTRONG 7(  %LECTRON SPIN ECHO #HAN -+ AND %.$/2STUDIESOFF THETYROSINERADICALSANDD THEMANGANESE

 CLUSTEROFF 0HOTOSYSTEM)))N-ATHIS0ED 0HOTOSYNTHESIS &ROM,IGHTTO"IOSPHERE 6OL PP¥+LUWER!CADEMIC 0UBLISHERS $ORDRECHT "USER#! 4HOMPSON,+ $INER"!AND"RUDVIG'7 %LECTRON TRANSFERREACTIONSINMANGANESE DEPLETED0HOTOSYS TEM))"IOCHEMISTRY ¥ #AMPBELL +!  #7ANDPU D LSED D %02STUDIESOFF 0HOTOSYS TEM)) AND D -N CONTAINING COMPLEXES4HESIS 5NIVERSITY OF F IA $AVIS #ALIFORN #AMPBELL +! 0ELOQUIN *- $INER "! 4ANG 8 3 #HISHOLM $! AND "RITT 2$  4HE TAU NITROGEN OF $ HISTIDINE ISTHEHYDROGENBONDDONORTOTHETYROSINERADICAL9$s OF 0HOTOSYSTEM))*!M#HEM3OC¥ #ANDEIAS,0 4URCONI3AND.UGENT*(! 4YROSINE9:AND 9$OFF 0HOTOSYSTEM))#OMPARISONOFOPT F ICALSPECTRATOTHOSE OFF TYROSINE OXIDIZED BY PULSED D RADIOLYSIS "IOCHIM "IOPHYS !CTA ¥ #HRISTEN ' AND D 2ENGER '  4HE ROLE OF HYDROGEN BONDS F HEMULTIPHASIC0s REDUCT FORT D ION BY9:IN0HOTOSYSTEM)) WITHINTACTOXYGENEVOLUTIONCAPACITY!NALYSISOFKINETIC($ ISOTOPEEXCHANGEEFFECTS"IOCHEMISTRY¥ #HRISTEN' 3EELIGER!AND2ENGER' 0s REDUCTION KINETICSANDREDOXTRANSITIONPROBABILITYOFTHEWATEROXIDIZ INGCOMPLEXASAFUNCT F IONOFF P(AND ($ ISOTOPEEXCHANGEIN SPINACHTHYLAKOIDS"IOCHEMISTRY ¥ #HU (! .GUYEN!0 AND D $EBUS 2*  !MINO ACIDD RESI D DUEST HATINmUENCETHE BINDINGOFMAN F GANESEORCALCIUMTO R ELICAL DOMAINSOFF THE$ 0HOTOSYSTEM))4HE LUMENAL INTERH POLYPEPTIDE"IOCHEMISTRY¥ #LEMENS+, &ORCE$!AND"RITT2$ !CETATEBINDINGATTHE 0HOTOSYSTEM))OXYGENEVOLVINGCOMPLEX!N3 STATEMULTILINE SIGNAL%3%%-STUDY*!M#HEM3OC¥ #OMMONER" (EISE**AND D 4OWNSEND* D  ,IGHT INDUCE D D PARAMAGNETISMINCHLOROPLASTS0ROC.ATL!CAD3C D I 53! ¥ #ONJ N EAUD D (AND -ATHIS0 4HEEFFECTSO F F FP(ONT HEREDUC D TIONS KINETICSOF0  F IN4RIS TREATED D CHLOROPLASTS"IOCHIM "IOPHYS!CTA¥ #UKIER2) 4HEORYANDSIMULATIONOFPROTON COUPLEDELEC TRONTRANSFER HYDROGEN ATOMTRANSFER ANDPROTONTRANSLOCATION INPROTEINS"IOCHIM"IOPHYS!CTA¥ $E 6 6ITRY # #ARLES # AND D $INER "!  1UANTITATION OF PLASTOQUINONE  IN 0HOTOSYSTEM)) REACTION CENTER PARTICLES #HEMICAL IDENTIlCATIONOFF THEPRIMARY QUINONE ELECTRONAC CEPTOR1!&%"3,ETT¥ $EBUS2* "ARRY"! "ABCOCK K '4AND -C)NTOSH , A 3ITE DIRECTEDMUTAGENESISIDENTIlESATYROSINERADICALINVOLVEDIN THEPHOTOSYNTHETICOXYGEN EVOLVINGSYSTEM0ROC.ATL!CAD 3CI53!¥ $EBUS2* "ARRY"! 3ITHOLE) "ABCOCK'4AND-C)NTOSH, B $IRECTED D MUTAGENESIS INDICATES THAT THE DONOR TO 0 IN 0HOTOSYSTEM))ISTYROSINE OFF THE$POLYPEPTIDE "IOCHEMISTRY ¥ $EKKER *0 "ROK K - AND 6AN 'ORRKOM (* A !BSORB R ANCE CHANGESOF : THECOMPONENTRESPONSIBLE FOR%02S F IGNAL )) FAST IN4RIS TREATED 0HOTOSYSTEM)) PARTICLES )N 3YBESMA # ED !DVANCESIN0HOTOSYNTHESIS2ESEARCH 6OL PP¥ -ARTINUS.IJHOFF$R7*UNK0UBLISHERS 4HE(AGUE $EKKER*0 6AN'ORKOM(* "ROK-AND/UWEHAND,B /PTICAL CHARACTERIZATION OFF 0HOTOSYSTEM)) ELECTRON DONORS "IOCHIM "IOPHYS!CTA¥

 $ELOSME2AND*O D LIOT0 0ERIOD FOUROSC F ILLATIONSINCHLO ROPHYLL A mUORESCENCE0HOTOSYNTH2ES¥ $INER"! !MINOACIDRESIDUESINVOLVEDINTHECOORDINA TIONANDASSEMBLYOFTHEMANGANESECLUSTEROF0HOTOSYSTEM)) 0ROTON COUPLEDELECTRONTRANSPORTOFTHEREDOX ACTIVETYROSINES AND ITSRELATIONSHIPTOWATEROXIDATION"IOCHIM "IOPHYS!CTA  ¥ $INER"!AND D "AABCOCK'4 K  3TRUCTURE DYNAM Y ICS ANDD ENERRGY CONVERSIONEFlCIENCY IN0HOTOSYSTEM)))N/RT$2AND9OCUM D #& EDS /XYGENIC0HOTOSYNTHESIS4HE,IGHT2EACTIONS PP ¥+LUWER!CADEMIC0UBLISHERS $ORDRECHT $INER"!ANDDE6ITRY# /PTICALSPECTRUMANDKINETICSOF THESECONDARYELECTRONDONOR : OF0HOTOSYSTEM)))N3YBESMA #ED !DVANCESIN0HOTOSYNTHESIS2ESEARCH 6OL PP¥ K UBLISHERS 4HE(AGUE -ARTINUS.IJHOFF$R7*UNK0U D .IXON0* %VIDENCE FOR$ ( F ISASTHE $INER"!AND PROTON ACCEPTOR IMPLICATED IN THE OXIDATION OF F REDOX ACTIVE TYROSINE9: OFF 03)))N'ARAB'ED 0HOTOSYNTHESIS-ECHA NISM AND D %FFECTS 6O F L )) PP ¥ +LUWER!CADEMIC 0UBLISHERS $ORDRECHT $INER"! .IXON0*AND&ARCHAUS*7 3ITE DIRECTEDMU TAGENESISOFPHOTOSYNTHETICREACTIONCENTERS#URR/PIN3TRUCT "IOL  ¥ $INER"! &ORCE$! 2ANDALL$7ANDD "RITT2$ (YDROGEN BONDING SOLVENTEXCHANGE ANDD COUPLEDPROTONAN D D ELECTRON F INTHEOXIDATIONANDD REDUCT D IONOFRE F DOX ACTIVETYRO TRANSFER SINE9: IN -N DEPLETED D CORE COMPLEXES OFF 0HOTOSYSTEM)) "IOCHEMISTRY¥ $INER"! 3CHLODDER% .IXON0* #OLEMAN7* 2APPAPORT& ,AVERGNE* 6ERMAAS7&*AND#HISHOLM$ 3ITE DIRECTED MUTATIONSAT$ (ISAND$ (ISOF0HOTOSYSTEM))IN 3YNECHOCYSTIS I 0##3ITESOFPRIMARYCHARGESEPARATIONAND CATIONAND D TRIPLETSTABILIZATION"IOCHEMISTRY¥ $INER"! "AUTISTA*! .IXON0* "ERTHOMIEU# (IENERWADEL 2 D HISHOLM$!  #OORDINATION "RITT2$ 6ERMAAS7&*AND# OFF PROTONAND ELECTRONTRANSFER F FROMT F HEREDOX ACTIVETYROSINE 9: OFF 0HOTOSYSTEM)) ANDD EXAMINATION OF F THE ELECTROSTATIC INmUENCEOFOXIDIZEDTYROSINE 9$s ( 0HYS#HEM#HEM0HYS n $IXON74AND-URPHY$ $ETERMINATIONOFTHEACIDITY CONSTANTSOFSOMEPHENOLRADICALCATIONSBYMEANSOFELECTRON SPINRESONANCE*#HEM3OC &ARADAY4RANS¥ $OLE & $INER "! (OGANSON #7 "ABCOCK K '4 AND "RITT 2$  $ETERMINATIONOFT F HEELECTRONSPINDENSITYONTHEPHE NOLICOXYGENOFT F HETYROSYLRADICAL OFF 0HOTOSYSTEM))*!M #HEM3OC¥ $ORLET0 6ALENTIN-$ "ABCOCK'4AND-C#RACKEN*, )NTERACTION OF 9:s WITH ITS ENVIRONMENT IN ACETATE TREATED 0HOTOSYSTEM))MEMBRANESANDREACTIONCENTERCORES*0HYS #HEM"¥ $ORLET 0 2UTHERFOR F D !7 ANDD 5N 3  /RIENTATION OFF THE TYROSYL $ PHEOPHYTINANION ANDD SEMIQUINONE 1!ns RADICALS IN 0HOTOSYSTEM)) DETERMINED BY HIGH lELDD ELECTRONPARAMAGNETIC RESONANCE"IOCHEMISTRY ¥ D 2ENGER' 4EMPERATURE DEPENDENCEOFF 0 %CKERT(*AND REDUCTION IN OXYGEN EVOLVING 03)) MEMBRANE FRAGMENTS AT DIFFERENTREDOXSTATES3IOFTHEWATEROXIDIZINGSYSTEM&%"3 ,ETT¥ %L +ABBANI/ #HANG#( 4IEDE$ .ORRIS*AND3CHIFFER- F 2HODO D BACTERSPH S AEROIDES D #OMP M ARISONOFF REACTIONCENTERSFROM AND 2HODO DOPSEUDOMONASVIRI D DIS/VERALL ARCHITECTUREAND PRO

#SVDF " %JOFS BOE 3 %BWJE
#SJUU TEIN PIGMENT INTERACTIONS"IOCHEMISTRY ¥ %VELO 2' (OFF !* $IKANOV 3! AND 4YRYSHKIN !-  !N %3%%- STUDY OF THE OXIDIZED ELECTRON DONOR OF PLANT 0HOTOSYSTEM))%VIDENCETHAT $ s ISANEUTRALTYROSINERADICAL #HEM0HYS,ETT¥ &ALLER0 $EBUS2* "RETTEL + 3UGIURA- 2UTH T ERFOR F D!7AN D D "OUS SAC! 2APID FORMAT F IONOFF THESTABLETYROSYL RADICAL IN D 3CI53!¥ 0HOTOSYSTEM))0ROC.ATL!CAD &ALLER0 2UTHERFOR F D!7AN D D $EBUS2*  4Y 4 ROSINE$OXIDA TIONATCRYOGENICTEMPERATUREIN0HOTOSYSTEM))"IOCHEMISTRY ¥ &ARRAR#4 'ERFEN'* 'RIFlN2' &ORCE$!AND"RITT2$ %LECTRONICSTRUCTUREOFTHE9$ TYROSYLRADICALIN0HOTOSYSTEM)) ! HIGH FREQUENCY ELECTRON PARAMAGNETIC RESONANCE SPECTRO SCOPICAND DENSITY FUNCT F IONAL THEORETICAL STUDY*0 Y HYS#HEM "  ¥ &ERREIRA+. )VERSON4- -AGHLAUOUI+ "ARB R ER*AND)WATA3 D  !RCHITECTURE OF F THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCE¥ &ORCE$! 2ANDALL$7 "RITT2$ 4ANG8 3AND$INER"!  (%3% %.$/2STUDYOFHYDROGENBONDINGTOTHETYROSINE RADICALS9$sAND9:sOF0HOTOSYSTEM))*!M#HEM3OC ¥ &OWLER#& 0ROTONEVOLUTIONFROM0 F HOTOSYSTEM))3TOI CHIOMETRY AND D MECHANISTICCONSIDERATIONS"IOCHIM "IOPHYS !CTA ¥ 'ARDNER +! AND D -AAYER *-  5NDERSTANDING # ( BOND OXIDATIONS(sAND (nTRANSFERINTHEOXIDATIONOFTOLUENEBY PERMANGANATE3CIENCE¥ 'ERKEN3 "RETTEL+ 3CHLODDER%AND7ITT(4 /PTICALCHAR R R ACTERIZATIONOFTHEIMMEDIATEELECTRONDONORTOCHLOROPHYLL A)) IN OXYGEN EVOLVING 0HOTOSYSTEM)) COMPLEXES 4YROSINE AS POSSIBLEELECTRONCARRIERBETWEENCHLOROPHY H LL A))AND D THEWATER OXIDIZINGMANGANESECOMPLEX&%"3,ETT¥ 'HANOTAKIS$& /-ALLEY0* "ABCOCK'4AN K D D9OCUM#&  3TRUCTUREAND INHIBITIONOFCOMPONENTSONT F HEOXIDIZINGSIDE OFF 0HOTOSYSTEM)))N)NOUE9 #ROFTS!2 'OV F INDJEE -URATA . 2ENGER'AND3ATOH+EDS /XYGEN%VOLVING3YSTEMOF 0HOTOSYNTHESIS PP¥!CADEMIC0RESS 4OKYO 'ILCHRIST -, *R "ALL *! 2ANDALL $7 AND "RITT 2$  0ROXIMITY OF THE MANGANESE CLUSTER OF 0HOTOSYSTEM)) TO THE REDOX ACTIVE TYROSINE9: 0ROC .ATL !CAD 3CI 53!  ¥ 'LASER- 7OLFFF # "UCHWALDD (%AND 7ITT(4  /NTHE PHOTOACTIVE CHLOROPHYLL REACTION IN SYSTEM )) OF F PHOTOSYN THESIS$ETECTIONOFF A FASTAN F D LARGECOMPONENT&%"3,ETT ¥ 'ULIN6) $IKANOV3! 4SVETKOV9$ %VELO2'AND(OFF!* 6ERYHIGHFREQUENCY'(Z %02OFTHEOXIDIZEDPRIMARY DONOROFTHEPHOTOSYNTHETICBACTERIA 2BSP S HAEROIDES 2  AND 2PSVIRIDIS AND D OFF9$SIGNAL )) OFP F LANT0HOTOSYSTEM))0URE !PPL #HEM¥ (ALES"*AND D $AS'UPTA!  3UPPOSITIONOFF THEORIGINOF SIGNAL )) FFROM RANDOM AND D ORIENTED D CHLOROPLASTS "IOCHIM "IOPHYS!CTA¥ (ALLAHAN"* .UGENT*(! 7ARDEN*4AND%VANS-#7 )NVESTIGATIONOFTHEORIGINOFTHE@3%02SIGNALFROMTHEOXY GEN EVOLVINGCOMPLEXOF0HOTOSYSTEM4HEROLEOFTYROSINE :"IOCHEMISTRY¥ (ARRIMAN! &URTHERCOMMENTSONTHEREDOXPOTENTIALSOF D TYROSINE*0HYS#HEM¥ TRYPTOPHANAND

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% (AUMANN - "OGERSHAUSEN / #HEREPANOV $ !HLBRINK 2 AND *UNGE7  0HOTOSYNTHETIC OXYGEN EVOLUTION ($ ISOTOPEEFFECTSANDTHECOUPLINGBETWEENELECTRONANDPROTON TRANSFER DURING THE REDOX REACTIONS AT THE OXIDIZING SIDE OF 0HOTOSYSTEM))0HOTOSYNTH2ES¥ D GE7  4Y 4 ROSINE : (AUMANN- -ULKIDJANIAN!AND*UN IN OXYGEN EVOLVING 0HOTOSYSTEM))! HYDROGEN BONDEDD TY ROSINATE"IOCHEMISTRY ¥ (AY A S!- 6ASSILIEV)2 'OLBECKK *(AND $EBUS2* 2OLE OFF $ (IS INPROTON COUPLEDD ELECTRONTRANSFERREACT F IONSIN 0HOTOSYSTEM))!CHEMICALCOMPLEMENTATIONSTUDY"IOCHEM ISTRY¥ (AYS!- 6ASSILIEV)2 'OLBECK*(AND$EBUS2* 2OLE OF$ (ISINTHEPROTON COUPLEDOXIDATIONOFTYROSINE9: IN MANGANESE DEPLETED 0HOTOSYSTEM)) "IOCHEMISTRY  ¥ (IENERWADEL 2 "OUSSAC! "RETON*ANDD "ERTHOMIEU# F INFRARE F D DIFFERENCE F STUDY OF F TYROSINE$ &OURIER TRANSFORM OXIDATIONAND D PLASTOQUINONE1! REDUCT D ION IN0HOTOSYSTEM)) "IOCHEMISTRY¥ (IENERWADEL2 "OUSSAC! "RETON* $INER"AND"ERTHOMIEU#  &OURIERTRANSFORMINFRAREDDIFFERENCESPECTROSCOPYOF 0HOTOSYSTEM))TYROSINE$USINGSITE DIRECTEDMUTAGENESISAND SPECIlC ISOTOPELABELING"IOCHEMISTRY¥ (OFBAUER 7 :OUNI ! "ITTL 2 +ERN * /RTH 0 ,ENDZIAN & UBITZ7 0HOTOSYSTEM))SINGLE &ROMME0 7I 7 TT(4ANDD ,U CRYSTALSSTUDIED BY %02SPECTROSCOPY AT'(Z4HETYROSINE RADICAL9$s0ROC.ATL !CADD 3CI 53!¥ (OGANSON#7AND"ABCOCK'4 %LECTRON TRANSFEREVENTS NEAR THE REACTION CENTER IN OXYGEN EVOLVING 0HOTOSYSTEM)) PREPARATIONS"IOCHEMISTRY¥ (OGANSON#7AND"ABCOCK'4 0ROTEIN TYROSYLRADICAL INTERACTIONSIN 0HOTOSYSTEM))STUDIED BYELECTRONSPINRESO NANCE AND D ELECTRON NUCLEAR DOUBL U E RESONANCE SPECTROSCOPY COMPARISONWITHRIBONUCLEOTIDEREDUCTASEAN D D INVITROTYROSINE "IOCHEMISTRY¥ (OGANSON#7ANDD "ABCOCK'4 K  !METALLORADICALMECHA NISMFORTHEGENERATIONOFOXYGENFROMWATERINPHOTOSYNTHESIS 3CIENCE¥ (OGANSON #7 ,YDAKIS 3IMANTIRIS . 4ANG 8 3 4OMMOS # 7 7ARNCKE+ "ABCOCK'4 $INER"! -C#RACKEN*AND3TYRING 3  ! HY H DROGEN ATOMABSTRACTIONMODEL FORT F HEFUNCT F ION OFF 9Z IN PHOTOSYNTHETIC OXYGEN EVOLUTION 0HOTOSYNTH 2ES  ¥ (OGANSON #7 3AHLIN - 3J 3 OEBERG " - ANDD "ABCOCKK '4  %LECTRONMAGNETICRESONANCEOFT F HETYROSYLRADICAL IN RIBONUCLEOTIDEREDUCTASEFROM %SCHERICHIACOLI*!M#HEM 3OC¥ (ULSEBOSCH2* VANDEN"RINK*3 .IEUWENHUIS3!- 'AST0 2AAP* ,UGTENBERG*AND(OFF!* %LECTRONICSTRUCTURE OFF THENEUTRAL TYROSINERADICAL IN FROZENSO F LUTION3ELECTIVE (  # AND / ISOTOPELABELING ANDD %02SPECTROSCOPY ATAND  '(Z*!M #HEM 3OC¥ D )NOUE9  0ARTIAL CHARACTERIZATION OF F THE )KEUCHI - AND IODINATION SITE IN $ PROTEIN OFF MANGANESE RETAINING AND MANGANESE DEPLETED 0HOTOSYSTEM)) MEMBRANES 0LANT #ELL 0HYSIOL¥ +AMIYA.AND3HEN* 2 #RYSTALSTRUCTUREOFOXYGEN EVOLV ING0HOTOSYSTEM))FROM4HERMOSYNECHOCOCCUSVULCANUS 4 AT ¯ RESOLUTION0ROC.ATL!CAD3CI53!¥ +ARGE- )RRGANG +$ 3ELLIN3 &EINAEUGLE2 ,IU" %CKERT(*

 D 2ENGER'  %FFECTSO F F HYDROGENDEUTE %ICHLER(*AND RIUMEXCHANGEONPHOTOSYNTHETICWATERCLEAVAGEIN03))CORE COMPLEXESFROMSPINACH&%"3,ETT¥ +ARGE - )RRGANG + $ AND 2ENGER '  !NALYSIS OF THE REACTIONCOORDINATEOFPHOTOSYNTHETICWATEROXIDATIONBYKI NETICMEASUREMENTSOFF NMABSORPTIONCHANGESAT DIFFERENT F T ER TEMPERATURESIN 0HOTOSYSTEM))PREPARATIONSSUSPENDED INEITH (/OR$/"IOCHEMISTRY¥ +LIMOV 66 AND D +RASNOVSKII !!  0HEOPHYTIN AS THE PRIMARYELECTRONACCEPTORIN0HOTOSYSTEMREACTIONCENTERS 0HOTOSYNTHETICA¥ + +OHL$(AND7OOD0- -OLECULARIDENTITYOF%32SIGNAL ))OBSERVEDINPHOTOSYNTHETICSYSTEMS4HEEFFECTOFHEPTANE EXTRACTIONANDRECONSTITUTIONWITHPLASTOQUINONEANDDEUTERATE Q D PLASTOQUINONE0LANT0HYSIOL¥ + HL $( 7RIGHT *2 AND +O D 7EISSMAN -  %LECTRON SPIN F DICALSDERIVED FROM F PLASTOQU Q INONE RESONANCESTUDIESOF FREERA ALPH AND GAMMA TOCOPHEROL AND D THEIRRELATIONSTOFREERA F DICALS OBSERVED INPHOTOSYNTHETICMATERIALS"IOCHIM"IOPHYS!CTA ¥ + +OULOUGLIOTIS$ 4ANG8 3 $INER"!AND"RUDVIG'7 3PECTROSCOPICEVIDENCEFORTHESYMMETRICLOCATIONOFTYROSINES $AND:IN0HOTOSYSTEM))"IOCHEMISTRY ¥ + +RAMER$- 2O FFEY2! 'OV F INDJEEAND D 3AYRE24 4HE!4 F OM #HL # AMYD M DOMONASREINHARD R TII THERMOLUMINESCENCE BAND FR AND D THEEFFECTSO F F FMUTA GENESISOF HISTIDINERESIDUESONT D HE DONOR SIDEOFT F HE0HOTOSYSTEM))$POLYPEPTIDE"IOCHIM"IOPHYS !CTA¥ +RISHTALIK ,)  %NERGETICS OF MULTIELECTRON REACTIONS 0HOTOSYNTHETIC OXYGEN EVOLUTION "IOCHIM "IOPHYS !CTA ¥ + +RISHTALIK,) !CTIVATIONENERGYOFPHOTOSYNTHETICOXYGEN EVOLUTION!NATTEMPTATTHEORETICALANALYSIS"IOELECTROCHEM "IOENERG¥ + HNE ( AND +UE D "RUDVIG '7  0ROTON COUPLED D ELECTRON F INVOLVING TYROSINE: IN0HOTOSYSTEM))*0HYS#HEM TRANSFER "  ¥ ,AKSHMI+6 %ATON33 %ATON'2 &RANK(!AND"RUDVIG'7  !NALYSISOFDIPOLARANDEXCHANGE)NTERACTIONSBETWEEN MANGANESEANDTYROSINE:INTHE39:sSTATEOFACETATE INHIBITED 0HOTOSYSTEM))VIA%02SPECTRALSIMULATIONSAT8 AND1 BANDS H HEM"¥ *0HYS# ,AKSHMI +6 %ATON33 %ATON'2AND D "RUDVIG '7 /RI ENTATIONOFF THETETRANUCLEARMANGANESECLUSTERAND D TYROSINE: INTHE/ EVOLVINGCOMPLEXOFF 0HOTOSYSTEM))!N%02STUDY OFF THE39:s3TATEINORIENTEDACETATE INHIBITED0HOTOSYSTEM)) MEMBRANES"IOCHEMISTRY¥ ,AVERGNE*AND*UNGE7 0ROTONRELEASEDURINGTHEREDOX CYCLEOFTHEWATEROXIDASE0HOTOSYNTH2ES¥ ,IND* 3HEN8 %RIKSEN4%AND-ERENYI' 4HEONE ELECTRON D IONPOTENTIAL OF SU F UBSTITUTED D PHENOXYLRADICALS INWATER REDUCT * !M #HEM3OC¥ -AC,ACHLAN$*AND D .UGENT*(! )NVESTIGATIONOFT F HE3 ELECTRONPARAMAGNETICRESONANCESIGNAL FROMT F THEOXYGEN EVOLV INGCOMPLEXOFF 0HOTOSYSTEM))%FFECTO F F INHIBITIONOFOX F YGEN EVOLUTIONBYACETATE"IOCHEMISTRY¥ -AMEDOV & 3AYRE 24 AND 3TYRING 3  )NVOLVEMENT OF HISTIDINE  ON THE $ PROTEIN IN ELECTRONPROTON TRANSFER REACTIONSONTHEDONORSIDEOF0HOTOSYSTEM))"IOCHEMISTRY  ¥ -AUZERALL $ AND D -ALLEY -  ,IGHT INDUCE D D INCREASE IN

 mUORESCENCE YIELD IN #HL # ORELLA ISCOMPLETEINNANOSECONDS 0HOTOCHEM0HOTOBIOL¥ -C#ONNELL (-  )NDIRECT HYPERlNE INTERACTIONS IN THE PARAMAGNETIC RESONANCE SPECTRA OF AROMATIC FREE RADICALS * #HEM0HYS¥ -C#ONNELL (- ANDD #HESNUT $"  4HEORY OF ISOTROPIC HYPERlNE INTERACTIONS IN P ELECTRON RADICALS * #HEM 0HYS  ¥ -ETZ*' .IXON0* 2OGNER- "RUDVIG '7ANDD $INER"! $IRECTED D ALTERATIONOFF THE$POLYPEPTIDEOFF 0HOTOSYSTEM)) %VIDENCETHATTYROSINE ISTHEREDOXCOMPONENT : CON NECTINGTHEOXYGEN EVOLVINGCOMPLEXTOTHEPRIMARYELECTRON DONOR 0"IOCHEMISTRY¥ -EYER" 3CHLODDER% $EKKER*0AND7ITT(4 /XYGEN EVOLUTIONAND# D HLA))0  NANOSECONDD REDUCT D ION KINETICS INSINGLE mASHESASAFUNCT F IONOFF P("IOCHIM "IOPHYS!CTA  ¥ -ICHEL (AND D $EISENHOFER* F  2ELEVANCEOFT F HEPHOTOSYN THETICREACTIONCENTERFROMPURP F LEBACTERIATOTHESTRUCTUREOF 0HOTOSYSTEM))"IOCHEMISTRY¥ .IEUWENHUIS3!- (ULSEBOSCH2* 2AAP* 'AST0 ,UGTENBURG* AND(OFF!* 3TRUCTUREOFTHE9$ TYROSINERADICALIN0HO TOSYSTEM))$ETERMINATIONOFTHEORIENTATIONOFTHEPHENOXYL F HEMETHY H LENEGROUP* RING BYENANTIOSELECTIVE DEUTERATIONOFT !M #HEM 3OC¥ D $INER "!  !NALYSIS OF F WATER OXIDATION .IXON 0* AND 3 NECHOCY CYSTIS MUTANTS CONSTRUCTURED IN THE CYANOBACTERIUM 3Y SP0##"IOCHEM3OC4RANS¥ .OGUCHI4 )NOUE9 AND4ANG 8 3  3TRUCTURAL COUPLING BETWEENTHEOXYGEN EVOLVING-NCLUSTERANDATYROSINERESIDUE IN 0HOTOSYSTEM)) AS REVEALED BY &OURIER TRANSFORM INFRARED SPECTROSCOPY"IOCHEMISTRY¥ .ORDLUND0 3 D 3JOEBERG"-ANDD %KLUNDD (  4HREE H DIMENSIONAL STRUCTUREOFT F HE FREERA F DICAL PROTEINOFF RIBONUCLEOTIDEREDUCTASE D .ATURE  ¥ /-ALLEY 0*  (YBRID DENSITY FUNCT F IONAL STUDIES OF F THE OXIDATIONOFF PHENOL IMIDAZOLEHYDROGEN BONDEDD COMPLEXES ! MODELFORTYROSINEOXIDATION INOXYGENICPHOTOSYNTHESIS* !M#HEM3OC¥ /-ALLEY 0* AND "ABCOCK '4  %02 PROPERTIES OF IM MOBILIZED QUINONE CATION RADICALS AND THE MOLECULAR ORIGIN OFF SIGNAL )) IN SPINACH CHLOROPLASTS "IOCHIM "IOPHYS!CTA H  ¥ /-ALLEY 0* "ABCOCK'4AN K D 0RINCE2# 4HECATIONIC PLASTOQUINONERADICAL OFF THECHLOROPLASTWATERSPLITTINGCOMPLEX (YPERlNE SPLITTING FFROM A SINGLE METHYL GROUP DETERMINES THE %02 SPECTRAL SHAPE OF SIGNAL )) "IOCHIM "IOPHYS!CTA ¥ /NO4AND)NOUE9 $ISCRETEEXTRACTIONOFTHECALCIUMATOM FUNCTIONALFOROXYGENEVOLUTIONINHIGHERPLANT0HOTOSYSTEM)) BYASIMPLE LOWP(TREATMENT&%"3,ETT¥ /NO4AND D )NOUE9 2EMOVAL OFF CALCIUM BYP(TREAT MENTINHIBITS3TO3TRANSITION INTHEPHOTOSYNTHETICOXYGEN EVOLUTIONSYSTEM"IOCHIM"IOPHYS!CTA¥ 0AGE ## -OSER ## #HEN 8 ANDD $UTTON 0,  .ATURAL ENGINEERING PRINCIPLES OF ELECTRON TUNNELLING IN BIOLOGICAL OXIDATION REDUCTION.ATURE¥ 0ECORARO6, "ALDWIN-* #AUDLE-4 (SIEH7 9AND,AW.!  !PROPOSALFORWATEROXIDATIONIN0HOTOSYSTEM))0URE !PPL #HEM¥

#SVDF " %JOFS BOE 3 %BWJE
#SJUU 0ELOQUIN*- #AMPBELL+!AND D "RITT2$  -NPULSED %.$/2 DEMONSTRATES THAT THE 0HOTOSYSTEM)) @SPLIT %02 SIGNAL ARISES FROM A MAGNETICALLY COUPLED MANGANO TYROSYL COMPLEX*!M#HEM3OC¥ 2APPAPORT&AND,AVERGNE* 0ROTONRELEASEDURINGSUC CESSIVEOXIDATIONSTEPSOFT F HEPHOTOSYNTHETICWATEROXIDATION PROCESS 3TOICHIOMETRIES AND D P( DEPENDENCE "IOCHEMISTRY  ¥ 2APPAPORT & AND D ,AVERGNE *  #OUPLING OF F ELECTRON AND PROTONTRANSFER F INTHEPHOTOSYNTHETICWATEROXIDASE"IOCHIM "IOPHYS!CTA¥ 2APPAPORT& "LANCHARD $ESCE-AND,AVERGNE* +INETICS OFELECTRONTRANSFERANDELECTROCHROMICCHANGEDURINGTHEREDOX TRANSITIONS OF THE PHOTOSYNTHETIC OXYGEN EVOLVING COMPLEX "IOCHIM "IOPHYS!CTA¥ 2AZEGHIFAR F D -2 +LUGHAMMER#AND0ACE2* D  %LECTRON PARAMAGNETICRESONANCE KINETICSTUDIESOFF THE3STATESINSPINACH THYLAKOIDS"IOCHEMISTRY ¥ 2IGBY 3%* .UGENT*(!AND/-ALLEY0*  4HEDARKSTABLE TYROSINE RADICAL OF 0HOTOSYSTEM  STUDIED IN THREE SPECIES USING %.$/2 AND %02 SPECTROSCOPIES "IOCHEMISTRY  ¥ 2ODRIGUEZ )$ #HANDRASHEKAR 4+ AND "ABCOCK '4  %.$/2CHARACTERIZATIONOFF WATERRDEUTERIUMOXIDEEXCHANGE INTHE $: RADICAL INPHOTOSYNTHESIS)N"IGGINS* ED 0ROG RESSIN0HOTOSYNTHESIS 6OL  PP¥-ARTINUS.IJHOFF 0UBLI U SHERS $ORDRECHT 2OFFE F Y 2! +RAMER$- 'OVINDJEEAND3A D AYRE24 A ,UME NALSIDEHISTIDINEMUTATIONSINTHE$PROTEINOF0HOTOSYSTEM)) AFFECT DONOR SIDE ELECTRON TRANSFER IN # #HLAMYDOMONAS REIN HARDTII R "IOCHIM"IOPHYS!CTA¥ 2OFFEY2! VAN7IJK+* 3AYRE24AND3TYRING3B 3PEC TROSCOPICCHARACTERIZATIONOFTYROS F INE :IN HISTIDINEMUTANTS OFF THE$PROTEIN IN 0HOTOSYSTEM))03)) IN #HL # AMYD M DOMONAS R HARD REIN R TII )MPLICATIONFORT F HESTRUCTURALMODEL OFT F HEDONOR SIDEOFF 03 ))*"IOL #HEM¥ 2 FmE36 $ONNELLY $ "LUNDELL4,AND 2U D .UGENT*(! ! THREE DIMENSIONALMODELOFTHE0HOTOSYSTEM))REACTIONCENTER OF 0ISUMSATIVUM0HOTOSYNTH2ES¥ 3APHON3AND#ROFTS!2 0ROTOLYTICREACTIONSIN0HOTOSYS TEM))!NEWMODELFORTHERELEASEOFPROTONSACCOMPANYING F H#¥ THE PHOTOOXIDATIONOFF WATER:.ATURFORSC D +LUG $2 3CHILSTRA-* 2APPAPORT& .UGENT*(! "ARNETT#*AND  0ROTONHYDROGENTRANSFERA F FFECTST F HE3 STATE DEPENDENT MICROSECOND PHASESOF0 F  REDUCT D ION DUR D ING WATERSPLITTING "IOCHEMISTRY ¥ 3CHLODDER% "RETTEL+ 3CHATZ'(AND7ITT(4 !NALY SIS OF THE #HL A)) REDUCTION KINETICS WITH NANOSECOND TIME RESOLUTIONINOXYGEN EVOLVING0HOTOSYSTEM))PARTICLESFROM 3YNECHOCOCCUS AT  AND  NM "IOCHIM "IOPHYS!CTA  ¥ 3CHLODDER % "RETTEL + AND D 7ITT (4  2ELATION BETWEEN D REDUCT D IONKINETICSOFP F HOTOOXIDIZED D CHLOROPHYLL A)) MICROSECOND 0  ANDP D HOTOSYNTHETICWATEROXIDATION"IOCHIM"IOPHYS !CTA¥ 3HINKAREV 60 AND 7RAIGHT #!  /XYGEN EVOLUTION IN PHOTOSYNTHESIS&ROMUNICYCLETOBICYCLE0ROC.ATL!CAD3CI 53!¥ 3JÚDIN- 3TYRING3 +ERMARK" 3UN,AND(AMMARSTRÚM,  0ROTON COUPLED D ELECTRON TRANSFER F FFROM TYROSINE IN A

$IBQUFS

5Z 5 SPTJOFT
:; BOE :% TYROSINE RUTHENIUM TRIS BIPYRIDINE COMPLEX #OMPARISON WITHTYROSINE:OXIDATIONIN0HOTOSYSTEM))*!M#HEM3OC ¥ 3 3JÚDIN- 3TYRING3 !KERMARK" 3UN,AND(AMMARSTRÚM,  4HE MECHANISM FOR PROTON COUPLED ELECTRON TRANSFER F FROMT YROSINEINAMODELCOMPLEXANDD COMPARISONSWITH 9: OXIDATION IN 0HOTOSYSTEM))0HIL 4RANS2OY O AL3OC,ONDON "  ¥ 3TUBB U E*AND D VANDER$ONKK 7!  0ROTEINRADICALSINENZYME CATALYSIS#HEM2EV¥ 3VENSSON" %TCHEBEST# 4UFFERY0 +AN0V 3MITH*AND3TYRING 3 !MODELFORTHE0HOTOSYSTEM))REACTIONCENTERCORE INCLUDINGTHESTRUCTUREOFTHEPRIMARYDONOR0"IOCHEMISTRY  ¥ 3ZALAI 6! +UHNE ( ,AKSHMI +6 ANDD "RUDVIG '7  #HARACTERIZATION OF F THE INTERACTION BETWEEN MANGANESE AND TYROSINE:INACETATE INHIBITEDD 0HOTOSYSTEM))"IOCHEMISTRY ¥ 4 KAHASHI9 AND 4A D 3ATOH +  )DENTIlCATION OFF THE PHOTO CHEMICALLYIODINATEDAMINOACIDRESIDUEON$ PROTEININTHE 0HOTOSYSTEM)) CORE COMPLEX BY PEPTIDE MAPPING ANALYSIS "IOCHIM"IOPHYS!CTA¥ 4 4ANG83 #HISHOLM$! $ISMUKES'# "RUDVIG'7AND$INER F ITE DIRECTEDD MUTANTSOF "!  3PECTROSCOPICEVIDENCE FROMS CYSTIS 0##IN FAVORO F F ACLOSEINTERACTIONBETWEEN 3 NECHOCY 3Y HISTIDINEANDD REDOX ACTIVETYROSINE BOTH OFF POLYPEP TIDE$OFT F HE0HOTOSYSTEM))REACTIONCENTER"IOCHEMISTRY ¥ 4 4ANG8 3 2ANDALL$7 &ORCE$! $INER"!AND"RITT2$B -ANGANESE TYROSINEINTERACTIONINTHE0HOTOSYSTEM))OXYGEN EVOLVINGCOMPLEX*!M#HEM3OC¥ 4ANG 8 3 :HENG - #HISHOLM $! $ISMUKES '# AND $INER 4 F INTHELOCALPRO "! A )NVESTIGATIONOFF THE DIFFERENCES TEINENVIRONMENTSSURROUNDINGTYROSINERADICALS 9:s AND9$s IN 0HOTOSYSTEM))USING WILD TYPEANDD THE$ 4Y 4 R0HEMUTANT OF 3YNECHOCY CYSTIS "IOCHEMISTRY ¥ 4 4OMMOS#AN D "AABCOCKK '4  /XYGENPRODUCT D IONINNATURE ! LIGHT DRIVENMETALLORADICALENZYMEPROCESS!CC#HEM2ES ¥ 4 4OMMOS#AND"ABCOCK'4 0ROTONANDHYDROGENCU R RENTS IN PHOTOSYNTHETIC WATER OXIDATION "IOCHIM "IOPHYS !CTA  ¥ 4 4OMMOS# $AV IDSSON, 3VENSSON" -ADSEN# 6ERMAAS7AND 3TYRING3  -ODIlEDD %02SPECTRAOF THETYROSINE$RADI CAL IN0HOTOSYSTEM)) INSITE DIRECTEDD MUTANTSOF 3Y 3 NECHOCY CYSTIS SP0##)DENTIlCATIONOFF SIDECHAINSINTHE IMMEDIATE VICINITY OF TYROSINE $ ON THE $ PROTEIN "IOCHEMISTRY  ¥ 4OMMOS # 4ANG 8 3 7ARNCKE + (OGANSON #7 3TYRING 3 4 -C#RACKEN* $INER"!AND"ABCOCK'4 3PIN DEN SITYDISTRIBUTION CONFORMAT F ION AND HY H DROGEN BONDINGOFT F HE REDOX ACTIVE TYROSINE 9: IN 0HOTOSYSTEM)) FFROM MULTIPLE ELECTRON MAGNETIC RESONANCE SPECTROSCOPIES )MPLICATIONS FFOR PHOTOSYNTHETIC OXYGEN EVOLUTION *!M #HEM 3OC  ¥ 4OMMOS # -C#RACKEN * 3TYRING 3 AND "ABCOCK '4  4 3TEPWISEDISINTEGRATIONOFTHEPHOTOSYNTHETICOXYGEN EVOLVING COMPLEX*!M#HEM3OC¥

 4OMMOS# 3KALICKY ** 0ILLOUD D $, 7AND!*AN D D $UTTON0,  $ENOVOPROTEINSASMODELSOFRADICALENZYMES"IO CHEMISTRY¥ 5N3 "RUNEL, # "RILL4- :IMMERMANN* ,AND2UTHERFORD!7  !NGULARORIENTATIONOFTHESTABLETYROSYLRADICALWITHIN 0HOTOSYSTEM)) BY HIGH lELDD  '(ZELECTRONPARAMAGNETIC RESONANCE0ROC.ATL !CAD 3C 53!¥ 5N3 4ANG 8 3AND D $INER"!  '(Z HIGH lELD D %02 STUDY OFF TYROSINE $s AND D TYROSINE :s INMUTANTSOFF 0HOTOSYS TEM))"IOCHEMISTRY ¥ 6ASS ) AND 3TYRING 3  P( $EPENDENT CHARGE EQUILIBRIA BETWEEN TYROSINE $ AND THE 3 STATES IN 0HOTOSYSTEM)) %S TIMATIONOFRELATIVEMIDPOINTREDOXPOTENTIALS"IOCHEMISTRY  ¥ 6ERMAAS7&* 2UTHERFOR F D!7AN D D D(ANSSON/  3ITE DI RECTED D MUTAGENESIS IN 0HOTOSYSTEM))OFF THECYANOBACTERIUM CYSTIS SP0##$ONOR$ISATYROSINERESIDUE D IN 3 NECHOCY 3Y D 3CI53!¥ THE$ PROTEIN0ROC.ATL !CAD D "RUDVIG'7 -ECHANISMOF 6RETTOS*3 ,IMBURG*AND PHOTOSYNTHETICWATEROXIDATION#OMBININGBIOPHYSICALSTUDIES OF0HOTOSYSTEM))WITHINORGANICMODELCHEMISTRY"IOCHIM "IOPHYS!CTA¥ 7ARDEN*4 "LANKENSHIP2%AND3AUER+ !mASHPHOTOLYSIS 7 F HEPHYSIOLOGICAL %32STUDYOFF 0HOTOSYSTEM))3IGNAL))VF T DONORTO 0  "IOCHIM "IOPHYS!CTA¥ 7 7ARNC KE+ -C#RACKEN*AND D "ABCOCK K '4  3TRUCTUREOF THE 9$ TYROSINE RADICAL IN 0HOTOSYSTEM)) AS REVEALED BY ( %LECTRON3PIN%CHO%NVELOPE-ODU D LATION %3%%- SPECTRO SCOPICANALYSISOFHYDROGENHYPERlNEINTERACTIONS*!M#HEM 3OC¥ 7ARNCKE + 4ANG 8 3 4OMMOS # "ABCOCK '4 $INER "! 7 AND -C#RACKEN *  3TATIC SIDE CHAIN CONFORMATIONAL DISTRIBUTION INTHE 9: TYROSINERADICAL INMANGANESE DEPLETED D 9$ 0HOTOSYSTEM)))N-ATHIS0 ED 0HOTOSYNTHESIS&ROM ,IGHT TO "IOSPHERE 6OL  PP ¥ +LUWER !CADEMIC 0UBLI U SHERS $ORDRECHT 7 7EAVER%#  0OSSIBLE INTERPRETATIONOFT F HESLOW DECAYI A NG %02SIGNALINALGALSUSPENSIONS!RCH"IOCHEM"IOPHYS ¥ 7 7ESTPHAL+, 4OMMOS# #UKIER2)AND"ABCOCK'4 #ONCERTEDHYDROGEN ATOMABSTRACTIONINPHOTOSYNTHETICWATER OXIDATION#URR/PIN 0LANT"IOL¥ 8IONG* 3UB U RAMANIAN3AND'OV D INDJEE  ! KNOW K LEDGE BASED F HE0HOTOSYSTEM))REACTIONCENTER THREE DIMENSIONAL MODELOFT OF #HLAMYD M DOMONASREINHARD R TII0HOTOSYNTH2ESn 9 RKES#4 "ABCOCK'4AN 9ER K D D#RO F FTS!2  !TRIS INDUCE D D CHANGE IN THE MIDPOINT POTENTIAL OF : THE DONOR TO 0HOTO SYSTEM)) ASDETERMINEDBYTHEKINETICSOFTHEBACKREACTION &%"3,ETT¥ 9 9OCUM#&AND"ABCOCK'4 !MINE INDUCEDINHIBITIONOF PHOTOSYNTHETICOXYGENEVOLUTION!CORRELATION BETWEENTHE F IGNAL ))FAN F D 0HOTO MICROWAVEPOWERSATURATIONPROPERTIESOFS SYSTEM)) ASSOCIATED D MANGANESE&%"3,ETT¥ 7 TT ( 4 +ERN * &ROMME 0 +RAUSS . 3AENGER7 :OUNI! 7I AND D /RTH0  #RYSTALSTRUCTUREOFF 0HOTOSYSTEM)) FROM F 3YNECHOCOCCUS ELONGATUS AT  ¯ RESOLUTION .ATURE  ¥

$IBQUFS
 5IF
$BUBMZUJD
.BOHBOFTF
$MVTUFS
0SHBOJ[BUJPO
PG
UIF
.FUBM
*POT 7JUUBM
,
:BDIBOESB

.FMWJO
$BMWJO
-BCPSBUPSZ SZ
1IZTJDBM
#JPTDJFODFT
%JWJTJPO
-BXSFODF
#FSLFMF Z Z /BUJPOBM
-BCPSBUPSSZ #FSLFMFZ
$"

6 $ 4" 4VNNBSZ   * *OUSPEVDUJPO  ** 0YJEBUJPO 4UBUFT
PG
UIF
.BOHBOFTF   "
.BOHBOFTF
93BZ
"CTPSQUJPO
/FBS
&EHF
4QFDUSPTDPQZ
9"/&4   # .BOHBOFTF
,` 93BZ
&NJTTJPO 4QFDUSPTDPQZ 9&4   $ 0YJEBUJPO
4UBUF
$IBOHFT
%VSJOH
44UBUF
5SBOTJUJPOT    4 UP
4 5SBOTJUJPO    4 UP
4 5SBOTJUJPO    4 UP
4 5SBOT 5 JUJPO   % 4VNNBSZ
PG
0YJEBUJPO
4UBUF
"TTJHONFOUT   *** 4USVDUVSF
PG
UIF
.BOHBOFTF $MVTUFS  "
)FUFSPHFOFJUZ
JO
UIF
.O.O
%JTUBODFT
BOE
4USVDUVSBM
*NQMJDBUJPOT   # 44UBUF
5SBOTJUJPOT
BOE 4USVDUVSBM
$IBOHFT    4 UP
4 .-4
5SBOTJUJPO    4 UP
4 H 5SBOTJUJPO    4 UP
4 5SBOT 5 JUJPO    4 UP
4{ 4:;r 5SBOTJUJPO   4 UP
4 5SBOTJUJPO   *7 4USVDUVSBM
3PMF
PG
UIF $BMDJVN $PGBDUPS   "
.BOHBOFTF
&YUFOEFE
93BZ
"CTPSQUJPO
'JOF
4USVDUVSF
&9"'4 BOE
$BMDJVN   #
4USPOUJVN
&YUFOEFE
93BZ "CTPSQUJPO
'JOF
4USVDUVSF
&9"'4
BOE
$BMDJVN  
*TPUSPQJD
4BNQMFT   
0SJFOUFE
4BNQMFT   0SJFOUBUJPO
PG
UIF
.BOHBOFTF
$PNQMFY   $
$BMDJVN
&YUFOEFE
93BZ "CTPSQUJPO
'JOF
4USVDUVSF
&9"'4   7 4USVDUVSBM
3PMF
PG
UIF $IMPSJEF $PGBDUPS  7*

.FDIBOJTN
PG
8BUFS 0YJEBUJPO
BOE 0 &WPMVUJPO  "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 4HE LIGHT INDUCE D D OXIDATIONOFF WATERTO/ ISCATALYZED BY AFOUR MAN F GANESEATOMCLUSTERASSOCIATEDD WITH 0HOTOSYSTEM))03)) 4HISCHAPTERSUMMARIZESONGOING INVESTIGATIONSOFF THEOXIDATIONSTATE THESTRUCTUREAND THE ASSOCIATED COFAC F TORS CALCIUMANDCHLORIDEOFTHECATALYTIC-NCLUSTERUSING8 RAYANDELECTRONPARAMAG D

NETICRESONANCE%02 SPECTROSCOPY-AN Y GANESE+ EDGE8 RAY A SPECTROSCOPY Y +` 8 RAY A EMISSIONSPECTROSCOPY 8%3 AND EXTENDED D 8 RAYABSORPTION lNESTRUCTURE%8!&3 STUDIESHAVENOTONLYDETERMINEDD THEOXIDA

%MAIL6+9ACHANDRA ,",GOV O

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © +L + UWER!CADEMIC0U D BLISHERS0RINTED D IN4 4HE .ET . THERL R ANDS D

7JUUBM , :BD : IBOESB



TIONSTATESANDD STRUCTURAL FEATURES F BUTALSOCHANGESTHATOCCURINOXIDATIONSTATEOFF THE-NCLUSTERAND IN ITS / FORMATION#OMBINING STRUCTURALORGANIZATIONDURINGTHEACCUMULATIONOFOXIDIZINGEQUIVALENTSLEADINGTO Q F IONWITH8 RAY A DIFFRACT F IONSTUDIES ANDD CONSISTENTWITH THEAVAILABLE%02DATA WE 8 RAY A SPECTROSCOPY INFORMAT HAVESUCCEEDED INLIMITINGTHERANGEOF LIKELYSTRUCTURESOFT F HE-NCLUSTER%8!&3STUDIESATTHESTRONTIUMAND CALCIUM+ EDGESHAVEPROVIDEDEVIDENCETHATTHECATALYTICCENTERISA-N#AHETERONUCLEARCOMPLEX"ASEDON THE8 RAY A SPECTROSCOPY DATA MODELS FORT F HESTRUCTUREANDD AMECHANISMFOR/ F  EVOLUTIONAREPRESENTED *
*OUSPEVDUJPO -OSTOFTHE / INTHEATMOSPHERETHATSUPP U ORTSLIFE D CYANO ON EARTH IS GENERATED BY PLANTS ALGAE AND BACTERIABYTHEPHOTO INDUCE D D OXIDATIONOFWATERTO F DIOXYGEN (/ A / En (



4HISREACTION ISCATALYZED BY A MANGANESECALCIUM W CH SEQUENTIALLY STORES FOU F R COMPLEX -N#A WHI OXIDIZINGEQUIVALENTSIETHE3N STATES WHEREN n THATAREUSED D TOOXIDIZETWOMOLECULESOFWATE F R TOMOLECULAROXYGEN+OKETA K L FORAREV F IEW SEE*OLIOT  4HE-NCOMPLEXISPARTOFTHEMULTI PROTEIN 0HOTOSYSTEM)) 03)) ASSEMBLY #HAPTERS W CH CONTAINS THE REACTION CENTER INVOLVED n WHI IN PHOTOSYNTHETIC CHARGE SEPARATION #HAPTER D AN ANTENNA COMPLEX OF F CHLOROPHYLL MOLECULES AND #HAPTER 4HEASSEMBLYALSOCONTAINSCYTOCHROME B #HAPTER ANDA&E QUINONEELECTRONACCEPTOR COMPLEX#HAPTER  8 RAY ABSORPTION SPECTROSCOPY 8!3 3AUER ET AL 2OBBLEEETAL  ANDELECTRONPARA MAGNETICRESONANCE %02 $ISMUKESAND D 3IDERER  -ESSINGER ET AL B "RITT ET AL  STUDIES HAVE EMERGED AS THE PRIMARY METHODS TO PROVIDESTRUCTURALAND D CHEMICAL INFORMAT F IONABOUT THE OXYGENEVOLVINGCOMPLEX/%# AND DEVISEA WORKINGMODELFORITS-NCLUSTER8!3HASBEENUSED TOEXAMINETHESTRUCTURAL ENVIRONMENTOFF METAL IONS INMANYPROTEINS #RAMER 9ACHANDRA   4HEENERGYOFTHEINCOMING8 RAYPHOTONSANDOF THEOUTGOING mUORESCENCE ISSPECIlCTOTHE-NATOM !BBREVIATIONS R #HL n CHLOROPHYLL % n ST MOMENT VALUE %02n ELECTRON PARAMAGNETIC RESONANCE %8!&3 n EXTENDED F IN 8 RAYABSORPTIONlNESTRUCTURE&4n&OURIERTRANSFORM)0%n mECTIONPOINTENERGY-,3nMULTILINE%02SIGNAL-N n CATA LYTIC MANGANESE CLUSTER /%# n OXYGEN EVOLVING COMPLEX 03))n0HOTOSYSTEM))3NnSTATESOFF THE/%#FORN F    OR  8!.%3 n 8 RAY A ABSORPTION NEAR EDGE SPECTROSCOPY 8!3n8 RAYABSORPTIONSPECTROSCOPY8%3n8 RAYEMISSION SPECTROSCOPY9$OX nOXIDIZEDFORMOFTHEREDOXACTIVETYROSINE ON$ 9:s nOXIDIZEDFORMOFTHEREDOXACTIVETYROSINEON$

HENCE OTHERMETALSORTHEPROTEINMATRIXNORMALLY CO PURIlEDD WITHTHE/%# INA03))PREPARATION DO NOTINTERFERE%LEMENT SPECIlCITYANDAPPLICABILITYTO A SPECTROS NON CRYSTALLINESAMPLES HAVEMADE 8 RAY COPYAUSEFU F LTECHNIQUEFORPRO F BINGTHESTRUCTUREOF THE-NCOMPLEXINTHECOMPLICATEDENVIRONMENTOF 03)) WHI W CH HASMANY N COMPONENTSSUCHASNON H HEME IRON CYTOCHROME CHLOROPHY H LLS THAT INTERFERE F WITH Q OTHERTECHNIQUES4HESTRUCTURALSTUDIESCANALSOBE PERFORME F D ON CRYSTALS OR ON FFROZEN SOLUTIONS AND SEVERAL OFT F HE 3 STATEINTERMEDIATESMENTIONEDD ABOVE HAVE BEEN STABILIZED AS FROZEN SOLUTIONS AND STUDIED BY 8 RAY A SPECTROSCOPY4 Y HE DIFFERENTRE F GIONSOFF THE 8 RAYSPECTRUMPROVIDECOMPLEMENTARYINFORMAT F ION 8 RAYABSORPTIONNEAREDGESPECTROSCOPY 8!.%3 YIELD INFORMAT F IONABOUTTHEOXIDATIONSTATESANDD SITE F HEABSORBI R NGATOMWHIL W EEXTENDEDD 8 SYMMETRYOFT RAYABSORPTIONlNESTRUCTURE%8!&3 ISSENSITIVE TODISTANCES NUMBERS ANDD ATOMICNUMBEROFF ATOMS AROUNDD THEABSORBI R NGATOM4HEMORERECENTLYUSED TECHNIQUEOF8 RAYEMISSIONSPECTROSCOPY8%3 HAS F IONREGARDINGTHE IDENTITYOF ALSOPROVIDED INFORMAT F PLEX INTHEVARIOUS THEOXIDATIONSTATESOF-NCOM 3 STATESANDTHECHANGESACCOMPANYINGTHE3 STATE ADVANCE #RITICAL QUEST Q IONSRELATEDD TOTHE PROCESSOF PHO TOSYNTHETIC WATER OXIDATION ARE I 7HAT ARE THE OXIDATIONSTATES ANDD STRUCTURALCHANGES INTHE-N COMPLEXASTHE/%#PROCEEDSTHROUGH THE 3 STATE CYCLEANDII 7HATISTHEMECHANISMBYWHICHFOUR ELECTRONS ARE REMOVED FFROM TWO WATER MOLECULES BY THE -N COMPLEX TO PRODUCE D AN / MOLECULE %LECTRONPARAMAGNETICRESONANCE%02 AND8 RAY A BETWEENTHESE SPECTROSCOPYSTUDIESANDD THEINTERPLAY TWOMETHODSHAVEPROVIDEDD SIGNIlCANT INSIGHTS INTO THESTRUCTUREANDTHEMECHANISMOFTHE /%#4HIS CHAPTER FFOCUSES ON THE APPLICATION OFF 8 RAAY SPEC TROSCOPICTECHNIQUESTORESO Q LVESTRUCTURAL QUEST Q IONS REGARDINGTHE-NCLUSTERINTHE/%# WITHEMPHASIS ONTHERESULTS FROMOUR F LABORATORY-AN Y NY EXCELLENT REVIEWSAREAVAILABL A E FORCOMPRE F HENSIVESURVEYSO I F THE FT -NCOMPLEXANDTHE/%# %02 AND8!3LITERATURE $EBUS 2UTHERFOR F D ETAL "RITT 

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ 9ACHANDRA ET AL   0ENNER (AHN  9 2OBBLEEETAL   **
0YJEBUJPO
4UBUFT
PG
UIF
.BOHBOFTF 4WOKEYQUESTIONSFORT F HEUNDERSTANDINGOFP F HOTO SYNTHETICWATEROXIDATIONAREI DOALLFOUROXIDIZ ING EQUIVALENTS NECESSARY TO OXIDIZE WATER TO / ACCUMULATEONTHEFOUR-N F IONSOFT F HE/%# ORDO SOMELIGAND CENTEREDOXIDATIONSTAKEPLACEBEFORE THEFORMAT F IONAND D RELEASEOFF / DUR D INGTHE 3 A ;3= A 3 TRANSITIONANDII WHATARETHEOXIDATIONSTATE ASSIGNMENTSFORTHE-NDURING3 STATEADVANCEMENT D EMISSIONSPECTROSCOPYOF-N F 8 RAY A ABSORPTIONAND D ANSWERSTOTHESETWO ALONGWITH %02HAVEPROVIDED PIVOTALQUESTIONS Q  "
.BOHBOFTF 9 9 3BZ
"CTPSQUJUJPO /FBS
&EHF 4QFDUSPTDPQZ
9"/&4

Z 8 RAYABSORPTIONNEAR EDGESPECTROSCOPY 8!.%3 ISTHEMOSTWELL KNOWNOFTHE8 RAYTECHNIQUESAND HAS BEENEXTENSIVELY USED D TO INVESTIGATETHEOXIDA TIONSTATESOFF REDOX ACTIVEMETALSIN METALLOPROTEIN ACTIVESITES8!.%3RESULTSFROMTHEEXCITATIONOFA SELECTRON+ SHELL TOAHIGHER BOUND D ORRBITAL &IG ,EFT E 4HE HIGHERTHEOXIDATIONSTATEOFF THEMETAL THE MORE POSITIVE THE OVERALL CHARGE OF THE ATOM AND HIGHERENERGY ISREQUIRED D TOEXCITEANELECTRONOUTOF ANORBI R TAL 3HULMANETAL  4HE lRST FORMA F LLY ALLOWEDELECTRICDI D POLETRANSITIONISTHESAPTRAN SITION$UETOTHESIZEOFF THEPORBI R TAL ITOVERLAPS WITHP ORBI R TALSOFT F HE LIGANDS EITHERTHROUGH mOR m / BONDING #ONSEQUENTLY THIS TRANSITION IS SENSITIVE TOTHEOXIDATIONSTATEAND D THELIGAND D ENVIRONMENTOF THEMETAL&ORCERTAINSYMMETRIESAROUND D THEMETAL THEFORMALLYELECTRIC DIPOLEFORBIDDENSAD D TRAN SITIONCAN BEOBSERVED ARROWPOINTSTO IT IN&IG - DDLE WHI -I W CH ISEXPANDED INTHEINSET OCCURRINGAT ALOWERENERGYTHANTHEMAINEDGETRANSITIONS2OE ETAL  4HISTRANSITION ISDUETOM D IXINGOFMETA F L D D AND PORRBITALS AND D GIVES INFORMAT F IONABOUTTHE LIGANDASWELLASABOUTTHEOXIDATIONSTATEANDSYM METRY OFF THE METAL COMPLEX 7ESTRE ET AL   4OINCREASETHESENSITIVITYOFF 8!.%3 ABSORPTION IS DETECTEDASANEXCITATIONSPECTRUMBYMEASURINGTHE +_ mUORESCENCEPTOS OFF THE-NATOMS*AKLEVIC ETAL   4HEPIONEERINGAPPLICATIONOF-N8!.%3TO03)) WASPERFORME F D BY +IRBY R ETAL  ONCHLOROPLASTS

 ANDD 'OODINETAL  ON03))PREPARATIONS INTHE EARLYS4HESESTUDIES ALTHOUGHEXTREMELYDIF F lCULTBECAUSEOFT F HELOW-NCONCENTRATION SHOWED THATTHE-N+ EDGE FROMT F HE3 STATEISSHIFTE F D TO HIGHERENERGYRELATIVETOTHE-N+ EDGEFROMTHE3 STATE$RAMATICIMPROVEMENTSINDETECTORTECHNOLOGY ANDD CRYOSTATCOOLINGCAPABILITIESHAVEMADE8!.%3 EXPERIMENTSPRACTICALUSINGCONCENTRATED03))PREPA RATIONS-C$ERMOTTETAL 'UILESETAL A $E2OSEETAL  ANDD THECOLLECTIONOFF 8!.%3 SPECTRAFROMDILUTE SINGLE mASHSATURABLE03))SAM PLESISNOWACHIEVABLE4HREE DIFFERENT F GROUPSHAVE INVESTIGATEDD THEOXIDATIONSTATESOFF -NFORT F HE3 3 3 AND3 STATESUSING 8!.%3ONSINGLE mASHSATU RABLE03))SAMPLES"ASEDD ONSHIFTS F INTHEABSORPTION K HEREOF 2OE F LOFSETA F L  PROPOSED EDGE ORLACKT THAT-NISOXIDIZEDDURINGTHE3 A 3 AND3 A 3 TRANSITIONS BUT ISNOTOXIDIZED DUR D INGTHE3 A 3 TRANSITION)NCONTRAST /NOETAL ANDD )UZZOLINO ETAL INTERPRETTHEIR8!.%3RESULTSTOINDICATE THAT-NISOXIDIZED DUR D ING EACH3 STATETRANSITION ALTHOUGH /NOETAL  REPORTEDD NOINDEPENDENT 3 STATEDETERMINATIONFORTHEIRSAMP M LESAND)UZZOLINO ETAL  HADD SIGNIlCANT3 STATE INHOMOGENEITY INTHEIRSAMPLES(OWEVER IMPROVEDD 8!.%3 DATA F FOR3 STATETRANSITIONS HAVESHOWNA -N+ EDGESHIFT OFON F LY E6 FORT F HE 3 TO 3 TRANSITION /NOETAL F LOFSETA F L  INDICATING  4HERESULTSOF2OE THEABSENCEOF-NCENTEREDOXIDATIONDURINGTHE 3 TO3 TRANSITION HASBEENREPRODUCE D D BY -ESSINGER ETAL USING BOTH 8!.%3&IG -IDDLE AND 2IGHT AND-N 8%3STUDIESSEEBELOW  # .BOHBOFTF ,` 9 9 3BZ
&N & JTT J JPO J 4

4QFDUSPTDPQZZ 9&4 4HERECENTSTUDIESBY-ESSINGERETAL HAVE COMBINED %02 8!.%3 AND 8%3 STUDIES TO AD DRESS THE QUESTIONS OF F OXIDATION STATE ASSIGNMENTS D INGTHE3STATECYCLE)NCONTRASTTO ANDD CHANGES DUR 8!.%3 +` 8%3DETECTSTHE8 RAYEMISSIONFROM THERELAXATIONOFF APELECTRONTOAS HOLE WHI W CH IS CREATED BYEXCITATIONOFF ASELECTRONINTOTHECON TINUUM&IG ,EFT F )NASIMPLIlEDMODEL TWOlNAL STATESEXISTDUETOACONSTRUCT D IVE+`  ORDESTRUCTIVE +`g SPIN EXCHANGEINTERACTIONBETWEENTH T EUNPAIRED ELECTRONSINTHEPANDDORBITALS4SUTSUMIETAL  4HE MAGNITUDE OFF THE EXCHANGE INTERACTION DEPENDS ON THE NUMBER OF F UNPAIREDD ELECTRONS IN THEDORBITAL)NTHEHIGHSPINCASE INCREASINGTHE OXIDATIONSTATEOFT F HEMETAL DECREASESTHENUMBEROF



7JUUBM , :BD : IBOESB

&IG ,EFT !SCHEMATICDIAGRAMFORTHEEXCITATIONANDEMISSIONPROCESSESINVOLVEDIN-N8!.%34HEWEAKSTODTRANSITIONIS & DIPOLE FORBIDDENANDTHEPRINCIPALTRANSITIONISFROMSTOAMOLECULARORBITALWITHPCHARACTER4OENHANCESENSITIVITY THE8 RAYABSORP TIONSPECTRAARECOLLECTEDD ASEXCITATIONSPECTRAUSING-N+_ PTOS mUORESCENCEDETECTION -IDDLE4OP O -N+ EDGE8!.%3SPECTRA OF mASH ILLUMINATED03))SAMP D LES0URE3 STATESPECTRABOTTOM WEREOBTAINED FROMT F HE mASHSPECTRATOP BY DECONVOLUTIONUSING D TRANSITION FORT F HE3 n3 STATES IS SHOWNINTHE INSET 4HE INmECTION MULTILINE%02SPECTRA4HEPRE EDGEREGIONPRINCIPALLY AS A D POINTENERGY )0% CHANGESFROM3 F U STANTIAL BUTAREMUCHSMALLERFORT F HE 3 TO 3 TRANSITION -I - DDLE  TO 3 AND 3 TO 3 TRANSITIONSARESUB "OTTOM3 STATE-N8!.%3DIFFERENCESPECTRAS F HOWTHATEACH TRANSITIONISUNIQUE4HEHORIZONTAL DASHED LINESSHOWTHEZEROVALUEFO F R EACHDIFFERENCESPECTRUM!DAPTEDFROM-ESSINGERETAL AND6ISSERETAL  2IGHT 3ECONDDERIVATIVESOFTHENORMALIZED D PURE3 STATE-N+ EDGESPECTRAOFTHE-N CLUSTEROF03))&ORCLARITYAVERTICALDASHEDLINEHASBEENDRAWNATTHEINmECTIONPOINTENERGY )0% OFTHE3 STATE4HECHANGEIN )0% 6 INE6 FOREACH3 STATEADVANCEISGIVENATTHERIGHTSHOWSTHESIGNIlCANTSHIFTSFORTHE3 TO3 AND3 TO3 TRANSITIONSBUTISMUCHSMALLERFORTHE3 TO3 TRANSITIONINDICATINGTHATA-NCENTEREDOXIDATIONISUNLIKELYFORTHE3 TO3 TRANSITION&ORREFERENCE T F HE+ EDGESPECTRUMOFF THE3 STATE ISPLOTTED D ASWELL DASHED LINE !DAPTED FROM-ESS F INGERETAL 

UNPAIRED D ELECTRONS CONCOMITANTLY THE SPIN EX CHANGE INTERACTION DECREASES!CCORDINGLY T Y HE+`  TRANSITIONSHIFTSTOA F HIGHERAND D THE+`|TRANSITION SHIFTSTOALOWERENERGY0ENGETAL  #OMPARED TOTHEPORBI R TALS THEPORBI R TALS HAVELESSOVERLAP WITHTHE LIGANDD ORRBITALS BECAUSETHEYARESMALLERAND MOREBURIEDWITHINTHEELECTRONICSHELLS4HEREFORE +` 8%3 ISLESSSENSITIVETOTHE LIGAND D ENVIRONMENT D TO 8!.%34HE +`  TRANSITION ISBETTER COMPARED D ANTHE +`| TRANSITION DUE TO A DIFFE F RENCE RESOLVEDTH INRELAXATIONPROCESSES(ENCE THE+`  TRANSITION ISUSED D ASA INDICATOROFF THEOXIDATIONSTATEOFF THE METAL ! MORE ACCURATE VIEW ON +` 8%3REQUIRESTHE Q LIGAND lELD D MULTIPLETFORMA F LISM4HISINDICATESTHAT THERE ISSOMEDEPENDENCEOFT F HELIGAND D ENVIRONMENT ON +` 8%3SPECTRA 3HOWNIN&IG -IDDLE AND INSETARETHE +` EMIS

SIONSPECTRAOF-N)6 / -N))) / AND-N)) / WHICH ILLUSTRATETHE FEATURESO F F +` EMISSIONSPECTRA ANDD THEIRSENSITIVITYTOTHEOXIDATIONSTATESOF-N!S F THE OXIDATIONSTATEOF-NINCREASESFROM-N)) TO -N))) TO-N)6 FEWERUNPA F IREDD D VALENCEELEC TRONSAREAVAILABLETOINTERACTWITH THEP HOLETHUS THEMAGNITUDEOFTHEPnDEXCHANGEINTERACTION BECOMESSMALLER LEADINGTOADECREASEINTHE+`| n+`  SPLITTING4HECONSEQUENCEISTHAT IFONE F FO CUSESONLYONTHEMOREINTENSE+`  EMISSION PEAK IT SHIFTSTO F LOWERENERGY AS-NISOXIDIZED7HEREASTHE +`EMISSIONSPECTRUM ISSENSITIVETOOXIDATIONSTATE THROUGHAPnDEXCHANGEINTERACTION THETECHNIQUE OFF 8!.%3SPECTROSCOPY ISSENSITIVETOOXIDATIONSTATE F HANISMCORE HOLESHIELDING THROUGH A DIFFERENTMEC EFFECTS4HISISWHY UPON-NOXIDATION +`  EMIS SION SPECTRA SHIFT F TO LOWER ENERGY Y WHILE 8!.%3

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ



&IG  +` SPECTRAOFF -N)) ))) AND )6 OXIDES ,EF &I EFT %NERGY LEVEL DIAGRAMSHOWINGTHEABSORPTIONSTOCONTINUUM AND D EMIS SIONPROCESSESPTOS INVOLVEDIN+` -N8%3)NASIMPLIlED D MODEL TWO lNAL SPINSTATESEXISTWITHEITHERACONSTRUCTIVE +`  ORDESTRUCTIVE+`g SPINEXCHANGEINTERACTIONBETWEENTHEUNPAIREDPANDDELECTRONS4HEMAGNITUDEOFTHEINTERACTIONDEPENDS ONTHENUMBEROFUNPAIREDDELECTRONS WHICHISRELATEDTOTHEOXIDATIONSTATEOFTHEMETAL!DAPTEDFROM-ESSINGERETAL AND 6ISSERETAL  -IDDLE #HANGESINTHE +`SPECTRUMDUETO-NOXIDATION4HEINSETSHOWSAPICTORIALREPRESENTATIONOFTHE +` mUORESCENCETRANSITION FFOR EACH OFTHE OXIDES +` 8%3ARISESFROMTHEEMISSIONOFAPELECTRONTOSHOLE WHICH ISFORMEDFOLLOWING G T 4HE3 3 3 3 3 3 AND 3 3 DIFFERENCE F HIGH RESOLUTION-N8 RAY+` EMISSIONSPECTRA4HE DERIVATIVE 8 RAYABSORPTION 2IGH SHAPESINDICATESHIFTS F INTHEENERGYOFT F HESPECTRA BETWEENTHE 3 AND 3 AND 3 AND 3 SPECTRA INDICATING OXIDATIONSOFF -N4HE3 3 DIFFERENCESPECTRA F DEMONSTRATESTHESIMILARITY OFT F HESPECTRA SUGGESTINGTHATTHISADVANCE DOESNOTINVOLVEA-NCENTERED D OXIDATION F INGERETAL  !DAPTED FROM-ESS

SPECTRASHIFTTO F HIGHERENERGY 5SINGAST MOMENTANALYSIS THEPOSITIONOFF THE MAIN +`  PEAKHASBEENCALCULATEDFOREACH3 STATE "ASED D ONTHESHIFTS OR F LACKT K HEREOF O F F THESTT MOMENTS % ITWASCONCLUDED D THAT-N ISOXIDIZED DUR D ING THE3 TO3 AND3 TO3 BUTNOTOXIDIZEDDURINGTHE 3 TO 3 TRANSITION4HERESULTS SHOWNASDIFFERENCE F SPECTRAIN&IG 2IGHT SHOWASHIFTINENERGYBETWEEN T THE3 AND3 STATESPECTRAANDBETWEENTHE3 AND3 STATESPECTRAANDSEENINTHEDERIVATIVE SHAPEDDIF F F FERENCESPECTRA(OWEVER T HEREISVERY LITTLECHANGE BETWEENTHE3AND3 STATES ASSEENINTHEDIFFERENCE SPECTRUM -ESSINGERETAL   $
0YJEBUJPO
4UBUF
$IBOHFT
%VSJOH 44UBUF 5 5SBOT JUJ UJPOT "ESIDESDETERMININGWHETHER-NISOXIDIZEDORNOT D INGTHE 3 STATETRANSITIONS BOTH8!.%3AND DUR D 8%3 ALSOALLOWONETOASSIGNTHEOXIDATIONSTATESOFT F HE -NATOMS4HISISOFTENDONE BYCOMPARISONTO-N MODEL COMPOUNDS

 4 UP
4 5SBOT 5 JUJUJPO -N+ EDGE 8!.%3SPECTRA&IG -IDDLE AN A D 2IGHTT ANDD THE+`  EMISSIONSPECTRA &IG 2IGHT I INDICATE F THEPRESENCEOFF -NOXIDATIONFROM-N ))) )6 TO -N))) )6 DURINGTHE3 TO 3 TRANSITION2OELOFSET W ERETHE 3 STATE AL -ESSINGERETAL  WH ISCHARACTERIZED BYTHEMULTILINE -,3 ORTHE G G G %02 SIGNAL #OLE ET AL  4HIS CONCLUSION IS CORROBORATED BY SEVERAL OTHERSPECTROSCOPICSTUDIES 3AYGINAND7 D 7ITT +RETSCHMANNETAL  3TYRING AND 2UTHERFORD  %VELO ET AL  $EKKER 3HARP  AND ISALSOCONlRMED BY%02SPECTROSCOPICMEASUREMENTS3 STATE%02 MULTILINESIGNALSIMULATIONS(ASEGAWAETAL  (ASEGAWAETAL  AND -N%.$/2SPECTROS COPYONTHE3 STATE 0ELOQUINETAL  AREMOST CONSISTENTWITHTHE OXIDATION STATES OF -N))) )6  HOWEVER OTHERSIMULATIONS :HENG ANDD $ISMUKES F IDATIONSTATESOFF -N))) )6 INTHE  PREFEROX 3 STATE )F F THE 3 STATE IS PROPOSEDD TO BE EITHER IN THE

7JUUBM , :BD : IBOESB

 -N))) )6 OR THE -N))) )6 OXIDATION STATES THENTHE OXIDATION STATES OF-NINTHE 3 STATE MUST BEEITHER-N))) )6 OR-N))) WHI W CHARE BOTH ONE ELECTRONREDUCT D IONSFROMT F HEOXIDATION STATE PRO POSALSFORTHE3 STATE!LTHOUGHTHEOXIDATIONSTATE F HE 3 STATE HAS BEENSUGGESTED BY A OFF -N))) FORT F FEWGROUPS :HENGAND D $ISMUKES +UZEK K AND 0ACE #HAPTER !HRLINGETAL THE 8!.%3 SPECTRUMORITS NDD DERIVATIVEOFTHE3 STATE &IGS -IDDLE AND2IGHT D SHOWSTHATITSEDGESHAPEISUN LIKETHEEDGESHAPEOBSERVEDFOR-N))) COMPLEXES 7HENTHE 8!.%3SPECTRUMOFT F HE3 STATE ORITS ND DERIVATIVE ISlTUSING-N))) AND-N)6 MODEL COMPOUNDS ITCANNOTBElTWELLUSINGONLY-N))) MODEL COMPOUNDS 2IGGS ET AL 9ACHANDRA ET AL   &URTHERMORE INSPECTION OFF THE ONLY AVAILABLE SET OF TETRANUCLEAR -N COMPLEXES WITH ALL /LIGATION FOUR F DISTORTED D -N))) )6 CUB U ANES SHOWS8!.%3 INmECTIONPOINTENERGY )0% VALUES AT^E6FORTHESECOMPLEXES#INCOETAL  CONSISTENTWITHTHE3 STATE8!.%3)0%OF F E6AND LOWERTHANTHE3 STATE8!.%3)0%OF F E64HESERESULTSARECONSISTENTWITHTHECONCLUSION 6 THAT THE OXIDATION STATES OFF -N IN THE 3 STATE ARE -N))) )6 NOT-N)))  !NOXIDATION STATEASSIGNMENTOF-N))) )6 IS ALSOCONSISTENTWITH RECENT +` 8%3EXPERIMENTSON THE3 STATE)NlTTINGTHE+`  EMISSIONSPECTRUMFOR F THE3 STATE "ERGMANNETAL FOUNDTHATTHE BESTlTTOTHEEXPERIMENTAL DATAWASOBTAINED D USING Q L AMOUNTS OF F THE +`  EMISSION SPECTRA FROM F EQUA -N))) AND-N)6 MODELCOMPOUNDSTHElTWAS SIGNIlCANTLYWORSE IFON F LY-N))) ORONLY-N)6 WAS USED FFOR THE lT "ERGMANN ET AL   )T IS THEREFOREMOSTLIKELYTHATTHEOXIDATIONSTATESFOR-N D -N))) )6 IN ARE-N))) )6 INTHE3STATEAND THE3 STATE  4 UP
4 5SBOT 5 JUJUJPO 4HE -N + EDGE 8!.%3 SPECTRA FOR THE 3 TO 3 TRANSITION DISPLAY A ED IN &IG  -IDDLE AND D THE ND DERIVATIVES &IG 2IGHT G SHOWACLEARSHIFTO F F  E6ASDETERMINEDBYTHEND DERIVATIVE)0%VALUES IN THE8!.%3EDGEPOSITION)NADDITION THE 33 +` 8%3DIFFERENCESPECTRUM S F HOWNIN&IG 2IGHT G IS DERIVATIVE SHAP A ED WHICHISINDICATIVEOF-NOXIDATION D INGTHISTRANSITION"ECAUSETHE3 STATE HAS-N DUR D ABOVE OXIDATIONSTATESOFF -N))) )6 AS DETAILED THECHANGES IN THE 8 RAY SPECTROSCOPIC DATA COULD BE DUETOE D ITHERA-N)) TO-N))) OXIDATIONORA

-N))) TO-N)6 OXIDATION 4HE lNDING THAT THE 3 3 8!.%3 DIFFERENCE W ATDIFFERENT F F FROM SPECTRUM&IG -IDDLE ISSOMEWH THE3 3 DIFFERENCESPECTRUMSUGGESTST F HATTHE3 TO 3 TRANSITIONMOSTLIKELYREmECTSA-N)) TO-N))) F HE8!.%3EDGEIS DIFFEREN F T OXIDATION4HESHAPEOFT F HE3 STATEAND 3 STATESPECTRATHISISSHOWN IN FORT PARTBYTHEPEAKATE6INTHE NDDDERIVATIVEOF THE3 STATE8!.%3SPECTRUM&IG 2IGH I T 4HIS IS SUGGESTIVE OFF THE PRESENCE OFF -N)) BASEDD ON COMP M ARISONTOTHENDD DERIVATIVESOF-N)) CONTAINING MODEL COMPOUNDS4HESIGNIlCANTLY LARGERSHIFT F IN THEND DERIVATIVE)0%VALUESFORTHE 3 TO3 TRANSITION COMPAREDTOTHE 3 TO 3 TRANSITION IS ALSO INDICATIVE FFOR A -N)) TO -N))) OXIDATION 4HIS IS BASED F HOMOLOGOUSSETSOFMO F DEL ONTHE lNDING THAT FOR COMPOUNDS THESHIFTSINTHE)0%VALUEFOR-N)) TO -N))) OXIDATIONSAREUSUALLY LARGERTHANTHOSESEEN F FOR-N ))) TO-N)6 OXIDATIONS /THERSPECTROSCOPICSTUDIES3AYGINAND7ITT  + +RETSC HMANNETAL 3TYRINGANDD 2UTHERFOR F D D  %VELOETAL  'UILESETAL A $EKKER 3HARP -ESSINGERETAL A B¯HRLING D ING ETAL  CONCURTHAT-NOXIDATIONOCCURSDUR THE3 TO3 TRANSITION4HEGREATERSPECTRAL WIDTHOF THE 3 STATE %02 MULTILINE SIGNAL COMPARED TO THE 3 STATE%02MULTILINESIGNAL HAS BEENATTRIBUTEDD TO THEPRESENCEOFF -N)) INTHE3 STATE-ESSINGERET AL A B¯HRLINGETAL  WHICHSUGGESTSA -N)) TO-N))) OXIDATIONFORT F HE 3 TO 3 TRANSI TION4HEPRESENCEOFF -N)) INTHE3 STATEMAYALSO EXPLAINWHY 9$OX CAN OXIDIZETHE 3 STATETOTHE3 STATE BUTNOTTHE3 STATETOTHE3 STATE 4HE +` 8%3AND8!.%3 D DIFFERENCES F PECTRA IN ADDITIONTOOTHERSPECTROSCOPICDATAARECONSISTENT WITH THEASSIGNMENTOFF THE 3 TO 3 TRANSITIONASA -N)) TO-N))) OXIDATION4HEREFORE T F HEOXIDA TIONSTATESOF-NINTHE 3 STATEARE PROPOSEDTOBE -N)) ))) )6 WHI W CH CHANGE UPON -N OXIDATION TO-N))) )6 INTHE3 STATE ALTHOUGH THEOXIDA TION STATES OF -N))) )6 FORTHE3 STATE CANNOT BE UNEQUIVOCALLY EXCLUDED 
4
UP 4 5SBOTJUJPO 4HE MOST CONTROVERSIAL 3 STATE TRANSITION HAS BEEN F ON THE 3 TO3 TRANSITION -OSTDEBATE HAS FOCUSED W ETHER THIS TRANSITION IS A -N CENTERED /NO ET WH AL )UZZOLINOETAL  ORLIGAND CENTERED 'UILESETAL B2OELOFSETAL -ESSINGER ETAL  OXIDATION)NADDITION AREDOX ISOMERISM

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ BETWEEN-NAND LIGANDSHASBEENPROPOSED FORT F HE 3 STATE2ENGER  )F-NWERETOBEOXIDIZED D INGTHE3 TO3 TRANSITION THEOXIDATIONWOULD DUR HAVETOBEA-N))) TO-N)6 TRANSITION!-N)6 TO-N6 TRANSITIONISUNLIKELYTOOCCURWITH-N))) STILLPRESENT INTHECOMPLEX GIVENTHEPROPOSED D RE ACTIVITYOFF THE-N6 ION-OREOVER -N6 HASA VERYDISTINCTIVE-N8!.%3SPECTRUMTHATHASNOT BEENOBSERVED IN03))PREPARATIONS69ACHANDRA D THAT THE UNPUBLI U SHED 4HUS IT WOULD BE EXPECTED 8!.%3 AND+` 8%3DIFFERENCESPECTRA ASWELLAS THEOBSERVED D SHIFTS F INTHE8!.%3)0%VALUESAND D THE +` EMISSIONSPECTRAST MOMENTVALUES % WOULD BEESSENTIALLYIDENTICALTOTHE3 TO 3 TRANSITION WHERE A-N))) TO-N)6 OXIDATIONALSOOCCURS(OWEVER F HE ASDESCRIBED BELOW THESPECTROSCOPICRESULTS FORT 3 TO3 TRANSITIONARECOMPLETELYDIFFERENTFROMTHOSE OFF THE 3 TO3TRANSITION 4HE 8!.%3 RESULTS FFROM &IG  PROVIDE STRONG SUPPORT FOR A LIGAND BASED OXIDATION OF THE /%# OCCURRING DUR D INGTHE 3 TO3 TRANSITION BASED D ONTHE SMALL E6 SHIFT F INTHE8!.%3 ND DERIVATIVE)0% VALUES4HE FACT THAT THE 3 3 8!.%3 DIFFERENCE SPECTRUM ISSIGNIlCANTLY DIFFERENT F F FROMT HE3 3 DIF F F FERENCESPECTRUM &IG -I - DDLE IS INCONSISTENTWITH A-N))) TO-N)6 OXIDATIONOCCURRINGDURINGTHE 3 TO3 TRANSITION)NSTEAD T D HE3 3 8!.%3 DIFFER F ENCESPECTRUMSHOWS HOWTHESHAPEOFT F HE8!.%3 EDGECHANGESBETWEENTHE3 AND 3 STATES WHICHIS CONSISTENTWITH A LIGAND BASED D OXIDATION 4HEREFORE F BASED D ONTHE8!.%3 DATA IT ISREASON ABLETOSUGGESTTHAT ALIGANDRADICALISFORMEDINTHE 3 STATEIN LIEUOFF -NOXIDATIONTHISWILL BEDENOTED BY-N))) )6 s4HISINTERPRETATIONISREINFORCE F D BY THE +` EMISSIONSPECTRAANDESPECIALLY THE+` 8%3 DIFFERENCESPECTRA F F FROM& IG 2IGHT I 4HEDIFFERENCE F SPECTRA SHOW THAT THE DERIVATIVE SHAPED DIFFERENCE F SPECTRUMTHATISEXPECTEDIF-NISOXIDIZED ASINTHE 3 TO3 AND D 3 TO 3 TRANSITIONS ISABSENTINTHE3 TO 3 TRANSITION)NADDITION THE%VALUEOFF  E6FORTHE3 STATEARGUESAGAINST-NOXIDATIONDUR INGTHE3 TO 3 TRANSITION BECAUSE IT IS INCONSISTENT WITHA-N)6 OXIDATIONSTATE WHI W CH ISREQUIRED F FORTHE 3 STATEIF-NISOXIDIZEDDURINGTHE3 TO 3 TRANSITION#OMPARISONOFT F HE3 STATE%VALUETO THE%VALUESFROM F DIFFERENTMONOMER F IC DIMERIC TRIMERIC ANDTETRAMERIC-N)6 MODELCOMPOUNDS WITH DIFFERENT F LIGANDS INCLUDING #Ln SHOWSTHATTHE 3 STATE%VALUE ISHIGHERTHANTHE%VALUEFOR F ANYOFTHE-N)6 COMPOUNDSSTUDIED)TISDIFlCULT TO EXPLAIN THIS RESULT UNLESS -N))) IS STILL PRES

 ENTINTHE3 STATE WHI W CHMEANSTHAT BASEDD ONTHE -N))) )6 REDOXSTATESDERIVEDFORTHE3 STATE A -N BASEDD OXIDATIONCANNOTOCCURDUR D ING THE3 TO 3 TRANSITION4HE %8!&3 STUDIES FROMT F HE 3 STATE DESCRIBEDBELOWALSOSUPPORTTHISASSIGNMENT U % 4VNNBSZ
PG
0YJEBUJPO
4UBUF
"TTJHONFOUT 4HE -N + EDGE ENERGY ANDD SHAPE DO NOT DEPEND SOLELYONTHEOXIDATIONSTATEOFF THE-NATOMS BUTALSO ONTHELIGANDENVIRONMENT4HISISCAUSEDBYMIXING OFT F HELIGANDD AND THE-NATOMICORBI R TALS)TISPOSSIBLE THATACHANGE INSTRUCTUREAND LIGANDD ENVIRONMENTWILL INmUENCETHEMAIN EDGEENERGYANDSHAPE4HEREFORE CAREMUSTBEEXERCISEDD WHENCORRELATING THE-N+ EDGEENERGIESTO-NOXIDATIONSTATESWITHOUTTAKING INTOACCOUNTTHENATUREOFTHELIGANDENVIRONMENT W ENSETSOFF COMPOUNDS 4HISISESPECIALLYAPROBLEMWH WITH T DIFFERENT F LIGANDSTRUCTURES D DIFFERENTNUC F LEARITIES AND DIFFERENT ANALYSIS METHODS TO DETERMINE EDGE F IDATION STATEINFORMAT F ION POSITIONSAREUSEDD TO INFEROX K D 0ACE ABOUTBIOLOGICAL SYSTEMS SUCH ASIN+UZEKAN  AND#ARRELLETAL (OWEVER NUMEROUS STUDIES HAVESHOWNTHATTHERE ISACLEARCORRELATION BETWEEN-NOXIDATIONSTATEANDD -N+ EDGEENERGY IFTHELIGANDENVIRONMENTSTAYSTHESAME IETHE)0% SHIFTSTOA F HIGHERENERGYUPONOXIDATIONWITHINSETS OF HOMOLOGOUSCOMPOUNDS ! DETAILEDD 8!.%3 AND8%3STUDYBY6ISSERET AL  OF HOMOLOGOUSDI — OXOANDD MONO — OXO BRIDGED BINUCLEARCOMPOUNDSINDIFFERENTOX F IDATION STATESSHOWSTHATINCONTRASTTO-N+ EDGE8!.%3 +`8%3SPECTRASHOWMUCH LESS DEPENDENCEON LI GANDD ENVIRONMENT4HESHIFTS F IN8%3SPECTRAARETHE SAMEFORA-N))) TO-N)6 OXIDATION IRRESPECTIVE F WHETHERTHE-N ISPARTOFA F DI ORR MONO — OXO OFW BRIDGEDD STRUCTURE6I 6 SSERETAL  7EHAVEALSO COMPLETEDA8%3STUDYOFHOMOLOGOUSSETSOFTRI AND TETRANUCLEAR -N COMPOUNDS IN DIFFERENT F OXIDATION STATESANDD WE HAVEFOUN F D THATTHE8%3SPECTRAARE MUCHLESSDEPENDENTONTHELIGATIONORSTRUCTUREAND AREPRIMARILY DEPENDENTONTHEOXIDATIONSTATEOFF -N 69ACHANDRA UNPUBLI U SHED  #ONSIDERINGBOTHTHELIMITATIONSANDSTRENGTHSOF THETWOMETHODSOFF 8!.%3AND 8%3 BY COMPARISON WITH NUMEROUS-NMODELSANDD THEKNOW K LEDGEFROM F %8!&3DATASEEBELOW WEHAVEPROPOSEDTHEOXIDA TIONSTATESSHOWN IN&IG&IGURESUMMARIZESTHE +`8%3ANDD 8!.%3 mASH PATTERNS FROMOURMOST F RECENTANDINOUROPINIONTHEMOSTCOMPREHENSIVE T ECONCLUSIONSAB A OUT-NOXIDATION STUDYY THATLEADSTOTH



: IBOESB 7JUUBM , :BD

&IIG  ,EF & EFT ! )NmECTION POINTENERGY )0% INE6OFF THE-N+ EDGEOF03))MEM F BRANESASA FUNCT F IONOFF THENUMBEROFAPP F LIED mASHES " 4HE lRSTMOMENTSOFF THE-N+` EMISSIONSPECTRA % ASAFUNCT F IONOF mASHNUMBER4HE-N+ EDGESHIFTSTO F HIGHERENERGYAS -NISOXIDIZED4HE lRSTMOMENTOFT F HE-N+` mUORESCENCESHIFTSTO F LOWERENERGYAS-N ISOXIDIZED4HECOMPLEMENTARYAND D MUTUALLY F INGRESULTSPROVIDEASTRONGCASE FOR-NOX F IDATION DUR D INGTHE3 TO3 AND 3 TO 3 ADVANCES BUTNO-N CENTERED D OXIDATIONATTHE REINFORC 3 TO3 ADVANCE 2IGHT4HE3 STATESCHEMEFOR/ EVOLUTIONANDTHEOXIDATIONSTATESTHATARECONSISTENTWITHTHE%02SIGNALSANDTHE 8 RAYABSORPTIONANDEMISSIONSPECTROSCOPYRESULTS!DAPTEDFROM-ESSINGERETAL 

STATES"OTH SPECTROSCOPIESSHOWACLEARSHIFT F INTHE SPECTRATOHIGHERENERGYFOR8!.%3SPECTROSCOPY AND LOWERENERGY FOR+ F ` 8%3 BETWEENTHESAMPLES GIVENAND D  mASHES INDICATINGTHEPRESENCEOFF -N OXIDATIONDURINGTHE3 TO 3 TRANSITION 4HE SMALL D mASHESINBOTH CHANGE BETWEENSAMPLES GIVENAND SPECTROSCOPIESPROVIDESSTRONGSUPPORTFORT F HE3 TO3 TRANSITIONPROCEEDINGWITHOUTA-N BASEDOXIDATION /NTHENEXTTRANSITION 3 A ;3= A 3 / ISRELEASED D SHIFT F INGTHEPOSITIONOFF THESPECTRAOFF SAMPLESGIVEN mASHESTOLOWERENERGYFORR 8!.%3SPECTROSCOPY F ` 8%34HESE mASHPATTERNS AND HIGHERENERGY FOR+ D OXIDATIONSTATESOFF THE AREEXPLAINED BYTHEPROPOSED -NCLUSTERINTHE 3 3 3 AND3 STATES ASSHOWN 4HESE OXIDATION STATES ARE 3 -N)) ))) )6 OR -N))) )6 3-N))) )6 3 -N))) )6 3 -N))) )6 s WHERE THE SUP U ERSCRIPT DOT REPRESENTS ANOXIDATIONTHAT ISNOT-NCENTERED ***
4USVDUVSF
PG
UIF
.BOHBOFTF
$MVTUFS %XTENDED8 RAYABSORPTIONlNESTRUCTURE%8!&3 F HEMODU D LATION SPECTRAATTHE-N+ EDGERESULTSFROMT

OFT F HEABSORPTIONCROSSSECTION BYTHECONSTRUCTIVE ANDDESTRUCTIVEINTERFERENCEOFTHES PHOTOELECTRON WAVEWITH THAT BACKSCATTERED FROMNE F IGHBORINGAT OMS 3AYERSETAL  4HE%8!&3MODU D LATIONS CAN BE THEORETICALLY MODELED TO DERIVE EXQUISITELY DETAILED INFORMAT F ION ABOUT THE DISTANCE FFROM -N TONEIGHBORINGATOMSTO^¯ OFTENTOANACCURACY ASPRECISEAS¯9ACHANDRA  (OWEVER DETERMINATIONOFT F HENUMBEROFSUC F H INTERACTIONS IS SUSCEPTIBLETOMANYUNCERTAINTIESANDD CANOFTEN F BEAS LARGEAS4HE %8!&3APPROACHHASBEENUSED TODETERMINETHE DISTANCES FROM-NAN F D IDENTIFYTHE BACKSCATTERINGATOMSOFF THE-NCLUSTERPRESENTINTHE /%#)TISNOTPOSSIBLETODISTINGUISHBETWEEN/OR ."UTITISOFTEN F FAC F ILETODISTINGUISH BETWEENLIGHT W CH AREPOTENTIAL LIGAND ATOMSSUCH AS. OR/ WHI ATOMSANDBACKSCATTERINGFROMAHEAVIERATOMSUCH AS-NOR#ATHATWOULD D ONLY BEPRESENTINAMULTI OR HETERO NUCLEAR-NCOMPL M EX4OINTERPRETTH T ERESULTSIN TERMSOFTHECOORDINATIONENVIRONMENTOFTHE-N IT HASBEENNECESSARYTOEXAMINEANUMBEROF INORGANIC K GENEROUSLYPROVIDED COMPLEXESOF KNOWNSTRUCTURE TO US BY OUR COLLABORATORS 'UILES ET AL A $E2OSEETAL #INCOETAL  4HE%8!&3

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ SPECTRUM ISUSUALLYPRESENTED D ASA &OURIERTRANSFORM F &4 WHICHISSIMILARTOARADIALDISTRIBUTIONFUNCTION WITHEACH &OURIERPEAKREPRESENT K INGSHELLSOFNE F IGH BORINGATOMS FROMT F HEABSORBI R NGATOM$ICHROISM RESULTINGFROMORIENTEDMEMBRANESCANBEUSEDTO DERIVEINFORMAT F IONABOUTTHEVECTORIAL DIRECTIONOF THEPARTICULARINTERACTION!TYPICAL&OURIERTRANSFORM F F FROMAN 3 STATEISSHOWNIN&IG 4O 4OP 9ACHANDRA F IENTED ETAL $E2OSEETAL  AND FROMOR 03)) MEMBRANES IN THE 3 STATE IN &IG  "OTTOM -UKERJIETAL  4HREEDISTINCT &OURIERPEAKS 4 AND DICHROISMISEVIDENT ARERESOLVED IN&IG 4OP INALLOFF THEPEAKS IN&IG "OTTOM /NTHEBASISOFTHELATESTlNDINGS WECONCLUDETHAT EACH-N ISSURROUNDED BYA lRSTCOORDINATIONSHELL OF/OR.ATOMSAT^TO^¯&OURIERPEAK) ASETOF-NATOMSAT^¯&OURIERPEAK)) AND ADDITIONAL-NAND#AAT^¯ &OURIERPEAK)))  F D 5SINGMODEL COMPOUND DATA ITWASSTRAIGHTFORWAR TOASSIGNTHETWOORTHREETO¯ -N -N VECTORS TODI + OXOOR HYDROXOBRIDGES BETWEENPAIRSOFF -N ATOMSANDTHE¯-N -NVECTORTOONEORTWO MONO + OXOBRIDGEDORSIMILARLONGERINTERACTIONS +IRBY R ET AL 9ACHANDRA ET AL   -C$ERMOTTETAL 'EORGEETAL 0ENNER (AHNETAL -AC,ACHLANETAL $E2OSEET AL -UKERJI R ETAL +USUNOKIETAL  2OBBLEE ET AL   &URTHERMORE STUDIES USING 3RTOREPLACE#AAND SUBSEQUENTLY DIRECTMEASURE MENTSUSING #A%8!&3 SHOWUNAMBIGUOUSLYTHE PRESENCEOFONEORTWO¯-N #AVECTORSWHERE THEBRIDGINGMOTIFCOULDALSOBEMONO — CARBOXYLATO ORMONO — HYDROXOSEEBELOW 4HEUSEOFF VECTOR TERMINOLOGYTODESCRIBETH T ESEINTERATOMIC INTERACTIONS ISPARTICULARLYAPPROPRIATEBECAUSEOFTHEDIRECTION INFORMAT F IONAVAILABLEFROM F DICHROISMMEASUREMENTS D SAMPLES &URTHERMORE ANALYSIS OFF THE ON ORIENTED %8!&3 LEADSTOTHECONCLUSIONTHATTHEDI ORMONO — OXOBRIDGEDD -NAND#AVECTORSARENOTCO D LINEAR THAT ISANYTHREEMETALATOMSINTHECLUSTERCANNOT BE LINEAR /NTHEBASISOFSUC F H DATAMANY N TOPOLOGICALTET RANUCLEAR-NSTRUCTURES ! THROUGH + COMPATIBLE WITHTHE OBSERVED %8!&3DATAHAVEBEEN PRESENTED $E2OSEETAL  &IG 4HESEMODELS HAVE BEENDESCRIBED INDETAIL $E2OSEETAL 2OB BLEEETAL  (OWEVER UNTILRECENTLYONLYONE D ASAWORRKING OFF THOSEOPTIONS ! WASWIDELY USED MODEL ALTHOUGHOPTIONS % & AND ' THENOMENCLA TUREWASINITIALLYINTRODUCEDIN$E2OSEETAL HAVE BEENSHOWNTO BEPREFERRE F D ONTHEBASISOFF %02



&IG IG 4Y 4 PICAL &OURIERTRANSFORMO F F THE%8!&3SPECTRUM FROM F A 3 STATESAMPLEFROMANISOTROPICSAMPLE 4O 4OP ANDORIENTED SAMPLE"OTTOM  4HETHREEMAIN&OURIERPEAKSLABELED) ))AND))) AREASSIGNEDTO^/LIGANDATOMSPER-NAT ¯AND ./ LIGANDATOMSPER-NATn¯&OURIERPEAK)  -N -N INTERACTIONSAT^¯&OURIERPEAK)) AND-NAND#AAT AND¯&OURIERPEAK))) RESPECTIVELY4HEDICHROISMOFTHE Y &OURIERPEAKS) ))AND D )))ISCLEARAND D CANBEUSED D TODETERMINE F THE VECTORS IN THE 03 )) MEMBRANE &OURIER THE ORIENTATION OF PEAK)) T K HAT HASBEENASSIGNED D TO-N -N INTERACTIONSAT^ ¯ ISMAXIMALWHENTHEE VECTOROFTHE8 RAYSISPARALLELTOTHE MEMBRANEPLANETHEANGLEONTHElGUREISBETWEENTHEMEMBRANE D THE 8 RAYE VECTOR !DAPTEDFROM9ACHANDRAETAL NORMAL AND  $E2OSEETAL AND-UKERJIETAL 

ANDD %.$/2 SIMULATIONS (ASEGAWA ET AL  0ELOQUINANDD "RITT  3OMEOFT F HESTRUCTURES ! THROUGH + ARELESSLIKELY3TRUCTURE # AND VARIATIONS CONSISTINGOFTWO F ISOLATED DI — OXO-Nn-NMOIETIES F D BY0ACEANDD CO WORRKERS BASEDD ONTHEIR AREPREFERRE %02SIMULATIONS3MITHAND0ACE  BUT # IS NOT WIDELY ACCEPTEDD ONTHEBASISOF%02S F IMULATIONS BY F D "R D ITT OTHERGROUPS "OUSSACANDD 2UTHERFOR ETAL 0ELOQUINETAL  OR%8!&3 DATA ,ATIMERETAL  )T ISALSOPHYSICALLY IMPOSSIBLE



: IBOESB 7JUUBM , :BD

&IIG 4OPOLOGICALMODELS FORT & F HE-NCOMPLEX IDENTIlEDD ASCOMPATIBLEWITH THE%8!&3DATABY $E2OSEETAL AND D 2OBBLEE ATAL 4HENUMBERSINPARENTHESISARETHENUMBEROFF SHORTTO ¯ -N -NVECTORSANDLONGTO¯-N -NVECTORS A DIFFRACTIONSTUDIES 4HEOPTIONS ' ) , AND - ALLHAVESHORTTO¯ -N -NVECTORSANDAREINBETTERAGREEMENTWITHTHE8 RAY OPTIONS % & AND ' HAVEBEEN IDENTIlEDASCOMPAT D IBLEWITH %02AND D %.$/2DATA/PTION ' ORA TOPOLOGICAL ISOMERSEEMSTO BETHE BESTMODELBYALLPRESENTLYAVAILABLECRITERIA8 RAYDIFFRACTION %8!&3AND%02

WITH FFOUR -N ATOMS TO OBTAIN THREE -Nn-N DIS TANCESSEEBELOW ATn¯WITHTWOSEPARATED DI — OXO-Nn-NMOIETIES3TRUCTURE ( WITHTHREE Y Y THE  ¯-Nn-NDISTANCESISUNLIKELY ASSHOWNB %8!&3SPECTRA FROMAS F IMILARSETOFF COMPLEXESnA SERIESOFDISTORTEDCUBANES#INCOETAL  4HE MORELIKELYCANDIDATESWERECONSIDERED D TOBE ! % & ' OR + ONTHEBASISOF%8!&3AND%02DATAONLY ' CONTAINEDD THREE -N -NDISTANCESAT^ ¯ WHILE THEOTHERSHADD TWOSUCH INTERACTIONS

"
)FUFSPHFOFJUZ
JO
U J UIF
.O.O %JTUBODFT
BO J E 4USVDUVSBM
*NQMJDBUJPOT !S MENTIONEDD ABOVE DETERMINING THE NUMBER OF -N BACKSCATTERINGATOMSCAN BEDIFlCULT4HENUM BEROFTHE-N -NAND¯VECTORSSHOWNIN PARENTHESIS IN&IG AREESPECIALLY IMPORTANTINTHIS ENDEAVORASTH T ESEDATADIRECTLY L LEADD ORPRECLUDECERTAIN ARRANGEMENTSOFTHE-NATOMSTHATCOMPRISETHE-N CLUSTER4HEDISTANCEOFF THE -N -NVECTORS AT^ AND¯ ISPARTICULARLYIMPORTANTBECAUSEITISEASILY T THEMOSTRELIABLYDETERMINABLEPARAMETERFROMTHE-N %8!&3SPECTRAOF03)))T F ISINFORMAT F IVETO FOCUSON F

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ



4 BLE #URVE lTTINGRESULTSFOR&OURIERPEAKAT^¯ THATISCHARACTERISTICOFF OXO BRIDGED 4A D -NMOIETIESFROMT F HE3 STATES 3 STATE 3 STATE

2EFERENCES F 4ABLE)))FROM'UILESETALA

2 ¯ 

2 ¯ 

. 

. 

.. . 

3 G G  STATE

4ABLE"FROM, F IANGETAL 











.( INHIBITED3 STATE

4ABLEFROM$AUETA F L 











& n INHIBITED 3 STATE

4ABLEFROM$E2OSEETAL











3 STATE

4ABLE"FROM, F IANGETAL 











3 STATE

4ABLEFROM2O F BBLEEETAL











4ABLEFROM2O F BBLEEETAL      3 STATE . AND . ARETHE NUMBER OF -N -N INTERACTIONS NORMALIZEDD TOONE-NINTHE-N COMPLEX ATTHETWO DISTANCES 2 AND 2 RESPECTIVELY Y !N . OFTRANSLATESTOTWO-N -NDISTANCESANDA.OFTRANSLATESTO-N -NDISTANCEATTHECORRESPONDINGDISTANCE4HE . .. . RATIO OFF  INTHE^AND^¯-N -NDISTANCESSHOWSTHEREAREATOTALOFSUCHSHORT-N -NDISTANCESINTHE /%#

THE^ ¯ -N -NDISTANCE7HENONLY ONE-N -N ¯DISTANCECANBEDERIVEDFROMTHEDATAASINTHE CASEOFTHE 3 AND3 STATES THENONEISCONSTRAINEDBY THEUNCERTAINTY INTH T ENUMB M EROFSUC F H INTERACTIONSTH T AT IS INHERENTTOTHE%8!&3METHODOLOGYIT ISATPRES ENTTWOORTHREESUCH-N -NINTERACTIONS(OWEVER WHENTHE DISTANCE DEGENERACY ISLIFTE F D AND THEREARE TWORESOLVABLEOR¯ -N -NDISTANCESTHEN ONE CAN DETERMINE THE NUMBER OF SUCH VECTORS TO A MUCH HIGHERACCURACY BECAUSEONECANONLY HAVEAN INTEGRALNUMBEROFEAC F HOFF SUCH INTERACTIONS )NTERESTINGLY WHENWEEXAMINEDSTATESTHATEXHIB ITED D SUCH HETEROGENEITY TY INTHE¯-N -NDISTANCES ITBECAMEQU Q ITECLEARTHATTHEREWAS DISTINCTPREFE F R ENCE FO F RTHE EXISTENCE OFTHREE SUCH -N -N VECTORS ASDESCRIBED BELOW SUMMARIZED IN4ABLE &IGURE  ,EFTT SHOWSTHE&OURIERTRANSFORMS F &4 OFT F HE K WEIGHTED%8!&3DATAFROMTHE 3 AND3 STATES 4HE &OURIERPEAK))WAS K ISOLATED D AND lTTOTHE%8!&3 Q ION WITH ONE OR TWO DISTANCES FFOR THE 3 AND EQUAT 3 STATES&IGURE 2IGHTT SHOWSTHEBESTlTSTOTWO -N -N DISTANCES FFOR THE 3 STATE AS A FUNCT F ION OF THENUMBEROFF EACH OFF THE-N -N INTERACTIONS4HE CONTOUR PLOT GRAPHICALLY ILLUSTRATES THAT THE GLOBAL MINIMUMATTWO-N -NINTERACTIONSAT^ ¯ AND ONE-N -NINTERACTIONAT^¯ISVERYWELLDElNED 2OBBLEEETAL   7 HAVE RE EXAMINED 7E D THE DATA FFROM THE 3 3 STATEINDUCE D D BY.(/(INCUB U ATION THE G3 G G  THE .( OR &nINHIBITED3 ANDTHE3 STATES WHERE WEHAVESHOWNTHATTHEREISDISTANCE HETEROGENEITY IN

THE -N -NVECTORS4HERESULTSOFF THISRE EXAMINA TIONAREGIVENIN4ABLE WHI W CH SHOWTHATNONEOFF THE 3 STATESTHATEXHIBITDISTANCEHETEROGENEITYAREBEST lT BYANEQUAL .VALUEFORBOTH-Nn-NSHELLSTHElT . RESULTS FROMT F HESE3 STATESARE INFACTMORECONS F ISTENT WITHA  .. . RATIO WHERE . CORRESPONDSTOTHE SHORTERDISTANCE)NEACH CASE THERATIOOFF THENUMBER F HETWO-N -N INTERACTIONSWAS^4HISLEADS OFT USTOTHINKTHATTHEREMAYBETHREE-NINTERACTIONS AT^ ¯ INTHE-NCLUSTERINITSNATIVESTATE ONEOF WHICH ISPERTURB R ED INTHE3 &n ANDD .( INHIBITED 3 ANDD THE G3 G  STATES ANDALLTHREEARE PERTURBED INTHE 3 STATE /NTHE BASISOFT F HESERE EVALUATIONSOFF THERESULTS FFROM THE 3 STATE AND EARLIER DATA IT IS IMPORTANT TOCONSIDERTHEOPTIONSTHAT INCLUDETHREE DI — OXO BRIDGEDD MOIETIESINTHE-NCLUSTER&IGURESHOWS SEVERALSUCHOPTIONS ' ) AND * THATWERE PROPOSED D TWO NEWER OPTIONS , AND - AMONG EARLIER AND SEVERAL OTHERSTHATCAN BECONCEIVED/PTIONS * AND ) ARELESSLIKELYSTRUCTURESBECAUSE * LACKS A -N -N INTERACTIONAT ¯ AND ) HASTWOSUCH INTERACTIONS F INORGANICCOMPOUND !U 4HE %8!&3DATA FROMAN GERETAL  WITHTHEMOTIFINOPTION * ISVERY DIFFERENT F F FROMT HATOBTAINED FROMA03))SAMP F LE6 9 HANDRA UNPUBLI 9AC U SHED /PTIONS ' , AND - ALL HAVE THREE DI — OXO BRIDGES AND ONE MONO — OXO BRIDGE THAT IS THREE  ¯ AND ONE  ¯ -N -N INTERACTIONS/PTIONS , AND - AREALSOSIMILARTOTHE STRUCTUREPROPOSEDONTHEBASISOFDENSITYFUNCTIONAL THEORYCALCULATIONSALTHOUGH #A ISNOT INCLUDED IN ,

7JUUBM , :BD : IBOESB



&IG  ,EF &I EFT &OURIERTRANSFORMSO F F HE3 DASHED LINE AND3 FT D  STATE SOLID LINE OFT F HE-N %8!&3SPECTRA4HEMAIN&OURIERPEAKSARE LABELED) ))AND)))4HEREARECLEARDIFFERENCESINAMPLITUDEANDPOSITIONOFTHESEPEAKSBETWEENTHE 3 AND3 STATES4HEPEAKLABELED ))ISFROM-N -NINTERACTIONSAT^ ¯ ANDINTHE 3 STATETHISVECTORISBESTlTTOTWODISTANCESATAND¯ 2IGHT 4HECONTOUR PLOTSHOWSTHElT QUALITY PARAMETER ¡ FORTHE3 STATEPLOTTEDVERSUSTHENUMBEROFTHETWO-N -NINTERACTIONS 4HEDASHEDLINESSHOWS THEDISTINCTMINIMUMATONE-N -NVECTORAT¯ANDTWO-N -NVECTORSAT¯!DAPTEDFROM2OBBLEEETAL  

AND - AND ISAN INTEGRALPARTOFT F HE3IEGBAHNMODEL 3IEGBAHN   )NADDITIONTOTHECONSTRAINTSIMPOSEDBYCONSIDER INGTHE %8!&3AND D %02DATA APRELIMINARY STRUCTURE D ON OFF THE /%# HAS BEEN RECENTLY REPORTED BASED 8 RAYCRYSTALLOGRAPHICDATAFROM03)):OUNIETAL D 3HEN &ERREIRAETAL  +AMIYAAND #HAPTERSn AND D A FURT F HERCRITERIONFORNARROW F ING THECHOICEOFFAVOREDCLUSTERGEOMETRIESCOMESFROM THESE8 RAY A CRYSTALLOGRAPHY DATAOFF 03))4HESEDATA AREMOSTCONSISTENTWITHAN/%#THAT ISASYMMETRIC ANDSHAPEDSOMEWHATLIKEA@94HEARRANGEMENT OFF THE-NATOMS HAS BEENDESCRIBED D ASPEAR SHAPED ANELONGATED D ELLIPSOID WITH ALL FOUR-N F LYINGAP PROXIMATELYINTHESAMEPLANE0RESENTLY ' , AND - AREQUALITATIVELY INAGREEMENTWITHTHEASYMMETRY SEEN INTHEELECTRONDENSITYOFF THE-NCLUSTER:OUNI ETAL  ANDSHOULDBECONSIDEREDASPOSSIBILI TIESFORATOPO F LOGICALMODELOFT F HE/%#BASED D ONTHE INSIGHTS DEVELOPED FFROM THE %8!&3 SPECTROSCOPY RESULTS WITHOPTION ' BEINGTHEBESTINTERMSOFBEING Y COMPATIBLEWITH THEMOSTCRITERIAELECTRONDENSITY %02 AND D %8!&3 (OWEVER AS CONlRMED BY -N AND3R%8!&3STUDIES,ATIMERETAL #INCO ETAL  THE/%# ISMOSTACCURATELY DESCRIBED D AS F #ASHOULD A-N#AHETERONUCLEARCLUSTERTHEREFORE BEINCORPORATEDINTOEACHOFTHEPROPOSEDSTRUCTURES SOTHATn-Nn#AVECTORSEXISTWHI W CHAREORIENTED CLOSETOTHEMEMBRANENORMAL &ERREIRAETAL  #HAPTER 

# 44UBUF
5SBOTJUJPOT
BOE 4USVDUVSBM
$IBOHFT !LTHOUGH%8!&3STUDIESMAYNOTBEABLETOUNIQUELY IDENTIFY THESTRUCTUREOFF THE-NCOMPLEX THEYARE INVALUABLE INUNDERSTANDINGTHECHANGESINSTRUCTURE IF ANY THAT MIGHT BE OCCURRING DURING THE 3 STATE TRANSITIONS!SNOTEDD ABOVE %8!&3 ISEXQUISITELY F SENSITIVE TO DISTANCES OFF NEIGHBORING ATOMS FROM -NANDHENCECAN PROVIDEANUNIQUEWINDOWINTO Q HOWTHE-NCLUSTERCHANGESAS ITPROCEEDSTHROUGH THECATALYTICCYCLE ASSUMMARIZED IN&IG4HESE CHANGESINSTRUCTUREPROVIDEARATIONALEFORTESTING VARIOUS MECHANISMS THAT HAVE BEEN PROPOSEDD AS DESCRIBED BELOW W  4 UP
4 .-4
5SBOTJUJPO 4 %8!&3STUDIESOFTHE3 STATE ANDTH D E 3 STATE CHARAC TERIZED BY THEMULTILINE%02SIGNALREVEALEDD THATTHE -Nn-NDISTANCESAREESSENTIALLYTHESAME^¯ AND  ¯ IN BOTHSTATES 4HIS DISTANCE IS CONSISTENT WITH THOSE FOUN F D IN NUMEROUS STUDIES OF DI AND MONO — OXO BRIDGEDD AND -N))) )6 ANDD -N)6 )6 COMP M LEXES/XIDATIONOFAMONO OR DI — OXOBRIDGED -NMOTIF FROM F ))) ))) TO ))) )6 ORFROM F ))) )6 TO )6 )6 HASMINIMALEFFECTONT F THE-N -NSEPARATIONIN MANY INORGANICMODELS7IEGHARDT 0ECORARO 0ECORAROANDD (SIEH   )NTERESTINGLY %8!&3STUDIESONORIENTEDD 03)) IN THE 3 AND3 STATESSHOWTHATTHEREMAYBE HETEROGENE ITYINTHE¯ VECTOR ACONTENTIONTHATISSUPPORTED

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ



&IG 3UMMARYOFTHECHANGESIN OXIDATIONSTATESAND-Nn-NDISTANCESATTO & D ¯ INTHENATIVE3 NATIVE3 MODIlED3 3 39:s NATIVE3 AND3 STATESOF03))ASDETERMINEDBY-N%8!&3

BYRELATED D STUDIESONMODIlED3 D  STATES4HIS -N -N VECTORHASBEENDETERMINEDTOBEATANAVERAGEANGLE R ET AL OFF ^  TO THE MEMBRANE NORMAL -UKERJI $AUETAL    4 UP
4 H

5SBOTJUJPO 4HEINEQUIVALENCEOFTHE Q DI — OXOBRIDGED-NUNITS BECOMESMOREEVIDENTINTHE3 STATETHATISPREPARED BYILLUMINATIONAT+AND D CHARACTERIZED BYTHE G G G%02SIGNAL,IANGETAL  3IMILARRESULTS WEREOBTAINED D WITH.( TREATED D AND ANNEALED D 3 STATE SAMPLES $AUETAL  AND IN&n TREATED D SAMPLES $E2OSEETAL  4HESCHEMEIN&IGSHOWS THATTHEREAREPATHS FROMT F HE-,33 STATETHAT LEAD D TO n TWOSTATESTHE 3 GSTATEINHIBITEDBY.( G G & OR s THE39: STATESNEITHEROFTHESESTATESCAN PROCEED TOTHEPHYSIOLOGICALLY RELEVANT3 STATE4HESESTATES AREDEPICTED IN&IGAS BRANCHINGAWAYFROMT F HE NORMAL PATHWAY LEADING TO THE 3 STATE BY PHOTON G STATE GENERATED ABSORPTION (OWEVER THE 3 G BY  NM ILLUMINATION AT  + "OUSSAC ET AL  CANPROCEEDTOAN3 STATE:IMMERMANNAND 2 HERFOR 2UT F D  D )NTHEMODIlED 3 STATES ONEOF THE -N -NDISTANCESINCREASESTO^¯ WHEREAS THEREISVERYLITTLECHANGEINTHEOTHERONEORTWO ¯ORTHE ¯ -N -NDISTANCE7ITHTHEDEGENERACY OFTHE¯LIFTED ITHASBEENPOSSIBLEBYSTUDYINGTHE DICHROISM OFTHE &OURIERPEAK TOASSIGNTHERELATIVE ORIENTATIONOFTHEAND¯ VECTORS $AUETAL  WHI W CHAREORIENTED D AT AND  RESPECTIVELY TO THE MEMBRANE NORMAL 4HE .( TREATED D AND &n TREATED D SAMPLES CANNOT

ADVANCE BEYONDD THEMODIlEDD 3 STATE 39:s STATE  )TISLIKELYTHAT &n OR.( PREVENTTHEOXIDATIONOF A LIGANDD ATOM THEREBY BLOCKINGSTRUCTURAL CHANGES FFROM OCCURRING THAT ARE NECESSARY FFOR THE FORM F A TIONOFTHE 3 STATE ANDTHUSINHIBITING/ EVOLUTION ACTIVITY)NT Y HECASEOFF .( ITISPROBABLY DUETOAN D AMIDO GROUP.(n REPLACEMENTOFF THEOXO BRIDGE INVOLVEDINOXIDATION4HEMODIlCATIONOFTHE-,3 SPECTRAUPONADDITIONOFF .( "ECKETA K L  AND %3%%-STUDIESUSING .( AND .( DEMONSTRATED THAT.(BECOMESALIGANDOF-N"RITTETAL   4HEASYMMETRYPARAMETERDERIVED FROM%3%%-RE F

SULTSSUGGESTEDD THATTHEAMIDOGROUPISLIKELYTO BEA BRIDGINGLIGAND"RITTETAL  )TISPROBABLETHAT THEOXO BRIDGETHATISDISPLACED BY.( ISOXIDIZED D INGTHE3 TO 3 TRANSITIONSEENEXTSECTION  DUR  4
UP 4 5SBOT 5 JUJUJPO %ARLIERR 8!3 DATAFROMTHE 3 STATESAMPLESPRODUCED BY ACRYOGENICDOUBL U E TURNOVERMETHOD INDICATED IN CREASEDDISORDERINTHEPEAKAT¯THATASTRUCTURAL CHANGETHATINVOLVEDTHELENGTHENINGOFTHE -N -N DISTANCEWASOCCURRING BETWEENTHE3 AND 3 STATES D INGTHE3TO 3 STATETRANSITION ANDD WASABSENTDUR 'UILESETAL B -ORERECENTDETAILEDANALYSIS F IVE3 STATESAMPLESCREATEDD UNDER OF DATA FROMNAT H IOLOGICALCONDITIONSWITHSATURATINGACTINICmASH PHYS ILLUMINATIONSHOWTHEREISASIGNIlCANTCHANGEINTHE ¯-N -NDISTANCESTHATCHARACTERIZETHEDI — OXOBRIDGEDD -N -NWITH ANINCREASEINTHESHORT -N -N DISTANCES FROM  ¯ TO ^ ANDD ^ ¯ ,IANG ETAL  



7JUUBM , :BD : IBOESB

&IG  ,EF &I EFT &OURIER lLTERED K SPACE%8!&3DATAFROM3 K K F LES4HEDIFFERENCES F INPHASE  DASHED LINE AND 3 SOLID LINE STATEOF03))SAMP FFREQUENCY AND D AMPLITUDESBETWEENTHERAW3 AND 3 STATE%8!&3SPECTRAAREVERYOBVIOUS INTHESESPECTRA 2IGH I T &OURIERTRANSFORM POWERSPECTRAOF 3 DASHED LINE AND3 D  SOLID LINE STATESOF03))4 F HEMAJ A OR&OURIERPEAKSARE LABELED D ) ))AND )))4HESPECTRAARE CLEARLY DIFFERENT F BETWEENTHE3 AND3 D  STATES4HERE ISAREDUCT D IONINAMPLITUDEINALLTHREEPEAKS INTHE 3 STATECOMPARED D TOTHE3 STATE -OREIMPORTANTLY PEAKS))AND)))AREATAGREATERAPPARENTDISTANCE2FORTHE3 STATECOMPAREDTOTHE3 STATEASSHOWN

4HE PERTURB R ATIONINTHE-N -N DISTANCESIS DIFFE F R ENTFROMTHATOBSERVEDINTHEMODIlED3 STATES %ACH OFF THEALTERED3 D  STATESGIVESAlTTHATISCONSISTENT WITHTHEALTERATIONOFONEO F F THE-N -N DISTANCES F FROM^TO¯ 5NLIKETHEMODIlED3 STATES THEPOSITIONOF &OURIERPEAK K )) FROMT F HENATIVE3 STATE F LONGERDISTANCEINTHE&4S &IG CLEARLYSHIFTSTOA WITH ONE ORTWO -N -N DISTANCES OF  ¯ AND ONE -N -NDISTANCEOF ¯ ,IANG ETAL  4HESE RESULTSUNDERSCORETHEPOINTTHAT DUR D INGTHE3TO 3 TRANSITIONUNDERPHYSIOLOGICALCONDITIONS ALLOFTHE DI — OXO BRIDGED D -N -N UNITS UNDERGO STRUCTURAL CHANGESTHATLEAD D TOAN INCREASEINTHE-N -N DIS TANCES!CHANGEINTHE-N -NDISTANCEFROM TO¯WASALSOOBSERVEDINSAMPLESTHATHADBEEN DEPLETED D OF #A BY.A#L TREATMENT -AC,ACHLANET AL  4HEBESTlTSFORTHE-N /.SHELLSHORTER -N / DISTANCE IS CHARACTERISTIC OF F -N / BRIDGING DISTANCESANDITINCREASESFROM¯INTHE3 STATE TO¯INTHE 3 STATE4HISISASIGNIlCANTCHANGE F HEINVOLVE AND ITPROVIDESADDITIONAL EVIDENCE FORT MENTOFT F HEBRIDGINGOXYGENATOMS DUR D INGTHE 3 TO 3 TRANSITION 4HERESULTSFROM F ISOTROPIC3 SAMPLESARESUPPORTED BY POLARIZED %8!&3 STUDIESONORIENTED D 03 )) INTHE NATIVE3 STATE 4HE DATA CONlRMTHATTHETWO -N -NVECTORSUNITSARENOTEQUIVALENT4HE POLARIZED %8!&3 DATA ARE DIFFERENT F FFROM THOSE OBSERVED IN THE3 STATE-UKERJIETAL $AUETAL   &OURIERPEAK))O K F 3 IS DICHROICAND ISREADILY RESOLVED

TO-N -NDISTANCESOF^¯AND^¯ EACHWITH ITSOWNDISTINCTPROJECTIONONTHEMEMBRANENORMAL &ERNANDEZETAL  3CHILLERETAL  HAVE REPORTEDD RESULTSONTHEORIENTEDD 3 STATE SHOWINGAN INCREASEINTHEAMPLITUDEOF&OURIERPEAK))THATIS INTERPRETED AS AN INCREASE IN THE NUMBER OF  ¯ -N -NINTERACTIONS INTHE 3 STATE(OWEVER ACLOSER EXAMINATIONOFTHEIR&4SINDICATESTHAT&OURIERPEAK)) OCCURSATALONGERDISTANCEAND IS BROADERCOMPARED TOTHATINTHE 3 STATE )NTHE 3 STATE IT ISSEENTHAT&OURIER PEAK)))AT ^¯ OCCURS AT A GREATER APPARENT DISTANCE THAN THATOFT F HE3 STATE INDICATINGALENGTHENINGOFF THETWO -N -N DISTANCES ORTHE -N -N ANDD -N #ADISTANCES COMPAREDD TOTHE 3 STATEBY  ¯ 3IGNIlCANTCHANGESAREOBSERVED INTHE-N -N DISTANCESINTHE3 STATECOMPAREDTOTHE3 ANDTHE3 STATES&IG 4HESECHANGESIN-N -NDISTANCESARE INTERPRETEDD ASCONSEQUENCESO Q F HEONSETOFF SU FT UBSTRATE WATEROXIDATIONINTHE3 STATE -N CENTERED OXIDATION ISEVIDENTDUR D INGTHE3 TO 3 AND3 D  TO3 TRANSITIONS $URINGTHE3 TO 3 TRANSITION WE PROPOSETHATTHE CHANGESIN-N -NDISTANCESARETHERESULTOFLIGAND T E FORMAT F IONOFF ANOXYL ORWATEROXIDATION LEADING TOTH RADICAL INTERMEDIATE FORME F D ATA BRIDGINGORTERMINAL POSITION4HEREACTIONOFTHEOXYLRADICALWITH/(n (/ ORANOXOGROUP DUR D ING THESUB U SEQUENT3 STATE F IONOF CONVERSION IS PROPOSEDD TO LEADD TOTHE FORMAT THE/ /BOND#HAPTER 

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ  4 UP
4{ 4:;r


5SBOTJUJPO 4HE OTHER UNPRODUCT D IVE STATE GENERATED FFROM THE 3 -,3STATEISDENOTED IN&IGASTHE 3| STATEOR 39:s STATE GENERATED IN #A DEPLETED SAMPLES4HE #A DEPLETED D SAMPLES ARE INACTIVE IN / EVOLUTION WHILEABROAD G  %02SIGNAL HASBEENOBSERVED INSUCHSAMPLES4HE G BROADENED%02SIGNAL HAS BEEN CONlRMED D TO ARISE FFROM THE TYROSINE9: RADICAL 'ILCHRISTETAL 0ELOQUINETAL  AND IT IS PROPOSED THAT THE SIGNAL IS BROADENED BY INTERACTIONWITH THESPINONTHE-NCLUSTER,AKSHMI ETAL   %8!&3 ANALYSIS FROM #A DEPLETED 3| STATE SAMPLES 39:s ,ATIMER ET AL  SHOWS THAT &OURIERPEAK K )) -N -NDISTANCEOF^¯ INTHE &4OF3| STATESAMPLES39:s WASINVARIANTRELATIVE TOTHATOFF THENATIVE3 STATESAMPLE4HE%8!&3lTS SHOWED THAT THE ^ ¯ -N -N DISTANCES DID NOT LENGTHENASOBSERVEDINTHENATIVE3 STATESAMPLES AND D AREESSENTIALLY UNCHANGED FROMT F HOSEOFT F HENA TIVE 3 STATE4HIS lNDINGISSURPRISING BECAUSETHE -N+ EDGESFROMTHESE#A DEPLETEDSAMPLESSHOWED A BEHAVIOR SIMILAR TO THE NATIVE 03)) IN THAT LITTLE F BSERVED INTHE3| TO3| OR39:s ORNOSHIFTWASO TRANSITION4HISDIFFERENCEBETWEENTHENATIVE 3 AND #A DEPLETED D 3| STATES INDICATETHATTHECOREDI — OXO BRIDGED D STRUCTUREISPROBABLY DISSIMILARINTHENATIVE 3 ANDTHE 3| 39:s STATES WITHTHESTRUCTUREINTHE 39:s STATERESEMBLING THENATIVE3 STATESTRUCTURE )TIS INTRIGUINGHOWTHETRANSFERO F FONEE F LECTRON FROM F THE -N CLUSTER ONTO 9: RESULT IN MAJOR CHANGES IN THE-N -N DISTANCESASISOBSERVED INTHENATIVE3 STATE4HEROLESOFT F HE 9: AND D THE-NCLUSTERINTHE PROCESSOFWATEROXIDATIONARECLEARLYDELINEATEDBY THECOMPARISONOFF THE 3 AND D THE3| OR39:s STATES )NSTEAD D OF THE-NCLUSTERJUSTPROVIDINGASCAFFO F LDING F FORWATEROXIDATION THISCOM PARISONSHOWSTHATTHE STRUCTURAL CHANGE INTHE-NCLUSTERINITIATED BYTHE TRANSFERO F FANE F LECTRONFROMT F HE-NCLUSTERTO9Z DUR D INGTHE 3 TO 3 TRANSITIONMIGHTPROVIDETHETRIGGER TOTHECHEMISTRYOFF THE FORMAT F IONOFT F HE/ / BOND D F ION OF F A LIGAND D RADICAL 4HE RESULTS VIA THE FORMAT SHOWTHATTHE -N CLUSTER IS INVO N LVEDD IN A MUCH MORE INTIMATEMANNERINTHECATALYSISTHANJUSTPROVIDING THEFRAMEWOR F RK4HE IMPLICATIONSTOTHEMECHANISM ARESIGNIlCANT#HAPTER  )N #A DEPLETED D SYSTEMS THE TYROSINE9: RADICAL ISSTABILIZED D AND THEOXIDATIONOFT F HE-N /%#AND THE CONCOMITANT CHANGES IN -N -N DISTANCES ARE PREVENTED)NTHE3 STATE WITH #APRESENT THEOXIDA

 TIONEQUIVALENTRESIDESONTHE-NCLUSTERLEADINGTO PROFOUNDCHANGESINTHE-N -NDISTANCES4HUS #A ISPROPOSEDD TOPLAY A ACRUCIAL ROLEINCONTROLLINGTHE REDOX POTENTIALANDD THUSTHECOURSEOFF THEMECHANISM OF WATER OXIDATION  4
UP 4 5SB S OTJUJP J O %ARLY %8!&3EXPERIMENTS BY'UILESETAL A WITH THE 3 STATE USED CHEMICAL TREATMENTS TO GET AROUNDTHEPROBLEMOFLOWCONCENTRATIONS(OWEVER THE 3 STATE GENERATED INTHISMANNERWASDESIGNATED AS 3 TO EMPHASIZE THAT IT IS GENERATED THROUGH CHEMICALTREATMENTANDISTHUSNOTANATIVE3 STATE !LTH T OUGH HAMPERED BY Y ALOWSIGNAL TO NOISERATIOAND THE UNCERTAINTYABOUT THERELATIONSHIP BETWEENTHE CHEMICALLYGENERATED 3 STATEANDTHENATIVE3 STATE THOSEEXPERIMENTSPROVIDEDD THElRSTEVIDENCE FROM F %8!&3 THAT HETEROGENEITY MAY EXIST IN THE ¯ -Nn-N DISTANCES IN THE 3 STATE IN THE FO F RM OF A D D AMPLITUDEOF &OURIERPEAKK )) INTHE3 STATE REDUCE RELATIVETOTHAT INTHE 3 STATE 2IGGS 'ELASCO ET AL A EXAMINED REDUCED F THE /%# GENERATED BY TREATMENT WITH 3 STATES OF .(/(ORHY H DROQUINONEANDD OBSERVEDD A DECREASE INTHEAMPLITUDEOFTHE¯-Nn-N&OURIERPEAK 4HISWASINTERPRETEDD ASAREDUCT D IONINTHENUMBEROF -Nn-NVECTORSINSTEADD OF THEAPP A EARANCEOF DISTANCE HETEROGENEITY Y 2ECENT %8!&3 EXPERIMENTS BY 2OBBLEE ET AL  SHOWTHAT INTHE 3 STATE HETEROGENEITYMOST LIKELYEXISTSINTHE¯-Nn-NDISTANCESWITHONE ORTWO-N -NDISTANCESAT¯ANDONE-N -N DISTANCEAT¯ASDESCRIBEDABOVE SEE&IG W WHICHCANBEEXPLAINEDTHROUGHTHE PROTONATIONOFA DI — OXO BRIDGEDD -Nn-NMOIETY TYANDORTH T EPRESENCE OFF -N)) #HAPTER  4HE PRESENCE OF DISTANCE HETEROGENEITY IN THE 3 STATE HAS BEEN EXPLOITED IN D WHOSERESULTSARESUG THECURVE lTTING PROCEDURE W GESTIVEOFF THEPOSSIBILITYTHATTHREEDI — OXO BRIDGED -Nn-NMOIETIESMAYEXISTINTHE/%#INSTEADOF THETWODI — OXO BRIDGEDD -Nn-NMOIETIESTHATARE WIDELY USED IN PROPOSEDD STRUCTURAL MODELS FFOR THE /%#SEEABOVE  *7 4USVDUVSBM
3PMF
PG
UIF $BMDJVN
$PGBDUPS !LONGWITH -NANDD #Ln #A ISANESSENTIALCOFACTOR F IN / EVOLUTION$EBUS #HAPTER VAN'ORKOM F SUPPRESSES AND 9OCUM  $EPLETING THIS COFACTOR

7JUUBM , :BD : IBOESB

 /%#ACTIVITY WHI W CHCANBERESTORED UPTO BY REPLENISHING WITH #A 0ARTIAL REACTIVATION UP TO F 3R TO #A DEPLETED  RESULTS FFROM ADDITION OF 03)) MEMBRANES 'HANOTAKIS ET AL  "OUS SACAND2UTHERFORD B ANDNOOTHERMETALIONS Y KINGTHISREQUIREMENTHIGHLY CANRESTOREACTIVITY MA F 'HANOTAKISETAL /NO SPECIlC FOR#AOR3R AND)NOUE   !LTHOUGH 3R REPLENISHESTHE#A DEPLETED D CENTERS TOASIMILAREXTENTASADDED #A THESLOWERKINETICS OFTHE /%#TURNOVERYIELDSANOVERALLLOWERSTEADY STATERATE "OUSSACAND D 2UTHERFOR F D A D  AT SATURATING LIGHT INTENSITIES3UB U STITUTIONOFF #AWITH 3RALSOALTERSTHE%02MULTILINESIGNAL-,3 FROM THE3 STATE GIVINGNARROWERHYPERlNESPLITTINGAND DIFFERENT F INTENSITYPATTERNS "OUSSACAND D 2UTHERFOR F D D B3IVARAJAETAL  -OSTRESEARCHERSAD DRESSING THESTOICHIOMETRYOFT F HE#ACOFACTOR F IN03)) F IONALWATEROXIDASEACTIVITY NOWCONCLUDETHATFUNCT  Q WHICHCANBEREMOVED REQUIRESONEESSENTIAL#A BY LOW P(CITRATEOR-.A#L WASH #AMMARATA D #HENIAE  +ATOHETAL /NOAND D )N AND OUE 3HENETAL !DELROTHETAL   )NHIGHERPLANTS ANOTHERMORETIGHTLY BOUND D #A IS D WITHTHE LIGHT HARVESTINGCOMPLEX ,(# ASSOCIATED )) ANDREQUIRESHARSHERTREATMENTSFORITSREMOVAL (ANAND D +ATOH #HENETAL   "
.BOHBOFTF
&YUFOEFE
93BZ Z "CTPSQUJPO 'JOF
4USVDUVSF
&9"'4
BOE
$BMDJVN & & 3EVERALINVESTIGATIONSHAVEINVOLVEDREMOVALOF#A D SU UBSTITUTIONOFVAR F IOUSMETALSINTOTHISBINDING AND SITE FO F LLOWED BY %8!&3STUDIESONTHE-NCLUSTER /NESETOFEXPERIMENTSUSING-N%8!&3ON3R REAC TIVATED03))MEM D BRANESWASINTERPRETED D TO INDICATE An¯DISTANCEBETWEENTHE#A3R ANDTHE-N CLUSTER,ATIMERETAL  4HISCONCLUSIONWAS BASED D ONTHEOBSERVATIONOF INCREASED D AMPLITUDEIN &OURIERPEAK)))AT¯UP U ONREPLACEMENTOF#AWITH 3R AHEAVIERATOMANDBETTER8 RAYSCATTERER!NALYSIS D 03)) MEMBRANE OFF %8!&3 SPECTRA FFROM PURIlED PREPARATIONSINDICATED D A-Nn#AINTERACTIONATSLIGHTLY L LONGERDISTANCE-AC,ACHLANETAL  ^  ¯  #A DEPLETION BY .A#L WASHING OFF 03)) MEM BRANESREMOVED D THEAND D  K$AEXTRINSICPROTEINS ANDLEDTOAREDUCEDAMPLITUDEFORTHIS¯FEATURE A SCATTERING ABILITY OF AND BECAUSEOFF THELOWER8 RAY .A THISRESULTWAS INTERPRETED D AS POSSIBLE.A SUB U STITUTIONFOR#A ATTHISDISTANCE-AC,ACHLANETAL  !NOTHER-N%8!&3STUDY 2IGGS 'ELASCOET D NOT DETECTANYCHANGES INTHE&OURIER AL B DID

PEAK AT  ¯ WHEN #A WAS REPLACED WITH 3R OR $Y IN03))REACTIONCENTERCOMPLEXESLACKINGTHE  ANDD  K$A EXTRINSIC POLYPEPTIDES HOWEVER IT WASPROPOSEDD THAT#AMIGHTBELINKEDD VIAA HY H DROGEN BONDTOTHEOXOLIGANDOFTHE-NCLUSTER # 4USPOUJVN
&YUFOEFE
93BZ
"CTPSQUJPO
'JOF 4
BOE
$BMDJVN 4USVDUVSF &9"'4 
*TPUSPQJD
4BNQMFT 4HE MOST COMMON APPROACH AS DESCRIBEDD ABOVE ,ATIMERETAL 2IGGS 'ELASCOETAL B WAS TO SUBSTITUTE OTHER METALS SUCH AS 3R FOR #A ANDD THENUSE-N%8!&3TO DETECTCHANGESINTHE CLUSTER )SOLATING THE -N #A OR 3R COMPONENT OF THE %8!&3 SPECTRUM FROM THE COMBINED %8!&3 F FROMA LL -N LIGANDD AND -N -N INTERACTIONSCAN BE DIFlCULT4HEREVERSEEXPERIMENTWH W EREONE PROBES FORBACKSCATTERINGFROM-NUSING#AOR3R%8!&3 #A3RCOFACTORPO F INT OF V F IEWFORNEAR F RBY -N ISAN ELEGANTALTERNATIVEAND ISMOREDElNITIVETHAN-N %8!&3RESULTS3UCHSTUDIESONBOTHISOTROPICAND D BRANES HAVEYIELDEDUNEQU D IVOCAL ORIENTED03))MEM F HEPROXIMITYOF#ATOT F HE-NCLUSTER EVIDENCE FORT SEEBELOW  3EVERAL FACTORS F FFAVOR 3R AS THE BETTER COFACTO F R F R 8!3 STUDY &IRST THE 8 RAY ENERGIES INVOLVED FO KE6FORTHE+ EDGE AREMOREPENETRATINGAND A ABSORPTION NOTATTENUATED BYAIR4HE HIGHER8 RAY CROSS SECTIONAND mUORESCENCEYIELD D OF 3RALSOMAKE THE EXPERIMENT PRACTICABLE 4HE 3R %8!&3 BASED TH 3RSU UBSTITUTED EXPERIMENTREQUIRES03))SAMPLESWITH F FOR#AW WHILEMAINTAINING/ EVOLVINGACTIVITYANDD A STOICHIOMETRYOF3RPER03)) TOFOCUSONTHEFUNC TIONAL COFACTOR F BINDINGSITE!LONG WITH REACTIVATED 3R 03)) AN INACTIVATEDD SAMPLECANBEPREPARED BY TREATING WITH HYDROXYLAMINE .(/( TO DISRUPT THE-NCLUSTERANDD SUPPRESSWATEROXIDASEACTIVITY 4AMURAAND# D HENIAE   "Y USING 3R %8!&3 ON ISOTROPIC 3R REACTIVATED 03))MEMBRANES THEPROXIMITY OF3R F AND IMPLICITLY #A TOWITHIN¯OFTHE-NCLUSTER#INCOETAL  HASBEENCONlRMED4HElNDINGWASBASEDON THEPRESENCEOFF ASECOND &OURIERPEAK PEAKK )) &IG  4O 4OP INTHE3R%8!&3 FROM F F FUNCT IONALSAMPLES APEAKTHATISABSENTFROMINACTIVE HYDROXYLAMINE K F FOUN D TOlT BEST TREATEDD 03))4HIS&OURIERPEAKWAS TOTWO-NAT^¯RATHERTHANLIGHTERATOMS# / 0 0 3 #L "OTHTYPESOFSAMPLESSHARESIMILARlRS T COORDINATIONSHELLSOFF OXYGEN0EAK) & K IG 4OP 4 

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ

 R ERnB n ACKSCATTERER!n" VECTORISALIGNEDD WITH ABSORB THEELECTRIClELD4HROUGHANALYSISOFTHEDICHROISM THEAVERAGEORIENTATIONq OFF THIS!n"VECTORRELATIVE TOTHEMEMBRANENORMAL ANDD THEAVERAGENUMBEROF SCATTERERSPERABSORBINGATOM. .IISO CANBEEXTRACTED #ONSTRAINTSONTHESTRUCTURAL MODELARETHENIMPOSED BYTHESEPARAMETERS 3R SUBSTITUTED03))SAMPLESMADEBYAPROCESSOF #A DEPLETION 3R REACTIVATION ANDD #HELEXTREATMENT TOREMOVEEXCESS3RWERE LAYEREDD ONTO mAT-YLARlLMS TOPRODUCEORIENTEDSAMPLES-UKERJIETAL  $AUETAL  4HE&OURIERTRANSFORMS F F FROMT HE POLARIZED3R%8!& D 3 SHOWEDD EXTREME DICHROISMIN &OURIERPEAK))&IG ,EFT .ONLINEARLEAST SQUARES T REGRESSIONANALYSISPRODUCE D D THESOLIDD CURVESHOWN IN&IG 2IGHTT ASTHEBESTlTOFTHEDATAPOINTS ANGLES FROM SIX SEPARATE SAMPLES AND THE RESULT T TRANS LATESTOn3Rn-NVECTORSWITH TH ANAVERAGEANGLE OF  OR  FORT F HEAVERAGERELATIVEANGLE BETWEENTHE 3Rn-NVECTORSANDTHEMEMBRANENORMALDEPENDING F LYSIS ONTHEMETHODD OFANA 
0SJFOUBUJPO
PG
UIF
.BOHBOFTF
$PNQMFY

&IIG  4O & 4OP &OURIERTRANSFORMS OF 3R %8!&3 FOR F INTACTAND INAC TIVE 3R SUB U STITUTEDD 03))SAMPLES#HELEX TREATED 4HEDOMINANT &OURIER0EAK K ) ISDUETO D LIGATING OXYGENSINTHE lRSTCOORDINA TIONSPHEREAND ISCOMMONTOBOTHSAMPLES4HETWO3R 03)) SAMPLESDIFFERMAINLYATTHE @2 ¯REGION WHERETHEINTACT SAMPLESEXHIBIT&OURIER0EAK))$ISRUPTIONOFTHE-NCLUSTERIN .(/( TREATEDSAMPLELEADSTOTHEABSENCEOF0EAK)) "OTTOM &OURIER TRANSFO F RM OF #A %8!&3 FROM #HELEX TREATED LAYERED SAMPLES WITH  #A03)) WITH / EVOLVING ACTIVITY ANDD AN 3 %02MULTILINESIGNAL4HE&4SSHOWTHEPRESENCEOFF ASECOND &OURIERPEAK INTHE#A03))SAMPLETHAT lTSTO#A -NTHAT IS ABSENT INTHECONTROLSAMPLEWH W ERETHE-NCOMPLEXHASBEEN DISRUPTED D WITH .(/(

 0SJFOUFE
4BNQMFT 4HE TECHNIQUE OF USING 3R %8!&3 HAS BEEN EX TENDED D TO USING POLARIZED D 3R %8!&3 ON LAY A ERED D SAMPLES TOPROVIDE IMPORTANTANGLE 3R SUB U STITUTED INFO F RMATION 0OLARIZED%8!&3INVOLVESCOLLECTING SPECTRA FOR F DIFFERENT F INCIDENT ANGLES e BETWEEN THE MEMBRANE NORMAL OFF THE LAYERED D SAMPLE AND THE 8 RAY ELECTRIC lELD VECTOR $ICHROISM IN THE %8!&3CANOCCUR DEPENDINGON HOWTHEPARTICULAR

4HEORIENTATIONDATAFROMT F HE3R%8!&3EXPERIMENTS CAN BE COMBINED WITHTHE DICHROISM DATA FROM -N %8!&3DATATOCALCULATETHEORIENTATIONOFTHE ¯ -Nn-NVECTOR4HE&OURIERPEAK INTHE-N%8!&3 W WHICHCONTAINSTHE-Nn-N¯ AND-Nn#A ¯ CONTRIBUTIONS ISDICHROIC WITHANAVERAGEANGLE F ITH RESPECTTOTHEMEMBRANENORMAL OF› W -UKERJIETAL  "YINCLUDINGTHE-N #AVEC TORAT  ANANGLEOF^ FORTHE¯-Nn-N VECTOR CAN BE CALCULATED 0REVIOUS POLARIZEDD -N %8!&3EXPERIMENTSON03))HAVESHOWNANGLESOF  AND FORTHE ¯-Nn-NVECTORS -UKERJ RI F LLOWSTHAT ETAL $AUETAL  4HUS IT FO ALL -Nn-N VECTORS LIE AT ROUGHLY THE SAME ANGLE ^  WITH RESPECTTOTHEMEMBRANENORMAL BUTARE NOTRESTRICTEDD TO BEING COLLINEAR BECAUSETHE03)) MEMBRANESAREORDEREDINONEDIMENSIONONLY4HE F HE8 RAY A DIFFRACT F IONSTUDIES ELECTRON DENSITY FROMT :OUNI ETAL  AREINAGREEMENTWITH SUCHAN ASSIGNMENT THE PLANE CONTAINING THE -N ELECTRON DENSITY ISAT^ TOTHEMEMBRANENORMAL $
$BMDJVN
&YUFOEFE
93BZ
"CTPSQUJPO
'JOF Z 4USVDUVSF
&9"'4

4 )NACOMPLEMENTARYANDDElNITIVEEXPERIMENT #A + EDGE%8!&3STUDIESHAVE BEENUSEDD TOPROBETHE



: IBOESB 7JUUBM , :BD

&IG  ,EF &I EFT &OURIERTRANSFORMSO F F 3R %8!&3 FROMOR F IENTED3R 03))SAMP D LESATTWOANGLES e 4HE DICHROISMISMOSTREADILY APPARENT IN&OURIERPEAK))2| ¯ WHICHISASSIGNEDTOBACKSCATTERINGFROM-N 2IGHT 4HEPOLARPLOTSHOWS.EXP lLLEDCIRCLES PLOTTEDVS THEANGLEOFDETECTION e4HESOLIDLINEISTHEBESTlTFROMWHICHWEOBTAIN .ISO NUMBEROF-NNEIGHBORSTO3RIS^ AND q ¾  THE ANGLETHE-N 3RVECTORMAKESWITHTHEMEMBRANENORMAL

BINDINGSITEOFTHENATIVECOFACTORFORANYNEARBY-N WITHIN^¯4HEUSEOF#A%8!&3SPECTROSCOPY HASPRODUCE D D RESULTSESSENTIALLYCONGRUENTWITH THOSE FFOUND BY OTHER INDEPENDENT METHODS 3R %8!&3 ON3R REACTIVATED03)) D #INCOETAL  AND D -N %8!&3ONSIMILARSAMPLES ,ATIMERETAL  BUTITFOCUSES F ONTHE NATIVE COFAC F TOR AND AVOIDSTHE T TREATMENTS INVOLVING#ADEPLETIONAND D 3RSUUBSTITUTION ,IKE THE EARLIER 3R %8!&3 THE #A %8!&3 STUDY HASFOCUSEDONTHE#ACOFACTOROF03))POISEDIN THE3STATE 4HISTECHNIQUE ISAMORESENSITIVEAND DIRECTPROBEOFF THE#A BINDINGSITEIN03))AND D THE#A %8!&3EXPERIMENTDIRECTLYPROBEDTHE#ACOFACTOR INASCLOSETOANATIVESYSTEMASPOSSIBLE F HE#A%8!&3 ISSHOWN IN&IG "OT 4HE&4OFT TOM AND THE SPECTRA ARE REMARKABLY SIMILAR TO THE &OURIER TRANSFORMS F OFF THE EARLIER 3R %8!&3 STUDY WITH3RSUB U STITUTED FOR#A4 F HElRST LARGEST &OURIER PEAKCORRESPONDSTOTHECOORDINATINGOXYGENATOMS CLOSEST TO #A )N CONTRAST TO THE CONTROL .(/( TREATED SAMPLE THE #HELEX TREATED D 03)) SHOWS A SECOND&OURIERPEAK7HEN&OURIERPEAK))ISISO LATED D AND SIMULATED D WITH POSSIBLESCATTERINGATOMS ITCORRESPONDEDBESTTO-NAT¯ RATHERTHANTO LIGHTATOM# /OR#L NEIGHBORS4HESERESULTSWERE CONSISTENTWITH THEEARLIER3R%8!&3STUDIES 4HERESULTSARESUMMARIZED INAMOTIFF SHOWN IN &IGANDTHISMOTIFDEPICTSTHE#ALINKEDTOTWO -NBYSINGLE / BRIDGES WHI W CHCANBESUPPLIED BY

PROTEINRESIDUESORWATER/NLYSINGLE OXYGENBRIDGES NOT BIDENTATE BRIDGES CAN PROVIDE THE REQUIRED ¯DISTANCEINDICATEDBYTHE#A%8!&3 lTTING 4HISBRIDGEMAYBEDERIVEDFROMCARBOXYLATELIGANDS ASPARTATE OR GLUTAMATE PROTEIN RESIDUES D PROTEIN BACKBONECARB R OXYL WATERORHY H DROXIDE "ECAUSE SIGNIlCANT INFORMATION ABOUT -Nn-N ANDD -Nn#A VECTOR ANGLES IS NOW AVAILABLE TOPO LOGICAL MODELSPREVIOUSLYDISCUSSEDD CAN BERElNED TO INCLUDE THE PRESENCE OF #A AND ACCOUNT FOR THE DICHROISMDATA&IG 7E HAVECHOSENTOMODIFY TWOOPTIONSOPTION & FROM& F IG WH W ERETHETWO DI — OXOMOTIFSAREFORMEDUSINGACOMMON-NATOM ANDD THE MONO — OXOMOTIF ISPLACEDD ATTHEENDD OF THE TRINUCLEARUNIT ANDD OPTION ' CANBETHOUGHTOFF ASAT THE CORNERS OFTHE BASE OF A TRIGONALPYRAMID WITH AN/ATOMATTHEAPEXANDD THREEADDITIONAL /ATOMS F FORM INGBRIDGESPAIRWISEAMONGTHE-NATOMSBE LOW THE BASE WITHTHE FOU F RTH-NLINKED D TO ONE OFTHE CORNER-NATOMSBYASINGLE/ ATOMBRIDGE/PTION &HASTWOTO¯-N -NVECTORSWHIL W E ' HAS THREE SUCH VECTORS 4HESE STRUCTURES ARE CONSISTENT WITH SIMULATIONSOF%02AN F D D%.$/2 DATAANDD ALSO QUALITATIVELYINAGREEMENTWITH THEREPORTEDD ELECTRON DENSITYOFTHE-NCLUSTER ESPECIALLYTHElRSTOPTION F R ' WITH #A FO )T HASBEENSPECULATEDD THAT#ACONTROLSSUB U STRATE WATERBINDINGTOTHECATALYTIC-NSITE#HENETAL  ANDD RECENT MECHANISMS HAVE SUGGESTEDD THE

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ

 OF F PHOTOSYNTHETIC / EVOLUTION IS A HETERONUCLEAR -N#ACLUSTERFORFURTHERDETAILS SEE#HAPTER "ARB R ERANDD )WATA  7 4USVDUVSBM
3PMF
PG
UIF
$IMPSJEF
$PGBDUPS 7

&IIG  4OP & 4 -ODELOF#A F BINDINGSITEOFT F HEOXYGEN EVOLVING COMPLEXIN03))&ROMTHERESULTSOFTHE#A%8!&3STUDIESON 03)) THE #ACOFACTORISLINKEDBYTWOSINGLE /BRIDGESTOTWO-N 4HEOXYGENSCANBEPROVIDEDBYWATER HYDROXYLORPROTEINRESIDUES CARBOXYLATE PHENOLATE /THERLIGANDSTO#AARENOTSHOWN4HE ARRANGEMENTSHOWN HERE IS NOT UNIQUEAS OTHERPLACEMENTS OF THETWO-NAROUNDD THE#AARECONCEIVABLE #INCOETAL   "OTTOM 2ElNED D MODELS FORT F HEACTIVESITEOFT F HE /%# IN03)) F IENTED3R SU D UBSTITUTED 4HESEMODELSCOMBINETHE lNDING FROMOR F IENTED 03))SAMPLESWITH PREVIOUSRESULTS FROM-N%8!&3ONOR 03))SAMPLES4HESEAREDERIVEDFROMCORESTRUCTURESTHATHAVE BEENDESCRIBEDINEARLIERSTUDIESANDTHEMODELSPRESENTEDHERE AREBASEDONOPTIONS & AND ' FROM&IG& WITH#AISAVARIATION ONAMODELORIGINALLYPROPOSEDBY3IEGBAHN  

CRUCIAL INVOLVEMENT OFF THE COFACTOR F !NANY N EV AND $ISMUKES  2ENGER  ,IMBURG ET AL  3IEGBAHN   4HE RESULTS -N %8!&3 3R%8!&3AND D #A%8!&3AREMUTUALLY CONSISTENT AND D CONVERGETOWARD THECONCLUSIONTHATTHE #ACO F FACTORISINTIMATELYSTRUCTURALLYLINKEDWITHTHE-N CLUSTERIN03))4AKENTOGETHER THETHREEMETHODS F LLINGEVIDENCETHATTHECATALYTICCENTER OFFERCOMPE

$ESPITEAMULTITUDEOFSPECTROSCOPICSTUDIES DElNI TIVESTRUCTURAL EVIDENCE FOR-N # F L LIGATION HASNOTYET BEENREPORTED4HERE ISONEFUNCT F IONAL#Ln PER03)) UNIT ,INDBERG AND!NDRÏASSON  /LESEN AND W ETHERCHLORIDE !NDRÏASSON  BUT ITISUNCLEARWH ISALIGANDD TOONEOFT F THE-NATOMSINANYOFT F THE3 STATES ,INDBERGETAL  3TEADY STATEKINETICSTUDIES INDICATETHEPRESENCEOFA F HALIDE BINDINGSITEONTHE -NCOMPLEX3ANDUS D KYAN K D 9OCUM 9OCUM ,INDBERGAND!NDRÏASSON  ANDACTIVITYIS INHIBI N TED BY Y SOMECOMPOUNDSWHI W CHH COMPETEWITH THTTHE #LnBINDINGSITE SUCHASmUORIDE3ANDUSKYAND9O CUM  PRIMARYAMINES3ANDUSKYAND9OCUM  ANDD ACETATE#LEMENSETAL  /NOETAL  HAVESHOWNTHAT#Ln DEPLETEDD 03)) PARTICLES INTHE3 STATE DO NOTHAVETHE USUAL MULTILINE %02 SIGNAL BUTRATHERHAVEASIGNAL AT G SIGNIFYING F D!SIMILAR%02 THATANALTERNATIVE3 STATEISFORME F ND SIGNALISOBSERVEDONTREATMENTWITH &n)T WAS FOU THATWH W ENTHE /%# ISINTHISALTERNATIVESTATE ITCAN NOLONGERADVANCETO HIGHER3 STATES(OWEVER THE %02MULTILINESIGNALISRESTOREDFOLLOWINGILLUMINA TIONANDD SUUBSEQUENTADDITIONOFF #Ln2ECENTSTUDIES INDICATETHATTHE PRESENCEOFF THE#LnISNECESSARYONLY F FORTHE 3 TO3 AND3 TO 3 TRANSITIONSOFTHE/%# F HECYCLECANPROCEED IN W ETHEEARLIERSTEPSOFT WHIL ITSABSENCE 7I 7 NCENCJ C USZETAL   4HESE STUDIES INDICATE THAT #Ln IS CLOSELY ASSOCIATED WITH THESTRUCTUREOFT F HE/%#ANDD THEMECHANISMOFF / EVOLUTION BUTITSDETAILEDD ROLEISASYETUNCLEAR &nPERTURBATIONOFTHE-Nn-NDISTANCESBYTREAT MENTWITH &n ISONEOFTHEMOSTDIRECTSTRUCTURALDATA AVAILABLETHAT IMPLIES HALIDEASA LIGANDD OF -N8!3 STUDIES OF &n INHIBITEDSAMPLESSHOWTHATONEOFTHE TWO¯-Nn-NDISTANCESISINCREASEDTO^ ¯ W CH ISSUGGESTIVEOF &n BINDINGTOTHE-NCLUSTER WHI $E2OSEETAL  4HERECENT%3%%-RESULTSFROM D LES #LEMENSETAL  ACETATETREATED03))SAMP ALSOPROVIDETHEMOSTDIRECTSPECTROSCOPICEVIDENCE TODATEFORTHEPRESENCEOFACHLORIDELIGANDTO-N 4HERE IS EVIDENCE FROM F %8!&3 STUDIES WITH ORIENTED 3 STATE SAMPLES INDICATING THE PRESENCE OF-N #LLIGATIONINTHE 3 STATE&ERNANDEZETAL F ISOTROPICSAMPLES INTHE  %8!&3RESULTS FROM 3 STATEHAVESHOWNTHATTHEREISCONSIDERABLECHANGE



: IBOESB 7JUUBM , :BD

& &IG 4HE&OURIERTRANSFORMO F F FANOR IENTED 3SAMPLE4HE-N -NVECTORSREPRESENTED BY&OURIERPEAKS LABELED D ))AND )))AREDICHROIC Y IERPEAK LABELEDD  ISDISCERNIBLETHATISNOTOBSERVED INOTHER3 STATESAND ITlTS BESTTO#L INDICATING THEPOSSIBLE )NTERESTINGLY A&OUR PRESENCEOFA#LLIGANDTO-NATADISTANCEOF¯&ERNANDEZETAL  INTHE3 STATE

IN THE -Nn-N DISTANCES AS THE SYSTEM ADVANCES F FROMT HE3 TOTHE 3 STATE ,IANGETAL  4HE TWO¯-Nn-NDISTANCESINTHE 3 STATE INCREASE TO^AND^ ¯ INTHE3 STATE7I 7 TH THEAIMOF DETERMININGTHERELATIVEORIENTATIONOFT F HEAND n ¯ -Nn-NVECTORS WEINITIATED 8!3STUD IESOFF ORIENTED 3 STATESAMPLES4HE&4IN&IG SHOWS THAT THE TWO -Nn-N VECTORS ARE DICHROIC )NTERESTINGLY MAYBEBECAUSEOFTHELENGTHENINGOF THE-Nn-NVECTORS ANEW&OURIERPEAK ISOBSERVED BETWEENTHElRSTAND D SECOND &OURIERPEAKS LABELED IN&IG#OMPARINGTHIS&4WITHTHATOFA-N BINUCLEARCOMPLEXWITHONE#LnASA TERMINAL LIGAND TOA-NATOMSHOWSTHATTHEPEAK K CORRESPONDINGTO #LBACKSCATTERINGINTHEMODELCOMPLEXISFOUNDAT APPROXIMATELYTHESAMEAPPARENTDISTANCEASTHENEW &OURIERPEAK INTHE03))SAMPLES &ERNANDEZETAL  )NOTHERMODELCOMPOUNDS THEAMPLITUDEOF THISPEAK INCREASESASTHERATIOOFF #L-NINCREASESAND THEPEAK ISABSENTWH W EN#Ln ISNOTPRESENTASALIGAND )NTHE3 STATESAMPLE THIS PEAKDOESNOTlTTOLOWER :ATOMSSUCH AS# . OR/4HE lT ISSIGNIlCANTLY BETTERFOR F #LBACKSCATTERINGAT^¯4HElTSFORTHE MODELCOMPOUNDAREVERYSIMILAR$ICHROISMSTUD IESON3STATESAMPLESSHOWTHATTHE&OURIERPEAK IS LARGERATTHE ORIENTATIONCOMPARED D TOTHE  ORIENTATION WHICHSUGGESTSTHATTHE -Nn#LVECTORIS MOREPARALLELTOTHEMEMBRANENORMAL

7*
.FDIBOJTN
PG
8BUFS 0YJEBUJPO
BOE
0 &WPMVUJPO 4HE MECHANISMS FFOR WATER OXIDATION ANDD OXYGEN EVOLUTION THAT HAVE BEEN PROPOSEDD CAN BE BROADLY DIVIDEDINTOFOURCATEGORIESINWHICHTHECHARACTER F HEOXYGENATOMSTHATULTIMATELY FORM F DIOXYGEN OFT IS DIFFERENT)NT F HESEFOURGROUPST F HEOXYGENATOMS COME FROM TWO TERMINAL OXYGENS BOUND TO TWO SEPARATE -N ATOMS ONE TERMINAL OXYGEN ANDD ONE OXYGENNOTBOUNDD TOTHE-NCLUSTER ONETERMINAL AND ONEBRIDGINGOXYGEN ORTWO— OXOBRIDGES%ACHOF F THESEMECHANISMS INVOLVES DISTINCTLY DIFFERENT-Nn/ BONDS FORME F D AND BROKENDUR D INGTHECATALYTICCYCLE SEE#HAPTERFORDETAILS  4HECONTRIBUTIONOFF 8 RAAY STUDIESTOTH T EMECHANISM W ETH T ERANOXYGENATOM OF / EVOLUTIONIS INRESOLVINGWH DERIVEDFROMABRIDGINGPOSITIONINTHE-NCLUSTERIS INVOLVED INTHEULTIMATEFORMAT F IONOF DIOXYGENAND IN F WHETHER -NCENTEREDD OXIDATION THEDETERMINATIONOFW OCCURSDURINGEACHSTAGEOFTHE3 STATETRANSITIONS /UR 8 RAY A SPECTRA ARE STRONGLY SUPPORTIVE OFF -N D ING THE 3 A 3 AND 3 A 3 CLUSTER OXIDATION DUR TRANSITIONS THEYDONOTSUPPORTASIMILAR-N CENTERED D ING 3 A 3 &URTHERMORE %8!&3 OXIDATION DUR ANALYSESSHOWCLEAREVIDENCEOFNON F DEGENERACY IN THETHREE¯VECTORLENGTHSIN3AND3 BUTNOT IN3 ANDD 3 ANDD SHOWSSIGNIlCANTSTRUCTURAL CHANGE

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ



&IG 3UMMARYOFCHANGESIN-NOXIDATIONSTATESAND-N -NDISTANCESDURINGPHOTOSYNTHETICWATEROXIDATIONUSINGOPTION & D ' IN&IG )NTHE3 A 3 TRANSITION ONE-N))) ISOXIDIZEDTO-N)6 ANDTHE-Nn-NDISTANCESDONOTCHANGE$URINGTHE3 A 3 TRANSITION THE OXIDIZINGEQUIVALENTISLOCALIZEDD ONTHE— OXO BRIDGE WHI W CHTRIGGERSTHEINCREASEIN -Nn-N DISTANCES)NTHE3 A ;3= A 3 TRANSITION A SHORT LIVEDPEROXO D INTERMEDIATEMIGHT BE FORME F D INTHE3 STATEA B C AND D AREMECHANISMS FOR/ / F BOND FORMAT F IONINTHE 3 STATE-ECHANISMS A B AND C AREWH W EREONEORTWOBRIDGING/ATOMSARE DIRECTLY INVOLVED IN/ / BOND FORMAT F IONAMOTIFF THATIS CONSISTENTWITH 8!3DATA-ECHANISM D WOULDD REQUIRELOCALIZATIONOFF ANOXIDIZING EQUIVALENTONANOXOBRIDGETHAT LATEREXCHANGESOR F BOND FORMAT F ION$URING THE 3 A 3 TRANSITION A-N)) A -N))) OXIDATIONORA-N))) REARRANGESTOATERMINAL / LIGAND BEFORE/ / TO-N)6 CONCURRENTWITHADEPROTONATIONOFAHYDROXYTOANOXOCAUSINGTHEDECREASEOFTHE-Nn-NDISTANCEISINDICATED&ORCLARITY  n 9: THECOFACTORS#A AND#L ANDTERMINAL-NLIGANDSARENOTSHOWN-Nn-NDISTANCESWEREDETERMINEDBY%8!&3SPECTROSCOPY!S MENTIONEDIN$E2OSEETAL #INCOETAL AND2OBBLEEETAL OTHERPOSSIBLETOPOLOGICALMODELSEXISTFORTHE/%# SIMILARMECHANISMSTHATCANBEPROPOSEDFOREACHOFTHEALTERNATIVETOPOLOGICALMODELSSHOULDBECONSIDEREDEQUALLYVIABLE

DURINGTHE 3 TO 3 TRANSITIONTHATISNOTACCOMPANIED D BYA-N CENTEREDOXIDATION4HE8 RAYSPECTROSCOPY F BRIDGING/ EVIDENCESUPPORTSTHEINVOLVEMENTOFA F HE FORMAT F IONOFT F HE/ / ATOM INTHEMECHANISMOFT BOND4HESEOBSERVATIONSMUSTBECONSIDEREDINANY POSTULATIONOFMEC F HANISTICASPECTSOFT F HESUCCESSIVE

STEPSINWATEROXIDATION /UR PROPOSED MECHANISM ILLUSTRATED IN &IG  USINGTHEPROPOSEDD MODEL ' WH W ERETHE/ / BOND IS FORME F D BETWEENONECRITICAL BRIDGINGOXYGENAND ANOTHER OXYGEN ATOM DERIVED FROM A BRIDGINGTER MINAL OXYGEN LIGANDD ORANEXOGENOUSOXYGENALSO

7JUUBM , :BD : IBOESB

 AVOIDSTHEFORMAT F IONOFT F HE/n/ BOND D UNTIL THEMOST OXIDIZEDSTATE3 ISREACHED&IG A B C  F IONS FORT F HE 3 STATEGENERATE/ WHI W CH !LL FOUROPT IS DERIVED FROM F INEQUIVALENTOXYGENATOMSINTHE3 STATE ASSHOWNBY //WATER EXCHANGEMEASURE MENTSONTHE3 STATE -ESSINGERETAL (ILLIER ETAL  )NADDITION ARECENT(/(/ &4)2 EXPERIMENTHASSHOWNTHEPRESENCEOFANEXCHANGE ABLE DI — OXO BRIDGEINTHE 3 AND D 3 STATES #HUET AL  4HISMECHANISMPRECLUDESTHEFORMAT F ION AND D RELEASE OF PEROXIDEOROTHEROXIDATION PRODUCTS OFF WATER INTHEEARLIER3 STATES THUSPREVENTINGTHE F HORTCIRCUITINGAND D AVOIDINGTHERISK SYSTEM FROM@S OF DAMAGING THEPOLYPEPTIDESOF03)) "DLOPXMFEHNFOUT 4HISWORKWASSUPPORTEDBYTHE.ATIONAL)NSTITUTESOF (EALTHGRANT'- ANDBYTHE$IRECTOR /FlCE F IC%NERGY R 3CIENCES $IVISION OFF 3CIENCE /FlCEOF"AS OFF %NERGY "IOSCIENCES 5NITED D 3TATES $EPARTMENT OF %NERGY UNDER CONTRACT $% !# 3& ) F L TO EVERY ONE OF F THE VERY TALENTED D AND AM GRATEFU MOTIVATEDGRA D DUATESTU D DENTSAND D POSTDOCS AND D MANY SABBATICALVISITORSTOTHE #ALVIN,ABTHAT)HAVEHAD THEPRIVILEGETOWORK RK WITHONTHEOXYGENEVOLVING COMPLEXOVERTHELASTTWO DECADESAND D MOREEACH ANDEVERYONEOFTHEMCANRECOGNIZESOMEASPECTOF THEWORK RK PRESENTED HERETHATTHEY WERERESPONSIBLEFO F R D THAT HASCONTRIBUTED D TOTHEPRESENTUNDERSTAND AND INGOFTHE-NCLUSTERINTHE /%#3PECIlCALLY THE CONTRIBUTIONSTOTHERESEARCH PRESENTED INTHISARTICLE BY2OEHL#INCO *OHN2OBBLEE *OHANNES-ESSINGER (ENK6ISSER 5WE"ERGMANN 0IETER'LATZEL #ARMEN &ERNANDEZ %LODIE !NXOLABÏHÒRE -ALLART 3HELLY 0IZARRO +AREN-C&ARLANE *UNKO9ANO 3TEVE#RA MERAND+EN3AUERISGRATEFULLYACKNOWLEDGED-EL +LEINSPROFOUN F D INmUENCEAND D CONTRIBUTIONSTOTHE %02AND8 RAYSPECTROSCOPYRESEARC D H PERVADESTHE WORKPRESENTEDINTHISCHAPTER)EXTENDMYTHANKS TOOURCOLLABORATORS0ROFS7(!RMSTRON F G '7 "RUDVIG '#HRISTOU 4#OLLINS * *'IRERD 2. D -UKHERJEE 6 , 0ECORARO AND +7IEGHARDT WHO HAVE GENEROUSLY PROVIDED D ALL THEINORGANIC-NCOM POUNDSTHATHASMADEALL THEDIFFERENCE F INANALYZING THEDATAFROM03))3YNCHROTRONRADIATIONFACILITIES WEREPROVIDED BY THE3TANFOR F D3 D YNCHROTRON2ADIA TION,ABORATORY 332, AND D THE!DVANCED D 0HOTON 3OURCE!03 BOTHSUPPORTEDBYTHE5NITED3TATES $EPARTMENT OF F %NERGY AND D THE .ATIONAL )NSTITUTES OFF (EALTH

3FGFSFODFT !DELROTH0 ,INDBERG R +AND!N D DRÏASSON,% D  3TUDIESOFF #A BINDINGINSPINACH0HOTOSYSTEM))USING #A"IOCHEMISTRY n ¯HRLING+! 0ETERSON3AND3TYRING3 4HE3 STATE%02 SIGNALFROMTHE-NCLUSTERIN0HOTOSYSTEM))ARISESFROMAN ISOLATED3šGROUN D D STATE"IOCHEMISTRYn D $ISMUKES'#  #ALCIUM INDUCES D BINDING !NANY N EV'-AND AND FORMAT F IONOFASP F IN COUPLED DIMANGANESE)) )) CENTER IN THE APO WATER OXIDATION COMPLEX OFF 0HOTOSYSTEM)) AS PRECURSORTOTHEFUNCT F IONALTETRA -N#ACLUSTER"IOCHEMISTRY n !UGER. 'IRERD* * #ORBELLA- 'LEIZES!AND:IMMERMANN * ,  3YNTHESIS STRUCTURE AND MAGNETIC PROPERTIES OF THE STABLE TRIANGULAR ;-N)6 /= CORE * !M #HEM 3OC  n "ECK K 7& $EPAULA*#AND D "RUDVIG '7  !MMONIABINDSTO T EMANGANESESITEOFF THE/ EVOLVING COMPLEXOFF 0HOTOSYSTEM TH )) INTHE 3 STATE*!M #HEM 3OCn "ERGMANN5 'RUSH -- (ORNE#2 $E-AROIS0 0ENNER (AHN *% 9OCUM#& 7RIGHT$7 $UBÏ#% !RMSTRONG7( #HRISTOU ' %PPLEY(*AND#RAMER30 #HARACTERIZATIONOFTHE-N OXIDATIONSTATESIN0HOTOSYSTEM))BY+` 8 RAYmUORESCENCE SPECTROSCOPY*0HYS#HEM"n "OUSSAC!AND D 2UTHERFOR F D !7 A 3 STATEFORMAT F IONAFTER F #A DEPLETIONINTH T E0HOTOSYSTEM))OXYGEN EVOLVING COMPLEX #HEM3CRIPT!n "OUSSAC!AND D 2UTHERFOR F D!7 D B .ATUREOFF THE INHIBITION OFF THEOXYGEN EVOLVING ENZYMEOFF 0HOTOSYSTEM)) INDUCE D D BY .A#L WASHING AND REVERSED BY THE ADDITION OF #A OR 3R "IOCHEMISTRYn "OUSSAC!AND2UTHERFORD!7 #OMPARATIVESTUDYOFTHE G%02SIGNALSINTHE3 STATEOF0HOTOSYSTEM))"IOCHIM H "IOPHYS!CTAn "OUSSAC! 'IRERD D * *AND 2UTTHERFOR F D!7 D  #ONVERSIONOFF THE SPINSTATEOFF THEMANGANESECOMPLEX IN0HOTOSYSTEM)) INDUCE D D BY NEAR INFRARE F D LIGHT"IOCHEMISTRYn "RITT2$ /XYGENEVOLUTION)N/RT$2AND9OCUM#& D EDS /XYGENIC0HOTOSYNTHESIS4HE,IGHT2EACTIONS PPn +LUWER!CADEMIC0UBLISHERS $ORDRECHT "RITT 2$ :IMMERMANN * , 3AUER + AND +LEIN -0  !MMONIA BINDS TO THE CATALYTIC MANGANESE OF THE OXYGEN EVOLVING COMPLEX OFF 0HOTOSYSTEM)) %VIDENCE BY ELECTRON SPIN ECHO ENVELOPE MODU D LATION SPECTROSCOPY Y * !M #HEM 3OCn "RITT2$ 0ELOQUIN*-AND D #AMPBELL+!  0ULSED D ANDPA D R ALLEL POLARIZATION%02CHARACTERIZATIONOFT F HE0HOTOSYSTEM)) OXYGEN EVOLVINGCOMPLEX!NN2EV"IOPHYS"IOMOL3TRUCT n #AMMARATA +6 AND #HENIAE '-  3TUDIES ON   K$A DEPLETED 0HOTOSYSTEM)) MEMBRANES 0LANT 0HYSIOL  n #ARRELL4' 4Y 4 RYSHKIN!-AND D $ISMUKES'# !NEVALUA TIONOFF STRUCTURAL MODELSFORT F HEPHOTOSYNTHETICWATER OXIDIZING COMPLEX DERIVED FFROM SPECTROSCOPIC AND D 8 RAAY DIFFRACT F ION SIGNATURES*"IOL )NORG #HEMn #HEN# +AZIMIR*AND#HENIAE'- #ALCIUMMODULATESTHE PHOTOASSEMBLYOF0HOTOSYSTEM))-N  CLUSTERSBYPREVENTING LIGATIONOFNONFUNCTIONALHIGH VALENCYSTATESOFMANGANESE "IOCHEMISTRYn #HU( ! 3ACKETT(AND D "ABCOCK K '4  )DENTIlCATIONOF

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ A-N / -NCLUSTERVIBRATIONAL MODEOFT F HEOXYGEN EVOLVING COMPLEXIN0HOTOSYSTEM))BYLOW FREQUENCY&4)2SPECTROS COPY"IOCHEMISTRYn #INCO2- 2OBBLEE*( 2OMPEL! &ERNANDEZ# 9ACHANDRA6+ 3AUER+AND+LEIN-0 3TRONTIUM%8!&3REVEALSTHE PROXIMITY TYOF CALCIUMTOTH T EMANGANESECLUSTEROFF OXYGEN EVOLV ING 0HOTOSYSTEM))*0HYS#HEM"n #INCO2- 2OMPEL! 6I 6 SSER( !ROMÓ' #HRISTOU' 3AUER + +LEIN -0 ANDD 9ACHANDRA6+  #OMPARISON OFF THE MANGANESE CLUSTER IN OXYGEN EVOLVING 0HOTOSYSTEM)) WITH DISTORTEDCUBANEMANGANESECOMPOUNDSTHROUGH8 RAYABSORP TIONSPECTROSCOPY)NORG#HEMn #INCO2- (OLMAN+,- 2OBBLEE*( 9ANO* 0IZARRO3! "EL LACCHIO% 3AUER+AND9ACHANDRA6+ #ALCIUM%8!&3 ESTABLISHESTHE-N #ACLUSTERINTHEOXYGEN EVOLVINGCOMPLEX OFF 0HOTOSYSTEM))"IOCHEMISTRY n #LEMENS+, &ORCE$!ANDD "RITT2$ !CETATE BINDING ATTH T E 0HOTOSYSTEM))OXYGENEVOLVING COMPLEX!N3 STATEMULTILINE SIGNAL%3%%-STUDY*!M# Y HEM3OCn #OLE * * 9ACHANDRA6+ 'UILES 2$ -C$ERMOTT!% "RITT 2$ $EXHEIMER3, 3AUER+AND+LEIN-0 !SSIGNMENTOF THEG%02SIGNALTOMANGANESEINTHE 3 STATEOFTHEPHOTO SYNTHETICOXYGEN EVOLVINGCOMPLEX!N8 RAYABSORPTIONEDGE SPECTROSCOPYSTUDY"IOCHIM "IOPHYS!CTAn H  #RAMER 30  "IOCHEMICAL APPLICATION OFF 8 RAAY ABSORP TION SPECTROSCOPY Y )N +ONINGSBERGER $# ANDD 0RINS 2 EDS 8 2AY A !BSORPTION 0RINCIPLES !PPLICATIONS 4ECHNIQUES OF 7 EY %8!&3 3%8!&3ANDD 8!.%3 PPn*OHN7IL 3ONS .EW9ORK $AU( !NDREWS*# 2OELOFS4! ,ATIMER-* ,IANG7 9ACHANDRA 6+ 3AUER+AND+LEIN-0 3TRUCTURALCONSEQUENCESOF AMMONIABINDINGTOTHEMANGANESECLUSTEROFTHEPHOTOSYN THETICOXYGEN EVOLVINGCOMPLEX!N8 RAYABSORPTIONSTUDYOF ISOTROPICAND D ORIENTEDD 0HOTOSYSTEM))PARTICLES"IOCHEMISTRY  n $EBUS 2*  4HE MANGANESE ANDD CALCIUM IONS OFF PHOTO SYNTHETIC OXYGEN EVOLUTION "IOCHIM "IOPHYS !CTA  n $EKKER*0 /PTICALSTUDIESONTHEOXYGEN EVOLVINGCOMPLEX OF 0HOTOSYSTEM)) )N 0ECORARO6, ED -ANGANESE 2EDOX %NZYMES PPn6#(0UBLISHERS .EW9ORK R ) ,ATIMER-* 9ACHANDRA6+ 3AUER+AND $E2OSE6* -UKERJI F HEMANGANESEOXYGEN EVOLV +LEIN -0  #OMPARISONOFT ING COMPLEX IN0HOTOSYSTEM))OFF SPINACH AND 3Y 3 NECHOCOCCUS SPWITHMULTINUCLEARMANGANESEMODELCOMPOUNDS BY8 RAY A ABSORPTIONSPECTROSCOPY*!M# Y HEM3OCn $E2OSE6* ,ATIMER-* :IMMERMANN* , -UKERJI) 9ACHANDRA 6+ 3AUER+AND+LEIN-0 &LUORIDESUBSTITUTIONINTHE -NCLUSTERFROM0HOTOSYSTEM))%02AND8 RAYABSORPTION SPECTROSCOPYSTUDIES#HEM0HYSn $ISMUK U ES'#AND3 D IDERER9 )NTERMEDIATESOFAPO F L LYNUC LEAR MANGANESE CLUSTER INVOLVED IN PHOTOSYNTHETIC OXIDATION OF WATER0ROC.ATL !CAD 3CI 53!n F D !7AND (OFFF !*  %02 %VELO2' 3TYRING 3 2UTHERFOR RELAXATION MEASUREMENTS OF 0HOTOSYSTEM )) REACTION CEN TERS ˆ INmUENCEOF3 STATEOXIDATIONANDTEMPERATURE"IOCHIM "IOPHYS!CTAn &ERNANDEZ# #INCO2- 2OBBLEE*( -ESSINGER* 0IZARRO3! 3AUER+ +LEIN-0AND9ACHANDRA6+ #ALCIUMAND CHLORIDECOFACTORSO F F HEOXYGENEVOLVINGCOMPLEX ˆ 8 RAY FT ABSORPTIONSPECTROSCOPYEV Y IDENCEFORA-N F N#A A #L HETERONUCLEAR

 CLUSTER )N 'ARAB ' ED 0HOTOSYNTHESIS -ECHANISMS AND %FFECTS 6OL)) PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT &ERREIRA + )VERSON4- -UGHLOUNI + "ARBER * AND )WATA 3  !RCHITECTURE OF THE PHOTOSYNTHETIC OXYGEN EVOLVING CENTER3CIENCEn 'EORGE'. 0RINCE2#AND D #RAMER30  4HEMANGANESE SITE OFF THE PHOTOSYNTHETIC WATER SPLITTING ENZYME 3CIENCE n 'HANOTAKIS $& "ABCOCK K '4 AND D9OCUM #&  #ALCIUM RECONSTITUTES HIGH RATES OF OXYGEN EVOLUTION IN POLYPEPTIDE DEPLETED 0HOTOSYSTEM)) PREPARATIONS &%"3 ,ETT  n 'HANOTAKIS$& "ABCOCK'4AND9OCUM#& 3TRUCTUREOF THEOXYGEN EVOLVINGCOMPLEXOFF 0HOTOSYSTEM))#ALCIUMAND LANTHANUMCOMPETE FORS F ITESONTHEOXIDIZING SIDEOFF 0HOTOSYS TEM))WHI W CH CONTROLTHE BINDINGOFF WATER SOLUBL U EPOLYPEPTIDES AND D REGULATETHEACTIVITYOFT F HEMANGANESECOMPLEX"IOCHIM "IOPHYS!CTAn 'ILCHRIST-, *R "ALL*! 2ANDALL$7AND"RITT2$ 0ROX IMITYOFTHEMANGANESECLUSTEROF0HOTOSYSTEM))TOTHEREDOX ACTIVETYROSINE9Z0ROC.ATL!CAD3CI53!n 'OODIN$" 9ACHANDRA6+ "RITT2$ 3AUER+AND+LEIN-0  4HESTATEOFMANGANESE F INTHEPHOTOSYNTHETICAPPARATUS D D CHANGESIN8 RAY AY ABSORPTION+ EDGE ENERGI R ES ,IGHT INDUCE OFF MANGANESEINPHOTOSYNTHETICMEMBRANES"IOCHIM"IOPHYS !CTAn 'UILES2$ 9ACHANDRA6+ -C$ERMOTT!% #OLE*, $EXHEIMER 3, "RITT 2$ 3AUER + AND +LEIN -0 A 4HE 3 STATE OF 0HOTOSYSTEM)) INDUCED BY HYDROXYLAMINE $IFFERENCES BETWEEN THE STRUCTURE OF THE MANGANESE COMPLEX IN THE 3 AND 3 STATES DETERMINED BY 8 RAY ABSORPTION SPECTROSCOPY "IOCHEMISTRYn 'UILES2$ :IMMERMANN*, -C$ERMOTT!% 9ACHANDRA D 6+ #OLE 7 EGHARDT+ "OSSEK K 5 3AUER+ *, $EXHEIMER3, "RITT2$ 7I AND D +LEIN-0B 4HE3 STATEOFF 0HOTOSYSTEM))$IFFE F R ENCESBETWEENTHESTRUCTUREOFF THEMANGANESECOMPLEX INTHE 3 AND3STATESDETERMINEDBY8 RAYABSORPTION SPECTROSCOPY "IOCHEMISTRYn (AN+ #AND+ATOH3 $IFFERENTLOCALIZATIONOFTWO#A IN SPINACHOXYGEN EVOLVING0HOTOSYSTEM))MEMBRANES%VIDENCE F INVOLVEMENTOFON FOR F LYONE#A INOXYGENEVOLUTION0LANT #ELL 0HYSIOL n (ASEGAWA+ +USUNOKI- )NOUE9AND/NO4 ! D  3IMULA TIONOF 3 STATEMULTILINE%02SIGNAL INORIENTED D 0HOTOSYSTEM)) F THEMANGANESECLUSTERIN MEMB M RANES3TRUCTURAL IMPLICATIONSFORT ANOXYGEN EVOLVINGCOMPLEX"IOCHEMISTRYn (ASEGAWA+ /NO4 ! )NOUE9AND+USUNOKI- 3PIN EXCHANGEINTERACTIONSINTHE3 STATEMANGANESETETRAMERIN PHOTOSYNTHETICOXYGEN EVOLVINGCOMPLEXDEDUCEDFROMG H  MULTILINE%02SIGNAL#HEM0HYS,ETTn (ILLIER7 -ESSINGER*AND D 7YDRZYNSKI 4  +INETIC DETERMI NATIONOFT F HEFASTEXC F HANGING SUB U STRATEWATERMOLECULEINTHE3 STATEOFF 0HOTOSYSTEM))"IOCHEMISTRYn )UZZOLINO, $ITTMER* $ÚRNER7 -EYER +LAUCKE7AND D $AU(  8 RAY ABSORPTION SPECTROSCOPY ON LAYERED 0HOTOSYS TEM))MEMBRANEPARTICLESSUGGESTSMANGANESE CENTEREDOXIDA TIONOFTHEOXYGEN EVOLVINGCOMPLEXFORTHE3 3 3 3 AND 3 3 TRANSITIONSOFTHEWATEROXIDATIONCYCLE"IOCHEMISTRY  n *AKLEVIC* +IRBY R *! +LEIN-0 2OBERTSON!3 "ROWN'3AND

 %ISENBERGER 0  &LUORESCENCE DETECTION OF F %8!&3 3ENSITIVITYENHANCEMENTFORDILUTESPECIESANDTHINlLMS3OLID 3TATE#OMMUNn *OLIOT0 0ERIOD FOUROSCILLATIONSOFTHEmASH INDUCEDOXYGEN FORMATIONINPHOTOSYNTHESIS0HOTOSYNTH2ESn D 3HEN*2 #RYSTALSTRUCTUREOFOX F YGEN EVOLV +AMIYA.AND ING 0HOTOSYSTEM))FROM F 4HERMOSY S NECHOCOCCUSVULCANUS AT ¯ RESOLUTION0ROC.ATL!CAD3CI53!n +ATOH 3 3ATOH + /HNO4 #HEN * 2 ANDD +ASHINO9  .UMBERS OFF CALCIUM IONS ASSOCIATEDD WITH OXYGEN EVOLVING 0HOTOSYSTEM))PREPARATIONSWITHDIFFERENTAFlNITIES)N"IGGINS *ED 0ROGRESSIN0HOTOSYNTHESIS2ESEARCH 6OL) PPn -ARTINUS.IJHOFF0UBLISHERS $ORDRECHT +IRBY*! 2OBERTSON!3 3MITH*0 4HOMPSON!# #OOPER32 AND D +LEIN -0  3TATEOFF MANGANESE INTHEPHOTOSYNTHETIC APPARATUS%XTENDEDD 8 RAAY ABSORPTIONlNESTRUCTURESTUD IESONCHLOROPLASTSAND DI — OXO BRIDGED DIMANGANESEMODEL COMPOUNDS*!M#HEM3OCn K " &ORRBUSH "ANDD -C'LOIN- #OOPERATIONOFF CHARGES +OK INPHOTOSYNTHETICOXYGENEVOLUTION)!LINEARFOURSTEPMECHA NISM0HOTOCHEM0HOTOBIOLn +RETSCHMANN( $EKKER*0 3AYGIN½AND7ITT(4 !N AGREEMENTONTHEQUATERNARYOSCILLATIONOFULTRAVIOLETABSORPTION CHANGESACCOMPANYINGTHEWATERSPLITTINGIN ISOLATEDD 0HOTO SYSTEM))COMPLEXESFROMT F HECYANOBACTERIUM 3Y 3 NECHOCOCCUS SP"IOCHIM"IOPHYS!CTAn +USUNOKI - 4AKANO 4 /NO 4 .OGUCHI 4 9AMAGUCHI 9 D )NOUE9  !DVANCEDD %8!&3STUDIESOF /YANAGI (AND THE 3 STATE MANGANESE CLUSTER IN PLANT 0HOTOSYSTEM)) )N -ATHIS0ED 0HOTOSYNTHESIS&ROM,IGHTTO"IOSPHERE 6OL)) PPn+LUWER!CADEMIC0UBLISHERS $ORDRECHT +UZEK$AND0ACE2* 0ROBINGTHE-NOXIDATIONSTATES INTHE/%#)NSIGHTSFROMSPECTROSCOP F IC COMPUTATIONALAND KINETICDATA"IOCHIM "IOPHYS!CTAn ,AKSHMI +6 %ATON33 %ATON'2 &RANKK (!AND "RUDVIG'7  !NALYSISOF DIPOLARANDD EXCHANGEINTERACTIONSBETWEEN MANGANESEANDD TYROSINE:INTHE39Zs STATEOFF ACETATE INHIBITED 0HOTOSYSTEM))VIA%02SPECTRALSIMULATIONSAT8 AND1 BANDS *0HYS#HEM"n ,ATIMER-* $E2OSE6* -UKERJI) 9ACHANDRA6+ 3AUER+AND +LEIN-0 %VIDENCEFORTHEPROXIMITYOFCALCIUMTOTHE MANGANESECLUSTEROFF 0HOTOSYSTEM))$ETERMINATIONBY8 RAY ABSORPTIONSPECTROSCOPY" Y IOCHEMISTRY n ,ATIMER-* $E2OSE6* 9ACHANDRA6+ 3AUER+ANDD +LEIN-0  3TRUCTURAL EFFECTSO F F CALCIUMDEPLETIONONTH T EMANGANESE CLUSTEROF0HOTOSYSTEM))$ETERMINATIONBY 8 RAY A ABSORPTION SPECTROSCOPY*0HYS#HEM"n ,IANG7 ,ATIMER-* $AU( 2OELOFS4! 9ACHANDRA6+ 3AUER +AND+LEIN-0 #ORRELATIONBETWEENSTRUCTUREANDMAG NETICSPINSTATEOFTHEMANGANESECLUSTERINTHEOXYGEN EVOLVING COMPLEXOFF 0HOTOSYSTEM))INTHE3 STATE$ETERMINATIONBY 8 RAY A ABSORPTIONSPECTROSCOPY" Y IOCHEMISTRYn ,IANG7 2OELOFS4! F #INCO 2- 2OMPEL! ,ATIMER -* 9U 9 7 7/ 3AUER+ + LEIN-0ANDD 9ACHANDRA6+ 3TRUCTURAL CHANGEOFF THE-NCLUSTERDUR D INGTHE3 A 3 STATETRANSITION OF THE OXYGEN EVOLVING COMPLEX OF 0HOTOSYSTEM)) $OES IT REmECTTHEONSETOFWATERSUBSTRATEOXIDATION$ETERMINATION BY-N8 RAYABSORPTIONSPECTROSCOPY*!M#HEM3OC n ,IMBURG* 3ZALAI6!ANDD "RUDVIG'7 !MECHANISTICAND STRUCTURALMODELFORTHEFORMATIONANDREACTIVITYOFA-N NV/

7JUUBM , :BD : IBOESB SPECIESINPHOTOSYNTHETICWATEROXIDATION*#HEM3OC $ALTON 4RANSn ,INDBERG+AND!NDRÏASSON,% !ONE SITE TWO STATEMODEL FORTHEBINDINGOFANIONSIN0HOTOSYSTEM))"IOCHEMISTRY n ,INDBERG + 7YD 7 RZYNSKI4 6ÊNNGÌRD D 4 AND !NDRÏASSON , %   3LOWRELEASEOFC F HLORIDE FROM F #L LABELED D 0HOTOSYS TEM))MEMBRANES&%"3,ETTn -AC,ACHLAN $* (ALLAHAN "* 2UFmE 36 .UGENT *(! %VANS -#7 3TRANGE27AND D (ASNAIN33  !N%8!&3STUDY OF THE MANGANESE OXYGEN EVOLVING COMPLEX IN PURIlED 0HOTOSYSTEM)) MEMBRANE FRACTIONS 4HE 3 AND 3 STATES "IOCHEM*n -AC,ACHLAN$* .UGENT*(! "RATT0*AND%VANS-#7 F F LCIUM DEPLETIONONTHE/ EVOLVING COMPLEX FCA 4HEEFFECTSO INSPINACH03))4HE3 3 AND 3 STATES AND D THE ROLE OFF THE D  K$AEXTRINSICPOLYPEPTIDES"IOCHIM"IOPHYS K$AAND !CTAn -C$ERMOTT!% 9ACHANDRA6+ 'UILES2$ #OLE*, $EXHEIMER 3, "RITT2$ 3AUER+AND+LEIN-0 #HARACTERIZATION OFTHEMANGANESE/ EVOLVINGCOMPLEXANDTHEIRON QUINONE ACCEPTORCOMPLEXIN0HOTOSYSTEM))FROMATHERMOPHILICCYA NOBACTERIUMBYELECTRONPARAMAGNETICRESONANCEAND8 RAY ABSORPTIONSPECTROSCOPY"IOCHEMISTRYn -ESSINGER* "ADGER-AND7 D 7YDDRZYNSKI4  $ETECTIONOF ONE SLOWLY EXCHANGINGSUB U STRATEWATERMOLECULEINTHE 3 STATE OF D 3CI53!n 0HOTOSYSTEM))0ROC.ATL!CAD -ESSINGER* .UGENT*(!AND%VANS-#7 D A $ETECTION OFAN%02MULTILINESIGNALFORTHE3 STATEIN0HOTOSYSTEM)) "IOCHEMISTRYn -ESSINGER* 2OBBLEE* 9U7/ 3AUER+ 9ACHANDRA6+AND+LEIN -0B 4HE3 STATEOFTHEOXYGEN EVOLVINGCOMPLEXIN 0HOTOSYSTEM)) ISPARAMAGNETIC$ETECTIONOFF AN%02MULTILINE SIGNAL*!M#HEM3OCn -ESSINGER* 2OBBLEE*( "ERGMANN5 &ERNANDEZ# 'LATZEL0 6 SSER ( #INCO 2- -C&ARLANE +, "ELLACCHIO % 0IZARRO 6I 3! #RAMER30 3AUER+ +LEIN-0AND D 9ACHANDRA6+ !BSENCEOF-N CENTEREDOXIDATIONINTHE3 A 3 TRANSITION )MPLICATIONSFORTHEMECHANISMOFPHOTOSYNTHETICWATEROXIDA TION*!M#HEM3OCn -UKERJI) !NDREWS*# $E2OSE6* ,ATIMER-* 9ACHANDRA6+ 3AUER+AND D +LEIN -0  /RIENTATIONOFT F HEOXYGEN EVOLV INGMANGANESECOMPLEX INA0HOTOSYSTEM))MEMBRANEPREPA RATION!N8 RAY A ABSORPTIONSPECTROSCOPYSTUDY" Y IOCHEMISTRY  n /LESEN+AND D !NDRÏASSON,% 4HEFUNCT F IONOFF THECHLO RIDE ION IN PHOTOSYNTHETIC OXYGEN EVOLUTION "IOCHEMISTRY n /NO4 !AND)NOUE9 $ISCRETEEXTRACTIONOFTHE#AATOM FUNCTIONALFOR/ EVOLUTIONINHIGHERPLANT0HOTOSYSTEM))BY ASIMPLE LOW P(TREATMENT&%"3,ETTn /NO4 !AND D )NOUE9  2OLESOF#A F  IN / EVOLUTION IN HIGHERPLANT0HOTOSYSTEM))%FFECTSO F F LACEMENTOFF #A SITE FREP BY OTHERCATIONS!RCH "IOCHEM"IOPHYSn /NO4 ! :IMMERMANN*, )NOUE9AND D 2UTHERFOR F D!7 D  %02EVIDENCEFORAMODIlED3 STATETRANSITIONINCHLORIDE DE PLETED0HOTOSYSTEM))"IOCHIM"IOPHYS!CTAn /NO4 ! .OGUCHI4 )NOUE9 +USUNOKI- -ATSUSHITA4AND /YANAGI( 8 RAYDETECTIONOFTHEPERIOD FOURCYCLING OFF THE MANGANESE CLUSTER IN PHOTOSYNTHETIC WATER OXIDIZING ENZYME3CIENCEn

$IBQUFS

.O $MVTUFS
BOE
9SBZ 4QFDUSPTDPQZ /NO4 ! .OGUCHI4 )NOUE9 +USUNOKI - 9AMAGUCHI(AND /YANAGI( 3TUDYOFTHEINTERMEDIATE3 STATESFORWA TER OXIDATION IN THE NORMAL AND #A DEPLETED PHOTOSYNTHETIC OXYGEN EVOLVING ENZYME BY MEANS OF mASH INDUCED 8 RAY ABSORPTION NEAR EDGE STRUCTURE SPECTROSCOPY "IOCHEM 3OC 4 ANS    4R 0ECORARO 6, ED  -ANGANESE 2EDOX %NZYMES 6#( R 0UBLI U SHERS .EW9ORK 0ECORARO6,AND D (SIEH7 9  4HEUSEOFMO F DELCOMPLEXES F ION OF F MANGANESE REDOX TO ELUCIDATE THE STRUCTURE AND FUNCT ENZYMES )N 3IGEL! AND 3IGEL ( EDS -ANGANESE AND )TS 2OLE IN "IOLOGICAL 0ROCESSES PP n -ARCEL $EKKER )NC .EW9ORK 0ELOQUIN*-AND"RITT2$ %02%.$/2CHARACTERIZATION OFF THEPHYSICAL ANDD ELECTRONICSTRUCTUREOFF THE/%#-NCLUSTER "IOCHIM "IOPHYS!CTAn 0ELOQUIN*- #AMPBELL+!ANDD "RITT2$ -NPULSED %.$/2 DEMONSTRATES THAT THE 0HOTOSYSTEM)) @SPLIT %02 SIGNAL ARISES FFROM A MAGNETICALLY COUPLEDD MANGANO TYROSYL COMPLEX*!M#HEM3OCn 0ELOQUIN*- #AMPBELL+! 2ANDALL$7 %VANCHIK-! 0ECORARO 6, !RMSTRONG7(AND"RITT2$ -N%.$/2OFTHE 3 STATEMULTILINE%02SIGNALOF0HOTOSYSTEM)))MPLICATIONS ONTHESTRUCTUREOFF THETETRANUCLEARCLUSTER * !M #HEM3OC  n 0ENG ' DE 'ROOT &-& (ÊMÊLÊINEN + -OORE *! 7ANG 8 'RUSH-- (ASTINGS*" 3IDDONS$0 !RMSTRONG 7( -ULLINS D #RAMER30 (IGH RESOLUTIONMANGANESE8 RAY A /#AND mUORESCENCE SPECTROSCOPY /XIDATION STATE AND SPIN STATE SENSITIVITY*!M#HEM3OCn 0ENNER (AHN *%  3TRUCTURAL CHARACTERIZATION OF THE -N SITE IN THE PHOTOSYNTHETIC OXYGEN EVOLVING COMPLEX 3TRUCT "OND  n 0ENNER (AHN *% &RONKO 2- 0ECORARO6, 9OCUM #& "ETTS 3$ AND D "OWLBY .2  3TRUCTURAL CHARACTERIZATION OF THE MANGANESE SITES IN THE PHOTOSYNTHETIC OXYGEN EVOLVING COMPLEXUSING8 RAY A ABSORPTION SPECTROSCOPY*!M# Y HEM 3OCn 2ENGER' -ECHANISTICANDSTRUCTURALASPECTSOFPHOTOSYN THETICWATEROXIDATION0HYSIOL0LANTn 2IGGS0* 9OCUM#& 0ENNER (AHN*%AND-EI2 2EDUCED DERIVATIVESOFT F HEMANGANESECLUSTERINTHEPHOTOSYNTHETICOXY GEN EVOLVINGCOMPLEX*!M#HEM3OCn 2IGGS 'ELASCO0* -EI2 9OCUM#&ANDD 0ENNER (AHN*% A 2EDUCE D D DERIVATIVESOFF THE-NCLUSTERINTHEOXYGEN EVOLVING COMPLEX OFF 0HOTOSYSTEM))!N %8!&3 STUDY Y *!M #HEM 3OCn 2IGGS 'ELASCO0* -EI2 'HANOTAKIS$& 9OCUM#&AND0ENNER (AHN*%B 8 RAYABSORPTIONSPECTROSCOPYOFCALCIUM SUBSTITUTED DERIVATIVES OF THE OXYGEN EVOLVING COMPLEX OF 0HOTOSYSTEM))*!M#HEM3OCn A SPEC 2OBBLEE*( #INCO2-ANDD 9ACHANDRA6+ 8 RAY TROSCOPY BASED D STRUCTURE OF THE -N CLUSTER ANDD MECHANISM OFF PHOTOSYNTHETIC OXYGEN EVOLUTION "IOCHIM "IOPHYS!CTA n 2OBBLEE*( -ESSINGER* #INCO2- -C&ARLANE+, &ERNANDEZ # 0IZARRO 3! 3AUER + AND9ACHANDRA6+  4HE -N CLUSTER IN THE 3 STATE OF THE OXYGEN EVOLVING COMPLEX OF 0HOTOSYSTEM)) STUDIED BY %8!&3 SPECTROSCOPY !RE THERE THREEDI — OXO BRIDGEDD -N MOIETIES INTHETETRANUCLEAR -N COMPLEX*!M#HEM3OCn

 2OE!, 3CHNEIDER$* -AY A ER2* 0YRZ*7 7IDOM*AND1UE , *R 8 RAYABSORPTIONSPECTROSCOPYOFIRON TYROSINATE PROTEINS*!M#HEM3OCn 2OELOFS4! ,IANG7 ,ATIMER-* #INCO2- 2OMPEL! !NDREWS *# 3AUER+ 9ACHANDRA6+AND+LEIN- /XIDATION STATESOFT F HEMANGANESECLUSTERDUR D INGTHEmASH INDUCE D D3 STATE D CYCLE OFF THE PHOTOSYNTHETIC OXYGEN EVOLVING COMPLEX 0ROC .ATL!CAD 3CI 53!n 2 HERFOR 2UT F D!7 : D IMMERMANN* ,AND D "OUSSAC! /XYGEN EVOLUTION)N"ARBER*ED 4HE0HOTOSYSTEMS3TRUCTURE &UNC TION AND -OLECULAR "IOLOGY PP n %LSEVIER 3CIENCE 0UBLISHERS"6 6 !MSTERDAM 3ANDUSKY0/AND9OCUM#& 4HECHLORIDEREQUIREMENTFOR PHOTOSYNTHETICOXYGENEVOLUTION&ACTORSAFFECTINGNUCLEOPHILIC DISPLACEMENTOFC F HLORIDE FROMT F HEOXYGEN EVOLVING COMPLEX "IOCHIM "IOPHYS!CTAn 3AUER + 9ACHANDRA6+ "RITT 2$ AND D +LEIN -0  4HE PHOTOSYNTHETICWATEROXIDATIONCOMPLEXSTUDIED BY %02AND Y)N0ECORARO6,ED -ANGANESE 8 RAY AYABSORPTIONSPECTROSCOPY 2EDOX%NZYMES PPn6#(0UBLISHERS .EW9ORK R 3AYERS $% 3TERN %! AND ,YTLE &  .EW TECHNIQUE FOR INVESTIGATING NONCRYSTALLINE STRUCTURES &OURIER ANALYSIS OF THEEXTENDED8 RAY ABSORPTIONlNESTRUCTURE0HYS2EV,ETT  n 3AYG A IN½AND7ITT(4 /PTICALCHARACTERIZATIONOFINTER MEDIATESINTHEWATER SPLITTINGENZYMESYSTEMOFF PHOTOSYN THESISˆPOSSIBLESTATESAND D CONlGURATIONSOFF MANGANESEAND WATER"IOCHIM"IOPHYS!CTAn 3CHILLER( $ITTMER* )UZZOLINO, $ÚRNER7 -EYER +LAUCKE7 3OLÏ6! .OLTING( &AND$AU( 3TRUCTUREANDORIEN TATIONOFTHEOXYGEN EVOLVINGMANGANESECOMPLEXOFGREEN ALGAEANDHIGHERPLANTSINVESTIGATEDBY8 RAYABSORPTIONLINEAR DICHROISMSPECTROSCOPYONORIENTED D 0HOTOSYSTEM))MEMBRANE PARTICLES"IOCHEMISTRYn D THEPHOTOSYNTH T ETIC 3HARP22 0ROTON.-2RELAXATIONDUETOT OXYGEN EVOLVINGCENTER)N0ECORARO6,ED -ANGANESE2EDOX %NZYMES PPn6#(0UBLI U SHERS .EW9ORK R 3HEN * 2 3ATOH + AND +ATOH 3  #ALCIUM CONTENT OF OXYGEN EVOLVING 0HOTOSYSTEM)) PREPARATIONS FROM HIGHER PLANTS %FFECTS OF .A#L TREATMENT "IOCHIM "IOPHYS !CTA  n 3HULMAN2' 9AFET9 % F ISENBERGER0AND D "LUMBERG7%  /BSERVATIONAND INTERPRETATIONOF8 RA F AY ABSORPTIONEDGESIN IRONCOMPOUNDSAND PROTEINS0ROC.ATL !CAD D 3CI53! n 3IEGBAHN0%- 4HEORETICALMODELSFORT F HEOXYGENRADI CALMECHANISMOFWATEROXIDATIONANDOFTHEWATEROXIDIZING COMPLEXOF0HOTOSYSTEM)))NORG#HEMn 3IVARAJA- 4SO*AND$ISMUKES'# !CALCIUM SPECIlCSITE INmUENCESTHESTRUCTUREANDACTIVITYOFTHEMANGANESECLUSTER F HOTOSYNTHETICWATEROXIDATION"IOCHEMISTRY RESPONSIBLE FORP  n 3MITH 0* AND D 0ACE 2*  %VIDENCE FFOR TWO FFORMS OF THE G GSIGNALINTHE3  STATEOFF 0HOTOSYSTEM))4WOMAGNETI CALLY ISOLATED D MANGANESE DIMERS"IOCHIM"IOPHYS!CTA n 3TYRING3!AND2UTHERFORD!7 4HEMICROWAVEPOWERSATU T RATIONOF3))SLOWVARIESWITHTHEREDOXSTATEOFTHEOXYGEN EVOLVING COMPLEXIN0HOTOSYSTEM))"IOCHEMISTRYn D #HENIAE '  %FFECTS F OF 0HOTOSYSTEM )) 4AMURA . AND EXTRINSIC PROTEINS ON MICROSTRUCTURE OF F THE OXYGEN EVOLVING

 COMPLEXAND ITSREACTIVITY TOWATERANALOGS"IOCHIM"IOPHYS !CTAn 4SUTSUMI+ .AKAMORI(AND)CHIKAWA+ 8 RAYMANGANESE +` EMISSION SPECTRA OF MANGANESE OXIDES AND MANGANATES 0HYS2EV"n A ÏHÒRE -ALLART% "ERG R MAN5 'LATZEL0 2OBBLEE 6 SSER( !NXOLAB 6I *( #RAMER30 'IRERDD * * 3AUER+ +LEIN-0ANDD 9ACHANDRA 6+ -N+ EDGE8!.%3ANDD +` 8%3 STUDIESOFTWO F F HREE DIFFEREN F T -N OXO BINUCLEARCOMPLEXES)NVESTIGATIONOFT OXIDATIONSTATESRELEVANTTOTHEOXYGEN EVOLVING COMPLEXOF 0HOTOSYSTEM))*!M#HEM3OCn 7 7ESTRE4% +ENNEPOHL0 $E7ITT*' (EDMAN" (ODGSON+/ AND3OLOMON%) !MULTIPLETANALYSISOF&E+ EDGES TODPRE EDGEFEATURESOFIRONCOMPLEXES*!M#HEM3OC  n 7IEGHARDT+ 4HEACTIVECENTERSINMANGANESE CONTAINING METALLOPROTEINSAND INORGANICMODELCOMPLEXES!NGEW#HEM D %NGL n )NT%D 7INCENCJ C USZ( VAN'ORK R OM(*AND9OCUM#& D  4HEPHO TOSYNTHETICOXYGENEVOLVINGCOMPLEXREQUIRESCHLORIDEFORITS REDOXSTATE3 TO3 AND3 TO3 TRANSITIONSBUTNOTFOR3 TO3 OR 3 TO3 TRANSITIONS"IOCHEMISTRYn 7 7INCENCJUSZ( 9OCUM#&ANDVAN'ORKOM(* 3 STATE DEPENDENCEOFF CHLORIDEBINDINGAFlNITIESANDD EXCHANGEDYNA D M ICS INTHEINTACTANDPO D LYPEPTIDE DEPLETEDD / EVOLVING COMPLEX OFF 0HOTOSYSTEM))"IOCHEMISTRY n 9 HANDRA 9AC D 6+ 8 RAY A AB A SORPTIONSPECTROSCOPYANDAPP D LICA TIONSINSTRUCTURAL BIOLOGY)N3AUER+ Y ED -ETHODSOFF %NZY MOLOGY 6OL PPn!CADEMIC0RESS 3AN$IEGO 9 9ACHANDRA6+ 3TRUCTUREOFTHEMANGANESECOMPLEXIN 0HOTOSYSTEM)))NSIGHTSFROM8 RAYSPECTROSCOPY0HIL4RANS 2 3OC,ONDON"n 9 HANDRA6+ 'UILES2$ -C$ERMOTT! "RITT2$ $EXHEIMER 9AC

7JUUBM , :BD : IBOESB 3, 3AUER+AND D +LEIN-0  4HESTATEOFMAN F GANESEIN THEPHOTOSYNTHETICAPPARATUS3TRUCTUREOFTHEMANGANESE COMPLEXIN0HOTOSYSTEM))STUDIEDUSING%8!&3SPECTROSCOPY 4HE3 STATEOFTHEOXYGEN EVOLVING0HOTOSYSTEM))COMPLEX FROMSPINACH"IOCHIM"IOPHYS!CTAn 9 HANDRA6+ 'UILES2$ -C$ERMOTT!% #OLE*, "RITT2$ 9AC D +LEIN -0  #OMPARISONOF $EXHEIMER3, 3AUER+AND T E3 AND 3 STATES OF T ESTRUCTUREOFF THEMANGANESECOMPLEX INTH TH A ABSORPTION THEPHOTOSYNTHETIC/ EVOLVING COMPLEX!N8 RAY SPECTROSCOPY STUDY" Y IOCHEMISTRY n 9 9ACHANDRA6+ $E2OSE6* ,ATIMER-* -UKERJI) 3AUER+AND +LEIN -0  7HERE PLANTS MAKE OXYGEN! STRUCTURAL MODEL FOR THE PHOTOSYNTHETIC OXYGEN EVOLVING MANGANESE CLUSTER 3CIENCEn 9 HANDRA6+ 3AUER+AND 9AC D +LEIN -0  -ANGANESECLUSTER IN PHOTOSYNTHESIS7HERE PLANTS OXIDIZE WATER TO DIOXYGEN #HEM2EVn 9 9OCUM#&  4HECALCIUMAND D CHLORIDEREQUIREMENTSFORP F HO TOSYNTHETICWATEROXIDATION)N0ECORARO6,ED -ANGANESE 2EDOX%NZYMES PPn6#(0UBLISHERS .EW9ORK :HENG-AND$ISMUKES'# /RBITALCONlGURATIONOFTHE VALENCEELECTRONS LIGANDlELDSYMMETRY ANDMANGANESEOXI DATIONSTATESOFTHEPHOTOSYNTHETICWATEROXIDIZINGCOMPLEX F HE 3STATEMULTILINE%02SIGNALS)NORG#HEM !NALYSISOFT  n :IMMERMANN*,AND D 2UTHERFOR F D !7 %LECTRONPARAMAG NETICRESONANCE PROPERTIESOFF THE3 STATEOFT F THEOXYGEN EVOLVING COMPLEXOFF 0HOTOSYSTEM))"IOCHEMISTRYn :OUNI! 7ITT (4 +ERN * &ROMME 0 +RAUSS . 3AENGER 7 AND/RTH0 #RYSTALSTRUCTUREOF0HOTOSYSTEM))FROM 3YNECHOCOCCUS ELONGATUS AT  ¯ RESOLUTION .ATURE  n

$IBQUFS
 5IF
$BUBMZUJD
.BOHBOFTF
$MVTUFS
1SPUFJO
-JHBUJPO 3JDIBSE +
%FCVT

%FQBSUNFOU
PG
#JPDIFNJTUSZ SZ
6OJWFSTJUUZ PG
$BMJGPSOJB3JWFSTJEF
3JWFSTJEF
$"
 64" 4VNNBSZ   *

*OUSPEVDUJPO  **

5IF
%
1PMZQFQUJEF   "
5IF
-PPQ
$POOFDUJOH )FMJDFT
"
BOE
#   
"TQBSUBUF

BOE
"TQBSUBUF
    "SHJOJOF
    (MVUBNBUF   
)JTUJEJOF   #
5IF
-PPQ
$POOFDUJOH )FMJDFT
$
BOE
%   
"TQBSUBUF
   (MVUBNBUF
   
)JTUJEJOF
  
(MVUBNJOF

1IFOZMBMBOJOF

(MVUBNJOF
 "TQBSBHJOF

BOE
)JTUJEJOF
   $
5IF
$BSCPYZM5FSNJOBM
%PNBJO   
)JTUJEJOF     (MVUBNBUF     "SHJOJOF
   
)JTUJEJOF   
"TQBSUBUF
   
5IF
$BSCPYZM5FSNJOVT
BU
"MBOJOF
  ***

5IF
$1
1PMZQFQUJEF  *7

$PODMVEJOH
3FNBSLT   "DLOPXMFEHNFOUT  3FGFSFODFT  

4VNNBSZ 7ATEROXIDATION IN0HOTOSYSTEM)) 03)) TAKESPLACEUNDERSEVERECONSTRAINTS"ECAUSELITTLESURPLUS FREEENER 7 F GY ISAVAILABLE THEMIDPO D INTPOTENTIAL OFT F HE-N  CLUSTERMUSTBETIGHTLYCONTROLLEDTOPREVENT D ITFROM F INCREAS INGSUBSTANTIALLYASOXIDIZINGEQUIVALENTSAREACCUMULATED)NADDITION THERELEASEOFTOXIC HIGHLYREACTIVE INTERMEDIATESOFWATEROX F IDATIONMUST BEMINIMIZED4OOPERATEUNDERTHESECONSTRAINTS THEREACTIVITY OFT F HE -N  #A 9: COMPLEX ISCAREFU F LLYCONTROLLED BY ITSPROTEINENVIRONMENT4HISCHAPTERDESCRIBESTHESITE DI RECTEDMUTAGENESISSTUDIESTHATHAVEBEENUNDERTAKENTOIDENTIFYTHEAMINOACIDRESIDUESTHATARERESPONSIBLEFOR F THISCONTROL4HESERESIDUES D INCLUDETHOSETHAT LIGATETHE-NAND# D A IONSANDD THOSETHAT INmUENCETHEELECTRON  AND PROTONTRANSFERREACT F IONSOF 9: AND D THE -N  #A METAL CLUSTER-OSTCHARACTERIZATIONSRELIED INITIALLYON NON INVASIVEMETHODSTHATWERECONDUCTEDINVIVO7ITHIMPROVEDD METHODSFORPURIFYING03)) CHARACTERIZA TIONS INCREASINGLY INVOLVE NEWER DEVELOPMENTS IN MAGNETIC RESONANCE VIBRATIONAL ANDD OPTICAL ABSORPTION F HEMUTAGENESISSTUDIESTHATARE DESCRIBED INTHISCHAPTER POSSIBLELIGANDSOFT F HE SPECTROSCOPY/NTHE BASISOFT

%MAILRICHARDDEBUS UCREDU

47YDRZYNS 4 D K KIAN D +3ATOH E  DS DS 0HOTOSYSTEM))4H 4 E,IGH G T $RIVEN7ATER0LASTOQUINONE/XIDORE D DUCTASE PPn D © 3PRINGER0RINTED D IN4 4HE.ETH T ERL R ANDS D



3JDIBSE
+
%FCVT

-N  CLUSTERWERE PROPOSED D TO INCLUDE$ !SP $ (IS $ 'LU $ (ISANDD $ !SP PLUS THECARBOXYL TERMINUSOFTHE$POLYPEPTIDEAT$ !LA0OSSIBLELIGANDSOFTHE#A IONWEREPROPOSED TOINCLUDE$ !SP $ !SPAND D $ !SP)NADDITION $ 'LUWASPROPOSEDTOPART D ICIPATE INA NETWORK RK OF HY H DROGEN BONDSTHAT FAC F ILITATESELECTRONTRANSFER F FROMT F HE -N CLUSTERTO9:% DUR D INGTHE HIGHER3 STATETRANSITIONSAND$ (ISWASPROPOSEDTOSERVEASTHE PROTONACCEPTORFOR9: 4HE RECENT  ¯ AND  ¯8 RAY A CRYSTALLOGRAPHICSTRUCTURALMODELSOF03))SUPPORTSOMEOFTHESEPROPOSALS BUTCONmICTWITHOTHERS MOSTNOTABLYTHELIGATIONOF# F A ANDTHEROLESOF$ 'LUAND D INTHE¯STRUCTURALMODEL THEROLEOFTHE CARBOXYL TERMINUSOFTHE$POLYPEPTIDE4WOSURPRISINGASPECTSOFTHESTRUCTURALMODELSARETHATONE-NION IS LIGATED BY BOTH$ !SPAND D $ 'LUAND D ANOTHERISLIGATED BY BOTH $ 'LUANDD $ (IS4HE PROPERTIESOFT F HE$ !SPAND D $ 'LUMUTANTSDIFFERMAR F RKEDLY AS DOTHEPROPERTIESOFT F HE$ 'LU AND$ (ISMUTANTS4HEMAINPOINTSOFAGREEMENTANDDISAGREEMENTBETWEENTHEMUTAGENESISSTUDIES D THERECENT8 RAY A CRYSTALLOGRAPHICSTRUCTURAL MODELSARE DISCUSSED AND *
*OUSPEVDUJPO 4HE / EVOLVINGCOMPLEX /%# IN0HOTOSYSTEM)) 03)) CONSISTSOFTHE-N  CLUSTER 9: ANDA#A ION !TTEMPTSTOIDENTIFY THE LOCATIONSOFT F HE-N  CLUSTERAND9 D : BEGAN INEARNESTAFTER+ F IMIYUKI 3ATOH AND COWORKERS DISCOVERED D THATTHE $$ HETERODIMER ISTH T ESITEOFC F HARG R ESEPARATIONBETWEEN0AND D 0HEO .ANBAAND3ATO D H /KAMURAETAL  4HIS SEMINALDISCOVERY COUPLEDWITHTHEDISCOVERYTHAT 9$% ISATYROSINERADICAL "ARRYAND D "ABCOCK  LED IMMEDIATELYTOTHE IDENTIlCATIONOF 9$ AS4YR OFTHE$POLYPEPTIDE$EBUSETAL A6ERMAAS ETAL  AND 9: AS4Y 4 ROFT F HE$POLYPEPTIDE $EBUSETAL B-ETZETAL  ;3YNECHOCYSTIS I NUMBERING IS USED HERE AND THROUGHOUT= 0ARALLELS BETWEENTHE$$HETERODIMERIN03))AND D THE,HETERODIMERINTHE BACTERIAL REACTIONCENTER"2# 4REBST  -ICHEL AND $EISENHOFER  LED TOTHEGENERAL BELIEFF THATTHE-N  CLUSTERWOULD BE LOCATED INTHELUMENAL DOMAINSOFT F HE$AND D $ POLYPEPTIDES!SSOONASAPPROPRIATEDELETIONSTRAINS F FlCIENTLY INTRODUC FORE D ING SITE DIRECTED D MUTATIONSINTO THE$AND D $POLYPEPTIDESOF 3YNECHOCYSTIS I SP0## WERECONSTRUCTED$EBUSETAL 6ERMAAS ETAL .IXONETAL B ALLCONSERVED!SP D !BBREVIATIONS R "2#nBACTERIALREACTIONCENTER#HL A n CHLORO PHYLL A #YT C nCYTOCHROME C$$nREACTIONCENTERCORE SUBUNITS $#-5 n    DICHLOROPHENYL N   DIMETHYLUREA %$4!nETHYLENEDIAMINETETRAACETICACID%.$/2nELECTRONNU CLEARDOUBL U ERESONANCE%02nELECTRONPARAMAGNETICRESONANCE %3%%-nELECTRONSPINECHOENVELOPEMODULATION%8!&3nEX TENDED8 RAYABSORPTIONlNESTRUCTURE&4)2n&OURIERTRANSFORM INFRARED /%# n OXYGEN EVOLVING COMPLEX 0n PRIMARY ELECTRONDONOROF03))03))n0HOTOSYSTEM))1! nPRIMARY PLASTOQU Q INONEACCEPTOROF03))3 F F HE/%# FORN F N nSTATESOFT     OR56nULTRAVIOLET6I 6 SnVISIBLE8!.%3n8 RAY A ABSORPTIONNEAREDGESTRUCTURE9$ nREDOXACTIVETYROSINEON $ 9: nREDOXACTIVETYROSINEON$

'LU (IS AND4YRRESIDUESINTHELUMENALDOMAINS OFT F HE$POLYPEPTIDEANDD ALL CONSERVED!SP ' D LU (IS !SN ANDD 'LNRESIDUES D INTHELUMENAL DOMAINS OF THE $ POLYPEPTIDE WERE TARGETED FOR EARLY RE VIEWS SEE$INERETAL .IXONETAL A 0AKRAS K IAND6ERMAAS $E D BUS  3YSTEMS FFOR INTRODUCING MUTATIONS INTO THE $ POLYPEPTIDE M DOMONASREINHARD R TII 2OFFE F YETAL  OF #HLAMYD -INAGAWAANDD #ROFTS 7 F HITELEGGEETAL  WEREDEVELOPEDSHORTLYTHEREAFTER!LLOFTHEMUTA GENESISSTUDIESOF03)) F HAVEBEENPERFORME F D WITH THECYANOBACTERIUM 3YNECHOCYSTIS SP0##AND THEGREENALGA #HLAMYDOMONASREINHARDTII 3OMEOFT F THE INITIAL CHARACTERIZATIONSOFS F ITE DIRECTED F D DON!SPO F THE$POLYPEPTIDE MUTANTS FOCUSE BECAUSETHISRESIDUEISLOCATEDONLYRESIDUESFROM 9: "OERNER ET AL  $INER ANDD .IXON  .IXON ANDD $INER   4HESE INITIAL CHARACTER IZATIONS SHOWED D THAT THIS RESIDUE IS CRUCIAL FO F R THE ASSEMBLYORSTABILITYOFT F HE -N  CLUSTER"OERNER ETAL .IXONANDD $INER  ANDD SERVESAS ACOMPONENTOFAHIGH AFlNITY-N)) BINDINGSITE $INERANDD .IXON .IXONANDD $INER  4HE F HESESTUDIESTOOTHERPOSITIONS INTHE EXTENSIONOFT $POLYPEPTIDEWASOFTENFRUSTRATEDBYTHEINSTABILITY F HE-N  CLUSTERINMANY N MUTANTS!CCORDINGLY Y OFT F FFORTWAS F DIRECTEDD ATCHARACTERIZING AGREATDEALOFE MUTANTS BY NON INVASIVE METHODS IN VIVO 4HESE D IMARILY ONMEASURING CHANGES IN METHODSRELIEDPR THE YIELD D OF CHLOROPHY H LL A mUORESCENCE THAT WERE CAUSEDBYSINGLEmASHES MULTIPLEmASHES ORSHORT F INUOUSILLUMINATION GIVENINTHEPRES PERIODSOFCONT ENCEORABSENCEOF$#-5 F .IXONANDD $INER  .IXONETAL B#HUETAL A$INER   F HE$ !SPMUTANTSBY 4HECHARACTERIZATIONOFT THESEMETHODSPROVIDEDD THEBASISFOREXTEN F DINGTHESE METHODSTOOTHERRESIDUESANDANUMBEROFCOMP M ENDIA WEREPUBLI U SHED .IXONANDD $INER #HUETAL

$IBQUFS

.VUBUJPOBM
4UVEJFT
PG 0 &WPMVUJPO



A B 4HE DEVELOPMENTOF IMPROVED D METHODSOF PURIFYING03))PARTICLESFROM 3YNECHOCYSTIS SP0## WITHCONVENTIONAL CHROMATOGRAPHY 4ANG AND $INER  AND D WITH METAL IONAFlNITYCHROMATOG RAPHY"RICKERETAL 2EImERETAL  HAS F ILITATED FAC D THEBIOPHYSICAL CHARACTERIZATIONOF INCREAS ING NUMBERS OFF MUTANTS WITH MAGNETIC RESONANCE %02 %.$/2 %3%%- VIBRATIONAL &4)2 AND 6 SOPTICALABSORPTIONSPECTROSCOPIES 566I -EANWHIL W E INPARALLEL TOTHEMUTAGENESISSTUDIES NUMEROUSCHEMICALMODIlCATIONSTUDIESWEREPROVID INGEVIDENCETHATTHE-N  CLUSTERISCOORDINATED BY BOTHCARB R OXYLATEAND HISTIDINERESIDUES D 4AMURAET AL 3EIBERTETAL 0RESTONAND3EIBERT A B"LUB U AUGHAND# D HENIAE -AGNUSON AND D !NDRÏASSON 4AMURAETAL 'HIRARDI ETAL A B /NTHEBASISOFCOMBINEDCHEMICAL MODIlCATIONAND PROTEASE DIGESTIONSTUDIES SPECIlC RESIDUES D $ (ISAND D $ (IS WEREPROPOSED ASLIGANDSTOTHE-N  CLUSTER0RESTONAND3EIBERT A'HIRARDIETAL B  -AGNETICRESONANCE F D AND ONT D HEBASIS STUDIESWEREALSO BEINGPERFORME OF%3%%-$E2OSEETAL :IMMERMANNETAL  AND%.$/2 D 4ANG ETAL A ANALYSES IT WASCONCLUDED D THATTHE-N  CLUSTERISLIGATED BYAT LEASTONENITROGENATOM3UBSEQUENT%3%%-STUDIES D D WITH T ;.=HISTIDINE LAB A ELED03))PART D ICLES CONDUCTE D THISCONCLUSIONTOSHOWTHATTHE -N  CLUSTER RElNED ISLIGATEDBYATLEASTONEHISTIDINERESIDUE4ANGET AL  AND D THATTHISRESIDUECOOR D DINATESTHE-N  CLUSTERWITH ITS ¡o o NITROGEN'ILCHRIST  !D DITIONALEVIDENCEFORCOORDINATIONBYHISTIDINEWAS PROVIDED BYAN&4)2STUDY OF ;.=HISTIDINE LABELED 03))PARTICLES.OGUCHI ETAL  

/NTHE BASISOFT F HESITE DIRECTEDD MUTAGENESISAND BIOCHEMICALSTUDIESTHATHADBEENCONDUCTEDBYLATE  MOSTORALL OFT F HEAMINOACID D RESIDUEST D HATLIGATE THE-N  CLUSTERWEREEXPECTEDD TO BECONTRIBUTED BY THE$POLYPEPTIDE&IG 4HERECENT¯AND ¯ 8 RAY A CRYSTALLOGRAPHIC STRUCTURAL MODELS OF 03)) "IESIADKAETAL &ERREIRAETAL  #HAPTER "ARBERAND)WATA HAVECONlRMEDTHIS EXPECTATION SHOWING THATTHE-N  CLUSTERISSUR ROUNDEDD MOSTLY BYRESIDUES D FORMT F HE$POLYPEPTIDE BUTALSOBYSOMERESIDUESFROM #0)NTHE¯ STRUCTURALMODEL ONLY ONE-NLIGAND #0 ' D LU ISPROVIDED BYAPOLYPEPTIDEOTHERTHAN$!CCORD INGLY THISCHAPTERFOCUSESONTHEEXTENSIVEMUTAGEN ESISSTUDIESTHATHAVEBEENDIRECTEDD ATTHELUMENAL DOMAINSOFF THE$POLYPEPTIDEAND DESCRIBESTHEFEW F MUTAGENESISSTUDIESTHATHAVEBEENDIRECTEDATTHE LARGE LUMENAL LOOPOF#0&OREAR F LIERREVIEWSON THISSUBJ U ECT SEE$EBUS  ANDD $INER  **
5IF
%
1PMZQFQUJEF "
5IF
-PPQ
$POOFDUJOH
)FMJDFT
"
BOE # 4HELOOPCONNECTINGHELICES!AND"CONTAINSSIX CONSERVED CARB R OXYLATE RESIDUES THREE CONSERVED TYROSINERESIDUES AN D D ONECONSERVED HISTIDINERESI DUE4HESEARE!SP !SP 'LU 4YR (IS 4 R 'LU !SP 'LU ANDD 4YR4HEMU 4Y TATIONS4YR0HE 4YR0HE 'LU'LN 'LU!SP !SP!SN !SP'LU 'LU'LN 'LU!SP ANDD 4YR0HECAUSEDD NO DISCERNIBLEALTERATIONSTO EITHERPHOTOAUTOTROPHICGROWTHOR/ EVOLUTION #HU

&IIG 3CHEMATICFO & F LDING PATTERNOFF THE LUMENAL DOMAINSOFF THE$POLYPEPTIDE!MINOACIDD RESIDUEST D HAT HAVE BEENFOUN F D TOINmUENCE D HATWEREIDENTIlED D ASPOSSIBLE LIGANDSOFF THE-N  CLUSTERBYSITE THEPROPERTIESOFF THE-N  CLUSTEROR9: ARE INDICATED2ESIDUEST DIRECTED D MUTAGENESISSTUDIESAREINDICATED BY lLLEDD CIRCLES-ODIlED FROM$E F BUS 

 ETAL A #ONSEQUENTLY ITWASCONCLUDED D THAT NEITHER4YR 4YR 'LU !SP 'LU NOR 4 RARECRUCIAL TOTHEMECHANISMOF / EVOLUTION 4Y #HUETAL A )NCONTRAST MOSTOFT F HEMUTATIONS THATWERECONSTRUCTEDAT!SP !SP 'LU AND (ISWERE DELETERIOUSTO/ EVOLUTION ATLEASTTO D $INER SOMEEXTENT.IXONETAL A.IXONAND #HUETAL A ANDHAVEBEENINVESTIGATED F HER4HECONSERVED FURT D RESIDUE!R D G HASALSO BEEN TARGETED ,IAND D "URNAP    "TQBSUBUF

BOE "TQBSUBUF
 !LL MUTATATIONS HAVE BEEN CONSTRUCTEDD IN 3YNECHO 3

CYSTIS SP 0##  .IXON ET AL A .IXON CY AND D $INER  #HU ET AL A !SPARTATE  HAS BEEN CHANGED TO 'LU !SN AND6AL!SPARTATE HASBEENCHANGED D TO'LU !SN !LA AND6A D L4HE D !SP'LU MUTANTS ARE !SP'LU !SP!SN AND STRONGLYPHOTOAUTOTROPHICTHEOTHERSAREWEAKLYSO D LUMUTANTS 4HE/%#SINTHE!SP'LUAND!SP' ARERELATIVELYUNIMPAIRED!SP'LUAND!SP' D LU CELLSCONTAINONLY^AND^THE03))CONTENT OFF WILD TY TYPECELLS RESPECTIVELY Y BUTEVOLVE/ AT^ THERATEOFW F ILD TYPECELLS4HE /%#S INTHEOTHER MUTANTSARECONSIDERABLYMOREIMPAIRED!SP!SN !SP!SN AND D !SP!LA CELLS CONTAIN n THE03))CONTENTOFF WILD TYPECELLS BUTEVOLVE/ AT ONLYnTHERATEOFWILD TYPECELLS4HE/%#S INTHE!SP6AL AND!SP6A D L MUTANTSAREEVENMORE IMPAIRED 4HE !SP!SN AND D !SP'LU MUTANTS EXHIBIT / mASH YIELDSTHATHAVENORMALPERIOD FOUR OSCILLATIONS 1IANETAL  (OWEVER !SP!SN D !SP!LACELLS LOSE/ EVOLVINGAC !SP!SN AND TIVITYRAPIDLYWHENILLUMINATEDWITHSATURATINGLIGHT #HUETAL A  /NTHE BASISOF #HL A mUORESCENCEANALYSESTHAT WERECONDUCTEDWITHINTACTCELLS ITWASCONCLUDED THATESSENTIALLY ALL OFT F HE03))REACTIONCENTERSINTHE D !SPMUTANTSCONTAIN INTACT PHOTOOXI !SPAND DIZABLE-N  CLUSTERSINVIVO#HUETAL A  F ITWASCONCLUDED D THATNEITHER!SPNOR 4HEREFORE !SP IS LIKELYTO LIGATETHEASSEMBLED -N  CLUSTER 4HISCONCLUSIONAGREESWITHTHERECENT¯AND¯ 8 RAY A CRYSTALLOGRAPHICSTRUCTURALMODELS"IESIADKA ETAL &ERREIRAETAL #HAPTER "ARB R E AND)WATA (OWEVER THERATEOF/ RELEASE IS SLOWED DRAMATICALLY IN!SP!SNAND!SP! D LACELLS F LD IN!SP!SNCELLSAND FO D  FO F LD IN!SP!LACELLS (UNDELTETAL A ANDISSLOWEDTOLESSEREXTENTS IN!SP'LUAND D !SP!SNTHYLAKOID D MEMBRANES

3JDIBSE
+
%FCVT 1IANETAL  3UB U SEQUENTANALYSESOF F !SP!SN 03)) PARTICLESSHOWEDTHATTHE3 A 3 AND3 A 3 TRANSITIONSARESLOWEDD APPROXIMATELY FO F LD D AND THAT THE3 A 3 TRANSITIONISSLOWEDD  FO F LD (UNDELTET AL A B INTHISMUTANT4HE3 A 3 TRANSITION ISALSOSLOWED IN!SP!SNAND!SP' D LUTHYLAKOID MEMBRANES BUTTO LESSEREXTENTS4HELIFET F IMESOF THE3 AND3 STATESARESHORTENEDIN!SP!SNAND D MEMBRANES1IANETAL  !SP'LUTHYLAKOID D OINT SUGGESTING THAT BOTH THE 33 AND 33 MIDP POTENTIALS ARE INCREASED IN THESE MUTANTS 3LIGHT INCREASES INTHE33 MIDPO D INTPOTENTIALWEREALSO F HL A mUORESCENCEANALYSES SUGGESTEDD ONTHEBASISOF# OFINTACTCELLS#HUETAL A 4HERATEOFELECTRON F FFROM9: TO0% ISUNCHANGED INAT LEASTTHE TRANSFER !SP!SNMUTANT(UNDELTETAL A  4HE 03)) REACTION CENTERS IN !SP!SN AND U STANTIALLY DIMIN !SP!LACELLSAPPEARTO HAVESUB ISHEDD AFlNITIES FOR F #A.EITH T ERMUTANTGREWPHOTOAU TOTROPHICALLYWHEN#A WASOMITTEDFROMTHEGROWTH MEDIUMANDD NEITHER3R NOR-G NOR.A WOULD SUB U STITUTE #HUETAL A  7HEN!SP!SNAND !SP!LACELLSWEREPROPAGATEDPHOTOHETEROTROPHI CALLY INTHEABSENCEOF#A F  LITTLEORNO / EVOLUTION WASOBSERVED THERATEOFF ELECTRONTRANSFER F FROM F 9: TO 0% SLOWEDDRAMATICALLY;ASISOBSERVEDIN03)) PREPARATIONSTHATHAVE BEENRIGOROUSLY DEPLETEDD OF #A IONS 3ATOH ANDD +ATOH  "OUSSAC ET AL  = AND 03)) BECAME VERY SENSITIVE TO LIGHT INDUCE D D DAMAGE #HUETAL A /NTHE BASISOF THESERESULTS ITWASPROPOSEDD THAT!SPANDD !SP INmUENCETHEBINDINGOF#A ANDMAYFORMPARTOFA #A BINDINGSITE#HUETAL A 3UB U SEQUENTLY Y IT WASPROPOSEDD THAT BINDING#A TOTHISSITE PROMOTES AREPOSITIONINGOFTHE # TERMINAL DOMAIN OFTHE $ POLYPEPTIDE DUR D INGTHELIGHT DRIVENASSEMBLY PHO TOACTIVATION OFT F HE -N  CLUSTER#HENETAL  #HAPTER )NAPPARENTSUPPORTOFTHISPROPOSAL AT LEASTONEUNSTABLE PHOTOACTIVATIONASSEMBLY INTERME DIATE ISDESTABILIZED DRAMATICALLY IN!SP!SNAND !SP'LU CELLS 1IAN ET AL   .EVERTHELESS D LIGATES#A INTHERECENT ¯ AND NEITHERRESIDUE ¯8 RAYCRYSTALLOGRAPHICSTRUCTURALMODELS"IESI ADKAETAL &ERREIRAETAL #HAPTER  F IONSAT!SPAND D !SPMUSTCAUSE 4HEREFORE MUTAT  STRUCTURALCHANGESTH T ATWEAKENTH T EAFlNITYFOR#A F F R FO ADISTANTSITE3IMILARMUTATION INDUCEDWEAKENING F HEAFlNITY OF03)) F F FOR# A HASBEENOBSERVEDD AT OFT !RG BELOW AND INTHE # TERMINAL DOMAINOFF THE $POLYPEPTIDE3ECTION))# 

$IBQUFS

.VUBUJPOBM
4UVEJFT
PG 0 &WPMVUJPO



J JOF
 J   "SHJO


(MVUBNBUF


4HE!RG'LU !RG'LN AND D !RG6AL MUTATIONS HAVE BEENCONSTRUCTED IN 3YNEC 3 HOCYSTIS SP0## ,IAND"URNAP  !LLTHREEMUTANTSAREPHOTO AUTOTROPHIC4HE!RG'LUAND D !RG'LNMUTANTS HAVE DECREASED D 03)) CONTENTS AND LIGHT SATURATED RATESOF/ EVOLUTIONCOMPAREDTOWILD TYPECELLS RKNESS AND LOSE / EVOLVING ACTIVITY RAPIDLY IN DARK !LLTHREEMUTANTSEXHIBIT/ mASH YIELDSTHATHAVE NORMAL PERIOD FOUR OSCILLATIONS )N THE !RG'LU AND D !RG'LNMUTANTS THEOSCILLATIONSARECHARAC TERIZED BY LARGERMISSPARAMETERSAND D THERATEOFF / RELEASEISSLOWEDTHREETOlVE FOLD/NTHEBASISOF #HL A mUORESCENCEANALYSES ITWASCONCLUDED D THAT THE3 3 MIDPO D INTPOTENTIALOFF THE-N  CLUSTERIS DECREASEDSLIGHTLYIN!RG'LUAND!RG'LNCELLS 0ARADOXICALLY T Y HE LIFET F IMEOFT F HE 3STATE ASMEA SURED D WITH / mASH YIELDS DECREASED D CONSIDERABLY IN !RG'LU THYLAKOID MEMBRANES AND DECREASED SLIGHTLY IN!RG'LNTHYLAKOID D MEMBRANES)TWAS D THATTHEACCELERATED DECAYOFF THE3 STATE SUGGESTED INTHELATTERMEASUREMENTSISCAUSEDBYACCELERATED ELECTRONTRANSFER F FROM F 9$ TOTHE 3 STATEOFT F HE-N  CLUSTER,I AND"URNAP  D  4HE 03)) REACTION CENTERS IN THE!RG'LU AND !RG'LN MUTANTS APPEAR TO HAVE SUBSTANTIALLY DIMINISHED D AFlNITIES FOR F #A.EITHERMUTANTGREW PHOTOAUTOTROPHICALLYWHEN#A WASREPLACEDBY.A INTHEGROWTH MEDIUM,I AND D "URNAP  4HE F HE LIGHT DRIVENASSEMBLYOFF THE QUANTUMYIELD FORT -N  CLUSTERISHIGHERIN!RG'LUTHANINWILD TYPE CELLS ALTHOUGHATLEASTONEUNSTABLE PHOTOACTIVATION D AS WAS OB ASSEMBLY INTERMEDIATE IS DESTABILIZED SERVEDPREVIOUSLYIN!SP!SNAND!SP'LUCELLS 3ECTION))!  4HE!RG'LU MUTATION GREATLY DESTABILIZES THE BINDINGOFTHEEXTRINSIC K$APOLYPEPTIDEAND TO A LESSEREXTENT DESTABILIZESTHEBINDING OFF #YT C )T WASSUGGESTED D THAT!RGFORMSASA F LT BRIDGETOTHE EXTRINSICK$APOLYPEPTIDEORTHATTHE!RG'LU D TOA LESSEREXTENT THE!RG'LNMUTA MUTATION AND TION CAUSESTRUCTURAL ALTERATIONSTHATINTERFEREW F ITH THE BINDING OF THE EXTRINSIC POLYPEPTIDES AND #A TO03)) ,IAND D "URNAP  )NTHERECENT8 RAY A D CRYSTALLOGRAPHICSTRUCTURALMODELS SEVERALRESIDUES OFTHEEXTRINSICK$APOLYPEPTIDEARELOCATEDVERY CLOSETO $ !RG"IESIADKAETAL &ERREIRA R ERAND D )WATA  ETAL #HAPTER "ARB

4HE'LU!SP 'LU'LN 'LU!LA AND' D LU,EU MUTATIONS HAVE BEENCONSTRUCTED IN 3YNEC 3 HOCYSTIS SP 0##.IXONETAL A.IXONAND$INER F HO #HUETAL A !LL FOURMUTANTSAREP TOAUTOTROPHIC ALTHOUGHONLY'LU!SPISSTRONGLY SO 4HE /%#INTHE'LU!SPMUTANTISESSENTIALLY UNIMPAIRED'LU!SPCELLSCONTAIN^THE03)) CONTENT OFF WILD TYPE CELLS ANDD EVOLVE / AT ^ THERATEOFWILD TYPECELLS4HE/%#SINTHEOTHER MUTANTS ARE CONSIDERABLY MORE IMPAIRED CELLS OF THESEMUTANTSCONTAINnTHE03))CONTENTOF WILD TYPECELLS BUTEVOLVE/ ATONLYnTHE RATEOFF WILD TYPECELLS /NTHE BASISOF #HL A mUORESCENCEANALYSESTHAT WERECONDUCTEDWITHINTACTCELLS ITWASCONCLUDED THAT ESSENTIALLY ALL OF F THE 03)) REACTION CENTERS IN THE 'LU MUTANTS CONTAIN INTACT PHOTOOXIDIZABLE -N  CLUSTERSINVIVO#HUETAL A 4HEREFORE ITWASCONCLUDEDD THAT'LU ISUNLIKELYTO LIGATETHE -N  CLUSTER4HISCONCLUSIONAGREESWITH THERECENT 8 RAYCRYSTALLOGRAPHICSTRUCTURALMODELS"IESIADKA ETAL &ERREIRAETAL #HAPTER )TWAS ALSOCONCLUDEDD THATTHE33 MIDPOINTPOTENTIALOF THE-N  CLUSTERISSLIGHTLYINCREASEDIN'LU,EU CELLS.IXONETAL A ANDD THATELECTRONTRANSFER F F FROM 1!%< TO1" ISSLOWEDD SLIGHTLYIN'LU'LNAND 'LU!LACELLS#HUETAL A  4HE'LU!SP 'LU'LN AND' D LU!LAMUTANTS W EN #A WAS OMITTED GREW PHOTOAUTOTROPHICALLY WH FFROMTHEGROWTHMEDIUM4HEREFORE ITWASCONCLUDED THAT'LUHASLITTLEORNO INmUENCEONTHE BINDING OF #A #HUETAL A   )JTU J UJEJJOF
 4HE(IS,EU (IS'LN AND(IS!SNMUTATIONS HAVE BEENCONSTRUCTED IN 3YNECHOCY CYSTIIS SP0## .IXONETAL A#HUETAL A2*$EBUS UNPUBLISHED !LLTHREEMUTANTSAREPHOTOAUTOTROPHIC Y SLIGHTLY IMPAIRED LIGHT SATURATEDD RATES ANDD EXHIBITONLY OF / EVOLUTION/NTHEBASISOF #HL A mUORESCENCE ANALYSESTHATWERECONDUCTEDWITHINTACTCELLS ITWAS CONCLUDEDD THATESSENTIALLY ALL 03))REACTIONCENTERS INALLTHREEMUTANTSCONTAININTACT PHOTOOXIDIZABLE -N  CLUSTERSINVIVO#HUETAL A2*$EBUS F ITWASCONCLUDEDD THAT(IS UNPUBLI U SHED 4HEREFORE ISUNLIKELYTOLIGATETHEASSEMBLED -N  CLUSTER 4HE (IS,EU MUTANT GROWS PHOTOAUTOTROPHI CALLYATNORMAL RATESWH W EN#A ISREPLACED BY 3R

 GROWSATAMUCHSLOWERRATEWH W EN#A ISREPLACED BY-G BUTFAILSTOGROWPHOTOAUTOTROPHICALLYWHEN #A ISREPLACED BY .A #HUETAL A 7HEN PROPAGATED D PHOTOHETEROTROPHICALLY INMEDIA HAVING .ASUBSTITUTEDFOR#A (IS,EUCELLSEXHIBITLOW D TOWILD TYPE RATESOFF / EVOLUTION COMPARED AND D SOMEW WHATALTERED #HL A mUORESCENCE PROPERTIES D OINT POTENTIAL SUGGESTIVEOFADECREASED33 MIDP #HU ET AL A  (OWEVER THE CELLS SHOW NO D THATTHE UNUSUAL LIGHT SENSITIVITY)TWASCONCLUDED (IS,EUMUTATIONINTRODUCESSTRUCTURAL PERTURBA  TIONSTHATWEAKENTHEAFlNITYOFF 03)) FOR#A F IONS #HU ET AL A BUT THAT THIS AFlNITY IS WEAK ENEDTOAMUCHLESSEREXTENTTHANIN!SP!SNAND !SP!LACELLS #
5IF
-PPQ
$POOFDUJOH
)FMJDFT
$
BOE
% 4HE LOOPCONNECTINGHELICES#AND D $CONTAINSTWO CONSERVEDCARBOXYLATERESIDUESANDONECONSERVED HISTIDINE RESIDUE4 D HESE ARE!SP 'LU AND (IS4HESE RESIDUES D HAVE BEEN TARGETED D EXTEN SIVELYBECAUSEOFTHECLOSEPROXIMITYINSEQUENCE 4 R AND BECAUSEEARLY MODEL OFF !SPTO 9: 4Y INGSTUDIESHADPRE D DICTED D THAT(ISWOULD INTERACT WITH9: $EBUSETAL A3VENSSONETAL   /THERRESIDUEST D HATHAVE BEENTARGETED INTHIS LOOPARE 'LN 0HE 'LN !SN AND D (IS  "TQBSUBUF
 !LL POSSIBLE MUTATIONS EXCEPT !SP0RO HAVE BEEN CONSTRUCTED IN 3YNECHOCY CYSTIS SP 0##  "OERNERETAL $INERAND D .IXON .IXON AND$INER #HUETAL A A2*$E BUS UNPUBLI U SHED 4HE!SP6ALMUTANT HASALSO BEENOBTAINED D ASASPONTANEOUS PSEUDOREVERTANTOF !SP!LACELLS#HUETAL A 4HE!SP(IS D IONS !SP!SN !SP4HR AND!SP0ROMUTAT HAVEBEENCONSTRUCTED IN #HLAMY L D DOMONASREIN HARD RDTII 7HITELEGGEETAL  4HE!SP'LUMUTANTIS PHOTOAUTOTROPHICAND D EVOLVES / AT^THERATE D !SP6AL OFF WILD TYPECELLS4HE!SP(ISAND MUTANTS ARE WEAKLY PHOTOAUTOTROPHIC AND EVOLVE / AT^AND D ^THERATEOFF WILD TYPECELLS RESPECTIVELY0HOTOAUTOTROPHICGROWTH ISABOLISHED INALLOTHERMUTANTS-OSTOFTHEMUTANTSHAVE03 )) CONTENTSTHATARESIMILARTOTHATOFF WILD TYPECELLS /NLY THE!SP0HE !SP4RP AND D !SP0RO MUTANTSCONTAINSHARPLYDIMINISHED03))CONTENTS !MONG THE NON PHOTOAUTOTROPHIC MUTANTS /

3JDIBSE
+
%FCVT EVOLUTION IS ABOLISHED IN !SP3ER !SP4HR !SP!LA !SP'LY !SP4YR AND!SP0RO A OLISHED IN!SP!SNAND!SP4RP D CELLS NEARLYAB CELLS AND DIMINISHEDD SU UBSTANTIALLY INALLOTHERS4HE !SP!SN MUTANT ASSEMBLES -N  CLUSTERS IN ONLY^OFF 03))REACTIONCENTERS4HE!SP!LA !SP3ER ANDD !SP4HR MUTANTS ASSEMBLE NO -N  CLUSTERS IN IN VIVO )NTHOSE!SPMUTANTSTHATEVOLVE / THEAS SEMBLED -N  CLUSTERSAPPEARTOFUNCTIONNORMALLY EXHIBITING / mASH YIELDSTHATHAVENORMALPERIOD FFOUR OSCILLATIONS (OWEVER MOST EXHIBIT SLIGHTLY DECREASED 33 MIDPOINT POTENTIALS AND LONGER 3 STATELIFETIMES4HEMUTANTSTHATEVOLVE/ DO NOTLOSE D ING ILLUMINATIONWITHSATURAT THISACTIVITYRAPIDLY DUR ING LIGHT UNLIKESOMEOFF THEMUTANTSTHATHAVE BEEN CONSTRUCTEDAT!SPAND!SP3ECTION))! ORIN THE # TERMINAL DOMAIN3ECTION))# /NTHE BASISOF #HL A mUORESCENCEANALYSESTHATWERECONDUCTE D D WITH INTACTCELLS ITWASALSOCONCLUDEDTHAT INTHE / EVOLV ING MUTANTS SIGNIlCANTPERCENTAGESOF03))REACT F ION CENTERS LACK -N  CLUSTERSINVIVO4HISPERCENTAGE WAS ESTIMATED TO BE n IN!SP'LU CELLS D L CELLS AND^ D ^IN!SP(ISAND!SP6A IN!SP,EU !SP)LE ANDD !SP!RGCELLS)N THESEMUTANTS THE-N  CLUSTERWASPROPOSEDTOBE Y UNSTABLEORTOBEASSEMBLED INEFlCIENTLY!RECENT %02 2 %3%%-STUDYHASSHOWNTHATTHE3 STATEAND 3 STATE MULTILINE%02SIGNALSOF!SP(IS03)) PARTICLESCLOSELYRESEMBLETHOSEOFF WILD TYPE03)) PARTICLES $EBUS ET AL   4HESE SIMILARITIES SUPPORTTHECONCLUSIONOFEARLIERSTUDIES.IXONAND $INER  #HU ET AL A7HITELEGGE ET AL  THATTHOSE-N  CLUSTERSTHATAREASSEMBLED IN!SP(IS CELLS FUNCTION NORMALLY4HE AMPLI TUDESOFF THE3 AND 3 STATEMULTILINE%02SIGNALS IN !SP(IS03))PARTICLESCORRELATEWITHTHELOWER / EVOLVING ACTIVITY OF!SP(IS CELLS AND 03)) PARTICLES nCOMPAREDD TOWILD TYPE SHOWING THAT INEFlCIENT ASSEMBLY OF F THE -N  CLUSTER NOT INSTABILITY ISTHEREASONTHATFEWER-N  CLUSTERS ARE PRESENTIN!SP(ISCELLS$EBUSETAL   /NTHE BASISOF #HL A mUORESCENCEMEASUREMENTS F R IT WAS CONCLUDEDD THAT THE RATE OF ELECTRON TRANSFE F FROM 9: TO0% ISSLOWEDD SLIGHTLY INMOSTOFT F HE !SP MUTANTS ALTHOUGH NOT IN!SP(IS CELLS ANDTOONLYASMALLEXTENTIN!SP6ALCELLS#HUET AL A A &URTHERMORE THE9:%9: MIDPO D INT POTENTIAL APPEAREDD TOBE INCREASED IN!SP3ERAND !SP!LA CELLS .IXON AND $INER  #HU ET F HEPROXIMITY OF!SPTO F AL A "ECAUSEOFT

$IBQUFS

.VUBUJPOBM
4UVEJFT
PG 0 &WPMVUJPO



9: 4YROFF THE$POLYPEPTIDE SUCH MUTATION N IRONMENTOF 9: MIGHT INDUCED ALTERATIONSTOTHE ENV NOT BEUNEXPECTED 4HE HIGH AFlNITY -N)) BINDING SITE THAT IS PRESENTIN-N DEPLETEDWILD TYPE03))PARTICLESIS ELIMINATED INAT LEASTTHE!SP!SN !SP!LA D !SP3ER MUTANTS .IXON AND D $INER  AND $INERAND.IXON  )NTHESEMUTANTS THE+ +F BINDING AND FOR D OXIDZING -N)) IONS IS INCREASED  TO FO F LD7HEN-N DEPLETED D WILD TYPE03)) PARTICLESAREILLUMINATEDINTHEPRESENCEOF-N)) IONS 9:% OXIDIZES-N)) TO-N))) AND D A PARALLEL POLARIZATION%02SIGNALOFF THE-N))) IONISOBSERVED #AMPBELLETAL  4HISSIGNALSHOWSHYPERlNE STRUCTURETHAT ISCONSISTENTWITH EITHERANELONGATED SIX COORDINATE OCTAHEDRAL -N))) GEOMETRY OR A lVE COORDINATESQUARE PYRAMIDAL-N))) GEOMETRY )N -N DEPLETED D !SP'LU AND D !SP(IS 03)) PARTICLES 9:% IS ALSO CAPABLE OF F OXIDIZING -N)) IONS .IXON AND $INER  $INER AND .IXON #AMPBELLETAL  (OWEVER A DIFFERENT F -N))) %02SIGNAL ISOBSERVED IN!SP(IS03)) PARTICLES ANDNO-N))) %02SIGNALISOBSERVEDIN !SP'LU03))PARTICLES#AMPBELLETAL   )NSTEAD T D HEILLUMINATED D !SP'LU03))PARTICLES EXHIBITAPERPENDICULARPOLARIZATION%02SIGNALTHATIS T AT!SP SUGGESTIVEOFF -N)6 4HESERESULTSSHOWTH F FORMSPARTO F HEBINDINGSITE FORT FT F HElRST-N)) ION THATISPHOTOOXIDIZEDDURINGTHELIGHT DRIVENASSEM BLY PHOTOACTIVATION OFF THE-NCLUSTERAND D THATTHE D HAT IS PRESENTATTHIS POSITION INmUENCESTHE RESIDUET COORDINATIONENVIRONMENTANDREDOXPROPERTIESOFTHE PHOTOOXIDIZEDD -N)) ION #HAPTER  )F!SP F LIGATESTHElRST-N)) IONTHAT ISPHO TOOXIDIZEDDURINGPHOTOACTIVATIONOFTHE-NCLUSTER IT WOULD D SEEM LOGICAL THAT THIS RESIDUE D WOULD D ALSO LIGATE THE ASSEMBLED -N  CLUSTER )NDEED MOST STUDIESOF!SPMUTANTSHAVESUGGESTEDTHISPOS SIBILITY(OWEVER Y IN APPARENT CONTRADICTION TOTHIS SUPP U OSITION !SP6AL CELLSAREPHOTOAUTOTROPHIC AND!SP,EU AND!SP)LE CELLS EVOLVE / AT F ILD TYPECELLS #HUETAL A  ^THERATEOFW .EITHER6AL NOR,EU NOR)LEWOULD BEEXPECTED D TO LIGATE-N/NEEXPLANATIONISTHAT!SP ALTHOUGH REQUIRED FORT F HEEFlCIENTPHOTOOXIDATIONOFF THE lRST -NIONTHAT ISLIGATED DUR D INGASSEMBLYOFF THE-N  CLUSTER DOESNOTREMAINASALIGANDTOTHE ASSEMBLED -N  CLUSTER (OWEVER IFF !SP DOES NOT LIGATE THEASSEMBLED -N  CLUSTER IT ISDIFlCULTTOEXPLAIN W WHYREPLACEMENTOFTHISRESIDUEWITH!SNISMORE DELETERIOUSTO/EVOLUTIONTH T ANREPLACEMENTWITH TH 6AL

,EU OR)LE4ORESOLVETHIS PARADOX ITWAS PROPOSED THAT6AL ,EU AND)LE BEINGBULKYANDHYDROPHOBIC R ATIONSTHATPERMITTHEMISSING CAUSESTRUCTURALPERTURB CARB R OXYLATEMOIETYTO BEREPLACED BYANOTHERRESIDUE D APEPTIDECARBONYLGROUP ORAWATERMOLECULE#HU ET AL A  #OMPENSATORY Y MUTATION INDUCED STRUCTURAL REARRANGEMENTS HAVE BEEN OBSERVED IN OTHERSYSTEMS #HUETAL A  2ECENTLY Y &4)2 AND %3%%- STUDIES WERE PER F FORME D TO DETERMINE IFF !SP LIGATES THE -N  CLUSTERAND IFSO IFITLIGATESTHE-NIONTHATUNDER GOESOXIDATIONDUR D ING THE3 A 3 3 A 3 OR3 A 3 TRANSITIONS"OTHSTUDIESWERECONDUCTEDWITH !SP(IS03))PARTICLES )NONE&4)2STUDY #HUETAL  A LOWFR F E QUENCYVIBRATIONALMODETHATAPPEARSAT^CMn INTHE 3 STATEINWILD TYPE03))PARTICLESSHIFTEDTO ^CMn IN!SP(IS03))PARTICLES &IG BOT TOMPANEL "ECAUSETHISMODEHAD BEENASSIGNED PREVIOUSLY TO A -N / -N STRUCTURAL UNIT #HU ET F FT F HISMODE IN!SP(IS03)) AL  THESHIFTO PARTICLESSHOWSTHAT!SPISSTRUCTURALLYCOUPLED TOTHE-N  CLUSTER4HIS&4)2STUDYALSOSHOWED THATTHE!SP(ISMUTATIONPRODUCESNOS D IGNIlCANT F ION  <  ALTERATIONSINTHEMID FREQUENCYREG CMn OFTHE3 MINUS 3 &4)2DIFFERENCESPECTRUM &IG TOPPANEL 4HE LACKO K F SUCHALTERATIONSSHOWS R OXYLATESTRETCHINGMODESOFF !SPARE THATTHECARB NOTCHANGEDDURINGTHE 3 A 3 TRANSITION ! MORE RECENT &4)2 STUDY HAS SHOWN THAT THE !SP(ISMUTATIONDOESNOTSIGNIlCANTLYALTERTHE MID FREQUENCYREGIONSOFANYOFTHE 3 A 3 3 A 3 OR3 A 3 &4)2DIFFERENCESPECTRA F $EBUSETAL F NONE OFF THESE3 STATETRANSITIONS  4HEREFORE SIGNIlCANTLYALTERSTHECARBOXYLATESTRETCHINGMODES F LIGATESTHE -N  CLUSTER OF!SP"ECAUSE!SP IN BOTH THE RECENT  ¯ AND  ¯ 8 RAY CRYSTAL LOGRAPHICSTRUCTURALMODELS"IESIADKAETAL  &ERREIRAETAL #HAPTER "ARB R ERANDD )WATA THESIMPLESTEXPLANATION FORT F HE &4)2DATAISTHATTHE -NIONTHATISLIGATEDBY!SPDOESNOTINCREASEITS CHARGEOROXIDATIONSTATE DUR D ING THE 3 A 3 3 A 3 OR3 A 3 TRANSITIONS4HISCONCLUSION HAS PROFOUN F D 

4HEAUTHORSOFANEAR F LIER&4)2STUDY OFF !SP'LU03))PAR TICLESCONCLUDEDTHATCARBOXYLATESTRETCHINGMODESOF!SPARE CHANGEDSIGNIlCANTLYDURINGTHE 3 A 3 TRANSITIONINWILD TYPE 03))PARTICLES3TEENHUISETAL  (OWEVER THE&4)2DIF F F FERENCESPECTRATHATAREREPORTEDIN3TEENHUISETAL ARE F PECTRATHATHAVE BEEN UNLIKETHE3 MINUS 3 &4)2 DIFFERENCES N OTHERLABORATORYAND D AREUNDOUB U TEDLY DOMINATED REPORTED BY ANY BYARTIFACTS F #HUETAL  



3JDIBSE
+
%FCVT

&IG 4HE3 MINUS 3 &4)2DIFFERENCESPECTRAOFWILD TYPEAND$ !SP(IS03))PARTICLESINTHEMID FREQUENCYTOPPANEL AND & LOW FREQUENCYBOTTOMPANEL REGIONSOFTHESPECTRUM.OTETHESIMILARITYOFTHEWILD TYPEAND!SP(ISSPECTRAINTHEMID FREQUENCY REGIONTOPPANEL .OTEALSOTHAT INTHELOWFREQUENCYREGIONOFTHESPECTRUMBOTTOMPANEL AMODEAT^CM Mn INTHEWILD TYPE n F INTHE!SP(IS03))PARTICLES4HISMODEHAS BEENASSIGNED D TOA-N / -NSTRUCTURAL UNITINTHE 03)) PARTICLESSHIFTSTO^CM -N  CLUSTER)NTHELOWERPANEL THEAMPLITUDEOFF THE!SP(ISSPECTRUMHASBEENMULTIPLIED BYA FACTORO F F TO FAC F ILITATECOMPARI SON-ODIlED FROM# F HUETAL  -ORERRECENT&4)2DATASHOWTHATTHE!SP(ISMUTATIONCAUSESNOSIGNIlCANTCHANGESTOTHE 3 MINUS 3 3 MINUS 3 OR3 MINUS 3 &4)2DIFFERENCESPECTRA F INTHEMID FREQUENC F YREGION $EBUSETAL A 

IMPLICATIONS FORT F HEMECHANISMOFF WATEROXIDATION 4HEPROXIMITYOF!SPTO9: TOTHE #AION AND TOAPUTATIVEPROTONTRANSFERPAT F HWAY A LEADING TOTHE PROTEINEXTERIORINTHE¯8 RAYCRYSTALLOGRAPHIC STRUTURAL MODEL &ERREIRA ET AL  #HAPTER  "ARB R ERAND D )WATA HAS LED D TOTHEIDEATHAT!SP LIGATES THE MOST CATALYTICALLY ACTIVE -N ION IN THE -N  CLUSTER2ECENTMECHANISTICPROPOSALSTHATARE BASEDONTHE ¯ STRUCTURALMODELPOSTULATETHATTHIS -NIONUNDERGOESOXIDATION DUR D INGONEORMOREOF THE3 A 3 3 A 3 OR3 A 3 TRANSITIONS-C%VOY D "RUDVIG -ESSINGER ,UNDBERGAND AND

3IEGBAHN  )NCONTRAST THE&4)2DATAIMPLY THATEITHER THEOXIDATIONOFTHIS-NIONOCCURS D ING THETRANSITORY3 A 3 TRANSITION OR ONLY DUR THEOXIDATIONINCREMENTSAND / FORMAT F IONCHEMISTRY THATOCCURDURINGTHECATALYTICCYCLEINVOLVEONLYTHE F HE-N  CLUSTER REMAINING -N  PORTIONOFT )N THE %3%%- STUDY $EBUS ET AL  THE TWO PULSE%3%%-SPECTRUMOFTHE'(Z3 STATE MULTILINE%02SIGNALOF F !SP(IS03))PARTICLESWAS F ILD TYPE03))PARTICLES SHOWNTORESEMBLETHATOFW )NPARTICULAR NOADDITIONALHISTIDYLNITROGENMODU D LATIONAPPEAREDD AT )H6@ FOXVWHUDQGG D FW\SHKHPHLVFRYDOHQWO\ DWWDFKHGG WR &\WF&\W E FRQWDLQVWKH E+ DQG E/KHPHVDQGWZR 84ELQGLQJ SRFNHWVGHQRWHG4R DQG4L7KH4R VLWH LV ORFDWHGG WRZDUGVWKH3VLGHRIWKHPHPEUDQHDQG WKH4L VLWHLVFORVHWRWKH1VLGH 7KHQHWUHDFWLRQFDWDO\]HGE\FRPSOH[,,,LVWKH UHGXFW G LRQRIWZRIHUUL&\W F WRWKHLU)H,, VWDWHDQG WKHR[LGDWLRQRIRQH84+ WR847KLVUHGR[ SURFHVV LVFRXSOHGWRDWUDQVORFDWLRQRIIRXUSURWRQVDFURVV WKHPHPEUDQH7KHVWUXFWXUDO EDVLVIRUWKHFDSDELOL D W\ RIFRPSOH[,,,WRWUDQVORFDWHWZRSURWRQVSHUVLQJOH HOHFWURQWUDQVIHUUHGIURP84+ WRIHUUL&\W F DULVHV IURPWKHVSDWLDORUJDQL]DWLRQRILWVUHGR[FHQWHUV LQWR D ELIXUFDWHGG HOHFWURQWUDQVIHUFKDLQFRQWDLQLQJRQH KLJKSRWHQWLDOEUDQFKDQGRQHORZSRWHQWLDOEUDQFK $FRPSOHWHF\FOHRIWKHHQ]\PHFRQWDLQVWKHIROORZ LQJVWHSV$IXOO\UHGXFH G G DQG SURWRQDWHG XELTXLQRO ELQGVWRWKH 4R SRFNHWDQGWUDQVIHUVRQHHOHFWURQYLD WKH KLJKSRWHQWLDO URXWH FRQVLVWLQJ RI WKH 5LHVNH LURQVXOIXU FHQWHU DQG G &\W F WR WKH ZDWHUVROXEO X H &\WF SURWHLQ$VHFRQGHOHFWURQLVGHOLYHUHGIURP WKHVHPLTXLQRQH4+s UDGLFDO IRUPHG LQWKH4R VLWH X  YLDWKHORZSRWHQWLDO EUDQFK EW\SH KHPHVWRDXEL

 TXLQRQHERXQGWRWKH 4L SRFNHW7KHIXOO\R[LGL]HG XELTXLQRQHLQWKH4R SRFNHWLVUHSODFHGE\ DXELTXLQRO X G WKHWZRVHSDUDWHHOHFWURQWUDQVIHUVDUHUHSHDWHG DQG JHQHUDWLQJDVHFRQGIHUUR&\WF PROHFXOHDQGDIXOO\ UHGXFHGXELTXLQROLQWKH 4L VLWH7KH4R DQG4L VLWHV G RQRSSRVLWHVLGHRIWKHPHPEUDQHDQG DUHORFDWHG WKHSURWRQLFUHDFWLRQVFRXSOHGWRWKH44+ UHGR[ SURFHVVHVJLYHULVHWRWKHQHWWUDQVORFDWLRQRISURWRQV DFURVVWKHPHPEUDQH ELOD\HU &\WF VKXWWOHVHOHFWURQVIURPFRPSOH[,,,WR FRP SOH[ ,9 F\WRFKURPH F R[LGDVH  ZKLFK FDWDO\]HV WKH ¿QDO VWHS LQ DHURELF UHVSLUDWLRQ E\ R[LGL]LQJ I IHUUR&\W F DQGUHGXFLQJ 2 WR +2)HUJXVRQ0LOOHU DQG%DEFRFN%DEFRFN 7KLVUHDFWLRQ UHTXLUHV D WRWDO RI IRXU HOHFWURQV DQG G IRXU SURWRQV DQGLPSRUWDQWO\WKHVXEVWUDWHSURWRQVDUHWDNHQXS H[FOXVLYHO\ IURP WKH 1VLGH RI WKH PHPEUDQH ,Q DGGLWLRQIRXUSURWRQVDUHSXPSHG G YHFWRULDOO\IURP G WKH WKH 1VLGHWRWKH 3VLGH %RWKWKH VXEVWUDWH DQG SXPSHGSURWRQVDUHWUDQVSRUWHGLQDXQLGLUHFWLRQDO PDQQHUDOWKRXJK WKHPHFKDQLVPIRUWKLVLVXQNQRZQ N ,QFRQWUDVWWRFRPSOH[,,,ZKLFKXVHVIUHHGLIIXVLRQ RIOLSLGVROXEOHTXLQRQHVIRUWKHSK\VLFDOWUDQVSRUWRI SURWRQVDFURVVWKHPHPEUDQHFRPSOH[,9WUDQVSRUWV WKHSURWRQVWKURXJKSURWHLQPHGLDWHGFKDQQHOV +LJKUHVROXWLRQ VWUXFWXUHV RI FRPSOH[ ,9 KDYH EHHQ REWDLQHG G IURP VHYHUDO VSHFLHV 6FKXOW] DQG &KDQ 7KHPDPPDOLDQHQ]\PHFRQWDLQV VXEXQLWVRIZKLFKLQWHUHVWLQJO\WKUHHDUHHQFRGHG \ E\ WKH PLWRFKRQGULDO '1$ 7KHVH WKUHH SURWHLQV FDUU\WKHUHGR[DFWLYHFHQWHUVDQGIRUPWKHHOHFWURQ DQG SURWRQWUDQVIHU XQLW RI WKH HQ]\PH %DFWHULDO FRPSOH[,9KDVWKUHHRUIRXUVXE X XQLWVPRVWRIZKLFK DUHKRPRORJRXVWRWKHWKUHHPDMRUSURWHLQVRIWKH PDPPDOLDQHQ]\PH7KHELQXFOHDUFRSSHU&X X$FHQWHU UHFHLYHVHOHFWURQVIURP&\W FDQG G WUDQVIHUVWKHPRQH DWWKHWLPH WR KHPH D ZKLFKLQWXUQUHGXFHVWKHEL QXFOHDUKHPH D&X% DFWLYH VLWH,QDGGLWLRQWRKHPH D DQG G WKHPRQRQXFOHDU&X% FHQWHUWKHDFWLYHVLWH FRQWDLQVDW\URVLQHWKDWKDVEHHQSURSRVHGWRIRUP DUDGLFDOGXULQJ FDWDO\VLV7\ 7 URVLQHUHGR[FKHPLVWU\ U LVDOVRHVVHQWLDOIRUSKRWRV\QWKHWLFZDWHUR[LGDWLRQ DV ZLOO EH GHVFULEHG EHORZ 
&MFDUSPO
BOE
1SPUPO
5SBOTGFST
JO
1IPUPTZ G ZO UIFTJT J -ANYOFF THEPROCESSESTHATOCCURINPHOTOSYNTHETIC ENERGYCONVERSIONAREANALOGOUSTOTHERESPIRATORY REACTIONS4HEUNIQUEEVENTSCATALYZED BYTHEPHOTO SYNTH T ETICORGANISMSARETH T ECAPTUREOF LIGHTQUANTAAND

#SJBO
3 (JCOFZ
BOE
$FDJMJB
5PNNPT THEPHOTOCHEMICALREACTIONSINWHICHTHEPHOTONENER R GIESARECONVERTEDINTOCHEMICALENERGYINTHEFORMOF D LANTS ALGAEANDP D HO CHARGE SEPARATEDSTATES'REENP TOSYNTHETIC BACTERIA CONTAIN BACTERIO CHLOROPHY H LLS " #HL CAROTENOIDSANDBILINMOLECULESTOCOLLECT THE SOLAR ENERGY Y 4HE EXACT PIGMENT COMPOSITION DEPENDSONTHEORGANISMANDD THEGROWTHCONDITIONS 4HE LIGHT HARVESTING ,( PIGMENTSHAVEIN GENERAL HIGH EXTINCTIONCOEFlCIENTSANDD ABSORB R ANCEMAXIMA F WAVELENGTHS WHI W CH ALLOW AT A VARIETY OF DIFFERENT THE PHOTOSYNTHETIC ORGANISMS TO EFlCIENTLY COLLECT LIGHTOVERA BROADSPECTRA D L RANGE4HEPIGMENTSARE BOUNDD TO PROTEINS THAT FFORM ANTENNA COMPLEXES WHICHAREHIGHLYORGANIZEDANDDYNAMICSTRUCTURES CONSTRUCTEDD TO EFlCIENTLY CAPTURE LIGHT QUANTA AND F FUNNE L THE EXCITATIONENERGY TO THE REACTION CENTER 2# !TOMICMODELSHAVEBEENOBTAINEDFORSEVERAL ANTENNASYSTEMS INCLUDINGTHEEXTERNAL,(COMPLEX ASSOCIATEDD WITH0HOTOSYSTEM))03)) +àHLBRANDT ETAL #HAPTER 'REENAND'ANTT#HAPTER (ANKAMERETAL ANDD THEANTENNAOFF PURPLEBACTERIA #OGDELLETAL   ! PHOTOSYNTHETIC 2# IS A TRANS MEMBRANE PRO TEINP  IGMENTCOMPLEX INWHI W CHTHE LIGHTENERGY TO CHEMICAL ENERGYCONVERSIONOCCURS2#SCONTAINELEC TRONICALLYCOUPLED" #HLMOLECULES WHICHTRAPTHE EXCITATIONENERGY DELIVERED BYTHEANTENNA5PONEX CITATION TH T EPHOTOCHEMICALLYACT L IVEPIGMENTSTRANSFE F R ANELECTRONTOANEARBYACCEPTOR!CHARGE SEPARATED F D WHICH ISSUB D W U SEQUENTLY STABILIZEDD VIAA STATEIS FORME SERIESOFF ELECTRON TRANSFERREACT F IONS4HESEEXERGONIC REDOXREACTIONSARECOUP U LEDTOPROTONTRANSFERSAND THECREATIONOFAPROTON F GRADIENTACROSSTHETHYLAKOID D DACCEPTOR D MEMBRANE4HEPHOTOCHEMICALLYREDUCE MOLECULE AND THE REDOX COFACTORS THAT PARTICIPATE IN THE SECONDARY ELECTRON TRANSFER F REACTIONS VARY BETWEEN DIFFERENT F SPECIES4HE COMPOSITION OFF THE REDOXCOFACTORSDIVIDESTHE2#INTOTWOMAJORGROUPS 0HOTOSYSTEM)03) ANDD THE2#S FOUN F D IN GREEN SUL F BACTERIAAND HELIOBACTERIAUSEIRON SULFURC FUR F LUSTERS ASTHETERMINALELECTRONACCEPTORS)NCONTRAST 03)) F LE BACTERIAAND GREENlLAMENTOUS ANDD THE2#SOFPURP BACTERIAEMPLOY BACTERIO PHEOPHYT H IN " 0HEO AND QUINONESASELECTRONACCEPTORS#HAPTER 2ENGER ANDD (OLWARTZ# L HAPTER 0ETROULEASAND#RO D F  FTS 3PECTROSCOPICANDD 8 RAYCRYSTALLOGRAPHICSTUDIES HAVEPROVIDEDDETAILEDKINETICANDSTRUCTURALINFOR MATION ON 2#S FFROM SEVERAL DIFFERENT F ORGANISMS !LLEN ANDD 7ILLIAMS  (ILLIER ANDD "ABCOCK (EATHCOTEETAL  &IGURESUMMARIZES THE PROTEINS ANDD PROSTHETIC GROUPS INVOLVED IN THE

$IBQUFS


%F /PWP 1SPUFJO
%FTJHO
JO
#JPFOFSHFUJDT



)LJ 6FKHPDWLFVXPPHU\RIWKHSURWHLQFRPSOH[HVSLJPHQWVDQGUHGR[FRIDFWRUVLQYROYHGLQSKRWRV\QWKHWLFHQHUJ\FRQYHUVLRQLQ ) G G V 7KHVROLGJUH\DUURZVVKRZWKHHOHFWURQÀRZZKLOHWKHVKDGHGDUURZLQGLFDWHVWKHVLWH GH WKHSXUSOHEDFWHULXP 5KRGREDFWHUVSKDHURL RIWUDQVPHPEUDQHSURWRQWUDQVSRUW7KH¿JXUHLVGHVFULEHGLQGHWDLOLQWKHPDLQWH[W

LIGHT DRIVEN GENERATION OF F A CHEMIOSMOTIC PROTON GRADIENT IN THE PURPLE BACTERIUM 2HODOBACTER S HAEROIDES4HE2#CONTAINSTHREESUBUNITS WHICH SP TOGETHERBINDS FOUR"# F HL A TWO"0HEO A TWO51 AND D A SINGLE NON HEME IRON 4HESE COFACTORS F ARE POSITIONEDSYMMETRICALLYAROUNDACENTRALTWO FOLD AXIS WHI W CH ISASTRUCTURAL THEMECOMMONTOALL 2#S D THAT IS BELIEVEDD TO 4HE2# ALSOCONTAINSACAROTENOID HAVEAPHOTOPROTECTIVEFUNCTIONANDALARGENUMBER OFF "#HLSAND D CAROTENOIDSAREBOUND D TOTHESURROUND INGANTENNASYSTEMS#OGDELLETAL  4WOOF THE2#CHROMOPHORESFORMA"#HLDIMER CALLEDTHE SPECIAL PAIR WHI W CH SERVES AS THE PHOTOCHEMICALLY ACTIVE ELECTRON DONOR IN THIS ORGANISM &OLLOWING ABSORPTIONOFANNMPHOTONBYTHESPECIALPAIR F D TOTHEACCESSORY"#HL TO ANELECTRONISTRANSFERRE THE"0HEO ONTOTHE lRST51ACCEPTOR1! AND FURT F HER TOTHESECOND51DENOTED1" 4HE LATTER IS REDUCED TWICE PROTONATED D AND THENREPLACED BYANOXIDIZED F HEMEMBRANE POOL4HE51( MOLECULES 51FROMT ARERE OXIDIZEDBYA#YT BC COMPLEXHOMOLOGOUS TOTHERESPIRATORYCOMPLEX)))4HEmOWOFE F LECTRONS THROUGH THEBIFURCATE F D ELECTRON TRANSFERPAT F HWAYOF THE BC COMPLEXGENERATESTHETRANS MEMBRANEPRO TON GRADIENT!SOLUBL U E C TYPE#YTCLOSESTHECYCLIC D INGTHE2#"#HLA  RADICAL ELECTRONmOW BYREDUC WITH ELECTRONS FROMTHE BC COMPLEX&IG  )NCHLOROPLASTSAND D CYANOBACTERIA 03)AND03)) D  D IVETHEREDUCT D IONOF.!$0 F WITH OPERATEINSERIESTODR ELECTRONSFROMWATER&IG 2ESEARCHONOXYGENIC PHOTOSYNTHESISHASRECENTLY ADVANCED D SIGNIlCANTLY WITH THE AVAILABILITY OF HIGH AND D MEDIUM RESOLU TION STRUCTURES FOR CYANOBACTERIAL 03 ) AND 03)) RESPECTIVELY &ROMMEETAL  *ORDANET

AL 2UTHERFOR F D AND &ALLER :OUNIETAL +AMIYAANDD 3HEN &ERREIRAETAL  #HAPTERSn 03))ISA0HEOQUINONETYPE2#AND THEREDOXREACTIONSTHATOCCURONTHEREDUC D ING SIDEOF THISENZYMEAREVERYSIMILARTOTHOSEDESCRIBEDD ABOVE F FORTHE2 # IN 2BSP S HAEROIDES 4HE REDOX COFAC F TORS ANDD REACTIONSTHATTAKEPLACEONTHEOXIDIZING SIDE OF03))ARE F HOWEVER UNIQUE4HEPRIMARYELECTRON DONORISAMULTIMERIC #HL A COMPLEXWITHABSORPTION MAXIMUMATNM#HAPTER &OLLOWINGPHOTOOXI DATION 0 ISRAPIDLYREDUCE D D BYATYROSINEDENOTED 9: #HAPTER 4HISRESIDUEISINTURNREDUCEDBYA -N  CLUSTER WHI W CH BINDSSUB U STRATEWATER#HAPTERS n  #ALCIUMANDD CHLORIDEAREREQUIRED FO F R CATALYSISANDAREMOSTLIKELYDIRECTLYASSOCIATEDWITH THEACTIVESITE#HAPTER !SECONDD REDOX ACTIVE TYROSINE 9$ AND#Y D T B SERVEASAUXILIARYELECTRON DONORSIN03))#HAPTER 4HEMULTIMERIC03)) COMPLEX ALSO CONTAINS A LARGE NUMBER OFF ,( PIG MENTSTH T ATAREASSOCIATEDD WITH T ITSINTEGRAL ANDD EXTERNAL ANTENNASYSTEMS#HAPTERSAND 4HEELECTRON TRANSFERC F HAIN IN03)) ISLINKEDD TO #YT B F VIATHE PLASTOQUINONEP  LASTOQUINOL 0101( POOL INTHE THYLAKOIDMEMBRANE&IG #YT B F ISSTRUCTURALLY LESSWELL CHARACTERIZEDASCOMPARE D D TOTHERESPIRATORY BC ENZYME.ONETHELESS THEELECTRON TRANSFERC F HAIN ANDTHECATALYTICCYCLEOF#YT B F AREMOSTLIKELYVERY SIMILARTOTHOSE DESCRIBEDD ABOVE FOR# F YT BC "ERRY ETAL  #YT B F REDUCESTHEPRIMARYELECTRON DONORIN03)VIAPLASTOCYANIN4HISSMALL SOLUBLE PROTEINCONTAINSASINGLECOPPERIONAND IS INSOME ORGANISMSREPLACED BYACYTOCHROME 4HECRYSTALSTRUCTUREOF03)ISATRIMERWITHEACH U UNITSANDD COFACTORS F MONOMERCONTAINING SUB



#SJBO
3 (JCOFZ
BOE
$FDJMJB
5PNNPT

)LLJ  6FKHPDWLFRYHUYLHZRIWKHSURWHLQFRPSOH[HVSLJPHQWVDQG ) G UHGR[FRIDFWRUV LQYROYHG LQSKRWRV\QWKHWLFHQHUJ\FRQYHUVLRQ LQ SODQWV JUHHQDOJDHDQGG F\DQREDFWHULD7KHVROLG JUH\ DUURZVVKRZWKHHOHFWURQ ÀRZZKLOHWKHVKDGHG G DUURZLQGLFDWHVWKHVLWHRIWUDQV PHPEUDQHSURWRQWUDQVSRUW7KH¿JXUHLVGHVFULEHGLQGHWDLOLQWKHPDLQWH[W

&ROMMEETAL *ORDANETAL #HAPTER 7ITT 4HE INTEGRAL ANTENNAOFF EACHMONOMERCONTA H INS D CAROTENOIDS4HE2#CORECON ABOUT#HL A AND TAINSSIX#HL A TWOPHYLLOQUINONES0H1 ANDTHREE ;&E3=CLUSTERS4HEPRIMARY DONORIN03)ISA#HL A PAIRWITH ABSORPTIONMAXIMUMATNMDENOTED 05P 5 ONPHOTOOXIDATION 0DELIVERSANELECTRON TOASECOND# D HL A SPECIESDENOTED ! AND FURT F HERTO F THE PHY H LLOQUINONES 4HE ELECTRON TRANSPORT ONE OF CHAIN ENDS WITHTHE IRON SULFU F R CLUSTERS DENOTEDD &8 &! AND D &"4HEELECTRONSARElNALLY TRANSFERRE F D FROM F 03)TO.! . $0 VIA FERRE F DOXINAND D AmAVOPROTEIN  $PGBDUPS
1SPQFSUJFT
BOE
UIF
'VODUJPO
PG
UIF 1SPUFJ 1S U JO
.BUSJY S )N SUMMARY Y THE RESPIRATORY AND PHOTOSYNTHETIC ENZYMESCONTAINSEVERAL GROUPSOFF COFACTORS0ROS F

THETIC GROUPS CONTAINING TETRAPYRROLIC MACROCYCLES F IONALLYPROMINENT (EMESAREINVOLVED INA AREFUNCT F IONS #HL AND D "#HL ARETHE VARIETYOF DIFFERENTREACT MAJOR ,( PIGMENTS IN PHOTOSYNTHETIC ORGANISMS AND D THE PRIMARY PHOTOCHEMISTRY INVOLVES " #HL AND " 0HEOMOLECULES!THIRDGROUPO D F COMMON ORGANICCOFACTORSISTHE mAVIN NUCLEOTIDES &OR EX AMPLE ANUMBEROF mAVOPROTEINS DONATEELECTRONS TO THE 5151( POOL OF F THE INNER MITOCHONDRIAL MEMBRANE&IG ,IPID SOLUBLEQUINONESRE Q PRESENT A FOURT F H GROUPOF IMPORTANTORGANICREDOXCOFACTORS F 0LASTOQUINONE PHY H LLOQUINONEAND D UBIQUINONE WHI W CH INSOMEORGANISMSAREREPLACEDBYMENAQUINONE ARE KEY PLAY A ERS INTHEREDOX DRIVENPROTON TRANSLOCATION

PROCESSES OCCURRING IN RESPIRATION AND PHOTOSYN THESIS&URTHERMORE AMINOACIDSARE BEINGUSEDD AS F 4 4YROS INESAREINVOLVED ENDOGENOUSREDOXCOFACTORS INPHOTOSYNTHETICWATEROXIDATIONANDTHISRESIDUEHAS ALSOBEENPROPOSEDTOPART D ICIPATEINTH T ECATALYTICCYCLE OFCOM F PLEX)6)NADDITIONTOHEME IRON ANUMBEROF REDOX ACTIVEMETALSAREINVOLVEDINBIOLOGICALENERGY TRANSDUCT D ION )RON SULFURC F LUSTERSAREESSENTIAL FORT F HE ELECTRON TRANSFERC F HAINS INCOMPLEX) ))ANDD )))OF THE RESPIRATORYCHAIN THEYSERVEASELECTRONACCEPTORSIN PHOTOSYNTHETIC2#SAND IN FERRE F DOXINS#OPPERCEN TERSARE FOUN F D INCOMPLEX)6AND INPLASTOCYANINAND A-N  CLUSTERISESSENTIALFORTHEACTIVITYOF03)) 'HVSLWH WKH UDQJH RI FKHPLFDO IXQFWLRQDOLW\ UHSUHVHQWHG E\WKHVHSURVWKHWLFJURXSVWKHSURWHLQ PDWUL[ KDV EHHQ UHFUXLWHG E\ HYROXWLRQ WR IXUWKHU FRQWUROFKHPLFDOUHDFWLYLW\$FOHDUH[DPSOHRIWKLV LV SURYLGHG E\ % &KO D ZKLFK SHUIRUPV HQHUJ\ WUDQVIHU ZKHQ ORFDWHG LQ WKH /+ FRPSOH[HV DQG HOHFWURQ WUDQVIHU ZKHQ VHUYLQJ DV D 5& SLJPHQW D W\WRPRGX G ODWHWKHNLQHWLF 3URWHLQVKDYHWKHFDSDELOL DQGWKHUPRG\QDPLFUHGR[SURSHUWLHVRIWKHLUFRIDF WRUVVXEVWDQWLDOO\)RUH[DPSOHW\URVLQHR[LGDWLRQLV DQLUUHYHUVLEOH SURFHVV LQVROXWLRQ7RPPRVHWDO  ,QFRQWUDVWWKHUHGR[F\FOHVRI&3++@ VKRZHGG WKDWLWDVVHPEOHV LQWRD SDUDOOHO IRXUKHOL[EXQGOHZLWKIRXUKHPHVERXQG LQ WKHFRUHDQGDFRIDFLDO&3 3 SDLUDWWKH1WHUPLQXVDV \ \UHQHZDVXVHG GHVLJQHG,QDPRUHUHFHQWVWXG\S WRPRGHO D % &KOVSHFLDO SDLU-RQHVHWDO  $ S\UHQH FKURPRSKRUH ZDV DWWDFKHG WR WKH 1WHU $ PLQXVRIDUHVLGXHSHSWLGHFRQWDLQLQJ /HXDQG ,OHUHVLGXHV G LQKHSWDG D DQG G SRVLWLRQVDQGUHVLGXHV ZLWKKLJKKHOLFDOSURSHQVLWLHVLQWKHPDMRULW\RIWKH UHPDLQLQJ SRVLWLRQV7KH SHSWLGH DOVR FRQWDLQHG D 7US LQDVROYHQWH[SRVHG KHSWDG I SRVLWLRQ,QDTXH 7U RXVEXIIHUWKHS\UHQHSHSWLGHKDVaVHFRQGDU\ VWUXFWXUH DQG H[LVWV LQ D FRQFHQWUDWLRQGHSHQGHQW PRQRPHUGLPHURFWDPHU HTXLOLEULXP /DVHU ÀDVK SKRWRO\VLVH[SHULPHQWVZHUHSHUIRUPHGXVLQJSHSWLGH VDPSOHVLQWKH +0UDQJHDWZKLFKWKHRFWDPHUVWDWH GRPLQDWHV 3KRWRR[LGDWLRQ RI WKH S\UHQH UHVXOWHG LQ WKH UHGXFWLRQ RI WKH H[WHUQDOO\ DGGHG HOHFWURQ DFFHSWRUPHWK\OYLRORJHQIROORZHGE\ IRUPDWLRQRI D VSHFLHV DVVLJQHG G WR D WU\SWRSKDQ FDWLRQ UDGLFDO $OWKRXJK WKH S\UHQHSHSWLGH DVVHPEO\ GHVFULEH DERYHGRHVQRWUHSUHVHQWDZHOOGH¿QHGV\VWHPLW VKRZVSURPLVHZLWK UHVSHFWWRLQWURGXF G LQJIXQFWLRQ LQWRSKRWRV\QWKHWLF5&LQVSLUHGGHVLJQV 7KHFRYDOHQWDWWDFKPHQWRIFRIDFWRUVDWWKHSHSWLGH G WRFRQVWUXFWPL[HGFR WHUPLQL SURYLGHVRQHPHWKRG I IDFWRUSURWHLQV,QDGGLWLRQ/\V&\VDQG+LVUHVLGXHV FDQEHXVHGIRUFRYDOHQWOLQNDJHRIYDULRXVFRIDFWRUV 7 7RVWX G\&KOSURWH  LQELQGLQJLQWHUDFWLRQV5DXHWDO  IRUPHGFRYDOHQWR[LPHERQGVEHWZHHQPRGL ¿HGO\VLQHVDQGDOGHK\GH JURXSVRQWKHWHWUDS\UUROH ULQJVRI=Q&KO GHULYDWLYHV)ODYLQVFDQEHDWWDFKHG WR &\V YLD DON\ODWLRQ DV RULJLQDOO\ GHVFULEHG E\ /HYLQH DQG .DLVHU   $ QRYHO ÀDYR&\W ZDV FRQVWUXFWHG E\UHGHVLJQLQJWKHSURWRW\SHGLVXO¿GH EULGJHG++LQWRDKHOL[ORRSKHOL[SURWHLQZLWK DXQLTXH&\VLQHDFKPRQRPHU)ODYLQDWWDFKPHQW I OORZHG E\KHPHVHOIDVVHPEO\SURYLGHG IR G WKHSKRWR

#SJBO
3 (JCOFZ
BOE
$FDJMJB
5PNNPT DFWLYDWDEOHKHPHÀ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c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c DQGcLQDVWHSZLVHPDQQHU(OHFWURQ _  WUDQVIHU UDWHV ZHUH PHDVXUHGG IRU WKH 5X,, _S VHULHV XVLQJ ODVHUÀDVK SKRWRO\VLV DQG SXOVHUDGL RO\VLVPHWKRGV $ ` YDOXHUHÀHFWLQJWKHHOHFWURQLF FRXSOLQJEHWZHHQWKHHOHFWURQGRQRUDQGDFFHSWRU G RI “ cn ZDV GHULYHG G IURPWKH NLQHWLF GDWD 7KHDXWKRUV FRQFOXGHGWKDWWKL G V YDOXH LV ZLWKLQWKH  ± cn UDQJH W\SLFDOO\ REVHUYHGG IRU QDWXUDO SURWHLQVDQGFRQVLVWHQWZLWKSUHGLFWLRQVPDGHXVLQJ WKHRUHWLFDOSDWKZD\ PRGHOVRIWKURXJKKHOL[HOHFWURQ WXQQHOLQJ7KLVZRUNN IXUWKHUYDOLGDWHVWKHXVHRIGH QRYRSURWHLQVWRVWXG\ELRFKHPLFDOIXQFWLRQ 2JDZDDQGFRZRUNHUVKDYHFRQVWUXFWHGUXWKHQLXP PRGL¿HGG WZRVWUDQGHGG FRLOHGG FRLOVWRVWXG\HOHFWURQ WUDQVIHUDFURVVDQRQFRYDOHQWLQWHUIDFHDQGPRUHUH \ \ \DORQ JKHOLFHV.R]ORYDQG2JDZD FHQWO\GLUHFWLRQDOO  GHVLJQHGG DQGG V\QWKHVL]HGG DQ DPSKLSKDWLF

$IBQUFS


%F /PWP 1SPUFJO
%FTJHO
JO
#JPFOFSHFUJDT

UHVLGXH SHSWLGH ZLWK D VLQJOH +LV LQ D KHSWDG I SRVLWLRQWRZKLFK5X,, ES\  RU5X,,, 1+  ZHUH DWWDFKHG 'LVVROYLQJ WKH PHWDOORSHSWLGHV LQ EXIIHU JLYHVDVWDWLVWLFDOPL[WXUHRIKRPRDQGKHWHURGLPHUV EXWDVQRWHGE\WKHDXWKRUVRQO\ WKHODWWHUVSHFLHV LVFDS D DEO D HRILQWUDPROHFXODUHOHFWURQWUDQVIHUDFURVV WKHKHOL[KHOL[LQWHUIDFH8VLQJWKHODVHUÀDVKTXHQFK PHWKRG&KDQJHWDO D¿UVWRUGHUUDWHFRQVWDQW WKDW ZDV LQGHSHQGHQW RI WKH SHSWLGH FRQFHQWUDWLRQ ZDVGHULYHG7KLVUDWHFRQVWDQWZDVDVVLJQHGWRWKH 5X,, 1+  WR5X,,, ES\  HOHFWURQWUDQVIH I UUHDF WLRQDFURVVWKHK\GURSKRELFFRUHRIFRLOHGFRLO GLPHU 7KLV LQLWLDO VWXG\ ZDV IROORZHG E\ PRUH GHWDLOHG FKDUDFWHUL]DWLRQVRIWKHPHWDOORSHSWLGHV.RUQLORYD HWDO DQG G DQ LQYHVWLJDWLRQRIWKHLQÀ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¿FLW\ ZDVGHVLJQHGXVLQJGLIIHUHQWOLJDQGVHWVDUUDQJHGLQ GLIIHUHQWFRRUGLQDWLRQJHRPHWULHVOLQHDUELV+LV YV WHWUDKHGUDO &\VWRSURYLGHVHOHFWLYLW\IRUKHPHDQG >)H6@$GGLWLRQDOO\WKHRUGHURIFRIDFWRULQFRUSRUD WLRQZDVLPSRUWDQWIRUDVVHPEO\7KH>)H6@ FOXVWHU ZDV LQVHUWHG ¿UVWIROORZHG E\WKH KHPHVEHFDXVHWKH LQFRUSRUDWLRQRIKHPH¿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



LPSRVHGE\WKHSURWHLQIROGPXOWLFRIDFWRUVLWHVFDQ EH DVVHPEOHG 7KHRUGHURIFRIDFWRUDGGLWLRQFDQEHXVHGG WRJHQHU DWHPL[HGFRIDFWRUSURWHLQVZKHQWKHOLJDQGW\SHDQG JHRPHWULFDODUUDQJHPHQWDUHVLPLODU++ZDV GHVLJQHGG WR ELQGG IRXU E KHPHV EXWLWKDVDOVR EHHQ XVHGDVDVFDIIROGWRVLPXOWDQHRXVO\ELQGWZRKHPH D DQGG WZR KHPH E FRIDFWRUV *LEQH\ HW DO   6WRLFKLRPHWULFDGGLWLRQRIWZRHTXLYDOHQWVRIKHPH E IROORZHGE\WZRHTXLYDOHQWVRIKHPH D UHVXOWV LQ WKHIRUPDWLRQRIWKHPL[HGFRIDFWRU>KHPH DKHPH E ++@6SHFWURVFRSLFPHDVXUHPHQWV GHPRQVWUDWH U WLV VLPLODUWRWKH EDFWHULDOR[LGDVH WKDWWKH FRQVWUXF &\WED'HWDLOHGHOHFWURFKHPLFDODQDO\VLVLQGLFDWHV WKDWWKHKHPH E FRIDFWRUV ELQGG WRWKH+SRVLWLRQV ZKLOH WKH KHPH D FRIDFWRUV ELQGG WRWKH + VLWHV 7KH VWDELOLW\RIWKHPL[HGFRIDFWRUFRPSOH[KDVEHHQDW WULEXWHGG WRWKHWLJKWELQGLQJRIKHPH D DQG E DQGG WKH FRUUHVSRQGLQJO\VORZRIINLQHWLFV ,,,3HUVSHFWLYH -ARK R EDD PROGRESSCONTINUESTOBEMADETOWARDSTHE ULTIMATEGOALOFDESIGNINGFUNCTIONALPHOTOSYNTHETIC F H!NUMBER ANDD RESPIRATORY BIOMIMETICSFROMSCRATC OF LINES OFF CHEMICAL AND BIOCHEMICAL RESEARCH ARE SYNERGISTICALLYMERGINGTOPROVIDETHENECESSARYFUN DAMENTALCONCEPTS FORCONSTRUCT F IONOFSUC F HSYSTEMS 3EVERAL DISTINCTAPPROACHESTOEACHOFF SEVERALCRITICAL AREASINTHEDESIGNOFBIOMIMETICSHAVEPROVENSUC CESSFU F L$ESIGNEDD PROTEINSCAFFO F LDSWH W OSESTRUCTURES MATCHT H HEINTENDED DESIGNAREKNOWNAN K D FUTUREPRO F G RESS PROMISESTO PROVIDEEVENMOREWITHCOFACTORS BOUND 2ATIONAL COMPUTATIONAL ANDD COMBINATORIAL D IN DESIGNSCONTAINING APPROACHESHAVEPROVIDEDPROTE MOSTOFTHECOFACTORSRELEVANTTOPHOTOSYNTHESISAND RESPIRATION (EME PHOTOACTIVE :N "#HL A mAVIN AMINO ACID PROTEIN RADICAL IRON SULFUR F CLUSTER AND COPPERSITESHAVEALLBEENASSEMBLEDIN DENOVO DE SIGNEDPROTE D INSCAFFO F LDS)NFACT F DESIGNSCONTAINING COMBINATIONSOFF THESECOFACTORSARE F DEMONSTRATING NOVELDESIGNCONCEPTSASWELLASFUNCTIONALPHOTO ACTIVATEDD ELECTRONTRANSFERREACT F IONS!LLOFF THESEARE NECESSARY STEPS IN THE CONSTRUCTIVE PROGRESSION OF D DESIGNOFPHOTOSYNTHETICANDRESPIRATORY THE DENOVO BIOMIMETICS3TARTING FROMT F HESIMPLICITYOFA F FO F LDED HELICAL PEPTIDESEQUENCE Q D DENOV O DESIGN HASASTRONG STARTTOWARDSTHEULTIMATEBIOLOGICALCOMPLEXITYOF THEPHOTOSYNTHETICANDD RESPIRATORYCOMPLEXES4HE F FUTUREC HALLENGE LIES IN FU F LlLLINGTHEPROMISEOF DE D

 NOVO DESIGN TO DELINEATE AND D EXPAND BIOCHEMICAL FUNCTION "DLOPXMFEHNFOUT 4HIS WORK WAS FUNDED BY THE .ATIONAL 3CIENCE &OUNDATION 'RANT#(% TO"2' ANDBY THE3WEDISH2ESEARCH #OUNCIL #4  3FGFSFODFT $OOHQ-3DQG: G :LOOLDPV-&  3KRWRV\QWKHWLFUHDFWLRQFHQWHUV )(%6/HWW± $Q¿QVHQ&% 3ULQFLSOHVWKDWJRYHUQWKHIROGLQJRISURWHLQ FKDLQV6FLHQFH± $Q¿QVHQ&%DQG6FKHUDJD+$ ([SHULPHQWDODQGWKHRUHWL FDODVSHFWVRISURWHLQIROGLQJ$GY3URW&KHP± G %UXQRUL 0  +HPRJORELQDQGG 0\RJORELQ $QWRQLQL (DQG LQWKHLUUHDFWLRQVZLWK OLJDQGV,Q)URQWLHUV LQ %LRORJ\9RO G 3XXEOLVKLQJ&R$PVWHUGDP 1RUWK+ROODQG %DEFRFN N *7  +RZ R[\JHQ LV DFWLYDWHGG DQG UHGXFH G G LQ G L86$± UHVSLUDWLRQ3URF1DWO$FDG6F %DODELQ ,$ DQG 2QXFKLF -1   '\QDPLFDOO\ FRQWUROOHG SURWHLQ WXQQHOLQJ SDWKV LQ SKRWRV\QWKHWLF UHDFWLRQ FHQWHUV 6FLHQFH± %DO]WHU/1LOVVRQ+DQG1LOVVRQ- 'H QRYR GHVLJQRI SURWHLQV²:KDWDUHWKHUXOHV"&KHP5HY± %HLQHUW++ROP5+DQGG 0QFN (  ,URQVXOIXUFOXVWHUV 1DWXUH¶V PRGXODU PXOWLSXUSRVH VWUXFWXUHV 6FLHQFH  ± %HQVRQ'(:L : V]06DQGG +HOOLQJD+: 5DWLRQDO GHVLJQ RI QDVFHQW PHWDOORHQ]\PHV 3URF 1DWO $FDG 6FL 86$  ± %HQVRQ'5+DUW%5=KX;DQG'RXJKW\0% 'HVLJQ V\QWKHVLVDQGFLUFXODUGLFKURLVPLQYHVWLJDWLRQRIDSHSWLGH VDQGZLFKHG G PHVRKHPH - $P &KHP 6RF± %HUU\($*XHUJRYD.XUDV0+XDQJ/DQGG &URIWV$5  6WUXFWXUHDQG G IXQFWLRQRIF\WRFKURPH EF FRPSOH[HV$QQX5HY %LRFKHP± %REURZVNL.3R]QDQVNL - +ROFPDQ-DQG: G :LHU]FKRZVNL ./   3XOVH UDGLRO\VLV VWXGLHV RI LQWUDPROHFXODU HOHFWURQ WUDQVIHULQPRGHOSHSWLGHVDQGSURWHLQV7US>1+s@±!7\U>2s@ UDGLFDOWUDQVIRUPDWLRQLQ+7US3UR Q7\U2+Q ±VHULHV RISHSWLGHV-3K\V&KHP%± %RORQ'1DQG0D\R6/ G  (Q]\PHOLNHSURWHLQVE\FRPSX WDWLRQDO GHVLJQ3URF1DWO$FDG6F G L86$± %UDXQ 3 2OVHQ -' 6WURKPDQQ % +XQWHU &1 DQG G 6FKHHU +  $VVHPEO\RIOLJKWKDUYHVWLQJ EDFWHULRFKORURSK\OO LQD PRGHOWUDQVPHPEUDQH KHOL[ LQLWVQDWXUDO HQYLURQPHQW - 0RO %LRO± %URPDQ/0DOPVWU|P%*$DVD5DQG9lQQJnUG7 4HE ROLEOFCOPPERINTHECATALYTICACTIONOFLACCASEANDCERULOPLAS MIN%LRFKLP%LRSK\V$FWD± %UXVVODQ-$DQG G 3HWHUVRQ03 7HWUDS\UUROHUHJXODWLRQRI QXFOHDUJHQHH[SUHVVLRQ3KRWRV\QWK5HV± %U\ U VRQ-:%HW]6)/X+66XLFK'-=KRX+2¶1HLO.7DQG 'H*UDGR:) 3URWHLQGHVLJQ$KLHUDUFKLFDSSURDFK

#SJBO
3 (JCOFZ
BOE
$FDJMJB
5PNNPT 6FLHQFH± &KDNUD N DEDUWW\$DQG G %DOGZLQ 5/  6WDELOL D W\RIDOSKDKHOLFHV $GY3URW&KHP± &KDNUDE D DUWW\$'RLJ$-DQG G %DOGZLQ5/ +HOL[FDSSLQJ SURSHQVLWLHVLQSHSWLGHVSDUDOOHOWKRVH LQSURWHLQV3URF1DWO $FDG6FL86$± &KDQJ,-*UD\+%DQG:LQNOHU-5 +LJKGULYLQJIRUFH HOHFWURQWUDQVIHULQPHWDOORSURWHLQV,QWUDPROHFXODUR[LGDWLRQ RI IHUURF\WRFKURPH F E\ 5XEE\ LP +LV  -$P &KHP 6RF± &KRPD&7/HDU-'1HOVRQ0-'XWWRQ3/5REHUWVRQ'(DQG 'H*UDGR :)   'HVLJQ RI D KHPHELQGLQJ IRXUKHOL[ EXQGOH-$P &KHP6RF± &RJGHOO5-,VDDFV1:+RZDUG7'0F/XV G NH\.)UDVHU1DQG3ULQFH60 +RZSKRWRV\QWKHWLFEDFWHULDKDUYHVW VRODUHQHUJ\-%DFWHULRO± &ROGUHQ&'+HOOLQJD+:DQG&DUDGRQQD-3 7KHUDWLRQDO GHVLJQDQGFRQVWUXFWLRQRIDFXERLGDOLURQVXOIXUSURWHLQ3URF 1DWO$FDG6FL86$± 'DKL\DW%,DQG G 0D\R6/ 'H QRYR SURWHLQ GHVLJQ)XOO\ DXWRPDWHG G VHTXHQFHVHOHFWLRQ6FLHQFH± 'DL 4+7RPPRV&)XHQWHV(-%ORPE P HUJ 05$'XWWRQ3/DQG : G $- 6WUXFWXUHRID GHQRY :DQ G R GHVLJQHG G SURWHLQPRGHO RIUDGLFDOHQ]\PHV-$P&KHP6RF± 'DXJKHUW\ 5*:DVRZLF]7*LEQH\ %5DQG'H5RVH9- 'HVLJQDQGVSHFWURVFRSLFFKDUDFWHUL]DWLRQRISHSWLGHPRGHOV IRU WKH SODVWRF\DQLQ FRSSHUELQGLQJ ORRS ,QRUJ &KHP  ± $E'RADO7& 7ASSERMAN:2AND D ,EAR*$  0ROTEIN DESIGN A MINIMALISTAPPROACH3CIENCEn $E'RADO7& 3UMMA#- 0AVONE6 .ASTRI&AND D ,OMBARDI!  $ENOVO DESIGNAND D STRUCTURAL CHARACTERIZATIONOFF PRO TEINSAND D METALLOPROTEINS!NNU2EV"IOCHEMn 'L%LOLR$- &UDQH%5:HKEL:$.LVHU&1 $EX2PDU00 &DUORV 50 5LFKDUGV -+ :LQNOHU -5 DQG *UD\ +%  3URSHUWLHVRISKRWRJHQHUDWHGWU\SWRSKDQDQGW\URV\OUDGLFDOV LQVWUXFWXUDOO\FKDUDFWHUL]HGSURWHLQVFRQWDLQLQJUKHQLXP, U RQ\O GLLPLQHV-$P&KHP6RF± WULFDUE 'ROSKLQ*7%ULYH/-RKDQVVRQ*DQG G %DOW]HU/ 8VHRI DURPDWLFDPLQRDFLGG UHVLGXHVWRUHVWU G LFWWKHG\QDPLFV LQWKH K\GURSKRELFFRUHRID GHVLJQHG KHOL[ORRSKHOL[GLPHU-$P &KHP6RF± (JJLQN//DQG+RREHU-. &KORURSK\OOELQGLQJWRSHSWLGH PDTXHWWHV FRQWDLQLQJ D UHWHQWLRQ PRWLI - %LRO &KHP  ± )HUJXVRQ0LOOHU6DQG%DEFRFN*7 +HPHFRSSHUWHUPLQDO R[LGDVHV&KHP5HY± )HUUHLUD . ,YHUVRQ 70 0DJKODRXL . %DUE U HU - DQG G ,ZDWD 6  $UFKLWHFWXUH RI WKH SKRWRV\QWKHWLF R[\JHQHYROYLQJ FHQWHU6FLHQFH± )ULHGULFK7DQG6FKHLGH' 7KHUHVSLUDWRU\ U FRPSOH[,RI EDFWHULDDUFKDHDDQGHXNDU\ U DDQGLWVPRGXOHFRPPRQZLWK PHPEUDQHERXQG PXOWLVXEXQLW K\GURJHQDVHV )(%6 /HWW ± )URPPH3-RUGDQ3DQG.UDX‰1 6WUXFWXUHRI3KRWRV\VWHP ,%LRFKLP %LRSK\V$FWD± )URPPH 3 .HUQ - /ROO % %LHVLDGND - 6DHQJHU::L : WW +7 . ‰1DQG .UDX G =RXQL$ )XQFWLRQDO LPSOLFDWLRQVRQWKH PHFKDQLVPRIWKHIXQFWLRQRI3KRWRV\VWHP,,LQFOXGLQJZDWHU R[LGDWLRQEDVHGRQWKHVWUXFWXUHRI3KRWRV\VWHP,,3KLORV7UDQV 5R\DO6RF/RQGRQ%± *HUHQ/+DKP6'XUKDP%DQG0LOOHWW) 3KRWRLQGXFHG

$IBQUFS


%F /PWP 1SPUFJO
%FTJHO
JO
#JPFOFSHFUJDT

HOHFWURQWUDQVIHUEHWZHHQF\WRFKURPH F SHUR[LGDVHDQG\HDVW D HOHG G DW &\V  ZLWK EURPRPHWK\O F\WRFKURPH F ODE PHWK\OELS\ULGLQH>ELVELS\ULGLQH @UXWKHQLXP %LRFKHPLVWU\ ± *KDGLUL056RDUHV&DQG& G KRL& $FRQYHUJHQWDSSURDFK WRSURWHLQGHVLJQ0HWDOLRQDVVLVWHGVSRQWDQHRXVVHOIDVVHPEO\ RIDSRO\SHSWLGHLQWRDWULSOHKHOL[EXQGOHSURWHLQ-$P&KHP 6RF± *LEQH\%5DQG'XWWRQ3/ 'H QRYR GHVLJQDQGV\QWKHVLVRI KHPHSURWHLQV,Q0DXN$*DQG6\NHV$*HGV $GYDQFHG,Q RUJDQLF&KHPLVWU\SS±$FDGHPLF3UHVV1HZ