Acta Biologica et Medica Germanica: Band 38, Heft 2/3 [Reprint 2022 ed.] 9783112650066

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ACTA BIOLOeiCA " ET MED1CA GERMANICA Scientific Conference CYtochrome P - 4 5 0 Stradurol ond Functionol Aspects

Band 38 Heft 2\3 • 1979 Seite 139-518 EVP 48, — M

ABMGAJ 38 (2/3) 1 3 9 - 5 1 8 (1979)

31002

Au fnahmebedingungen 1. Die ACTA BIOLOGICA E T MEDICA GERMANICA, Zeitschrift f ü r funktionelle Biowissenschaften, publiziert Arbeiten aus den Fachgebieten Biochemie, Molekular- u n d Zellbiologie, Physiologie (einschließlich Pathophysiologie), Pharmakologie u n d I m m u n biologie. E s werden n u r Arbeiten angenommen, die nicht an anderer Stelle m i t demselben Inhalt veröffentlicht oder zur Veröffentlichung angeboten werden. Der Autor verpflichtet sich nach Annahme, die Arbeiten a n keiner anderen Stelle zu veröffentlichen. 2. Die Arbeit m u ß wissenschaftlich wertvoll sein. Bestätigungen bekannter Tatsachen, Versuche u n d Beobachtungen ohne positives Ergebnis werden, wenn überhaupt, n u r in kürzester F o r m aufgenommen. Nicht aufgenommen werden Polemiken und spekulative Arbeiten, falls sie nicht wesentlich neue Gesichtspunkte enthalten. Die Arbeiten sollen den Charakter wissenschaftlicher Originalarbeiten haben. Als solche gelten alle Mitteilungen, die zur vorwärtsführenden Erweiterung des Erkenntnisstandes auf den genannten Fachgebieten führen. Originalarbeiten sollen 20 Manuskriptseiten nicht überschreiten. Kurzmitteilungen werden bei der Drucklegung zeitlich bevorzugt; sie dürfen 5 Manuskriptseiten nicht überschreiten. Als Kurzmitteilung gelten solche Arbeiten, in denen über neue Ergebnisse berichtet wird, ohne Details einer Originalarbeit zu enthalten. Besonders aktuelle Untersuchungsergebnisse können in kurzer F o r m (bis 4 Seiten) im Offsetverfahren publiziert werden, wofür reproduktionsreife Manuskripte erforderlich sind. In F o r m von Übersichtsarbeiten (Reviews) werden Artikel entgegengenommen, die zu aktuellen Gebieten einen Überblick geben, in d e m F a k t e n dargestellt, besprochen u n d kritisch bewertet werden. 3. Die Arbeiten müssen so kurz als möglich abgefaßt werden und in einem druckreifen Zustand geschrieben sein. Einleitung (Problematik), Methodik, Befunde und Diskussion sollen deutlich in Erscheinung treten. Der Arbeit ist eine Zusammenfassung der wesentlichsten Ergebnisse voranzustellen, wobei bei deutschsprachigen Manuskripten auch eine englische Übersetzung notwendig ist. Arbeiten werden in Deutsch, Englisch und Russisch angenommen. Die Manuskripte sind in zweifacher Ausfertigung einzureichen. Bei Manuskripten in deutscher Sprache ist die Schreibweise des „ D u d e n " verbindlich; bei eingedeutschten Wörtern ist die ,,K-Z"Schreibweise anzuwenden. Von den Abbildungen sind 2 Kopien sowie 1 Satz reproduktionsreife Vorlagen beizufügen. Genaue Hinweise zur Manuskriptgestaltung sind von der Redaktion der Zeitschrift a n zufordern u n d unbedingt einzuhalten. Manuskripte, die diesen Bedingungen nicht entsprechen, gehen unbearbeitet zur Revision an den Autor zurück. 4. Die Arbeiten werden im Sofortumbruch gesetzt; Korrekturen in F o r m von Streichungen bzw. Zusätzen sind daher in der Umbruchkorrektur nicht zulässig. 5. Manuskripte sind an die Redaktion der ACTA BIOLOGICA E T MEDICA G E R M A N I C A , DDR-1115 Berlin-Buch, Lindenberger Weg 70, zu senden. 6. Von jeder Originalarbeit werden kostenlos 80 Sonderdrucke geliefert. Chefredaktion/Herausgeber

Zeitschrift ACTA BIOLOGICA E T MEDICA GERMANICA Herausgeber: Prof. Dr. R. Baumann, Prof. Dr. H. Dutz, Prof. Dr. A. Graffi, Prof. Dr. F. Jung, Prof. Dr. O. Prokop, Prof. Dr, S. M. Rapoport. Verlag: Akademie-Verlag, DDR-108 Berlin, Leipziger Straße 3—4; Femruf 223 6221 oder 223 6229; Telex-Nr. 1144 20; Bank: Staatsbank der DDR, Berlin, Kto.-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. Heinz Bielka, Prof. Dr. Werner Scheler. Anschrift der Redaktion: DDR-1115 Berlin-Buch, Lindenberger Weg 70. Fernruf: 569 78 51, App. 222. Veröffentlicht unter der Lizenznummer 1318 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckerei „Thomas Müntzer", DDR-582 Bad Langensalza. Erscheinungsweise: Die Zeitschrift erscheint monatlich. Die 12 Hefte eines Jahrganges bilden einen Band. Bezugspreis je Heft 3 5 , - M (Preis für die DDR: 24,— M); Bandpreis 4 2 0 , - M) zuzüglich Versandspesen (Preis für die DDR 288,— M). Bestellnummer dieses Heftes: 1053/XXXVIII/2/3. Urheberrecht: Alle Rechte vorbehalten, insbesondere die der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgend ein anderes Verfahren — ohne schriftliche Genehmigimg des Verlages reproduziert werden. © 1979 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 50117

ACTA BIOLOGICA " [T mi riunii • I L I

I V I • I V I L

1 1 1 1 1 I I U I U ^ l

^

funktionelle Biowissenschaften

• • r n H I M HI 1 | f •

UEnMANIbA

=

:

R. Baumann • H. Dutz A. Graffi . F. Jung O. Prokop • S. M. Rapoport Unter Mitarbeit von: H. Ambrosius • H. Ankermann G. Dörner • H. Drischel H. A. Freye • H. Frunder E. Goetze • H. Hanson E. Hofmann • F. Klingberg W . Köhler • F. Markwardt H. Matthies • W. Oelßner G. Pasternak • A. Schellenberger E. Schubert • G. Sterba. A. Wollenberger

Band 57 1978

AKADEMIE-VERLAG BERLIN

ACTA BIOLOGICA ET MEDICA GERMANICA Zeitschrift für funktionelle

ISSN 0001-5318 Herausgeber: R. Baumann, H. Dutz, A. Graffi, F . Jung, O. Prokop, S. M. Rapoport Chefredaktion: H. Bielka, W. Scheler

Biowissenschaften

Band 38

Proceedings of the Scientific Conference Cytochrome P-450 Structural and Functional Aspects July 9—13, 1978

Eberswalde, GDR

Edited by M . J . C o o n , I . C. G u n s a l u s , S. M a r i ^ i ó , a n d K .

10 Acta biol. med. germ., Bd. 38, Heft 2—3

Ruckpaul

1979

Heft 2—3

Zeitschrift ACTA BIOLOGICA ET MEDICA GERMANICA Herausgeber: Prof. Dr. R. Baumann, Prof. Dr. H. Dutz, Prof. Dr. A. Graffi, Prof. Dr. F. Jung, Prof. Dr. O. Prokop, Prof. Dr. S. M. Rapoport. Verlag: Akademie-Verlag, DDR-I08 Berlin, Leipziger Straße 3—4; Fernruf 2236221 oder 2236229; Telex-Nr. 114420; Postscheckkonto: Berlin 5021. Bank: Staatsbank der DDR, Berlin, Kto.-Nr. 6836-26-20712. Chefredaktion: Prof. Dr. Heinz Bielka, Prof. Dr. Werner Scheler. Anschrift der Redaktion: DDR-1115 Berlin-Buch, Lindenberger Weg 70. Fernruf: 56978 51, App. 222. Veröffentlicht unter der Lizenznummer 1318 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckerei „Thomas Müntzer", DDR-582 Bad Langensalza. Erscheinungsweise: Die Zeitschrift erscheint monatlich. Die 12 Hefte eines Jahrganges bilden einen Band. Bezugspreis je Heft 35,— (Preis für die DDR; 24,— M): Bandpreis 420,— M zuzüglich Versandspesen (Preis für die DDR 288,— M). Bestellnummer dieses Bandes: 1053/XXXVII. Urheberrecht: Alle Rechte vorbehalten, insbesondere die der Obersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. © 1978 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 50 117

I N H A L T S V E R Z E I C H N I S

Heft 1 Biochemie/Molekularbiologie Zur Charakterisierung der Ca-Aufnahme durch eine Mikrosomenfraktion aus glatter Gefäßmuskulatur 1 — 12 B . B I E N W A L D U. P. H E I T M A N N : Wechselwirkung von Uranylionen mit anorganischer Pyrophosphatase aus Bäckerhefe 13 — 17 N . H Ö S E R , H . D A W C Z Y N S K I , K. W I N N E F E L D U. R . D A R G E L : Kontrolle des mitochondrialen Mg + -Efflux 19—29 ++ H . S C H A L L E R , G. L E T K O u. W. K U N Z : Einfluß von Mg -Ionen auf die Eigenschaften von Rattenherzmitochondrien in Abhängigkeit von der Präparation 31-38 D. L U P P A , S . H Ö N I C K E , D. R E I S S I G U. F . M Ü L L E R : Der Einfluß des Alloxandiabetes auf die (Na+ + K + )-aktivierbare ATPase in der Bürstensaummembran der Rattendünndarmmukosa 39—47 H . R E M K E , W. S C H E L L E N B E R G E R , T. M O T H E S U. F. M Ü L L E R : Zum Mechanismus der Na + -abhängigen Monosaccharidresorption: Kompartimentierung des resorbierten N a + u n t e r in vitro-Bedingungen 49—57 T H . G E I E R , M. G L E N D E U. J . G . R E I C H : Bedeutung der Bindung von 2,3-Diphosphoglyzerat und ATP an Hämoglobin für die Glykolyse in Erythrozyten: Aktivierung der Hexokinase durch 2,3-Diphosphoglyzerat unter intrazellulären Bedingungen 59—72 H . S E I M u. R . D A R G E L : Induktion mitochondrialer Volumenänderungen durch homologe O-Acylcarnitin-Isomere 73 — 82 R . G R O S S E , K. E C K E R T , J . M A L U R u. K. R . H . R E P K E : Analyse funktionsbezogener Wechselwirkungen von ATP, Na + und K + mit der (Na + K + )-ATPase. Inaktivierungsstudien mit einem Thiolreagenz 83 — 96 R. B O H N E N S A C K U. W . K U N Z : Ein mathematisches Modell für die Regulation der oxydativen Phosphorylierung bei intakten Mitochondrien 97 — 112 K . G R A D E , R. E C K E L U. R. L I N D I G K E I T : Transkriptionskapazität von Rattenleberkernen und von isoliertem Chromatin bei verschiedenen Ammoniumsulfat-Konzentrationen 113 — 120 U . K L I N N E , D . E H L E R S U. G . B A R T S C H :

Zellbiologie Trennung von Knochenmarkzellen normaler und anämischer Kaninchen im isopyknischen Dextran-Dichtegradienten

J . G R O S S , R . S T A A K U. S . R O S E N T H A L :

121 — 125

Physiologie/Pathophysiologie M. K S E L I K O V Ä , M. F R E I O V Ä u. I . P O T M E S I L O V Ä : Der Einbau von [14C]-L-Leuzin in Milz- und Lebergewebe von Ratten, die durch Äthylpalmitat „chemisch splenektomiert" wurden 127 — 130 G R I M M E R , R . M O L L E R , D. G M Y R E K U. J . G R O S S : Bilirubin-UDP-Glukuronyltransferase-Aktivität im Leberhomogenat menschlicher Feten 131 — 135

V . ÖEBESTFK,

I.

Inhaltsverzeichnis

IV

C. P F E I F F E R , J . G Ü N T H E R , W. I. K A P E L ' K O U. F. Z. M E E R S O N : Über Veränderungen im Verhältnis von Kontraktionshöhe und -folge beim Rattenmyokard durch Kalzium und physisches Training

137-145

Pharmakologie J . Z A U M S E I L U. W . K L I N G E R : Einfluß von Phenobarbital und Chlorpromazin auf Gallenfluß und Gallensäurenausscheidung bei männlichen Wistarratten

A.BARTH,

147-154

Immunologie G . K Ü C K E N , D . M Ü C K E , J . B R O C K U. W . S T R A U B E

Serum mittels Immunosorbentien

: Absorption von Anti-Plazenta-

Kurzmitteilungen Zellbiologie. G . CSABA, V . LÄSZLÖ U. S . D A R V A S : Die Wirkung der Antagonisten des H x - und H 2 - Rezeptors auf Tetrahymena Zellbiologie. F . S C H O B E R : Darstellung der neurosekretorischen hypothalamorhombenzephalen Verbindung bei der Ratte durch retrograden axonalen Transport von Meerettich-Peroxidase Physiologie. D . U N G E R U. H . W A R Z E L : Stimulator mit drahtloser Energieübertragung Biochemie. H . - J . B Ö H M E , R . F R E Y E R , P . R E T T E R R A T H , W . S C H E L L E N B E R G E R U. E. H O F M A N N : Kinetische Untersuchung der Wechselwirkung von CibacronBlau F3G-A mit Hefe-Phosphofruktokinase Buchbesprechung Molekularbiologie. P . P L I E T Z , H. D A M A S C H U N , G . D A M A S C H U N u. K . D . S C H W E N KE : Bestimmung der Quartärstruktur der 11 S Pflanzenglobuline mit Hilfe der Röntgen-Kleinwinkelstreuung Biochemie. W . - H . S C H U N C K , P . R I E G E , R . B L A S I G , H . H O N E C K u. H . - G . M Ü L L E R : Zytochrom P-450 und Alkanhydroxylaseaktivität in Candida guilliermondii

155 — 160

161-163 165-167 169-172 173-177 178 K 1 - K 2

K 3 - K 7

Heft 2 Biochemie H. G Ö B E L , H. W A N D , A. G A B E R T U. W . B O C K : Synthese und Eigenschaften trägerfixierter Enzyme. X. Kovalente Bindung von Polygalakturonase an wasserunlösliche Träger O. A S P E R G E R , H.-P. K L E B E R u. H. A U R I C H : Zytochrom-Zusammensetzung von Acinetobacter calcoaceticus J . F U S K A , A. F U S K O V Ä U. A. J U R Ä S E K : Die in vitro-Wirkung von , due to permanent charges, is perturbed with respect to the bulk concentration: [Ha =

[H+t] e ^ k T

(1)

thus P&H, in =

out +

0.43ey'IKT

(2)

where "in" and "out" refer respectively to the local and bulk concentrations, and e is the unit charge. When y> < 0, the local concentration of protons "seen" by any ionizable group in the field is much higher than the bulk concentration. Coun-

146

R . L A N G E , G . H U I B O N H O A , V.

D E B E Y , I . C . GUNSALTJS

Fig. 2. Combined effects of KC1 and pa.H on the spin state of F e 3 + • R H at —40 °C. The mixed solvent contains 3 mM camphor, 25 mM Na buffer (acetate, cacodylate, or phosphate), and varying concentrations of KC1. XHS — mol fraction of high spin species, a) paH profiles at different K + concentrations; b) ionic strength profiles a different pan's. XHS = 0.5 is indicated by a dotted line

tenons attracted by the charges form an ion "cloud" which decreases the value of \p. In a first approximation [13]: y = A — B log I

(3)

If one assumes that the low spin fraction is proportional to the protonated fraction A~ + H + ) of an ionizable group influenced by an electrostatic potential, log HS/LS = log [A-]/[AH] = paH, to- pK0 = paH, out + OA5ey>lkT~pK0

(4)

and the apparent pK (pKJ of the transition is related to its intrinsic pK(pKe) by the relation: pKa = pKe—OA^eyi/kT = pK— 0.43e(A — Blog I)jkT

(5)

The direction of variation in pK with log I in the present case shows clearly that the electrostatic field is created by negative charges, possibly carboxyl groups. The very low isoionic point of cytochrome P-450 (pi = 4.67) also indicates that the protein is negatively charged in the paH range of stability. The unique specific effects of K + could result from a higher affinity for the negatively charged residues, i. e. the K + ions come into direct contact with the polyanionic groups rather than forming an ion "cloud" in their vicinity; log I in equation 5 would thus be replaced by log K + [14].

Thermodynamic-kinetic analysis of the heme pocket of P-450

147

Fig. 3. Relation of pKa with log I (or log K + ). a) Data of Fig. 2 expressed as pKa versus ( ) log I, and ( ) log (K + ). Temperature - 4 0 °C. b) Identical experiments at — 20°C .Solvent as in Fig. 2. Ionic strength made up with NaCl (o)or spermidine (•)

paH> ¡J, calibration

Since at high ionic strength paHin, 4= paH< out, the measurements of i f , indicate the internal paH. The calculation of paHin for three experimental conditions is given in Fig. 4. The figure reveals also that the physico-chemical environment can impart within the heme pocket extreme proton concentrations without deleterious effects on the protein, whereas in the bulk medium such acidity would render the protein totally inactive. Similarly, slight modulations in the external environment provide a sensitive and versatile fine regulation of the internal conditions. For example a shift of K + from 0 to 5 mM, paH 7.33 yields to apa H< ^ increase of 0.85. Furthermore identical Ke — and thus paHtiu — can be attained by adjusting the cation concentrations in general, or K + or H + . Factors other than proton and cation concentrations could be included in the same graph; for example, an increase in camphor concentration in the millimolar range, and well above the dissociation constant for the first catalytic site, decreases the high spin fraction, whereas ethylene glycol has the opposite effect [10]. The equilibrium constant can be adjusted again by changing other variables. Functional consequences

Variations of the spin state are related to paH> to and are of functional consequence to other properties such as redox potential [15] and the spontaneous autoxidation

148

R . LANGE, G . H U I BON HOA, P . D E B E Y , I . C. GUNSALUS

rate of the ternary oxygenated compound [1]. The suggested mechanism of formation of an "active oxygen", that is a proton assisted catalytic cleavage of Fe 2+ bound dioxygen [3, 4], is also visualized as dependent on the local (and perhaps temporary) concentration of protons. Also relevant are the observed effects of the putidaredoxin binding on the substrate adduct of the ferric cytochrome, namely a decrease in Ke, with concomitant increase in AH (Fig. 5). Both effects are qualitatively similar to those caused by a decrease in paHt ^ or cation concentration, that is an increase in the internal proton concentration. This in turn would lead to a higher probability of a proton mediated splitting of dioxygen. Evidence is becoming available that the environment — including inducers, substrates, and effectors — modulate the spin equilibrium of the ferric state in other cytochrome P-450 systems, from microbial or mammalian sources (mitochondria or microsomes), whethersolubilizedor native in the intact membrane [16—20]. A recent paper by KUMARI et al. [21] emphasizes the influence of external and internal factors on the in vivo spin state, and their importance to the level and specificity of the hydroxylating activity.

Fig. 4. Calibration of Kt = HS/LS in terms of p&H, in. Experimental data of Fig. 2. The straight line is obtained from the experimental values at high ionic strength (200 mM KC1). The pa.H, in for three other experimental points is calculated using this calibration (see equations in the text)

T h e r m o d y n a m i c - k i n e t i c a n a l y s i s of t h e h e m e p o c k e t of P - 4 5 0

149

F i g . 5. T e m p e r a t u r e d e p e n d e n c e of t h e Ke = H S / L S of t h e t e r n a r y P d — F e 3 + • R H Complex. M i x e d s o l v e n t = 1 / 1 (v/v) 100 m M K + p h o s p h a t e , p H 7, a n d e t h y l e n e glycol, c o n t a i n i n g 100 m M KC1 a n d 3 m M c a m p h o r . D o u b l e c o m p a r t m e n t cells a r e t h e r m o s t a t e d a t t h e s a m e t e m p e r a t u r e (above o r b e l o w 0 °C). P u t i d a r e d o x i n (Pd) a n d F e 3 + • R H a r e i n t w o d i f f e r e n t c o m p a r t m e n t s of t h e reference cell, a n d a r e i n t h e s a m e c o m p a r t m e n t of t h e s a m p l e cell. F e 3 + • R H = 9.7 (xM, P d = 45 (JIM; 5 m m light p a t h ; t e m p e r a t u r e as i n d i c a t e d . B a s e line b e f o r e a d d i t i o n of P d

Kinetics of spin state modulation Spin state response rates were examined by stopped flow at subzero temperatures by following the approach to a new equilibrium after rapid variations in the concentrations of external modulators such as K + or H + . £

The forward and reverse reaction rates (LSi==^HS) were calculated from the measured values of kohs — (kx + and Ke = kjk-i. Table 1 summarizes the first order rate constants and obtained at —17 °C by various K+ and H + jumps. Both processes are found to be perfectly first order monophasic, and much

150

R . LANGE, G. H u i BON HOA, P . D E B E Y , I. C. GUNSALUS

slower than is characteristic for purely electronic transitions. Furthermore kobs is very similar for paH jumps from 7.5 to 5 and 7.5 to 6, or, on the other hand, for K + jumps from 0 to 5 mM and 0 to 10 mM. These results strongly suggest that the rate limiting steps are not the binding of K + or H + ( O H - ) ions to specific site(s) on the protein, but conformational changes followed by much faster electronic transitions. These would presumably occur as responses to modulation of the electrostatic potential by external factors. The kinetic rate constants depend, however, on the physico-chemical parameters of the protein environment, that is the H + concentration for K + jumps, and the K + concentration for H+ jumps. Compare for example the rate constants on stopped flow jump from paH 5 to 7.5 with KCl at 0 and 10 mM (Table 1). On the other hand, kohs is identical for identical final states, as for example a 5 to 7.5 paH Table 1 Reaction rates for spin state changes at — 17 °C in hydro-organic medium Experimental conditions P&H

0 0 0 0 0

7-5

5

pa.H jumps 5 6 S

[ K + ] jumps

7-5 7.5 7-5

S mM 10 mM 5 mM 10 mM 25 mM

Constants

Ke

ÄOBS (S"

5-9 10.3 0.2 0.4 0.75

12 12.7 2.1 3-4 3-3

3.15 3.15 9-5

2.5 2.8

h (s-1) 10.3 11.6

0.34 0.95 1.4

(s-1) 1.7 1.1 1.8

2.4 1.9

[K + ] 0 mM 10 mM

10

1.9 2.1 9

0.6

0.7 1

jump at 10 mM K + or a 0 to 10 mM K + jump at paH — 7.5- These would seem to reflect therefore the same conformational change, and the forward and reverse rate constants can be considered to relate more to the local environment of the heme than to the external physico-chemical conditions. Finally these studies provide a first approximation to the velocity of conformational changes in the protein which accompany the spin transition of the ferric iron, i. e. 30 to 80 s _ 1 at 4 °C in aqueous or hydro-organic solvent. Furthermore they suggest conformational relaxations as rate limiting processes for individual steps of the catalytic redox cycle. Conclusions The present results suggest several conclusions. (1) "The ligand field inside the cytochrome P-450 heme pocket is close to the crossing over of the spin pairing" [20], so that substrate binding is sufficient to reverse the spin state and confer to the heme a very high sensitivity to variations in environmental parameters. (2) The existence of negative charges in the vicinity of the heme could provide the

Thermodynamic-kinetic analysis of the heme pocket of P-450

151

proton concentration necessary to "activate" the oxygen. (3) The sensitivity of the heme iron spin state on external physico-chemical parameters can be explained by changes of the heme microenvironment via modulation of the electrostatic potential. (4) The variations of spin state involve conformational changes of the protein, possibly behaving as rate limiting step of individual reactions. Acknowledgements We want to express our gratitude to Dr. P. Douzou for constant interest and stimulating discussions, and to Dr. C. B A L N Y for helpful advices. This work was founded by grants from the Délégation Générale à la Recherche Scientifique et Technique (Contrat N° 75-7-0745), the Institut National de la Santé et de la Recherche Médicale (Unité 128), The D.R.M.E. (Contrat N° 77/1117) and the Fondation pour la Recherche Médicale Française. References [ 1 ] G U N S A L U S , I . C . , J . R . M E E K S , J . D . L I P S C O M B , V. D E B R U N N E R , and E. M U N C K in: cular mechanisms of oxygen activation. O . H A Y A I S H I (Ed.). Academic Press, New

London 1974, p. 559

MoleYork,

[ 2 ] SLIGAR, S . G.: Biochemistry 1 5 , 5 3 9 9 ( 1 9 7 6 ) [ 3 ] U L L R I C H , V . , H. H. R U F , and P . W E N D E : Croat, chem. Acta 49, 2 1 3 ( 1 9 7 7 ) [4] G R O V E S , J. T., G . A. M C C L U S K Y , E. W H I T E , and M . J. C O O N : Biochem. biophys. [5] [6] [7]

[8] [9] [10] [11] [12] [13] [14]

[15]

Res. Commun. 81, 154 (1978) G U N S A L U S , I . C . , and G . C . W A G N E R : Meth. Enzym. 51, 1 6 6 ( 1 9 7 7 ) Hui B O N H O A , G „ and P . Douzou: J . biol. Chem.'248, 4 6 4 9 ( 1 9 7 3 ) M A U R E L , P . , F . T R A V E R S , and P . Douzou: Anal. Biochem. 57, 555 ( 1 9 7 4 ) Hui BON HOA, G., and P. Douzou: Anal. Biochem. 51, 127 (1973) L A N G E , R . , G . H U I B O N H O A , P . D E B E Y , and I . C . G U N S A L U S : Eur. J . Biochem. 77, 479 (1977) L A N G E , R . , C . B O N F I L S , and P . D E B E Y : Eur. J . Biochem. 79, 623 (1977) G O L D S T E I N , L . , Y . L E V I N , and E . K A L T C H A L S K Y : Biochemistry 3, 1 9 1 3 ( 1 9 6 4 ) Douzou, P., and P. M A U R E L : Proc. natn. Acad. Sci. U.S.A. 74, 1 0 1 3 ( 1 9 7 7 ) T R A U B L E , H . , M . T E U B N E R , P . W O O L L E Y , and H . E I B L : Biophys. Chem. 4, 3 1 9 ( 1 9 7 6 ) L E R O Y , J. L . , and M . G E R O N : Bioplymers (in press) SLIGAR, S . G.: Fedn Proc. Fedn Am. Socs exp. Biol. 36, 663 (1977)

[ 1 6 ] T A K E M O R I , S . , K . S U H A R A , S . H A S H I M O T O , M . H A S H I M O T O , H . SATO, T . G O M I , a n d

M.

Biochem. biophys. Res. Commun. 63, 5 8 8 ( 1 9 7 5 ) T A K E M O R I , S . , H. SATO, T . G O M I , K. S U H A R A , and M. K A T A G I R I : Biochem. biophys. Res. Commun. 67, 1151 (1975) H A U G E N , D . A . , and M . J . C O O N : J . biol. Chem. 251, 7 9 2 9 ( 1 9 7 6 ) A P P L E B Y , C. A . , and R. M . D A N I E L in: Oxidases and redox systems. T. E. K I N G , H . S. M A S O N , and M . M O R R I S O N (Eds). University Park Press, Baltimore 1973, P- 515 R E I N , H . O . R I S T A U , J . F R I E D R I C H , G . - R . J Â N I G , and K . R U C K P A U L : Croat, chem. Acta 49. 251 (1977) KATAGIRI:

[17] [18] [19] [20]

[21]

KUMARI, K . ,

M.

SATO,

H.

KON,

and D. W.

NEBERT:

J. biol. Chem. 253, 1048 (1978)

Discussion U L L R I C H : Can you distinguish between the endogenous low spin form and a form which has bound a camphor molecule by the carbonyl group to the ferric heme ? At high concentrations this may occur, since from model systems it is evident that a coordination of a carbonyl group may occur resulting in low spin spectra similar to those of low spin cytochrome P-450. D E B E Y : This cannot be excluded.

152

R . LANGE, G . H U I B O N H O A , P . D E B E Y , I . C . GUNSALUS

MARiCid: Proton magnetic relaxation measurements of solubilized microsomal P-450 in 20% ethylene glycol reveal a substantial immobilization of solvent around the solute.* This is expected to be even more pronounced for a 50% ethylene glycol solution. Could it have any relevance for the interpretation of your experiments ? D E B E Y : Your measurements have shown t h a t the P - 4 5 0 heme is quite accessible to glycerol; this is probably also true for ethylene glycol. We find also a solvent effect on the spin state which could be relevant to your result. But I do not think it changes basically the phenomena we observe since we find qualitatively identical results in purely aqueous supercooled water droplets. E S T A B R O O K : Have you attempted any other physical method, such as equilibrium dialysis, to confirm your conclusions about spin state changes ? In other words do you consider conditions where substrate is bound to the low spin form or a high spin state without substrate bound etc. etc. I t seems to me that we are going to confuse the literature as well as ourselves with the complexities of low spin form camphor bound, the low spin form without camphor, the high spin form without camphor bound, one molecule of camphor bound to the high spin form, two molecules of camphor bound to the high spin form etc. etc. I believe some other physical methodology should be used to resolve these proposed multiple states. D E B E Y : We did not do other measurements than kinetics of camphor binding. At low temperature one can measure directly the binding and dissociation rate constants for camphor under various conditions. Thus one can deduce the dissociation constant; it varies slightly with the experimental conditions, including temperature,but remains in the 10—20 (JIM range. Since the spin equilibrium studies are performed with 700 (iM or 3 MM camphor, we believe t h a t we are always dealing with the camphor bound enzyme. Furthermore it has been shown in aqueous medium t h a t both camphor-free and -bound can be found in a t least two spin states. + S C H L E Y E R : At what K do you begin to see deviations from linearity (kinetics, e.g. kobs) ?

Above 200 mM KC1; then the electrostatic field is practically cancelled. RUF: TO the kinetic trace of the p H -jump experiment: 1) Why does the trace rise at the beginning ? 2) Can the apparent fast phase be greatly underestimated by this dead time ? 3) How large was the absorbance difference from steady state measurements ? D E B E Y : 1) There is a fairly large dead time of the mixing, at low temperature, of two solutions of very different buffer concentration (different diffraction coefficients). 2) There is no apparent fast phase. The semi-log exploitation of the kinetics appears monophasic and its extrapolation to time t — 0 correlates well with the initial absorbance. We do not know, however, if other phenomena occur during the dead time. 3) The absorbance difference observed after paH or K + jumps is of the order of 0.1 to 0.2. DEBEY:

* To be published in the proceedings of this conference.

Acta biol. med. germ.. Band 38, Seite 153—162 (1979) The University of Texas, Department of Biochemistry Dallas, T X 75235, USA

Cytochrome P-450Cam and putidaredoxin interaction during electron transfer J . A . PETERSON a n d D . M . MOCK

Summary Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s _ 1 at 25 °C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 6 4 5 nm absorbance band of high-spin Fe(III) cytochrome P - 4 5 0 O a m ; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin and Fe(IIl) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the E P R signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P - 4 5 0 . The mechanism of the loss of the EPR signal of cytochrome P - 4 5 0 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation. Introduction

Cytochrome P-450 (P-450) is an iron-protoporphyrin IX containing monooxygenase which functions in a cyclic maimer to catalyze the oxidation of a wide variety of hydrophobic compounds [1,2]. During the catalytic cycle of this class of heme containing proteins, two electrons are required for the activation of dioxygen (Oa) with the insertion of one of these oxygen atoms into the substrate while the other atom is reduced to water [3, 4]. Anaerobic titration experiments with sodium dithionite have shown that this class of hemeproteins accepts these two electrons in two one-electron steps [5—7]. In the case of bacterial cytochrome P ^ O , ^ (P^SOcam), the proximal electron donor is putidaredoxin [8] a 2-iron, 2-sulfur, iron sulfur protein which is a one-electron carrier [9]. An understanding of the catalytic mechanism and control of this important enzyme system is dependent upon a knowledge of the relative rates of the various steps of this cyclic reaction. The first step of this ordered reaction cycle is substrate binding which has a second Presented at the Scientific Conference 'Cytochrome P-450: Structural and Functional Aspects' (held in Eberswalde-GDR July 9—13, 1978) by R. W. ESTABROOK. Correspondence concerning this paper should be addressed to J . A. PETERSON, Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, T X 75235, USA

154

J . A . P E T E R S O N , D . M . MOCK

order reaction rate constant of 15.8 • 10* M _1 s- 1 [10] at 25 °C and pH 7.0 in the presence of 0.1 M potassium chloride. Oxygen binding has also been established to be rapid with a second order reaction rate constant of 7.7 • 10® M -1 s - 1 at 4 °C [11]. Since these reactions, under most conditions, are very rapid with respect to catalytic turnover, they will not usually be important in metabolic control of this cycle. Thus, control must be exerted at the introduction of one of the two electrons required for this reaction [12]. In preliminary experiments to determine the kinetics of reduction of the camphor complex of Fe(III) P-450cam by reduced putidaredoxin, the reaction seemed to be zero order with respect to reduced putidaredoxin concentration [12]. This data was interpreted as indicating that a complex was formed between putidaredoxin and the cytochrome prior to intracomplex electron transfer which was proposed to be the rate limiting step in this reduction process. This communication clearly establishes that Fe(III)-cytochrome P-450cam and reduced putidaredoxin form a one-to-one complex which then decays in a first-order manner to give Fe(II)cytochrome P-450cam and oxidized putidaredoxin. Materials and methods ¿-Camphor and all other s t a n d a r d chemicals used were of reagent grade and were obtained from either Sigma or B a k e r Chemical Companies. T h e gases and cryogenic supplies were of t h e prepurified grade and were obtained f r o m Linde Co. The buffer used for all experiments consisted of morpholinopropane sulfonic acid (MOPS) titrated t o pH. 7.4 with Tris base. The potassium chloride concentration in t h e buffer was maintained uniformly a t 0.1 M because of t h e effect of salt on b o t h camphor binding b y P - 4 5 0 c a m [5, 1 3 ] a n d t h e i n t e r a c t i o n of P - 4 5 0 c a m a n d p u t i d a r e d o x i n [ 1 4 ] .

P-450cam was purified f r o m Pseudomonas p u t i d a (ATCC 17453) which h a d been grown on dcamphor as t h e carbon source. The purification procedure used was described b y O ' K E E F F E et al. [15] and results in preparations which have a p u r i t y index of a t least 1.3 (ratio of absorbance a t 392 t o 280 nm) indicating t h a t t h e preparation was a t least 90% pure [15, 16]. All P-450cam samples were a t least 95% in t h e high spin form in t h e presence of 1 mM ¿-camphor and 0.1 M KC1 as judged b y optical absorbance spectra a t room temperature (24 °C) [15] a n d had no significant amounts of P-420. The concentration of P-450cam was determined f r o m t h e absorbance of either t h e Fe(III)-camphor bound form a t 392 n m (e — 104 m M - 1 c m - 1 ) or t h e carbonyl complex of t h e Fe(II) form a t 446 n m (e = 120 m M - 1 c m - 1 ) [5, 15]. T h e concentration determined b y either of these two methods agreed within 2 % in all cases. Putidaredoxin was purified as a by-product of P-450cam preparation b y washing t h e first DEAE-cellulose column with 20 mM MOPS, pH. 7 A, 0-5 M KC1, a f t e r t h e P-450cam h a d been eluted. T h e eluted putidaredoxin was concentrated by ultrafiltration and dialyzed. The remainder of t h e purification was similar t o t h a t described previously [17]. The material used for these studies was a t least 90% pure as judged b y t h e ratio of absorbance a t 455 t o 280 n m which was 0.45 [17]. T h e NADH-putidaredoxin reductase was recovered during t h e purification of P-450Cam as a crude fraction during t h e elution of t h e Sephadex A-50 column and was n o t f u r t h e r purified. This reductase preparation did not contain measurable quantities of either P-450cam or putidaredoxin. The r a t e of reduction of P-450cam b y putidaredoxin was measured with a computerized stopped-flow spectrophotometer which has been previously described [18, 19]. T h e mixing dead-time of t h e instrument has been established to be 3.5 ms under t h e conditions of these experiments. T h e space around t h e reaction chamber of t h e stopped-flow a p p a r a t u s was continually flushed during these experiments w i t h d r y prepurified nitrogen t o aid in maintaining anaerobiosis. T h e temperature of t h e reaction mixture was measured with a HewlettPackard electronic t h e r m o m e t e r with a-thermocouple bonded t o t h e mixing chamber of t h e apparatus. T h e t e m p e r a t u r e of all kinetic experiments was maintained a t 25 ± 0 . 5 °C.

Cytochrome P-450-putidaredoxin interaction

155

The freeze-quenching experiments were performed with an apparatus which was similar to one previously described [20]. The reaction solutions were mixed with a four jet tangential mixer and then sprayed at high velocity into a solution of isopentane which had been cooled to —140 °C. The mixing and freezing dead-time of this device, which is the time required to cool the reaction solution to the temperature at which no further reaction takes place, had been established to be less than 5 ms [20] and our control experiments confirmed these results. The length of time between mixing and freezing was varied by changing the length of tubing between the mixing chamber and the spray nozzle. The reaction solutions were maintained in gas-tight syringes in a temperature regulated water bath which was continuously bubbled with prepurified nitrogen to assist in maintaining anaerobiosis. As an additional precaution to exclude oxygen from the reaction mixture, the space between the spray nozzle and the isopentane was continuously flushed with high purity helium. Anaerobiosis is necessary in these experiments to prevent oxidation of reduced putidaredoxin by molecular oxygen [14] as well as to prevent the reoxidation of the reduced P-450Cam which is a product of the reaction. Precautions which were taken to insure anaerobiosis included the use of prepurified gases which were additionally bubbled through scrubbing towers which contained F I E S E R ' S solution [21]. An oxygen scavenger system (glucose, 1 2 0 mM; glucose oxidase, 0.1 mg per ml; and catalase, 3000 units per ml) was also included in all anaerobic solutions [22]. The EPR spectra were recorded with a Varian model E-4 E P R spectrometer (Varian Instrument Co.) connected to a PDP-11 minicomputer (Digital Equipment Corp.). Spectral manipulations including scaling, baseline correction, and integration were performed by the software of the minicomputer. The amount of P-450cam or reduced putidaredoxin present in the E P R samples was quantitated by measuring the peak-to-peak height of separate spectral features of the species of interest rather than by double integration. Control experiments have established that under these conditions the line shapes of the E P R signals of P - 4 5 0 c a m and putidaredoxin do not change significantly [14]. Thus, the amount of each of these species in an experiment can be determined by the peak height (g — 1.94 for reduced putidaredoxin and either g = 8.0 or 2.24 for Fe(III) cytochrome P-450cam. respectively). Results and discussion The reduction of Fe(III)-cytochrome P ^ O ^ by reduced putidaredoxin was difficult to measure by conventional spectrophotometry because both of these proteins are colored and the absorbance spectra change as a function of oxidation state. However, the Fe(III)-camphor bound form of P-450cam has a unique absorbance maximum at 645 nm which is not present in any other form of the enzyme [5]. In addition, both the reduced and oxidized forms of putidaredoxin exhibit very weak absorbance in the 645 nm region [9]. The major drawback to determinations using this wavelength is that the change in molar absorptivity of P-450 c a m on reduction is quite small {Ae = 3.6 m M - 1 c m - 1 ) necessitating the use of high concentrations (50 fiM). Another problem associated with determining the kinetics of reduction of P-450eam is the source of reduced putidaredoxin. The NADH-specific putidaredoxin reductase isolated from this organism was chosen to reduce the putidaredoxin because the reaction was enzymatic and because the rate could be controlled so that re-reduction of putidaredoxin, after electron transfer to P^SOoam, was small. The time course of reduction of P-450 c a m as measured by the loss of absorbance at 645 nm by a two-fold molar excess of reduced putidaredoxin is shown in Fig. 1. Since the ratio of the concentration of the reactants was only two, classical chemical kinetics predicts that this bimolecular reaction should exhibit second order kinetics but instead the reduction of P ^ O ^ m was clearly first order with a rate constant of 33 s _ 1 - The simplest interpretation 11 Acta biol. med. germ., Bd. 38, Heft 2—3

156

J . A . PETERSON, D . M. MOCK

Fig. 1. Kinetics of reduction of P-450cam at 645 nm.The absorbance at 645 nm was followed with the stopped-flow spectrophotometer following the mixing of P-450cam and reduced putidaredoxin. The final concentration of P-450cam was 10 ¡xM and putidaredoxin was 20 (xM in carbon monoxide saturated buffer. The temperature was 25 °C. The curve shown is the average of 5 experiments

of this data was that there was the rapid formation of a complex between P-450cam and reduced putidaredoxin with subsequent rate limiting intracomplex electron transfer resulting in reduction of P-450 and loss of the 645 nm absorbance maximum associated with the high-spin Fe(III) enzyme as shown in equation 1: £ + ++ + P d f 4 ^ £ + + + - P d f — - £ + + - P d o x «-1

(1)

where E+++ and E++ represent the Fe(III) and Fe(II) forms of P^CUm, respectively. Pd r and Pd ox represent the reduced and oxidized forms of putidaredoxin. This kinetic relationship can be restated simply as shown in equation 2: (2)

A complete study of the kinetics of reduction of P - 4 5 0 c a m as measured by the loss of absorbance at 645 nm was difficult because of the low molar absorptivity which hinders an investigation over a wide range of putidaredoxin/P-450cam concentrations and ratios. The molar absorptivity of the 446 nm band of the carbonyl complex of Fe(II) P-450cam is very large thereby increasing the sensitivity of the assay procedure. The correction for the absorbance increase at this wavelength due to the oxidation of putidaredoxin is negligible. Previous experiments have established that the combination of carbon monoxide with P - 4 5 0 c a m in the presence of an atmosphere of carbon monoxide is fast when compared to the rate of reduction of P-450cam [23] as expressed in equation 3: • P d ^ + CO

• CO • Pd ox

(3)

i. e., k3 • [CO] > k2. Fig. 2 shows the change in absorbance at 446 nm which occurs during the reduction of P^SOcam by putidaredoxin in the presence of an atmosphere of carbon monoxide. In agreement with the results presented above, the reaction was seen to be clearly first-order even though the reactants were present in nearly the same concentration. Thus, the rate of reduction of P ^ C W could be measured by following the absorbance changes associated with either the loss of the high-spin Fe(III) form (645 nm) or the production of the carbonyl complex of the Fe(II) form (446 nm). In an attempt to analyze the kinetics of reduction of P-450cam by reduced putidaredoxin, the ratio of these proteins was varied over a wide range to determine the effect on the rate of reduction. Table 1 is a matrix of the apparent first-order

157

Cytochrome P-450-putidaredoxin interaction

rate constants obtained in a series of experiments similar to that described above and in Fig. 2. In any given experiment, the amount of P ^ C W which was reduced was dependent on the ratio of putidaredoxin to P^Ocam. If the ratio was greater than one all of the P-450 was reduced; however, if the ratio was less than one, the fraction of P-450 reduced was the same as the ratio. This is a thermodynamically reasonable result because the reduction potential of P-450 is — 170mV [5, 24] and its subsequent reaction with carbon monoxide will make its reduction even more favorable. The reduction potential of putidaredoxin is —240 mV [24] and because of the difference in reduction potential between these two proteins, should

Fig. 2. Kinetics of reduction of P-450cam at 446 nm. P-4S0Cam (S |*M) and putidaredoxin were mixed in the stopped-flow spectrophotometer at 25 °C in the presence of carbon monoxide saturated buffer. The formation of the carbonyl complex of Fe(II) P-450cam was followed at 446 nm. The figure contains the absorbance measurement ( ) as well as a first order plot ( ) in order to demonstrate that the reaction is first order Table 1 Matrix of the apparent first order decay constant, k2. Each matrix element represents the k2 value measured at the corresponding value of P-450cam concentration (rows) and putidaredoxin concentration (columns). All values are final concentrations in |xM for reactions at 25 °C P-450

il»

Pd 1

2

5

10

25

1

12.2 12.7

18.0 190

30.0

31.3 31.5

n.d.

2

23-1 23-4

18.0 20.7

27.8

34.3 33.9

35-4

5

27-1 28.9

28.6

25-9 23-3

31.3

31.9 32.6

10

36.6

36.5

33-3

30.3

31.8 36.6

25

n.d.

32.7

31.0

30.6

33.8

158

J . A . PETERSON, D . M . MOCK

result in the reduction of approximately 90% of the P-450 even in the absence of carbon monoxide. An examination of the first row of this table illustrates the effect of increasing the concentration of putidaredoxin while holding the concentration of P-450cam constant. The rate constant increases and reaches a maximum of approximately 31 s _ 1 . An examination of the first column reveals similar behavior as a function of the concentration of P-450oam. The rate increases and reaches a maximum which is similar to that measured for the first row with putidaredoxin in 25 fold molar excess. Further examination of this matrix of values reveals that it is symmetric about the diagonal for equal concentrations of P ^ O , ^ and reduced putidaredoxin. This is best illustrated for the third column with a constant concentration of putidaredoxin (5 ¡xM). Increasing the concentration of P-450cam first causes a slight decrease in the apparent first order rate constant and then an increase in this value. The point of minimum rate is the one-to-one complex. A subsequent publication will discuss in detail this complex kinetic pattern but the major point to be made by this table is that the apparent first order rate constant reaches a limiting value of approximately 33 s - 1 at high concentration of either one or both of the reactants. This type of kinetic behavior is characteristic of a system in which two components rapidly react to form a complex prior to a rate limiting step. The rapid reaction in this case would be the formation of a reduced putidaredoxin-Fe(III) P-450cam complex which subsequently undergoes intracomplex electron transfer. Since this was quite exciting a second analytical technique was utilized to confirm these results. Both camphor-bound Fe(III) P^Ocam [5, 25] and reduced putidaredoxin [9] are paramagnetic and, therefore, lend themselves to an analysis of the kinetics of reduction using E P R spectroscopy of freeze-quenched samples [20]. The E P R signal of reduced putidaredoxin is the most readily measured of this pair and, therefore, the kinetics of the loss of this signal in the freezequenched samples was measured as a function of time after mixing of the two proteins. For this experiment the conditions were similar to those described earlier for the stopped-flow spectrophotometric experiments with the exception that the final concentration of reduced putidaredoxin and Fe(III) P^SOcam was 50 ¡xM each. The time between mixing and freezing was varied as described in „Materials and methods" and a first-order plot of the loss of the g = I.94 signal versus time (Fig. 3) shows that the reaction obeys first-order kinetics and has an apparent rate constant of 30—35 s - 1 . This value is in excellent agreement with the value obtained with the stopped-flow spectrophotometric experiments. The original concentration of both P ^ O c m and reduced putidaredoxin were identical within experimental error (±5%), resulting in complete oxidation (loss of E P R signal) of reduced putidaredoxin after long reaction times. Control experiments in which either Fe(III) P-450cam or reduced putidaredoxin were freeze-quenched in the absence of the other have shown that the process of freeze-quenching has no significant effect on their E P R spectra [14]. A remarkable finding, shown in Fig. 4, was made when the temperature of the sample in the E P R spectrometer was lowered to 10 °K to measure the E P R signal of high-spin Fe(III) P^SOcam- The middle trace (P-450) is of a sample of Fe(III) P-450cam which was rapidly frozen in the absence of reduced putidaredoxin and shows the EPR signal at g — 8 characteristic of camphor-bound high-spin Fe(III) P-450cam. In addition, the signal

Cytochrome P-450-putidaredoxin interaction

159

gaS

I

P-¥50+/SP

A^ifi I

•8 0

10

20 30 Jme (ms)

Fig. 3 Fig. 4 •

ISP

kO

350

750

1350 3200 Field (gauss)

3500

Fig. 3. First order plot of the oxidation of reduced putidaredoxin as measured by EUR spectroscopy. The amount of reduced putidaredoxin was determined from a freeze-quenching experiment with quantitation by EPR. The logarithm of the fraction of reduced putidaredoxin remaining gives a straight line within the error of the method. The slope of the line gives a first order decay constant, k2, of approximately 33 s _ 1 Fig. 4. EPR signals of P-450Cam and the complex at 10 °K. The top spectrum is of a sample of P-450cam and putidaredoxin which was quenched after 16 ms. The middle spectrum is a control sample which was quenched under conditions identical to the top experiment except without putidaredoxin. The P-450cam concentration was adjusted to give the concentration which should remain at 16 ms based on stopped-flow experiments and/or the measured disappearance of putidaredoxin

of a low-spin form of camphor-bound Fe(III) P-450cam» which is evident at this temperature, is seen at g = 1 .97 in the right-hand panel. The concentration of Fe(III) P-450cam used for the control experiment was 25 [xM so that the magnitude of the signal could* be directly compared with the magnitude of the P ^ O , ^ remaining in the mixed sample (P-450 + ISP) (50% reduced in about 16 ms). The spectrum of the reduced putidaredoxin-cytochrome P ^ O , ^ mixture is shown at the top of Fig. 4 and it can be seen that the magnitude of the signals attributable to Fe(III) P~450cam are greatly attenuated when compared to the expected size. This attenuation occurs within the first 16 ms and represents an interaction of the reduced putidaredoxin with Fe(III) P-450cam- Since both P-450cam and reduced putidaredoxin were initially the same concentration, the immediate loss of the EPR signal of high-spin Fe(III) P-450cam while the signal of reduced putidaredoxin disappears with the expected kinetics is direct evidence for the formation of a Fe(III)-P-450cam-putidaredoxin complex prior to intra-complex electron transfer. At this point it would be pure speculation to discuss the mechanism whereby the EPR signal of Fe(III) P-450cam is lost on complex formation. A close examination of the signals of the complex of reduced putidaredoxin with Fe(III) P-450cam revealed however that the mechanism of loss of the P-45CW signal on complex

160

J . A . PETERSON, D . M . MOCK

formation was not simply dipolar coupling of these two paramagnetic centers. The reasoning behind this conclusion is that only the P-450cam signal is affected by complex formation and if the loss of signal was due to dipolar coupling between these two paramagnetic centers, both signals would be expected to be effected. Other explanations which relate to chemical or physical interaction between these two oxidation-reduction centers prior to electron interchange await further experimentation. References [1] E S T A B R O O K . R . W . , A . H I L D E B R A N D T , H . R E M M E R , J . B . SCHENKMAN, O . ROSENTHAL, a n d D. Y. COOPER in: Biochemie des Sauerstoffs. B. H E S S and H J . S T A U D I N G E R (Eds).

Springer, Berlin 1968, p. 142—177 I. C., T. C. P E D E R S O N , and (1975) M A S O N , H . S., W. L . R O W L K S , and E .

[2] GUNSALUS,

S. G . SLIGAR:

[3]

PETERSON:

A. Rev. Biochem.

J . Am. ehem. Soc.

44,

77,

377—407

2914—2915

(1955)

[4] H A Y A I S H I , O., M. K A T A G I R I , and S. R O T H B E R G : J . Am. ehem. Soc. 7 7 , 5450—5451 (1955) [ 5 ] P E T E R S O N , J . A.: Archs Biochem. Biophys. 1 4 4 , 678—693 (1971) [ 6 ] P E T E R S O N , J . A . , R . E . W H I T E , Y . Y A S U K O C H I , M . L . COOMES, D . H . O ' K E E F F E , R . E . E B E L , B . S . S . M A S T E R S , D . P . B A L L O Ü , and M . J . C O O N : J . biol. Chem. 2 5 2 , 4 4 3 1 — 4 4 3 4 (1977)

[ 7 ] C O O P E R , D . Y „ M . D . C A N N O N , H . S C H L E Y E R , and O . R O S E N T H A L : J . biol. 4 7 5 5 - 4 7 5 7 (1977) [ 8 ] K A T A G I R I , M., B. N . G A N G U L I , and I. C. G U N S A L U S : J . biol. Chem. 2 4 3 ,

Chem.

252,

3543—3546 (1968) [ 9 ] T S A I , R. L.: Ph. D. Dissertation, The University of Illinois, 1 9 6 9 [10] G R I F F I N , B. W., and J . A. P E T E R S O N : Biochemistry 1 1 , 4740—4746 (1972) [ 1 1 ] P E T E R S O N , J . A., Y. I S H I M U R A , and B . W . G R I F F I N : Archs Biochem. Biophys. 1 4 9 , 1 9 7 - 2 0 8 (1972) [ 1 2 ] P E T E R S O N , J . A . , and D . M . M O C K in: Cytochromes P - 4 5 0 and b5. D . Y. C O O P E R , O . R O S E N T H A L , R . S N Y D E R , and C. W I T M E R (Eds). Plenum Press, New York 1 9 7 5 , p. 3 1 1 to 3 2 4 [ 1 3 ] L A N G E , R., C. B O N F I L S , and P . D E B E Y : Eur. J . Biochem. 7 9 , 6 2 3 — 6 2 8 (1977) [14] M O C K , D. M . : Ph. D. Dissertation, The University of Texas, Health Science Center a t

Dallas 1978

[15]

O'KEEFFE,

[16]

GUNSALUS,

D. H., R. I. C . , and

E . EBEL,

and J . A. P E T E R S O N : Meth. Enzym. 5 2 , 151 — 157 (1978) Meth. Enzym. 5 2 , 1 6 6 — 1 8 8 ( 1 9 7 8 )

G . C. W A G N E R :

[17] D E R VARTANIAN, D . V . , W . H . ORME-JOHNSON, R . E . H A N S E N , H . B E I N E R T , R . L . T S A I , J . C. M . T S I B R I S , R. C. BARTHOLOMAUS, and I . C. G U N S A L U S : Biochem. Biophys.

[18] [19]

Res. Commun. 26, 5 6 9 - 5 7 6 (1967) P E T E R S O N , J . A., and D. M . M O C K : Anal. Biochem. 6 8 , 545—553 (1975) P E T E R S O N , J . A., R . E . E B E L , and D. H . O ' K E E F F E : Meth. Enzym.

(1978) [ 2 0 ] BALLOU,

D. P., and G. P A L M E R : Anal. Chem. 4 6 , 1 2 4 8 — 1 2 5 3 L. F . : J . Am. chem. Soc. 4 6 , 2639—2647 (1924)

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FIESER,

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PETERSON, J . A . , R . E . E B E L , D . H . O ' K E E F F E , T . MATSUBARA, a n d R . W . ESTABROOK:

J . biol. Chem. 251, 4010—4016 (1976) [23] P E T E R S O N , J . A., and B. W . G R I F F I N : Archs Biochem. Biophys. [24]

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J . D . LIPSCOMB, P . D E B R U N N E R , a n d E . M U N C K

Molecular Mechanism of Oxygen Activation. O. New York 1974, p. 5 5 9 - 6 1 3

HAYAISHI

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H . BEINERT:

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Cytochrome P-450-putidaredoxin interaction

161

Discussion In the putida system the cytochrome/putidaredoxin complex decay is rate limiting (1st order) in the reduction reaction. We have done similar experiments of the liver system with the cytochrome/reductase complex. Have you any idea about the respective steps in the complex decay ? (electron transfer, conformational change, etc) ? E S T A B R O O K : The liver microsomal cytochrome P-450 system is much more complex in its interaction than is the bacterial system. Obviously in both cases there will have to be conformational changes associated with the electron transfer process. As yet we do not understand what these conformational changes are. R U C K P A U L : Did your experiments in changing the K + concentration have any information to prove that the shift in the spin equilibrium — Dr. Debey has spoken about — is of biological importance ? + E S T A B R O O K : The question of the effect of K on the "spin equilibrium" is an interesting one because this is something which we initially observed in 1969 and have recently begun to study again. I t is difficult to decide whether the effect of K + on camphor binding has any physiological significance. Under experimental conditions which may be considered to approximate cellular salt concentrations small changes in salt do not have a significant effect on the K e q for camphor binding to P-450camU L L R I C H : A question concerning the quenching of the E P R signal of P - 4 5 0 in the complex with putidaredoxin. How was oxygen excluded which could form the diamagnetic FeOa complex and what was the signal height of the reduced putidaredoxin in a control ? E S T A B R O O K : Oxygen was carefully excluded from the reaction mixture but for our purposes it would not have been necessary for the following reasons: 1) oxygen will only bind to the Fe(II) form of the enzyme and neither the Fe0 2 or Fe(II) form have an E P R signal; and 2) the loss of the putidaredoxin signal, a measure of electron transfer, obeyed the expected kinetics, indicating that the unexpected loss of the E P R signal of P - 4 5 0 was not due to either reduc'tion or oxy-complex formation. The magnitude of the E P R signal of the putidaredoxin control was used as the reference and the decay of the signal in the sample obeyed the expected kinetics. D E B E Y : 1) If I understood your slide, the first order rate constants for P-450 reduction depend on the Pd and P-450 concentration. This is not compatible with a fast formation of a di-enzyme complex and a rate limiting electron transfer. Does it mean that the di-enzyme complex formation rate is of the same order of magnitude as the electron transfer ? 2) Do you find identical rates in the presence and absence of CO ? These rates should not be identical since the small AE'° between Pd and P-450 yields a substantial reverse electron transfer rate from P-450 to Pd. . E S T A B R O O K : 1) The dependence of the rate of reduction of P-450 on the concentration of putidaredoxin and P-450 was not the same as a simple bimolecular reaction but was as described in equation 1 in the paper. The simplest interpretation is that a t low concentrations of either Pd or P-450 the rate of formation of the complex was only a few fold larger than the rate of the decay of the complex. In such a system there will be a lag in the attainment of the linear rate of reduction. Thus, in the low concentration experiments, the lowered rate is a reflection of this lag. A more complete discussion of this type of kinetics is presented in Dr. Mock's dissertation. 2) The rates are not exactly identical in the presence and absence of CO but they differ in a direction opposite from what would be predicted if CO was "pulling" the reaction by becoming a ligand of P-450 i.e., in the absence of CO reaction. With respect to your comment about the redox potentials for P-450 and putidaredoxin (see manuscript) the AE'° is sufficiently large to cause all ( > 90%) of the P-450 to be reduced under the conditions described here. G U N S A L U S : Comment and question: As you will know, the rate of each reaction, the steady state concentrations and stable intermediates and the demonstration that the Pd • P-450cam complex, demonstrated by Sligar et al; is indeed an obligatory intermediate in the final reaction were reported at Berlin in 1976 and Primosten. The redox rate, pH 7.0, 50 ¡iM potassium phosphate, camphor 300 |xM, 20 °C is 41 s- 1 . Therefore, your now reported value of 31 s _ 1 agrees with earlier data. This may indicate the sample of P-450cam either lacks some heme or is not quite pure! BLANCK:

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Do your experimental data quantitate the expected Pd oxidized and P-450 reduced ? The increase in P-450 low spin fraction on addition of Pd confirms the Lipscomb data especially the g — 2.45/1.97 signal. E S T A B R O O K : With respect to your first comment I am aware of the report of Pederson in 1976 but we are following up on our work which was presented in Philadelphia in 1974 and published in Cytochromes P-450 and bs edited by D. Y. Cooper et al, pp. 311 — 324, Plenum Press, New York 1975With respect to your second comment: As we reported in the symposium referenced above, the rate will be a complex function of salt, buffer and enzyme concentrations and therefore since our experimental conditions are somewhat different, I don't think that the rates are directly comparable. With respect to your third comment about Lipscomb's data — You have misunderstood our results. The increase in low spin signal which Lipscomb reports is probably an artifact due to salt effects. Under the conditions of this experiment, there is no increase in the magnitude of the low-spin form of cytochrome P-450 on combination with putidaredoxin. L A S S M A N N : I am interested to know more details of the technical realization of your mixedfreezing system because we have built also a stopped-flow-EPR device for radical kinetics and conformational kinetics of spin-labeled proteins in the liquid state. What kind of syringe driving mechanism did you use and by what is the dead time determined ? E S T A B R O O K : The freeze-quench device which we have used to prepare our samples for E P R spectroscopy is not really a stopped-flow device. The design of the apparatus is described in detail in: 1 ) B A L L O U , D. P. in: Ph. D. Dissertation, The University of Michigan, Ann Arbor Michigan. 2) B A L L O U , D. P . , and P A L M E R , G., Anal. Chem. 4 6 , 1248—1253 (1974) The driving device was a motor driven cam which pushed a ram which in turn pushed the syringes. The dead time for mixing and freezing has been determined to be less than ~ 7 ms, by following the reaction by metmyoglobin with azide. This reaction can be followed optically as well as by E P R spectroscopy.

Acta biol. med. germ., Band 38, Seite 163—175 (1979) The University of Texas, Health Science Center, Department of Biochemistry, Dallas, Texas, USA

Spin state transitions of liver microsomal cytochrome P-4501,2 J . W E R R I N G L O E R , S . KAWANO, a n d R . W . E S T A B R O O K

Summary Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation. Introduction

A critial prerequisite for understanding the cyclic function of cytochrome P-450 is the identification and the quantitation of its various intermediates generated during the activation of molecular oxygen. The presumed sequence of cytochrome P-450 reactions leading to substrate hydroxylation is considered to be initiated by the interaction of the substrate with the hemeprotein in its ferric state [1]. The resulting low-spin to high-spin transition of the hemin iron was found to be associated with characteristic changes in the optical spectrum [2—4]. These optical changes can in turn be used as a criterion for the extent of occurrence of such reactions. The present study describing the spectrophotometric analysis of high spin cytochrome P-450 was undertaken to characterize the ferric hemeprotein in the native environment of the microsomal membrane and to provide an additional tool for the elucidation of the principle rate determining step(s) in the sequence of microsomal electron transport reactions. Materials and methods Male Sprague-Dawley rats (Charles River CD., 250—300 g body weight) were maintained on Purina chow and water ad lib. The regimen of treatment with inducing agents was as follows: phenobarbital sodium (80mg/kg, i.p., daily over a period of 4 days), pregnenolone-16acarbonitrile (100 mg/kg, p.o., daily over a period of 4 days), /S-naphthoflavone (80 mg/kg, i.p., over a period of 3 days), and 3-methylcholanthrene (40 mg/kg, i.p., over a period of 3 days). The animals were starved for 18 h prior to sacrifice on the day following the last Supported in part by a research grant from the National Institutes of Health (NIGMS16488) 2 Presented by J . W E R R I N G L O E R at the Scientific Conference 'Cytochrome P-450: Structural and Functional Aspects' (held in Eberswalde-GDR, July 9—13, 1978) 1

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J . W E R R I N G L O E R , S . KAWANO, R . W . ESXABROOK

treatment. The livers were perfused with 0.15 M saline and homogenized in 0.25 M sucrose. The microsomal fractions were obtained by differential centrifugation as previously described [5]. The pellets were suspended to give a protein concentration of approximately 30 mg per ml in 0.25 M sucrose containing 50 mM Tris-chloride, pH. 7.5, and were used within 24 h of their preparation. For the spectrophotometric analyses described, microsomes were diluted to protein concentrations ranging between 1 and 2 mg per ml. The buffer medium consisted of 50 mM Tris-Hepes, pH 7.5, 150 mM KC1, and 10 mM MgCl2. The difference spectra were recorded using an Aminco DW-2 spectrophotometer with the cell compartment maintained at 25 °C. The chemicals used in these studies were purchased as follows : NADPH and NADH, chromatopure, (PL-Biochemicals), ethylmorphine (Merck Chemical Co.), androstanedione, 3-methylcholanthrene (Sigma Chemical Co.), /9-naphthoflavone (Aldrich Chem. Co.), phénobarbital sodium (J. T. Baker Co.). The following compounds were obtained as generous gifts from pharmaceutical companies: pregnenolone-16 a-carbonitrile, benzphetamine (The Upjohn Co.), hexobarbital, sodium (Sterling-Winthrop), SKF 525-A (Smith Kline and French Co.), metyrapone (Ciba-Geigy). Results and discussion

The interrelation between spin state and spectral properties of ferric cytochrome

P-450

Difference spectrophotometry has been successfully applied to evaluate spin state transitions of ferric cytochrome P-450 as they occur in the presence of substrates or upon other appropriate manipulations [4, 6—10]. The characteristic features of the difference spectrum observed upon low spin to high spin transitions of the hemin iron are readily apparent when one compares the absolute spectra of the purified hemeprotein in its low spin and high spin state, respectively (cf. Fig. 1). The absorbance maximum of the gamma-band of cytochrome P-450 is hypsochromically shifted from about 418 nm to 392 nm giving rise to the appearance of the classic Type I spectral change [11] when the difference in absorbance between the high spin and low spin forms of the pigment is analyzed in the Soret region. The markedly attenuated alpha-absorbance band observed in the absolute spectrum of high spin cytochrome P-450 is reflected in the formation of a trough at about 5 70 nm in the difference spectrum. Further, so-called charge transfer absorbance bands [12], termed 'D' and 'E' [13], become visible at about 645 nm and in the 500 nm region using either difference or absolute spectrophotometry. The intensity of the beta-band, although obscured in the presence of the ' E ' -charge transfer absorbance band, does not appear to be altered significantly. In general, the spectral changes observed upon the conversion of low spin cytochrome P-450 to its high spin state are consistent with those shown to be characteristic for spin state transitions of other ferric hemeproteins [12, 13]. Quantitative aspects of spin state transitions of microsomal cytochrome P-450 Since the extinction coefficients for the absolute spectra of the low spin and high spin forms of bacterial and mammalian cytochrome P - 4 5 0 are in close agreement [ 1 4 — 1 7 ] , a generally applicable extinction coefficient can be calculated for the Type I spectral change, e. g., for the difference in absorbance between the peak at about 388 nm and the trough at about 421 nm. Applying such an extinction coefficient, estimated by PETERSON and his coworkers to be approximately 110 • mM - 1 • c m - 1 [9], one can calculate that almost 40% of the liver microso-

165

Cytochrome P-450 spin state

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moves nearer to that of the male. In contrast to the marked effect seen in the females, hypophysectomy of the animals had no significant effect on hepatic androstenedione metabolism. Daily oestradiol benzoate treatment (1 (ig, once a day) of male animals caused a dramatic change in hepatic steroid metabolism after 14 days of treatment. The 5 «-reductase activity was greatly increased (408% of the normal male; p < 0.001) and the 17-hydroxysteroid oxidoreductase, 6/5- and l6a-hydroxylase activities decreased {p