Basis and Mechanisms of Regulation of Cytochrome P–450 [Reprint 2021 ed.] 9783112563328, 9783112563311

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Frontiers in Biotransformation Vol. 1

Frontiers in Biotransformation

Volume 1

Basis and Mechanisms of Regulation of Cytochrome P-450 Edited by Klaus Ruckpaul and Horst Rein

Akademie-Verlag Berlin 1989

Gesamt-ISBN 3-05-500456-6 Vol. 1-ISBN 3-05-500457-4 Erschienen im Akademie-Verlag Berlin, Leipziger Straße 3—4, Berlin, D D R - 1 0 8 6 © Akademie-Verlag Berlin 1989 Lizenznummer: 202 • 100/486/88 Printed in the German Democratic Republic Gesamtherstellung: V E B Druckhaus „Maxim Gorki", 7400 Altenburg Lektor: Christiane Grunow Gesamtgestaltung: Martina Bubner LSV 1315 Bestellnummer: 7637949 (9090/1) 06400

Preface

An increasing number of papers in the field of biotransformation has led to a real need for review articles to keep abreast of the new information. This flow of papers implies that the frontier of our knowledge is moving forward rapidly, requiring a continuous re-evaluation of existing data and accepted principles. Consequently this series has been planned to publish overviews of the current state-of-the-art by experts actively working in the field. The editors do not aim to produce a textbook with well established data, rules and principles but rather to focus on the borderline of our knowledge by presenting innovative results, controversial ideas and aspects, and so to stimulate the progress of understanding. The pivotal role of cytochrome P-450 in biotransformation is well established, but biotransformation involves more enzymes than only cytochromes P-450. Therefore, to provide the reader with an in-depth presentation of a well defined topic, biotransformation has been chosen as the more appropriate subject for this series rather than the narrower field of cytochrome P-450 research. Biotransformation as an essential biological process has developed into a research field with an interdisciplinary character in which the biochemist, biophysicist, pharmacologist, toxicologist, biologist and other bioscientists are all interested. The editors will keep this in mind. On the other hand the broad scope of the series will not interfere with the interest of readers with specialized interest because each volume will focus on a distinct topic such as regulation, induction or protein/lipid interaction. Besides new results in basic research concerning theory, models, updating of thermodynamic and physicochemical data for better understanding of, for example, reaction mechanisms, structure/activity relationships and kinetically based interdependences, the editors intend also to follow carefully the progress that molecular biology has brought about, and will continue to, in classical biotransformation research and likewise to cover recent developments in which the role of cytochrome P-450 in integrated biological regulation processes has become evident. But there are also close relationships between biotransformation and environmental pollution problems, for example, food additive toxicity, occupational

I

diseases and toxicity of environmental pollutants. Clearly there is increasing interest in application of knowledge from biotransformation research. In addition to applications in toxicology, another growth area will undoubtedly be biotechnology based on progress in molecular biology and protein engineering. This series hopes to contribute to a successful linkage between basic research and such applications by providing up-to-date reviews of these topics. Klaus Horst

II

RUCKPAUL REIN

Preface

C ontents

Introduction K . Ruckpaul and H. Rein

IV

Chapter 1 Regulation Mechanisms of the Activity of the Hepatic Endoplasmic Cytochrome P-450 K . Ruckpaul, H. Rein, and J. Blanck

1

Chapter 2 Catalytically Active Metalloporphyrin Models for Cytochrome P-450 D. Mansuy and P. Battioni

66

Chapter 3 Structural Multiplicity and Functional Versatility of Cytochrome P-450 F. P. Guengerich

101

Chapter 4 Multiple Activities of Cytochrome P-450 A. I. Archakov and A. A. Zhukov

151

Chapter 5 The Hormonal and Molecular Basis of Sexually Differentiated Hepatic Drug and Steroid Metabolism in the Rat E. T. Morgan and J.-A. Gustafsson Chapter 6 Evolution, Structure, and Gene Regulation of Cytochrome P-450

176

. . . .

195

0 . Gotoh and Y . Fujii-Kuriyama List of Authors

244

Subject Index

246

III

Introduction K . RTTCKPAUL a n d H . R E I N

Scientific knowledge — as a simplified rule — passes from the discovery of a new phenomenon (fact, subject, reaction) through its disintegrative analytical phase, to the integration of resolved detailed d a t a and on into a more or less reconstituted system of mutual relationships. These general characteristics m a y similarly refer to cytochrome P-450 research which started about t h i r t y years ago. Now it is one of the most intensively studied enzymes. I n the last decade alone, more t h a n 10,000 papers relating to cytochrome P-450 have been published. This abundance of information requires a comprehensive review to condense t h e d a t a to the very essentials. I n the beginning, cytochrome P-450 investigations mainly dealt with physiological, pharmacological and toxicological aspects. These comprised studies on its occurrence in different species and tissues together with analyses of its localization within the cell. Finally, examinations of its implications in detoxifying and toxifying processes, including the responce t o inducers and the quantitation of t h e degree of induction. The analytical phase is characterized by the isolation and purification of the essential components of the different cytochrome P-450 systems, and the determination of their molecular weights, their spectral and other physicochemical properties. I t is f u r t h e r characterized by studies on the broad substrate specificity of the catalytic activity and the analysis of t h e different pathways of oxygen activation. This period led to the recognition of the reaction cycle and the detection of t h e functional versatility and the structural multiplicity including sequencing of the primary structure of cytochrome P-450. The fulllength (or nearly full-length) cDXA and/or amino acid sequences for about 80 gene products have currently been analysed. One of the high-lights in this period was the analysis of the 3-dimensional structure of cytochrome P-450cam in 1985. The phase of a comprehensive treatise is characterized by an evaluation of accumulated analytical data, the integration of which permits deeper insights into the complex relationships which constitute the structural and more so the functional integrity of cytochrome P-450 systems. Molecular biology and its novel techniques have become of special importance by accelerating the

process, thus enabling the alignment of different complete primary structures and the quantitation of overall homologies among these structures resulting in the statement t h a t cytochromes P-450 exhibit a gene superfamily which — dependent on the inducer — can be divided into families and subfamilies. I n addition, new experimental approaches have become available to investigate comparative and integrative considerations at different levels of integration. Problems inherent to this stage include: — which structural features of the multitude of compounds t h a t interact with cytochromes P-450 determine their capability to control oxygenatic and oxidatic pathways of the enzyme? — how is the catalytic activity of the complex enzyme system regulated? — a t what level of cytochrome P-450 biosynthesis is the induction process regulated in dependence on different inducers — and what consequences result with regard to active interference? — to what extent is hormonal control of importance for the induction of sex specific cytochromes P-450, what is triggering t h a t process and by what route is it accomplished? The first volume of 'Frontiers in Biotransformation', following the considerations outlined above, is intended to review selected topics which all contribute to the concept of 'Regulation'. Regulation is conceivable at different levels of integration. At t h e molecular level interactions can be amended by low molecular effectors (substrate, cosubstrate) and by high molecular effectors (reductase, cytochrome b 5 ). The sum of the interactions results in changes of either conformation, redox behaviour and/or turnover rates of cytochrome P-450. Due to t h e enzyme's membrane bound character the surrounding phospholipids provide a matrix which itself has been proposed as regulating enzymatic activity by affecting the e "-transfer through influencing the stability of the cytochrome P-450/reductase-complex and the organization of the enzyme system within t h e matrix. Finally, the physiologically most important regulation proceeds a t the cellular level. Induction, inducibility as well as sex, species and tissue specificity determine via the isozymic distribution and the enzyme concentration t h e extent of toxification and detoxification. A reliable comprehensive review naturally requires much accumulated data. Looking at the current state-of-the-art for various cytochromes P-450 it can be seen t h a t the level of knowledge achieved t o date differs greatly. As compared to the remarkable progress in the elucidation of structural and functional details in cytochrome P-450cam and the mammalian cytochromes P-450, t h e analytical period of microbial and plant cytochromes has just begun. This implies t h a t a sufficient a m o u n t of d a t a necessary for a comprehensive review of those cytochrome P-450 systems is still missing. Due to its pharmacological and toxicological origin it is not surprising t h a t the liver system and especially t h a t of the endoplasmic reticulum has been most

Introduction

V

intensively studied. Consequently most papers published so far are concerned with this system. T h a t emphasis is likewise reflected in the articles of volume 1. The concentration on the endoplasmic system, however, implies correspondingly less research on other systems. An extrapolation of mechanisms found valid for onedistinct system has therefore to be carefully carried out taking into account the biochemical diversity of cytochrome P-450 dependent monooxygenatic systems so far characterized (soluble — membrane b o u n d ; endoplasmic — mitochondrial). The present volume is introduced b y an article which is mainly concerned with regulation of the liver exoplasmic cytochrome P-450 LM2 (cytochrome P-450 I I B1 according to recently published recommendations for a unified nomenclature). Several considerations favour this chapter as a n i n t r o d u c t o r y one. (1) Studies on an isolated cytochrome P-450 isozyme which represents a simplified system are suited to the elucidation of principle mechanisms of action and regulation. (2) A remarkable amount of data on cytochrome P-450 I I B1 has been accumulated which provides a reliable basis for such a review. (3) Cytochrome P-450 I I B l , due to its involvement in biotransformation reactions of a huge number of exogenous substrates, drugs and environmental contaminants, is of high relevance with regard to pharmacological and toxicological implications. I n the following articles the scope outlined above is detailed, starting with a comprehensive contribution on model systems in chapter 2. In general, utilization of models has proved an appropriate means to convert complex biological systems into relevant subjects which can be experimentally examined. Therefore, metalloporphyrins are surveyed, the aim being to study the physicochemical, geometrical and electronic requirements for a catalytically-active metalloporphyrin and to derive conclusions on the chemical reactivity and functional diversity of the heme group in the enzyme. Moreover, the studies on models of cytochrome P-450 also represent a possible line which m a y initiate f u t u r e developments of engineered synthetic enzymes having optimized functions. A comprehensive review which is aimed a t elucidating regulation phenomena has to consider the structural suppositions which are necessary for the catalytic function. Therefore, structural multiplicity which at least in p a r t originates from the existence of isozymes is an essential for understanding the functional versatility and — based on these structure/function-relationships — to understand further the molecular basis of regulation. The data accumulated so far are summarized in chapter 3. Cytochrome P-450-catalyzed reactions depend on the enzyme's capability to activate and split molecular oxygen by insertion of electrons. This unique function brings about functional versatility in t h a t the insertion of a different number of electrons leads to differential degrees of oxygen activation, generating either an oxygenatic or an oxidatic pathway. Due to their reactivity, uncoupled activated oxygen species are of toxicological importance. Besides direct toxicological effects on cytochrome P-450, the formation of activated

VI

Introduction

oxygen species represents an important side effect of monooxygenatic reactions, the regulation of which is discussed in chapter 4. A more complex mechanism of cytochrome P-450 regulation proceeds a t the cellular level. I t is hormonal in nature and involved in sexually differentiated steroid drug metabolism. Great efforts in the last 5 years have revealed an immense progress in insight, not only into the molecular basis of t h e sex-specific isozymes b u t also into the regulation of their biosynthesis. The detailed knowledge about t h a t differentiation m a y be assumed to have practical pharmacological consequences on sex-differentiated biotransformation recativities in the near f u t u r e (chapter 5). Molecular biology and especially genetic engineering will allow not only new experimental approaches to the analysis of structure/function relationships by site directed mutagenesis but also the construction of arteficial (e.g. chimeric) cytochromes P-450. By way of these new techniques the molecular basis of genetic relatedness of structural multiplicity and the mechanisms of its regulation have become available. F u r t h e r these investigations include studies on the phylogenetic relations of evolutionarily distant cytochromes P-450. .Based on the results derived therefrom, their application to clinical problems m a y become relevant. The articles of this volume, being mainly concerned with endoplasmic cytochrome P-450, present comprehensive information on t h e principles of regulation. Therefore they contribute knowledge and strategies for integrational t r e a t m e n t of cytochrome P-450 systems outside this selection. Following these lines, data will be elaborated which may prove the general validity of t h e outlined regulation principles as challenging task for f u t u r e progress.

Introduction

VII

Chapter 1 Regulation Mechanisms of the Activity of the Hepatic Endoplasmic Cytochrome P-450 K . RITCKPAUL, H . R E I N , a n d J . B L A N C K

1.

Introduction

3

2. 2.1.

Regulation at the molecular level The importance of the axial heme iron ligancls for the hemoprotein function The relation between spectral changes and spin state The spin equilibrium of cytochrome P-450 The significance of the spin equilibrium in molecular regulation . . The redox potential of cytochrome P-450 The relation between redox potential and spin state The spin/redox couple as regulator of the reduction reaction . . . The control function of the substrate Active site characteristics Structure/activity relationships The regulation of alternate pathways of oxygen activation . . .

6

2.2. 2.3. 2.4. 2.4.1. 2.4.2. 2.4.3. 2.5. 2.5.1. 2.5.2. 2.6. 3. 3.1.

3.4.3. 3.4.4. 3.5. 3.6.

Regulation at the membrane level Lipid composition and protein constituents of the endoplasmic reticulum Membrane fluidity and mobility of membrane components . . . . Structural peculiarities of protein/lipid interactions Functional aspects of the component interactions in the membrane Characteristics of the cytochrome P-450 reduction Regulation of the electron transfer by reductase/cytochrome P-450 interaction Phospholipid control of electron transfer Electron transfer and substrate conversion Cytochrome b 5 dependent regulation Isozyme interactions and protein compartmentation

4. 4.1.

Regulation at the cellular level Historical background of induction

3.2. 3.3. 3.4. 3.4.1. 3.4.2.

7 9 11 16 16 18 20 22 22 24 26 29 29 30 32 34 35 37 38 41 43 47 47 49

1

4.2. 4.3. 4.4.

Classification of inducers and mechanisms of induction Relations between induction and chemical carcinogenesis Polymorphism of induction

5.

Concluding remarks and outlook

53

6.

References

55

2

. . . .

49 51 52

1.

Introduction

Hardly any other field of enzymology has developed as dynamically as t h a t of cytochrome P-450 dependent monooxygenases. The reason for the extent and intensity of this progress is the key function of this enzyme system in the metabolism of endogenous substrates (steroids, f a t t y acids) as well as in t h e biotransformation of xenobiotics (drugs, environmental contaminants). However, cytochrome P-450 research is following t h e same general approach used to investigate other complex biological systems, based on an enhanced understanding of molecular mechanisms and structures. Studies on cytochrome P-450 dependent monooxygenase systems over the last decade have resulted in the elucidation of biosynthetic p a t h w a y s of various important classes of biologically active compounds (steroids, prostaglandins and vitamin D) and their regulation. I n view of the increasing exposure to pollutants in the highly developed industrialized countries, chemical carcinogenesis has become of increasing importance. For m a n y carcinogens t h e incidence of cancer is closely linked to the extent of activation and so the toxicity of xenobiotics is regulated by the activity of cytochrome P-450. The elucidation of this relationship has led to the study of toxicology at the molecular a n d cellular level. Recent results using molecular biology have allowed this enzyme system to be applied for diagnostic, therapeutic and biotechnological purposes. I n contrast to this remarkable progress, t h a t molecular biology has already introduced into cytochrome P-450 research, analysis of the regulation mechanisms of its activity has shown less spectacular development. The biological importance of cytochrome P-450 and its ubiquity, however, demands a n understanding of such mechanisms in detail. The purpose of the present survey is to bring together relevant results, to assess t h e m critically and to deduce new insights. Cytochrome P-450 dependent monooxygenases belong to the class of oxygenactivating enzymes which are known to transfer electrons to molecular oxygen thereby catalyzing biological oxidations. Biological oxidations in general proceed via two different mechanisms. One mechanism in which hydration and hydrolysis are involved is catalyzed b y dehydrogenases. I n this case the oxygen atom is derived from a water molecule. The other mechanism which involves the activation of molecular oxygen, is catalyzed b y a class of enzymes which can be divided into oxidases and oxygenases. Oxidases transfer electrons f r o m a substrate to oxygen, thereby oxidizing the substrate. Depending on the numAbbreviations used: PC: phosphatidylcholine; P E : phosphatidylethanolamine; PS: phosphatidylserine; PI: phosphatidylinositol; PA: phosphatidic acid; DLPC: dilauroyl PC; DMPC: dimyristoyl PC; DPPC: dipalmitoyl PC; DOPC: dioleoyl PC; P A H : polycyclic aromatic hydrocarbons; A H H : aryl hydrocarbon hydroxylase; PCB: polychlorinated biphenyl; P B B : polybrominated biphenyl; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; PCN: pregnenolone-16a-carbonitriIe; B N F : /S-naphthoflavone; 3-MC: 3-methylcholanthrene.

Regulation Mechanisms of Cytochrome P-450

3

ber of electrons which are transferred to oxygen, either superoxide anion, hydrogen peroxide or water is formed. Oxygenases, on the other hand, transfer oxygen to a substrate after reductive splitting of molecular oxygen ( H A Y A I S H I , 1974). The electrons for this activation process are provided by the N A D P H (NADH)-dependent cytochrome P-450 reductases (Fig. 1).

Oxygen A c t i v a t i n g

Enzymes

Oiidaje j

a) S H ; + V 2 o ?

—

S t Hz0

b) S H ; +

—

S + H702

0;

S + 0 j + H,X -

c) SH + 0j — S + OftH* a)

SO + U H j O ( X = e x t e r n a l electron

p-diphenol oxidase ( l a c c a s c )

tryptophan

o-diphenol oxidase (catecholase)

(tryptophan pyrrolase,

c y t o c h r o m e oxidase, a s c o r b a t e oxidase

E.C. 1.13.1.12 )

2.3-dioxygenase

p y r o c a t e c h a j e (E.C. 1.13.1.1.) b)

0-amino acid

oxidase

e-transfer flayoproteins c)

xanthine o x i d a j e

l-lysine monooxygenase

donor) mixed - f u n c t i o n monooxygenases ( microsomes, mitochondria, microorganisms )

Fig. 1. Oxygen activating enzymes.

The basic mechanism of monooxygenases consists in the reductive cleavage of molecular oxygen and the splitting of bonds in the substrate for the insertion of oxygen, e.g. a C-H bond. From a thermodynamic point of view oxygenation reactions in general should proceed spontaneously, because the dissociation energy for 0 2 (about 460 kJ/mol) and t h a t of the C-H bond of the substrate (about 420 kJ/mol) is more t h a n compensated b y formation of the product bonds. The overall reaction (1) in the living cell can be formally described in terms of several intermediate steps ( 2 ) — ( 5 ) ( J U N G and R I S T A U , 1 9 7 8 ) RH

+ 02 + NADPH + H+ —- ROH + H20 + NADP-

N A D P H + H.+ --> H 2 + NADP+ 02(g)

->20 ( g )

(1)

AGl =

19.3 kJ/mol

(2)

AG2 =

460.5 kJ/mol

(3)

H2(g)

+ o(g, - H A , ,

AG, = - 4 7 0 . 1 kJ/mol

(4)

RH ( 1 )

+ o ( g ) -•» ROH ( 1 )

AGt = - 3 9 3 . 9 kJ/mol

(5)

AG = - 3 8 4 . 2 kJ/mol R H = cyclohexane. Reactions with molecular oxygen, however, are kinetically restricted. The low reactivity of the oxygen biradical is due to the peculiar electronic confi-

4

K . RUCKPAUL; H . R E I N

guration of the molecule. The triplet ground state is characterized by the following electron configuration: ( — K f

(1 +

Kt)

(13)

(1 +

and K.. =

K,

(14)

i.e. t h e l a t t e r e q u a t i o n relates t h e substrate-induced modification of K 1 t o t h e s u b s t r a t e affinities of t h e high-spin a n d low-spin conformers. T h e c o n s t a n t s Kx — Kt as determined for cytochromes P-450 f r o m different sources are shown in Table 1.

Table 1. Constants of spin and substrate-binding equilibria of several cytochromes P-450 according to the four-state coupling model Source of Substrate cytochrome P-450

K!

P-450 LM2

benzphetamine

0.08

0.4 370.0

70.0

P-450 PB-B benzphetamine

0.07

0.6 250.0

29.0

P-450 CAM

0.08

camphor

K2

15.0

KJ

K4

(¡Am)

((Am)

9.0

0.05

References

RISTAU et al. (1979) GIBSON

(1984)

and

TAMBURINI

SLIGAR (1976)

T h e difference in t h e s u b s t r a t e affinity between t h e two spin conformers is most pronounced in cytochrome P-450 CAM, which is characterized by a substrate-induced high-spin shift of more t h a n 9 0 % ( S L I G A R , 1 9 7 6 ) . I n agreement

14

K . RUCKPAUL; H . REIN

with this large effect, the substrate affinities differ by a factor of about 200. On the other hand smaller substrate-induced high-spin shifts of cytochrome P-450 LM2 agree with smaller asymmetric substrate binding; for example benzphetamine exhibits an approximately five-fold higher affinity to the highspin conformer of cytochrome P-450 LM2 than to the low-spin conformer (RISTAU et al., 1978). In general, from the four-state model of the enzyme, it follows that the higher affinity of type I substrates to the high-spin conformer is the reason for the spin equilibrium shift towards the high-spin state, and conversely, the observed low-spin shift induced by inverse type I substrates is to be interpreted as a higher affinity of the substrates to the low-spin conformer. The existence of "type O spectra" (PIBBWITZ et al., 1982a) is explicable by the four-state model, too. Despite the high substrate affinity (Ks) determined from the EPR spectrum, an optical substrate difference spectrum is not observed because the respective substrate has the same affinity to both spin conformers. The study of the temperature dependence of Kx — Kt allowed the determination of the thermodynamic parameters of the respective partial reactions for cytochrome P-450 LM2 (RISTAU et al., 1978) (Table 2). Table 2. Equilibria a n d t h e r m o d y n a m i c constants of t h e t h e r m o d y n a m i c model for t h e interrelationship between substrate-binding (benzphetamine) a n d spin equilibrium of P-450 LM2

Equilibria

2

=

K (20°C)

JH (kj-mol1)

JS ( J • mol" 1 • K - 1 )

[P-450 h s ] [P-450 l s ]

0 0g

-44.3 ±0.8

-130 ± 5

- 6 . 3 ± 0.8

[P-450 h s • S] [P-450 ] s • S]

0 39

_

-121 ± 5

- 2 . 5 ± 0.8

K3 = l P ' 4 5 0 ' s ] tS] [P-450 l s • S] [P-450 h s ] [S] [P-450 h s • S]

3 g 0

JG ( k J • mol" 1 )

0.37 mM

31.4 ± 2 . 1

171 ± 9

- 1 8 . 8 ± 2.5

0.07 mM

26.3 ± 2.1

171 ± 9

- 2 3 . 8 ± 2.5

The low-spin/high-spin transition shows remarkably high entropy values which are a typical for pure spin state equilibria (GOODWIN, 1976) and denoted as configuration entropy in metal complexes (KONIG and RITTEE, 1 9 7 4 ) . Caused by the large difference in thermodynamic freedom between the lowspin and high-spin states, this "excess" entropy is especially large in metallo-

Regulation Mechanisms of Cytochrome P-450

15

proteins. The observed excess entropy for the spin state transition of cytochrome P-450 LM2 could originate from cooperative breaking of van der Waals heme/protein contacts (RISTAU et al., 1979) and/or release of water molecules from the active site. Such an assumption agrees with the interpretation of X-ray data for cytochrome P-450 CAM by POULOS et al. (1986), who suggested that the displacement of the active site water molecules, including the iron-linked water, shifts both the redox potential to more positive values and the spin equilibrium towards the high-spin state. Release of water molecules or at least structural changes of iron-linked water at the spin transition (REIN and RISTAU, 1978) should be important for the polarity of the heme environment, which determines the redox potential (KASSNER, 1972). The entropy controlled heme/protein interaction, which in agreement with the low energy barrier between the two spin states should be connected with only small local conformational changes in the protein, could represent the molecular basis for the observed spin dependent redox properties of cytochrome P-450.

2.4. The significance of the spin equilibrium in molecular regulation 2.4.1. The redox potential of cytochrome P-450 Cytochromes P-450 are characterized by high negative oxidation-reduction potentials without pronounced isozymic or species differences (Table 3). This property is related to the presence of both the thiolate ligand and the local nonpolar heme environment. Substrate binding induces a shift of the negative redox potential to more positive values. This was established at an early date for cytochrome P-450 CAM. Concomitantly with a strong ( ~ 9 0 % ) high-spin shift in camphor binding, the redox potential of this microbial cytoc h r o m e P - 4 5 0 is c h a n g e d f r o m — 3 0 3 m V t o — 1 7 3 m V (SLIGAR, 1976).

In

contrast the redox potential of cytochrome P-450 LM2 is altered less on substrate binding, with corresponding smaller substrate-induced spin shifts. SIES and KANDEL (1970) observed a small change of 16 MV to more positive values in microsomal cytochrome P-450 with hexobarbital. GUENGERICH et al. (1975) did not find any significant change in the redox potential of phenobarbital-induced liver cytochrome P-450 upon binding of benzphetamine, which in the absence of substrate exhibits a redox potential of —330 MV. SLIGAR et al. (1979) measured the redox potential of isolated and partially purified P-450 from uninduced rat liver, both in the presence and absence of type I substrates. A redox potential of — 300 mV was found (relative to the standard hydrogen electrode) in the absence of substrates. Contrary to earlier measurements, the addition of hexobarbital or benzphetamine to the partially purified P-450 increased the redox potential distinctly to —237 inV and —225 MV, respectively.

16

K . RUCKPAUL; H. REIN

Table 3. Oxidation-reduction potentials of several cytochromes P-450

Source of P - 4 5 0

Induction/Isozyme

Pseudomonas putida

Rabbit liver

Experimental conditions

camphor

microsomes, phénobarbital induced

without substrate

E0' (mV)

References

-303

SLIGAR, 1 9 7 6

-173

— 3 4 0 WATERMAN a n d MASON, 1 9 7 2

benzphetamine

P-450 LM2 and microsomes, phénobarbital induced

soluble P-450 LM2 and in vesicles

P-450 LM4

pH 7 . 4

— 3 3 0 GTJENGERICH e t

al., 1 9 7 5

-~ — 3 4 0 BACKSTROM e t a l . , 1983

— 3 6 0 HUANG e t a l . , 1986

R a t liver

partially purified, uninduced

— substrate + hexobarbital + benzphetamine

— 3 0 0 SLIGAR e t a l . ,

microsomes, phénobarbital induced

without substrate

-362

P-450pb_b

+ DLPC

-319

+ DLPC, + benzphetamine + DLPC, + cyclohexane from low-spin band from high-spin band + DLPC + DLPC, ethylmorphine

-311

—237

1979

-225

-306 — 300

GUENGERICH,

-286

1983*

-336 -346

* selected values

Regulation Mechanisms of Cytochrome P-450

17

Table 3 — Continued Source of P-450

Induction/Isozyme

Bovine adrenals

mitochondrial P-450, without external partially purified substrate P-450„,

P-450 nß

Experimental conditions

low spin (predominantly) high spin (predominantly) low spin + cholesterol, pH 7.4

E„'

References

(MV)

— 4 0 0 COOPER e t al. 1970

— 4 1 2 LIGHT a n d ORMEJOHNSON, 1981 — 297 -305

pH 7.0

- 2 8 4 LAMBETH a n d PEMBER, 1 9 8 3

pH 7.5, + desoxycorticosterone

- 2 8 6 HUANG e t al., 1986

2.4.2. The relation between redox potential and spin state T h e t r e a t m e n t of t h e redox equilibrium of cytochrome P-450 requires consideration n o t only of t h e valence states of t h e iron b u t also of t h e substrate-binding a n d t h e spin equilibrium. Hence a t h e r m o d y n a m i c three-dimensional interaction model for cytochrome P-450 CAM was presented b y SLIGAR (1976) which involves t h e linkage between s u b s t r a t e binding, spin a n d redox states. This complete coupling model, however, can be simplified as follows. At least in cytochrome P-450 LM2 a n d related phenobarbital-inducible low-spin isozymes, substrate-binding affects only t h e spin equilibrium, a n d substratespecific conformational changes of t h e enzyme can be neglected. This is in accordance with a weak nonspecific e n z y m e / s u b s t r a t e interaction (MISSELWITZ et al., 1976, 1977), which is reflected in almost unchanged CD-spectra in t h e Soret region of solubilized cytochrome P-450 f r o m phenobarbital-induced r a b b i t liver on s u b s t r a t e binding (REIN et al., 1976b). Based on these results t h e t h e r m o d y n a m i c " c u b e " model of SLIGAB (1976) can be simplified to a p l a n a r scheme where P-450 (Fe 3 + ) a n d P-450 (Fe 2 + ) denote either substrate-free or s u b s t r a t e - s a t u r a t e d ferric a n d ferrous cytochrome P-450, respectively, K a ° a n d K ^ are t h e spin equilibrium constants for b o t h valence states, K b h s a n d K b l s t h e redox equilibrium constants for high-spin a n d low-spin cytochrome P-450, respectively (Scheme 2)

18

K . RUCKPAUL; H . R E I N

Scheme 2 P-450 (Fe 3 + ) h s

P-450 (Fe 3 + ) l s

P-450 (Fe 2 + ) h s

±e

" x P-450 (Fe 2 + ) l s ±e~

Using this approach an effective redox equilibrium constant Kbets derived R

eft =

[P-450(Fe 2 + ) h s ] +

[P-450(Fe 2 + )i s ]

[P-450(Fe ) ] +

[P-450(Fe 3 + ) l s ]

b

3+ hs

can be

A further simplification can be introduced due to the fact that the ferrous cytochrome P-450 obviously exists only in the high-spin state (8 = 2) as demonstrated by several experimental approaches (Mossbauer spectroscopy, SHARROCK et al., 1 9 7 3 ; magnetic susceptibility, CHAMPION et al., 1 9 7 5 ; magnetic circular dichroism spectra, DAWSON et al., 1 9 7 8 ) . The omission of the P-450 (Fe 2 + ) l s term in equation (15) leads to [P-450(Fe 2 + ) h s ] [P-450(Fe 3 + ) h s ] + [P-450(Fe 3 + ) l s ]

=

=

XBHS

K

0

—

(17)

= Kh^oc

(18)

where « represents the high-spin fraction of the ferric cytochrome P-450, a = [P-450 (Fe 3 + ) h s ]/[P-450 (Fe 3 + ) h s ] + [P-450 (Fe 3 + ) l s ], The redox potential of cytochrome P-450 can now be calculated by use of the Nernst equation after introduction of the derived relationships HT 111* RT Em = — In Khe" = — In Kb** + — In « T

T

T

(19)

A linear dependence of the redox potential Em on the logarithm of the high-spin fraction R O H + H 2 0 + NADP+

(24)

- > 2 0 2 - + NADP+ + H+

(25 a)

- » HOOH + NADP+

(25 b)

2 H20 + 2NADP

+

ROH + XOH

(25 c) (26)

The oxidase reactions can be differentiated according to the number of electrons transferred to oxygen, resulting in the formation of either superoxide anion (1 electron, equation 25a), hydrogen peroxide (2 electrons, equation 25b) or water (4 electrons, equation 25c). The possibility that cytochrome P-450 can function as a peroxidase (equation 26) was initially proposed by H R Y C A Y and O ' B R I E N (1972) and has been supported by the detection of a free radical species formed on interaction of cumene hydroperoxide with cytochrome P-450, comparable to that observed for compound I of peroxidase or the higher valence state of iron for hemoglobin and myoglobin ( R A H I M T U L A et al., 1974). This so-called shunt mechanism of cytochrome P-450 is of no physiological importance. Nothing is known about its regulation except that cytochrome P-420 exhibits no peroxidase activity. Taking into account potential biological and particularly toxicological implications of reactive oxygen metabolites, the oxidase activity of cytochrome P-450 deserves special consideration with regard to regulation mechanisms. In considering what factors regulate the oxidase and the monooxygenase pathways three main questions must be answered (for more details see Chapter 4): (1) By which mechanism is the formation of the activated oxygen species determined? (2) What role does the substrate play in regulating both pathways? (3) How much does isozyme specificity influence substrate control? In 1957 G I L L E T T E et al. observed the formation of H 2 0 2 by rabbit liver microsomes during demethylation of monomethyl-4-aminoantipyrine in the presence of N A D P H . This finding led to efforts to explain the discrepancy between the N A D P H oxidized on the one hand and the sum of oxygen consumed and product formed on the other, and so to clarify the mechanism for generation of hydrogen peroxide during N A D P H oxidation. An understanding of this mechanism required the identification of which activated oxygen species produced the hydrogen peroxide. The detection of a ferrous oxygenated cytochrome P-450 complex ( I S H I M U R A et al., 1971; T Y S O N et al., 1972; E S T A B R O O K et al., 1971) stimulated further research on this point. Experimental results from several groups yielded two differing conclusions. The main difference between these lay in whether the superoxide anion was assumed to act

Regulation Mechanisms of Cytochrome P-450

27

as one-electron reduced source for the generation of the hydrogen peroxide (ESTABROOK a n d WERRINGLOER, 1 9 7 7 ; KUTHAN a n d ULLRICH, 1 9 8 2 ) o r t h e

second electron reduction step occurred with conversion of the ferrous dioxygen/ cytochrome P-450 complex to a peroxide anion complex of ferric cytochrome P - 4 5 0 (HEINEMEYER e t al., 1980).

Based on the measured stoichiometry of 0 2 ~ to H 2 0 2 (close to 2 : 1 in the absence of any substrate) and the finding that substrates can modify the rates of decomposition of the ferrous dioxygen complex of cytochrome P-450 and thereby the formation of H 2 0 2 via 0 2 , KUTHAN and ULLRICH (1982) drew the conclusion that protonation and subsequent release of a two-electron reduced oxygen species does not represent a significant pathway for the formation of H 2 0 2 . Due to dissociation and dismutation, the superoxide anion radical disproportionates according to equation (14) into hydrogen peroxide and oxygen 202- + 2 H + ^ H 2 0 2 + 02

(27)

Several authors have proved either the influence of the substrate on the generation of activated oxygen species or the importance of isozymes in this process by investigating reconstituted systems (NORDBLOM and COON, 1977; MORGAN e t a l . , 1 9 8 2 ; OPRIAN e t a l . , 1 9 8 3 ; GORSKY e t a l . , 1 9 8 4 ) . T h e s e s t u d i e s

revealed that in the absence of substrates that can be hydroxylated different isozymes generate different amounts of H 2 0 2 . The relation between the oxidase and the monooxygenase pathways in an individual isozyme is further regulated by the respective substrate. What structural or physicochemical peculiarities of the substrate or the isozyme are decisive in determining which pathway the reaction follows is unknown as yet. A recent paper by PARKINSON et al. (1986) on an induced uncoupling by alkylation of Cys-292 by means of 2-bromo-4'nitroacetophenone in cytochrome P-450 c suggests a conceivable mechanism by which a substrate may regulate different pathways. In addition to the formation of H 2 0 2 there exists a four-electron reduction pathway leading to water. In the presence of pseudo substrates (perfluoroalkanes) which have been shown not to be hydroxylated (STAUDT et al., 1974) the formation of H 2 0 2 was not enhanced. Instead, on the basis of a ratio of NADPH oxidation to oxygen consumption of 2:1, water was assumed to be the final reduction product of dioxygen via the oxidatic pathway. From studies on microsomes without exogenous substrates and a ratio NADPH oxidized/ H202

g e n e r a t e d g r e a t e r t h a n 2 , ZHUKOV a n d ARCHAKOV ( 1 9 8 2 )

interpreted

their results likewise as four-electron reduction of 0 2 to H 2 0. Irrespective of isozyme specific H 2 0 2 generation, GORSKY et al. (1984) observed an increased NADPH/0 2 ratio with a stoichiometry of about 2 in accordance with the proposed water formation. OPRIAN et al. (1983) examined the dependence of water formation on the ratio P-450/oxygen. At a four-fold molar excess of cytochrome P-450 LM4 over oxygen, formation of water occurs, whereas at a two-fold molar excess of P-450 LM4 over oxygen, H 2 0 2 is formed, indicating an increased NADPH consumption per oxygen under oxygen deficiency. To what

28

K . RIJCKPAUL; H. REIN

extent t h a t pathway accounts for the consumption of cellular energy remains an open question. Proof t h a t water is the product requires confirmation b y experiments with 1 8 0 2 . The toxicological relevance of such oxygen activating pathways has been shown recently by GOND EE et al. (1985). These authors studied the susceptibility of responsive and non-responsive mice towards oxygen radicals and H 2 0 2 in the presence of high oxygen pressure. Such strains which are susceptible to hepatic microsomal enzyme induction by aromatic hydrocarbons were more sensitive to toxic effects of 100% oxygen exposure t h a n were genetically unresponsive mice, despite the fact t h a t high oxygen concentrations also induce antioxidant enzymes such as catalase and superoxide dismutase in several species. Non-responsive mice survived significantly longer with less lung damage. The authors concluded t h a t such genetic differences in susceptibility to oxidative stress may have long-term implications in therapeutics and patient care in humans. The capacity of cytochrome P-450 to convert not only a great variety of compounds via detoxifying and toxifying pathways b u t also to produce toxic activated oxygen species via alternate pathways, demands réévaluation of t h a t functional versatility.

3.

Regulation at the membrane level

The polysubstrate monooxygenase system of the mammalian liver cell is located in the endoplasmic reticulum. The successful reconstitution of a n enzymically active monooxygenase system (Lu et al., 1969a, b ; L u and LEVIN, 1974) showed t h a t phospholipids are required for the catalytic activity (STROBEL et al., 1970). The structural peculiarities of the endoplasmic reticulum therefore will exert a functional control upon the enzyme system (INGELMAN-SUNDBERG, 1 9 8 6 ) .

3.1. Lipid composition and protein constituents of the endoplasmic reticulum The endoplasmic reticulum is composed of a variety of lipid species. About 55% PC, 2 0 - 2 5 % PE, 5 - 1 0 % PS and PI, respectively, and 4 - 7 % sphingomyelin are the main constituents ( D E P I E R R E and DALLNER, 1 9 7 5 ) . The f a t t y acid content comprises mainly the 16:0, 16:1, 18:0, 18:1, 18:2, 20:4, 22:6 compounds (LEE and SNYDER, 1973), where the first number refers to t h e number of carbon atoms and the second to the number of double bonds in the f a t t y acid. The structural organization of the membrane lipids provides a highly ordered matrix for the incorporation of proteins. I n general, the solubility of lipids in water is low. Therefore they tend to associate into supramolecular aggregates, the critical micelle formation concentration (CMC) lying in t h e

Regulation Mechanisms of Cytochrome P - 4 5 0

29

micromolar range. The association and its specificities depend on the chain lengths of the fatty acids. About 60—70% by weight of the endoplasmic reticulum consists of protein and 30—40% of phospholipid. The protein moiety is composed of about 10% cytochrome P-450 which increases to about 2 0 % after phénobarbital induction. The cytochrome P-450/lipid molar ratio amounts to about 1:200 to 1:120 (DEPIERRE

a n d DALLNER,

1 9 7 5 ; D E P I E R R E a n d ERNSTER,

1975,

1977).

The

stoichiometry of reductase and cytochrome P-450 was determined with a molar ratio of 1 : 1 5 independent of induction ( S H E P H A R D et al., 1 9 8 3 ) . Both proteins are attached to the membrane of the endoplasmic reticulum by hydrophobic anchors which comprise 1 0 % of the flavoprotein molecule ( B L A C K et al., 1 9 7 9 ) and one or two transmembranal segments of the cytochrome P - 4 5 0 molecule (SAKAGUCHI et al., 1 9 8 1 ; D E L E M O S - O H I A R A N D I N I et al., 1 9 8 7 ; N E L S O N and S T R O B E L , 1 9 8 8 ) contrary to previous suggestions assuming at least 8 transmembranal segments ( B A R - N U N et al., 1 9 8 0 ) . With respect to the lateral distribution of both proteins within the membrane bilayer, a macroscopic asymmetry has been excluded ( S C H U L Z E and S T A U DINGER, 1 9 7 5 ) . The formation of domains of cooperating proteins, however, was proved ( S C H U L Z E et al., 1 9 7 2 ) . B y means of ferritin-labelled antibodies, a cluster-like association of cytochrome P - 4 5 0 ( M A T S U U R A et al., 1 9 7 8 ) and of reductase (MORIMOTO et al., 1 9 7 6 ) within microsomes has been detected. Generally a heterogeneous distribution of the membrane proteins across the cytoplasmic surface of the membrane must be assumed (MORIMOTO et al., 1 9 7 6 ; D E P I E R R E a n d E R N S T E R , 1 9 7 7 ; MATSUURA e t a l . ,

3.2.

1978).

Membrane fluidity and mobility of membrane components

The interaction of the components of the monooxygenase system depends strongly on the fluidity of the membrane matrix, which is characterized by a phase transition from the gel state into the fluid liquid-crystalline state of the constituting lipids. This process is characterized by the phase transition temperature Tc, which increases with increasing chain length of the fatty acid and with its degree of saturation (CHAPMAN, 1 9 7 5 ; L I G H T E N BERG et al., 1 9 8 3 ) . The functional significance of unsaturated fatty acids results from their ability to decrease Tc and thus increase the membrane fluidity. The constitutive membrane lipids of the monooxygenase system are mainly in the fluid state at the mammalian physiological temperature, and calorinietric investigations have proved the absence of any phase transition in that range ( M A B R E Y et al., 1977).

The mobility of the membrane lipids is rather high. The lateral diffusion coefficients Dh are 10" 7 — 10~ 9 cm 2 s _ 1 (Kuo and WADE, 1979). Data for reconstituted vesicles irrespective of their cytochrome P-450 or reductase loading are within this range ( S C H W A R Z et al., 1984). The transversal exchange in phospholipid bilayers, on the other hand, proceeds on a time scale of minutes

30

K . RUCKPATTL; H . R E I N

or even days (ROTHMAN and DAWIDOWICZ, 1975). The latter changes can therefore be eliminated as being rate-determining in the catalytic process of the enzyme system, even though the incorporation of cytochrome P - 4 5 0 enhances the transversal lipid transfer (BOSTERLING and TRUDELL, 1982 a ; BARSUKOV e t a l . , 1 9 8 2 ) .

The lateral diffusion of the membrane proteins is likewise rapid. I n DMPCreconstituted vesicles the diffusion coefficient of cytochrome P - 4 5 0 at 25 °C amounts to Z)L = 10" 8 cm 2 s - 1 , and similar results were obtained with egg yolk PC (Wu and YANG, 1984). These data are consistent with the respective parameters of other membrane proteins (CHERRY, 1979). A local lateral diffusion coefficient £>l 1oc of 10~ 9 cm 2 s _ 1 has been determined b y means of rotation investigations for cytochrome P - 4 5 0 and a cytochrome P-450/reductase complex in reconstituted lipid vesicles (GUT et al., 1982; KAWATO et al., 1982). These data have been used to derive a collision rate of the electron exchange proteins within vesicular systems of around 10 3 s 1 (KAWATO et al., 1982; W u and YANG, 1984). Since the electron transfer from the cytochrome P - 4 5 0 reductase towards cytochrome P - 4 5 0 proceeds within seconds (DUPPEL and ULLRICH, 1 9 7 6 ; PETERSON e t al., 1 9 7 6 ; TANIGUCHI e t al., 1 9 7 9 ) , a diffusion l i m i t a t i o n of

t h a t reaction seems rather unlikely, though the entropy factor may decrease the effective collision rate by orders of magnitude, as is usual for macromolecules. A limitation is obvious from studies on cytochrome P - 4 5 0 reduction in D M P C - r e c o n s t i t u t e d s y s t e m s (TANIGUCHI et al., 1 9 8 0 ; SMETTAN e t al., 1 9 8 4 ) .

Below the phase-transition temperature (23°C), the rate constant of the rapid cytochrome P - 4 5 0 reduction is significantly decreased, in correspondence with a decrease in the lateral diffusion coefficient of more than one order of magnitude (Wu and YANG, 1984). Microsomal incorporation or removal of cholesterol by means of vesicle exchange techniques (ARCHAKOV et al., 1983), on the other hand, showed that the observed changes in viscosity of the membrane did not alter the characteristics of the NADPH-dependent cytochrome P - 4 5 0 reduction, whereas the NADH-dependent reduction of cytochrome b 5 was significantly affected. The rotational diffusion of the microsomal proteins indicates the formation of cytochrome P - 4 5 0 oligomers and of complex formation with reductase as well, which would facilitate the electron transfer process (PETERSON et al., 1976). First investigations by transient dichroism methods (RICHTER et al., 1979; MCINTOSH et al., 1980), or with delayed fluorescence polarization techniques (GREINERT et al., 1979), indicated a significant rotational mobility of cytochrome P - 4 5 0 in both microsomes and in reconstituted proteoliposomes. Studies on the absorption anisotropy after flash photolysis of the CO complex of cytochrome P - 4 5 0 yielded a mean rotational relaxation time for rat liver microsomes of 120 ¡J.S at 20 °C, and for PC/PE/PS-(orPA-)reconstituted vesicles of 95 ¡AS (KAWATO et al., 1982). W i t h decreasing lipid/protein ratio, cytochrome P - 4 5 0 was gradually immobilized. Coreconstitution of equimolar amounts of cytochrome P - 4 5 0 and reductase resulted in a significant decrease in the relaxation time towards 40 ¡j.s. This value has been taken to indicate the for-

Regulation Mechanisms of Cytochrome P-450

31

mation of a monomolecular 1:1 complex between these proteins (GUT et al., 1982). Similar measurements using eosin isothiocyanate covalently bound to rabbit liver cytochrome P-450 LM2 incorporated into PC/PE/PA-reconstituted vesicles yielded a rotational relaxation time of 1 1 0 ¡AS at 2 5 °C (GREINERT et al., 1979). Using eosin maleimide and rat liver cytochrome P-450, similar values of 5 0 — 1 0 0 [xs were determined (KAWATO et al., 1 9 8 2 ) . Thus there is reasonable agreement with respect to different species and different methods which indicates the formation of both cytochrome P-450 aggregates and cytochrome P-450/reductase complexes. Similar characteristics of the protein association in membranes have been determined by applying saturation transfer E P R . Labelling of sulfhydryl groups of rabbit liver microsomal cytochrome P-450 with a spin-labelled maleimide revealed an effective rotational correlation time of 480 as at 20 °C (SCHWARZ et al., 1982b). The corresponding value for the rotation of cytochrome P-450 LM2 in reconstituted vesicles was 180 ¡JLS (SCHWARZ et al., 1982a). The correspondence of the data is evident, while differences may be due to varying experimental conditions. The association of the membrane proteins depends not only on the lipid/ protein ratio but also on the conformational state and the degree of immersion of the proteins in the membrane. Thus substrate binding increases the rotation time of cytochrome P - 4 5 0 , while reduction leads to a decrease ( G R E I N E R T et al., 1982 a, b). In general, the relatively high values of the cytochrome P-450 rotation time indicate a significantly lower protein mobility in the lipid matrix than in aqueous solution, which reflects not only the increased viscosity of the medium but also a protein association. For cytochrome P-450 LM2 in buffer solution a correlation time of 220 ns has been determined (SCHWARZ et al., 1982b). As compared to a theoretical value of the monomer in water solution of 21 ns the tenfold increase is consistent with a hexameric aggregate of cytochrome P-450 LM2, as supported by different methods (COON et al., 1976). Considering the increased viscosity of the lipid membrane, a correlation time for the monomer of 21 [zs was calculated, consistent with corresponding data for other membrane proteins (Cherry, 1979). On the basis of the experimental data of 50—100 ¡AS for cytochrome P-450 in microsomes and reconstituted proteoliposomes, an association of 6—12 molecules into clusters is suggested to represent the structural basis of cytochrome P-450 function in vesicles.

3.3.

Structural peculiarities of protein/lipid interactions

The structural features of the membrane-bound cytochrome P-450 system constitute a variety of intermolecular interaction facilities, which allow a multitude of regulation mechanisms on a molecular level. Whereas the protein/protein interaction mainly concerns reactions between monomeric species but modified by protein/protein association, the protein/lipid interaction is mainly based on

32

K . RTTCKPAUL; H . REIN

supramolecular lipid structures which are formed above the CMC. That refers to biological cytochrome P-450 systems in the endoplasmic reticulum or its biochemical equivalent, the microsome, and equally to reconstituted vesicle systems. DLPC, on the other hand, which is most frequently used in reconstitution experiments, was shown to be active already below the C M C (MIWA and Lu, 1981). Moreover, this lipid forms vesicles above the CMC as shown by electron microscopy ( H A U S E R and BARRATT, 1 9 7 3 ) , but no vesicle formation was observed for the protein-containing reconstituted system (AUTOR et al., 1973). Reconstitution experiments with this lipid, therefore, should contribute to discriminate molecular and supramolecular lipid effects. First insights into the mechanism of cytochrome P-450/lipid interactions were obtained by various spectroscopic techniques. CHIANG and COON ( 1 9 7 9 ) observed that in the presence of phospholipids the ellipticity of the peptide chromophore of cytochrome P-450 in the far UV region increases, indicating an increase in the helical content of the protein. Derivative spectroscopic analyses revealed that both phospholipids and detergents provoke a tyrosine signal on interaction with cytochrome P - 4 5 0 (RITCKPAUL et al., 1 9 8 0 ) . In contrast, other amphiphilic or hydrophobic compounds such as fatty acids and alkanes, did not elicit that signal. The spectral changes can be explained by an increase of the nonpolar character of the immediate environment of the tyrosine residues. This may be caused by either a conformational change of the protein or by a change in its surrounding milieu, thus influencing tyrosine residues at the surface of the macromolecule. The latter seems more likely because of the relatively low specificity of the effect, as well as the absence of a signal in the presence of the nonmicellar partners of interaction. The investigations by CHIANG and COON ( 1 9 7 9 ) , however, and measurements by ARCHAKOV (1982) point to at least some additional conformational changes of cytochrome P - 4 5 0 in the interaction with lipids. CHIANG and COON ( 1 9 7 9 ) further observed that in the near U V region (Soret band) in the presence of DLPC a cytochrome P-450 difference spectrum is formed, which indicates a shift towards the high spin state of the cytochrome P-450 spin equilibrium. A similar result could be seen for partially purified solubilized rat cytochrome P-450 which on addition of a microsomal lipid extract exhibited a spin shift from 17% to 5 3 % highspin content at 20 °C; the addition of oleic acid resulted in a shift towards 4 0 — 4 5 % high-spin (GIBSON et al., 1 9 8 0 ) . The incorporation of cytochrome P-450 LM2 into preformed vesicles similarly provoked a high-spin shift which was favoured by negatively charged lipids ( R U C K P A U L et al., 1 9 8 2 ) . Triton N-101 and Tween 20 proved unable to effect that conversion. Likewise proteoliposomes, obtained by cholate gel filtration (SCHWARZE et al., 1 9 8 5 ) or octylglucoside dialysis (PETZOLD et al., 1 9 8 5 ) exhibited the lipid-induced spin shift. The interaction of cytochrome P-450 with substrates or reductase also results in a high-spin shift of the spin equilibrium. B y means of this common effect, the mutual interaction of substrates, reductase, and lipid can be analysed and even quantitated ( F R E N C H et al., 1 9 8 0 ) . The binding of D L P C to cytochrome

Regulation Mechanisms of Cytochrome P-450

33

P-450 LM2 exhibits biphasic binding characteristics, with an apparent dissociation constant for the high-affinity site of 3 — 6 ¡xM, and of the low-affinitv site of 50—70 [xM. Benzphetamine or reductase addition favoured the lipid binding. The lipid, on the other hand, increased the benzphetamine and reductase affinities towards cytochrome P-450. The apparent K u = 180 (j.M for benzphetamine in the presence of DLPC was decreased on addition of reductase to 110 ¡AM, and likewise the respective reductase parameter KD = 115 [J.M was decreased to 42 (JLM. Thus the results indicate a mutually favoured binding to cytochrome P-450. These findings provide evidence that conformational and reactivity variations are induced in cytochrome P-450 on interaction with lipids. On the other hand, rather less is known about the interaction of lipids with the reductase. The likely reason is that only the hydrophobic portion of the flavoprotein (6.1 kDa) would be directly involved, whereas communication to the hydrophilic head ( 7 1 . 2 kDa) ( B L A C K et al., 1 9 7 9 ; B L A C K and COON, 1 9 8 2 ) and thus to the main part of the molecule, would be less affected. The corresponding interaction could therefore lie below the limit of detection. Nevertheless, by means of CD measurements in the 200—300 nm range, an increase in the helical content of the protein from 28% without lipid up to 41% on interaction with lipid has recently been observed; DLPC, D L P E , DLPS proved less effective, but DMPC and DPPC were highly active (MAGDALOU et al., 1 9 8 5 ) .

3.4.

Functional aspects of the component interactions in the membrane

The results discussed so far refer mainly to structural alterations of the protein moiety in interaction with lipids. The functional implications and the mechanisms of these protein/protein- and protein/phospholipid-interactions will be dealt with now. First of all, the successful reconstitution of the enzyme system proved the requirement for phospholipid to produce the functional activity, especially under physiological stoichiometry of the components (Lu and COON, 1 9 6 8 ; S T R O B E L et al., 1 9 7 0 ; Lu and W E S T , 1 9 7 2 ) . The extraction of lipids by organic solvents, on the other hand, decreased the catalytic activity of cytochrome P - 4 5 0 ( V O R E et al., 1 9 7 4 ) . The investigations showed a correlation between catalytic activity and the NADPH-dependent cytochrome P-450 reduction in that the rate of electron transfer decreased on delipidation, due to an alteration in the contribution of the fast and slow partial reaction to the biphasic process. Further correlations between cytochrome P-450 reduction and substrate conversion or NADPH oxidation ( I M A I et al., 1 9 7 7 ; I Y A N A G I et al., 1981) suggest an important regulatory role in catalysis for the first electron transfer process. The second electron transfer reaction or even a step beyond further controls the catalytic activity of the enzyme system. It is generally accepted that late steps in the catalytic cycle represent the rate limitation (MATSUBARA e t a l . , 1 9 7 6 ; I M A I e t a l . , 1 9 7 7 ; ISHIMURA, 1 9 7 8 ; TANIGUCHI e t a l . ,

1979; WHITE

34

and

COON,

1 9 8 0 ) . The cytochrome P - 4 5 0 substrate binding

K . RUCKPAUL; H . R E I N

reaction as well as t h e 0 2 binding process, on t h e other hand, h a v e been shown t o be not rate-limiting ( B L A N C K et al., 1 9 7 6 ; I M A I et al., 1 9 7 7 ) . The regulation of t h e catalytic function b y b o t h electron transfer steps obviously depends on p H . A r a t e limitation by t h e first step a t low p H changes t o t h e second transfer with increasing p H ( W E R R I N G L O E R and K A W A N O , 1980b; RUF and EICHINGER, 1982). Studies on protein/lipid interaction in electron transfer t h u s have already to start with studying t h e first electron transfer and its regulation per se. This step m a y f u r t h e r reflect common structural features with respect t o both transfer processes. 3.4.1.

Characteristics of the cytochrome

P-450

reduction

The general p a t t e r n of cytochrome P-450 reduction in t h e first electron transfer process is of a biphasic reaction, composed of two independent first-order partial reactions. This was observed in early investigations on microsomes, in solubilized systems, and in reconstituted systems (reviewed b y B L A N C K et al., 1984a). The lipid component can be effectively s u b s t i t u t e d b y detergents (LIT et al., 1974a; I N G E L M A N - S U N D B E R G , 1977a); these systems also exhibit biphasic kinetics in t h e cytochrome P-450 reduction process (TANIGUCHI et al., 1979; R U C K P A U L et al., 1982). Although as m a n y as four partial reactions could be observed a t r a t microsomes (RUF, 1980), most studies of cytochrome P-450 reduction have used a biphasic approximation. I n microsomes about 50—70% of t h e total cytochrome P-450 is reduced in t h e rapid partial reaction under anaerobic conditions. The slow p a r t i a l reaction differs in its specific r a t e c o n s t a n t b y a factor 10—20 (GILLETTE et al., 1973; M A T S U B A R A et al., 1976; P E T E R S O N et al., 1976; B L A N C K et al., 1979a). T h e fast reaction obviously represents t h e physiologically relevant process. The relative a m o u n t s of t h e two partial reactions differ depending on t h e stoichiom e t r y of t h e constituting components, on t h e substrate, on t h e aggregation state of t h e system, and on t h e presence or absence of oxygen. The biphasic reaction p a t t e r n of t h e N A D P H - d e p e n d e n t cytochrome P-450 reduction may be described b y 4 interaction models (Fig. 6): 1. The electron transfer between reductase and cytochrome P-450 proceeds via statistical collision of t h e randomly distributed proteins. T h e increase in r a t e constant with increasing relative reductase content a n d decreasing relative lipid a m o u n t is t a k e n to indicate r a t e limitation b y t h e lateral mobility of t h e membrane. The biphasic characteristics of cytochrome P-450 reduction, however, is not explained by this r a n d o m distribution model ( Y A N G , 1975, 1977; D U P P E L and U L L R I C H , 1976; Y A N G et al., 1977, 1978). 2 . A more likely interpretation of t h e cytochrome P - 4 5 0 specificity is provided b y t h e cluster model ( F R A N K L I N and E S T A B R O O K , 1 9 7 1 ; S T I E R and S A C K MANN, 1 9 7 3 ; P E T E R S O N et al., 1 9 7 6 ; S T I E R , 1 9 7 6 ) . According t o this concept cytochrome P - 4 5 0 is assumed t o homologously associate for t h e m a i n p a r t and moreover to be capable of a f u r t h e r heterologous association with reductase. The cytochrome P-450/reductase clusters formed would t h u s

Regulation Mechanisms of Cytochrome P-450

35

Interaction model

Random distribution

Cluster formation

Redox state control

Spin state control

Specificity

•• • • •o • • o• ©loo oo oo o o®o o oo oo •• ••

oo oo

P : localization

P : cluster random

R : high potential low potential

P : high spin low spin

Fig. 6. Model approaches of the reductase/P-450 interaction in the first electron transfer process (P-450 reduction). (Abbreviations cf. p. 37).

represent a translation-free electron transfer system with a more favourable cytochrome P-450 reduction than with a randomly distributed portion of cytochrome P-450, which, being structurally impaired, would exhibit the slow partial reaction. Alternatively, reductase-free cytochrome P-450 associates (rotamers) are proposed to interact with randomly distributed reductase and the biphasic reaction characteristics is suggested to be an expression of negative kinetic cooperativity in the cytochrome P-450 oligomers (GREINERT et al., 1982a, b; STIER et al., 1982). An additional reductase association at the microsomal stoichiometrv is open as yet. At stoichiometric excess of reductase, on the other hand, a dissociation of the cytochrome P-450 associates and the formation of 1 : 1 binary complexes between both proteins could be evidenced (GUT et al., 1982). 3. A differentiation of the cytochrome P-450 reduction by reaction with different redox states of the reductase is further proposed (OPRIAX et al., 1979). This redox state model is based on the dependence of the phase distribution of the reduction process on the N A D P H concentration. This model, however, must be questioned because of the maintained biphasicity of the reaction in the presence of excess NADPH and reductase. 4. The sequential spin state model considers a specific reactivity of the highspin and the low-spin component of the cytochrome P-450 population. The high-spin fraction is assumed to exhibit the rapid partial reaction, the lowspin fraction, on the other hand, is considered to be reduced via a ratelimiting relaxation into the high-spin state conformation (BACKES et al.,

36

K . RUCKPAUL; H . R E I N

1980, 1 9 8 2 ; TAMBURINI e t al., 1984). T h e m e a s u r e m e n t of t h e r e d u c t i o n

kinetics in the high-spin band at 650 nm directly proved the favoured electron transfer towards that component (BACKES et al., 1985). However, the relaxation of the spin equilibrium has been measured to proceed in the nanosecond range (ZIEGLER et al., 1982, 1983), thus excluding rate limitation by this step. In consequence, an additional consecutive conformational change, being spectrally inactive, is proposed to represent the rate-limiting step in the sequential spin state model (TAMBURINI et al., 1984). For further treatment of the cytochrome P-450 reduction at the membrane level a threefold control by — substrates via the spin equilibrium, — reductase interaction, — phospholipid interaction, has been differentiated. Substrate control represents a rather molecular phenomenon with respect to cytochrome P-450. This regulation therefore has been discussed in section 2 of this chapter. Main topics of the following treatment are thus the intermolecular component interactions of the cytochrome P-450 system. 3.4.2. Regulation of the electron transfer by reductase/cytochrome P-450 interaction The reductase/cytochrome P-450 interaction in general is determined by the stoichiometry of both proteins, which in microsomes is about one order of magnitude in favour of cytochrome P-450 (P/R 1 > 10). Thus the reductase must cycle throughout the cytochrome P-450 population because of an as yet unproved intermolecular electron transfer between cytochrome P-450 molecules. Consequently the reductase would represent the "enzyme" for the "substrate" cytochrome P-450 in the reaction. This implies a Michaelis-Menten type mechanism. B y means of detergent disintegration of the enzyme system and variation of the R / P stoichiometry, a monophasic first order process has been observed (BLANCK et al., 1 9 7 9 a , b ; ROHDE et al., 1983)

Rred + Pox

x

^

X

RredPox

7 Rox^red

x

^

X

Rox + Pred

(28)

in which the formation of the donor/acceptor complex R re dP 0 x is followed by the rate-limiting electron exchange (k) and the dissociation of the produccomplex RoxPrcd- The rate of electron transfer may be regulated by intramolecular transfer steps in either protein, or by the intermolecular transfer. 1

Abbreviations used in section 3.4: P : cytochrome P-450; R : NADPH-dependent cytochrome P-450 reductase; R P : complex between R and P (molar ratio: 1 : 1 ) ; P o x , P r e i j, R o x , R re .

3.0

1.0

0 Fig. 7. Anaerobic NADPH-dependent P-450 LM2 reduction in reconstituted vesicles. Dependence of the rate constant k a p p of the rapid partial reaction on the protein stoichiometry R / P e ( ( . (For details cf. BLANCK et al., 1984b).

With reconstituted cytochrome P-450 LM2 systems, evidence for the same mechanism has been obtained. The rapid partial reaction of the biphasic firstorder cytochrome P-450 reduction process which would predominate in the catalytic process, exhibited a corresponding stoichiometric profile with clear saturation characteristics (BLANCK et al., 1984b). Kinetic titration of the RPcomplex formation within liposomes prepared by use of a microsomal lipid mixture (MIC) revealed a dissociation constant K w = 0.048 [J.M in the presence of benzphetamine (Fig. 7). 3.4.3. Phospholipid control of electron transfer

With regard to the phospholipid specificity of cytochrome P-450 reduction, similar characteristics were observed with different liposomal preparations. The reaction kinetics depend on the specific lipid, with a dissociation constant KRp = 0.47 |AM for DOPC and KRP = 0.051 ¡xM for a 3:1 (w:w) mixture of DOPE/PS. This indicated that negatively charged lipids favour the formation of the electron exchange complex (Fig. 7) (BLANCK et al., 1984b).

38

K. RUCKPAUL; H. REIN

A complex formation between the two proteins in the presence of DLPC can be directly observed by difference spectroscopy with dissociation constants KKp = 0.12 (xM in the absence of substrate and KRI, = 0.04 ¡xM in the presence of benzphetamine ( F R E N C H et al., 1 9 8 0 ) . The rate constant k, on the other hand, turned out to be independent of the lipid species ( B L A N C K et al., 1984b). This result agrees with the observation of unchanged redox potentials of cytochrome P-450 in the presence of different phospholipids ( G U E N G E R I C H et al., 1975 ; B Â C K S T R Ô M et al., 1983 ; G U E N G E R I C H , 1983).

80 %

| 60 Ss W

/*

/ /

IHIC

/

'

A *

20

0

/

A

\

I DOPE/PS J-

/

'

IDOPC

1.0

R/P

-

2.0

Pig. 8. Anaerobic NADPH-dependent P-450 LM2 reduction in reconstituted vesicles Extent of the rapid partial reaction g>1 (%) on the protein stoichiometry R / P . (For details cf. BLANCK: et al., 1984b).

The regulation of cytochrome P-450 reduction by phospholipids is further reflected in the specificity of the fractional distribution of both partial reactions. With increasing acidity of the headgroups of the lipids, as indicated by the increased electrophoretic mobility of the vesicles ( I N G E L M A N - S U N D B E R G et al., 1983), the physiologically relevant rapid cytochrome P-450 reduction process is favoured ( I N G E L M A N - S U N D B E R G et al., 1983; B L A N C K et al., 1984b) (Fig. 8). Thus the role of phospholipid may be further understood as a control of the functional association of cytochrome P-450 molecules to form cluster-like structures as a structural basis for efficient electron transfer. The charge dependence of the lipid control in the cytochrome P - 4 5 0 reduction process suggests charge-dependent electrostatic interactions between the electron exchanging proteins. Such interactions have been observed in the cytochrome b5 system ( L O V E R D E and S T R I T T M A T T E R , 1 9 6 8 ; D A I L E Y and S T R I T T M A T T E R , 1 9 7 9 ) , between cytochrome b 5 and cytochrome P - 4 5 0 ( J A N S -

Regulation Mechanisms of Cytochrome P-450

39

SON et al., 1985; T A M B U R I N I and S C H E N K M A N , 1986), and in the cytochrome P-450 system, as well ( B E R N H A R D T et al., 1983, 1984; M A K O W E R et al., 1984). Chemical modification of the N-terminal amino group and of a functionally linked lysine of cytochrome P-450 LM2 resulted in restricted electron transfer and catalytic activity. High ionic strength in general perturbs electron exchange ( B E R N H A R D T et al., 1984). A negatively charged vesicle surface may therefore interact with complementary groups on the cytochrome P-450 molecule or, on the other hand, on the reductase, as deduced for the membraneneighbouring part of the amino acid sequence which connects the hydrophilic head and the membrane-bound anchor of the molecule ( B L A C K and COON, 1982). These electrostatic interactions would improve recognition and ordering or improve alignment of the interacting proteins.

The endogenous interaction of reductase and cytochrome P-450 at physiological stochiometry obviously requires a further mode of protein association, with at least a homologous aggregation of cytochrome P-450 molecules to build up functional clusters. The distintegration of cytochrome P-450 systems by means of different detergents ( B L A N C K et al., 1979a; I N G E L M A N - S U N D B E R G , 1977b; D E A N and G R A Y , 1982; R O H D E et al., 1983; W A G N E R et al., 1984; D A V Y D O V et al., 1985) significantly decreases the electron transfer rate and catalytic activity. Homologous protein association by a predominantly electrostatic interaction seems less plausible. The control function of the lipid seen at the supramolecular level points to further regulation mechanisms. The phospholipid seems to provide the structural features for protein association into functional clusters which leads to the formation of a catalytically active reductase/ cytochrome P-450 1:1 complex. That control must provide — the correct local concentration of particular phospholipids to provide conditions for partitioning of cytochrome P-450, — the correct association of cytochrome P-450, based on lipid specific association constants, but with high mobility of the cytochrome P-450 molecules to allow rapid successive reductase/cytochrome P-450 interaction in an active 1:1 complex. Protein-induced phase separations of membrane lipids have been the subject of several investigations ( G R E I N E R T et al., 1982a; S T I E R et al., 1982; B A Y E R L et al., 1984, 1985, 1986). B y means of 31 P-NMR and photoselection techniques it was shown that a non-lamellar lipid phase — absent in protein-free liposomes — is formed in liver microsomes and reconstituted vesicles. This phase is suggested to be formed by the lamellophobic P E ( G R E I N E R T et al., 1 9 8 2 A ; S T I E R et al., 1 9 8 2 ) which, like P A and P S , forms the hexagonal form I I (LuzZATI et al., 1 9 6 8 ) if there is an unsaturated fatty acid located in position 2 of the glycerol. The wedgeshaped P E structure is proposed to adapt cytochrome P - 4 5 0 specifically to the bilayer membrane by a half-micelle structure of this lipid. Correlation of cytochrome P - 4 5 0 and the P E content in microsomes has been reported for the increased level of protein and lipid in male rats ( B E L I N A et al., 1 9 7 4 ) , in age dependence ( K A M A T A S K I et al., 1 9 8 1 ; S T I E R et al., 1 9 8 2 )

40

K . RUCKPAUL; H .

REIN

and on drug induction ( S T I E R et al., 1 9 8 2 ) . The high transverse molecular mobility ( V A N D E N B E S S E L A A R et al., 1 9 7 8 ) is suggested to depend on the perturbation of the endoplasmic reticulum bilayer membrane ( S T I E R et al., 1982).

The microsomal reductase participates in the cytochrome P - 4 5 0 / P E interaction. The isotropic 3 1 P-NMR signal disappears after trypsin treatment, and the lateral diffusion coefficient decreases in parallel from 10~ 7 cm 2 s" 1 to 10~ 8 cm 2 K 1 at 37°C ( B A Y E R L et al., 1984). The cleavage of the hydrophilic part of the protein molecule leaves a hydrophobic anchor part which evidently cannot generate the interaction signal. In further investigations by use of hexane phosphonic acid diethyl ester as a molecular 3 1 P-NMR probe the specific interaction of cytochrome P-450 and reductase with P E was confirmed ( B A Y E R L et al., 1985). Recently, by means of nonionic detergents, a preferential removal of PC over P E from microsomes without significant effects on the functional properties of the enzyme system at the PC solubilization level has lent further support to a specific protein/PE interaction ( B A Y E R L et al., 1986). The acidic phospholipid PA likewise appears to show a specific cytochrome P-450 interaction. B y means of the cholate dialysis technique vesicles of PC/ PA were reconstituted with cytochrome P-450 LM2. In the absence of the protein a phase separation of the lipid into domains of gel phase which contained enriched PA, and a surrounding fluid matrix of mainly PC was observed. The PA phase transition at Tc disappeared on cytochrome P-450 incorporation, indicating a preferential PA/cytochrome P-450 interaction ( B O S T E R L I N G et al., 1981). A specific PS/cytochrome P-450 interaction is not yet proven. The catalytic activity of reconstituted vesicles containing PS is significantly increased in comparison to egg yolk PC or DOPC systems ( I N G E L M A N - S U N D B E R G and J O H A N S SON, 1980b; I N G E L M A N - S U N D B E R G et al., 1981). The increase in catalytic activity of reconstituted vesicle systems with the acidity of the lipid headgroup would indicate the charge to be more significant than the lamellophobic molecule shape ( K I S S E L et al., 1979; I N G E L M A N - S U N D B E R G and J O H A N S S O N , 1980b; I N G E L M A N - S U N D B E R G et al., 1981).

3.4.4. Electron transfer and substrate conversion The cytochrome P-450 reduction reaction and the catalytic activity show similar dependences on spin equilibrium, R/P-stoichiometry, and lipid specificity. Quantitative relations between cytochrome P-450 reduction and substrate conversion V m a x have been derived recently ( S C H W A R Z E et al., 1985), and have been confirmed by several detailed investigations. The substrate conversion is preceded by the introduction of the second electron, which is widely accepted to represent the rate-limiting step in the overall reaction. The reaction scheme under anaerobic conditions may thus

Regulation Mechanisms of Cytochrome P-450

41

be approximated by R + P

RP

RP-

RP~

) R + P

(30)

That equation considers reductase R and cytochrome P to build up the 1:1 interaction complexes R P , R P " , R P which contain cytochrome P-450 up to a twofold reduced state. The approximation further proposes rapid kinetics with respect to the preequilibration step (K R ?) and the decay of the two-electron reduced complex R P to release products, H 2 0 , and oxidized protein (substrate, products, H 2 0 omitted for simplicity). It is further proposed that in excess of NADPH the electron transfer towards free reductase is not rate limiting. According to the scheme the reductase is cycling between R P and R P " thus leading to the steady state equation jfc, [RP] = (&_, + k2) [ R P ]

(31)

for the catalytic process. The substrate conversion rate F m a x is then given by F m a x = k2 [ R P " ] = k2

K_i -(- tC2

[RP]

(32)

This equation indicates a linear dependence of F m a x on the interaction complex R P , as shown for the apparent rate constant of cytochrome P-450 reduction, DP ( B L A N C K et al., 1984b). Therefore titrating R P in dependence on the ratio R / P by means of the substrate conversion rate F m a x must likewise reveal the dissociation constant K R P in the investigated system, as shown by D I G N A M and S T R O B E L (1977), V E R M I L I O N and COON (1978), M I W A et al. (1979), O P K I A N et al. (1979), F R E N C H et al. (1980), M I W A and L U (1981, 1984) and M U L L E R E N O C H et al. (1984). The importance of a proper structural association and alignment of the electron exchange proteins (reductase and cytochrome P-450) in the catalytic process has been recently supported by the isolation of a cytochrome P-450 monooxygenase in Bacillus megaterium which in a single polypeptide chain (119 kDa) contains 1 mol each of FAD and FMN per mol of heme. This selfsufficient monooxygenase exhibits the highest catalytic activity for a cytochrome P-450 system yet reported (4 600 nmol of fatty acid hydroxylated per nmol of cytochrome P-450 and min) ( N A R H I and F U L C O , 1986). The regulation of F m a x according to equation (32) depends on the rate-limiting constant k2 of the second electron transfer and on the steady state concentration of the one electron-reduced intermediate 1:1 complex of reductase and cytochrome P-450 (RP"). The quantity of that complex depends on the spin state of the cytochrome P-450 population through the rate constant of the first electron transfer klt on the rate constants k j and k2, and on the amount of the oxidized 1:1 complex of reductase and cytochrome P-450 (RP). The latter

42

K . RUCKPAUL; H . REIN

quantity is determined by the respective concentrations of reductase and cytochrome P-450 and by the lipid controlled dissociation constant of the complex. Thus equation (32) comprises the rate-limiting control of the second electron transfer as well as the spin state, the reductase/cytochrome P-450, and the phospholipid control of the first transfer reaction. For microsomes as well as for cytochrome P-450 LM2 reconstituted systems with a series of benzphetamine analogues, kx increases linearly with the high-spin amount A of cytochrome P - 4 5 0 (BLANCK et al., 1 9 8 3 ; SCHWARZE et al., 1 9 8 5 ) . The transformation of this spin state regulation towards F m a x is elucidated by the modified equation (33) F m a x = k2 [ R P ] = k2

k]

7°nSt\

(33)

kt was proposed to be the only rate constant which depends on the spin state of a stoichiometrically determined system. Because k1 > k2 the variable regulation term of F m a x amounts to k^/(k1 + k^), and the dependence of Fmax o n therefore must exhibit hyperbolic characteristics, a linear dependence at i , < k_i, and saturation behaviour at kt > k A. The respective pattern of the benzphetamine series indicated an approximate k, < system, that was supported by an independent proof of kl ~ k^ ( R U F and EICHINGER, 1 9 8 2 ) . Since the variable regulation term reflects the amount of the intermediate ternary complex of cytochrome P-450, substrate, and oxygen, that quantity must increase with k^ Experimental evidence for that relation has been pres e n t e d (IYANAGI e t a l . , 1 9 8 1 ) .

3.5.

Cytochrome b5 dependent regulation

The regulation of electron transfer and thus of the catalytic activity of cytochrome P-450 systems as treated so far involves the interaction of the three main components of the liver microsomal monooxygenase system. The cytochrome P-450 system in the endoplasmic reticulum, however, exhibits an additional interaction with the NADH-dependent cytochrome b 3 electron transfer chain, which may be involved to generate a synergistic enhancement of the enzymatic activity (CONNEY et al., 1957; NILSSON and JOHNSON, 1963; COHEN and ESTABROOK, 1971a, b, c; HILDEBRANDT and ESTABROOK, 1971; CORREIA and MANNERING, 1973a, b; ARCHAKOV and D E V I C H E N S K Y , 1975; ARCHAKOV et al., 1975a, b). Cytochrome b 5 contributes the second electron to the oxyferrous complex of some cytochrome P-450s, whereas the first electron is predominantly provided by the cytochrome P-450 reductase (WHITE and COON, 1980), as indicated by the facts that: — the NADPH-dependent reduction of microsomal cytochrome P-450 is not accelerated by addition of N A D H (HILDEBRANDT and ESTABROOK, 1971),

Regulation Mechanisms of Cytochrome P - 4 5 0

43

— t h e addition of cytochrome b 5 antibodies does not d i s t u r b t h a t reaction (NOSHERO a t al., 1980),

— t h e N A D H - d e p e n d e n t reduction of cytochrome P-450 is relatively slow ( ~ 1 0 % ) c o m p a r e d to t h e N A D P H transfer chain reaction ( A R C H A K O V et al., 1 9 7 5 a ; M I Y A K E et al., 1975). T h e d o n o r f u n c t i o n of cytochrome b 5 is not obligatory (Lu et al., 1974b, c; I M A I a n d S A T O , 1977). The protein exhibits a s u b s t r a t e specificity: cytochrome b 3 antibodies inhibit t h e monooxygenation of 7 - e t h o x y c o u m a r i n , benzo[a]pyrene a n d b e n z p h e t a m i n e to different e x t e n t s b u t do n o t inhibit t h e conversion of aniline (NOSHIRO et al., 1979). F u r t h e r m o r e , isozyme specificities were observed in reconstituted systems (JANSSON a n d SCHENKMAN, 1973, 1977; H R Y C A Y and E S T A B R O O K , 1974), a n d an obligatory r e q u i r e m e n t for a d i s t i n c t cytochrome P-450 isozyme could even be shown (SUGIYAMA et al., 1979, 1980; M I K I et al., 1980). T h e electron transfer between cytochrome b 5 a n d cytochrome P-450 has been t h e subject of several more recent investigations ( W E R R I N G L O E R a n d K A W A N O , 1980a; B O N F I L S et al., 1981; N O S H I R O et al., 1981; P O M P O N a n d COON, 1984), which enabled t h e integration of t h e cytochrome b 5 -dependent reactions into a general reaction scheme for t h e catalytic f u n c t i o n of t h e cytochrome P-450 system in t e r m s of regulation specificities. The kinetic studies so far lend f u r t h e r s u p p o r t to t h e rate-limiting role of t h e second elect r o n t r a n s f e r to t h e terminal protein, which m a y interfere with synergistic effects of t h e cytochrome b 5 t r a n s f e r chain. Of t h e proteins of t h e cytochrome b 5 chain, the FAD-containing cytochrome b 5 reductase ( S T R I T T M A T T E R a n d V E L I C K , 1 9 5 6 a, b) a n d cytochrome b 5 appear t o be r a n d o m l y distributed in t h e endoplasmic reticulum a n d to be functionally controlled b y translational diffusion in t h e two dimensional m e m b r a n e p l a n e ( R O G E R S a n d S T R I T T M A T T E R , 1 9 7 4 ) . T h e p e r t u r b a t i o n b y cholesterol of cytochrome b 5 reduction s u p p o r t s t h a t result ( A R C H A K O V et al., 1 9 8 3 ) . T h e r a n d o m lateral distribution of t h e proteins within t h e m e m b r a n e lipid allows t h e potential complexation of cytochrome b 5 with cytochrome P-450. I n t h e presence of D L P C a 1 : 1 complex is formed, as indicated b y a n increase in t h e high-spin c o n t e n t of cytochrome P - 4 5 0 L M 2 ( B O N F I L S et al., 1 9 8 1 ) . T h e a p p a r e n t dissociation c o n s t a n t K D is 2.3 ;xM, a n d decreases in t h e presence of b e n z p h e t a m i n e t o 0.4 fxM. C o m p l e m e n t a r y studies proved t h e stabilization of t h e s u b s t r a t e complex of cytochrome P-450 with b e n z p h e t a m i n e b y cytochrome b 5 , t h e respective dissociation c o n s t a n t s being 220 ¡XM a n d 50 ¡¿M. The investigations d e m o n s t r a t e d an obligatory role of lipid for m a x i m a l interaction of t h e c o m p o n e n t s a n d different binding sites on t h e c y t o c h r o m e P-450 molecule for c y t o c h r o m e b 5 a n d for b e n z p h e t a m i n e . The m u t u a l facilitation of t h e compounds t o w a r d s complex f o r m a t i o n parallels t h e corresponding observation for c y t o c h r o m e P - 4 5 0 ( F R E N C H et al., 1 9 8 0 ) . The results for t h e cytochrome P-450/cytochrome b 5 couple support conformational changes inducing interactions (HLAVICA, 1984): CD measurements in t h e far U V region revealed

44

K . RUCKPAUL; H . R E I N

concomitant conformational changes in one or both proteins, and. the dissociation constant determined was KB = 3.2 ¡xM. Further support for 1 : 1 complex formation has been presented ( I N G E L M A N - S U N D B E R G et al., 1980; C H I A N G , 1981; B E N D Z K O et al., 1982; B O S T E R L I N G and T R U D E L L , 1982b; V A T S I S et al., 1982; TAMBURINI a n d GIBSON, 1 9 8 3 ; INGELMAN-SUNDBERG a n d JOHANSSON, 1 9 8 0 a,

1984; T A N I G U C H I et al., 1984). Phospholipid is also required for an appropriate interaction to take place ( I N G E L M A N - S U N D B E R G and J O H A N S S O N , 1980a). Whereas binary complex formation between cytochrome b 5 and cytochrome P-450 is a prerequisite for an effective second electron transfer, the catalytic action, on the other hand, requires the additional involvement of the cytochrome P-450 reductase. The ternary cytochrome P-450 reductase/cytochrome P-450/cytochrome b 3 complex has been shown to be the catalytically active species, with the component in deficit being rate limiting ( I N G E L M A N - S U N D B E R G et al., 1980; I N G E L M A N - S U N D B E R G and J O H A N S S O N , 1984). The interactions of the proteins in and between the two electron transfer chains are obviously electrostatic in nature. They control the coupling of cytochrome b 3 reductase/cytochrome b 3 ( L O V E R D E and S T R I T T M A T T E R , 1 9 6 8 ; D A I L Y and S T R I T T M A T T E R , 1 9 7 9 ) , of cytochrome P - 4 5 0 reductase/cytochrome b 5 ( D A I L Y and S T R I T T M A T T E R , 1 9 8 0 ) , of cytochrome P-450/cytochrome b 3 ( T A M B U R I N I et al., 1 9 8 5 ) , and of cytochrome P - 4 5 0 reductase/cytochrome P - 4 5 0 (cf. above). B y means of specifically reconstituted bilayer systems from purified components of rabbit liver, a systematic study of the first electron transfer towards cytochrome P-450 was performed. The investigations revealed that the electron transport via cytochrome P-450 reductase represents the effective reaction : the rate constants of the NADPH- and the NADH-supported processes were 1.1 s 1 and 0.5 s ~ r e s p e c t i v e l y ( T A N I G U C H I et al., 1984). The rapid NADH-supported cytochrome b 5 reductase/cytochrome b 3 transfer, the rate constant of which was determined as 30 s" 1 , was ineffective because of the slow cytochrome b5/cytochrome P-450 transfer at 0.046 s l . Thus the observed cytochrome b 3 synergism must be attributed to the second electron transfer process (Fig. 9). The second electron is transferred towards the one-electron reduced cytochrome P-450 species in the 1 : 1 complex with reduced cytochrome b 3 , the dissociation constant of which is K^ = 7.5 ¡xM, falling to 2.2 ¡¿M in the presence of benzphetamine ( B O N F I L S et al., 1981). The photoreduced cytochrome P-450 after 0 2 binding shows first order rate constants for the electron transfer from cytochrome b 3 (as measured by reoxidation of either cytochrome b 5 or cytochrome P-450) of 2.5 s _ 1 and 4—7 s" 1 in the presence of the substrate. These data in the order of the rate of first electron transfer between cytochrome P-450 reductase and cytochrome P-450 at reductase saturation ( B L A N C K et al., 1984b) indicate the competence of the second electron transfer in cytochrome b 5 -dependent synergism of cytochrome P-450 driven reactions. The reoxidation of the cytochrome P-450/oxygen complex can proceed alternatively by donation of an electron to the oxidized cytochrome b 3 (POM-

R e g u l a t i o n M e c h a n i s m s of C y t o c h r o m e P - 4 5 0

45

PON and COON, 1984). The multiphasic reaction exhibits rate constants consistent with the electron acceptance process. Thus in general the decay of the reduced cytochrome P-450/oxygen complex is increased by interaction with cytochrome b 5 . A specificity has been observed with respect to distinct cytochrome P-450 isozymes: cytochrome P-450 LM4 preferentially donates an electron to cytochrome b5, whereas cytochrome P-450 LM2 prefers to accept an electron from cytochrome b 5 . Reversible electron exchange between cytochrome P-450 LM2 and cytochrome b5 in a covalent complex is proposed to occur in the catalytic cycle: the first electron input generates ferrous cytochrome P-450, followed by immediate transfer of this electron to the oxidized cytochrome b 5 ; after a further reduction of the ferric cytochrome P-450 and binding of molecular oxygen, the ferrous cytochrome b5 returns the electron to the oxyferrous cytochrome P-450 (TAMBURINI and SCHENKMAN, 1987). NADPH

*Rp-45o - * - P - 4 5 0 (1.1 s ' 1 ) I >Rb

5

*b5

(0.47s-1)

>b5

(very s l o w )

^-450-^-450 I— NADH

*Rb5

b 5 —» P - 4 5 0 ( 0 . 0 4 6 s - 1 )

(0.5s"1)

> b5

(0.15s-1)

b5

P-450(0.046s-1)

*b5

(30s-1)

b5

P-450"( 2-7s-1)

Fig. 9 . Electron transfer reactions (according to BONFILS et al., 1981; TANIGUCHI et al., 1984).

Besides the electron carrier role of cytochrome b5 in cytochrome P-450 catalyzed reactions the former protein may further exert an effector function. The decay of the cytochrome P-450 ternary complex has been shown to be favoured in the presence of cytochrome b 5 but with no evidence of a redox coupling of the two proteins (GUENGERICH et al., 1976). Moreover, in a reconstituted bypass system, the cumene hydroperoxide-supported conversion 4-chloroaniline is modified by cytochrome b 5 addition: the apparent KD values of the substrate and of the oxygen donor complex as well, are decreased, and the rate of complex formation is increased; the temperature function of the ternary complex formation by reaction of substrate-bound cytochrome P-450 with the oxygen donor, furthermore, is significantly altered (HLAVICA, 1984). The obligatory cytochrome b5 activation of one distinct P-450 isozyme points further to the effector action of that protein by means of the induction of conformational changes in the terminal hemoprotein oxygenase (SuGIYAMA e t a l . , 1 9 7 9 , 1 9 8 0 ; MIKI e t a l . , 1 9 8 0 ) .

46

K . RUCKPAUL; H . REIN

The control of the oxidase pathway of the monooxygenase system additionally may depend on cytochrome b 5 . Since the oxidative generation of H 2 0 2 proceeds mainly by dissociation of the 1-electron reduced cytochrome P-450/ oxygen complex and subsequent dismutation ( H I L D E B R A N D T and R O O T S , 1 9 7 5 ; HILDEBRANDT e t al., 1 9 7 5 ; KUTHAN e t al., 1 9 7 8 ; KUTHAN a n d ULLRICH,

1982), the pathway. line with observed,

amount of the latter species in the steady state would determine t h a t Decreased H 2 0 2 generation at low p H and at low ionic strength, in a decreased steady state level of oxycytochrome P-450 could be due to an increased reductive action of cytochrome b 5 (INGELMANS U N D B E R G , 1980a; W E R R I N G L O E R and K A W A N O , 1980a; N O S H I R O et al., 1981). The partition between productive and abortive pathways in dependence on cytochrome b 5 has been dealt with recently by P O M P O N ( 1 9 8 7 ) . Cytochrome b 3 could be shown to increase effectively product formation and to decrease H 2 0 2 and H 2 0 generation. A steady state kinetic model with two branch points for water, hydrogen peroxide and product formation is proposed which allows to predict cytochrome b- effects. 3.6. Isozyme interactions and protein compartmentation The functional interaction of the cytochrome P - 4 5 0 monooxygenase system with components of the cytochrome b 5 system is evidently based on protein/ protein interactions. A further interaction may be generated in microsomes by isozyme/isozyme interactions or even by interactions with the multitude of other membrane proteins. Recent investigations on the microsomal metabolism of warfarin demonstrated specific mutual inhibitions of rat microsomal isozymes induced by different agents, which could not be compensated for by additional reductase ( K A M I N S K Y and G U E N G E R I C H , 1 9 8 5 ) . Moreover, an isozyme specificity was exhibited with respect to the reductase interaction. The cluster hypothesis of reductase/cytochrome P - 4 5 0 interaction mentioned earlier has been further p u t into question by recent results on the regiospecific hydroxylation of steroids ( E S T A B R O O K et al., 1 9 8 5 ) . By means of differential inhibition it was shown that specific reactions may proceed independently of each other, thus exhibiting the failure of a competition for reductase in molecular deficit. The findings are discussed as being due to a structural hetero-distribution of the interacting proteins. The heterogeneously composed lipid matrix can obviously provide the structural basis for a specific localization or compartmentation of the membrane proteins.

4.

Regulation at the cellular level

Cellular regulation of cytochrome P-450 activity is characterized by a high degree of complexity and is mainly concerned with induction. Induction in mammals is subject to various influences such as biosynthesis and turnover of

^Regulation Mechanisms of Cytochrome P-450

47

cytochrome P-450 as well as metabolic a n d diffusional processes. Tissue, sex a n d age dependences have been observed. I n d u c e r s of cytochrome P-450 m a y also initiate induction of post-oxidative enzymes. Moreover, regulation a t t h e cellular level includes inducibility a n d t h u s genetic p o l y m o r p h i s m can result in individual differences with regard to b i o t r a n s f o r m a t i o n of xenobiotics. A t h o r o u g h discussion of all these subjects would exceed t h e e x t e n t of this article. H o r m o n a l control of cytochrome P-450 is discussed b y MORGAN a n d GTJSTAFSSON later in this volume, a n d induction will be dealt with extensively in t h e following volume of this series. This section, therefore will be limited to some selected general aspects of induction of cytochrome P-450, as t h e most i m p o r t a n t e n z y m e in t h e control of b i o t r a n s f o r m a t i o n of xenobiotics a t t h e cellular level. D u e to t h e capability of cytochrome P-450 to t r a n s f o r m xenobiotics t o inactive p r o d u c t s or to convert t h e m — dependent on their chemical n a t u r e — t o reactive intermediates, induction of cytochrome P-450 has become of increasing i m p o r t a n c e with regard t o biological consequences of biotransformation. T h e r a t e a n d p a t h w a y s of xenobiotic b i o t r a n s f o r m a t i o n decisively dep e n d s on t h e concentration a n d t h e p a t t e r n of cytochrome P-450 isozymes in different tissues. 61 different cytochrome P-450 enzymes (55 endoplasmic, 3 mitochondrial a n d 3 soluble in origin) have been recognized recently (cf. ESTABROOK a n d VERECZKEY, 1985). M a n y of t h e m occur as isozymes firmly evidenced as distinct gene p r o d u c t s b y protein a n d D N A sequencing which recently has been reviewed b y BLACK a n d COON (1986). Therefore cellular regulation of cytochrome P-450 a c t i v i t y essentially dep e n d s on regulation of induction. Available i n f o r m a t i o n strongly implies t h a t induction is controlled transcriptionally whereas its m o d u l a t i o n proceeds a t t h e level of expression. Tissues are characterized b y different activities of expression which even exist among differently localized hepatocytes. Significant effects on t h e expression of certain cytochrome P-450 genes have been observed with endotoxins, interferons a n d steroid hormones (WOLF, 1986). Additionally, activity of inducible a n d constitutive isozymes can be regulat e d by exogenous substances a n d steroid hormones acting as positive or negative effectors. T h u s flavones stimulate t h e b i o t r a n s f o r m a t i o n of zoxazolamine in vivo (LASKER e t al., 1982). F l a v o n e a n d 7,8-benzoflavone s t i m u l a t e t h e catalytic a c t i v i t y of cytochrome P-450 LM3c a t t h e b i o t r a n s f o r m a t i o n of benzo(a)-pyrene. F o r nomenclature of cytochrome P-450 isozymes t h e reader is referred t o GUENGERICH (chapter 3, t h i s volume). I t has been shown t h a t certain metabolites of progesterone m a y influence t h e 6/?-hydroxylase a c t i v i t y of cytochrome P-450 LM3b b o t h negatively a n d positively proving t h e competitive inhibition of t h e 6/?-hydroxylation as most effective kind of influence. Like cytochrome P-450 LM3b, t h e LM3c f o r m shows homotropic cooperativity in t h e biotransformation of steroids t h u s indicating a n allosteric regulation (JOHNSON et al., 1983). The induction process m a y be connected with p r o m o t o r releasing enhancer f u n c t i o n s (JONES et al., 1986).

48

K . RITCKPAUL; H. REIN

4.1.

Historical background of induction

The induction of drug metabolizing enzymes was discovered in the early 50's by MILLER et al. (1952) performing investigations on chemical carcinogenesis. These studies were designed to investigate the role of biotransformation of dimethylaminoazobenzene-induced carcinogenesis, as initially described by GYORGY et al. (1941). RICHARDSON and CUNNINGHAM (1951) reported that liver tumors develop much slower in rats which were treated simultaneously with 3'-methyl-4-dimethylaminoazobenzene and 20-methylcholanthrene than in those rats which were treated with the azo compound only. Since an increased oxidative demethylation from methylated amino azo dyes was found in liver homogenates from rats and mice after pretreatment with aromatic hydrocarbons (BROWN et al., 1954) the lower carcinogenic effect of two simultaneously administered carcinogens could be explained by an increased activity of the biotransformation system. The mechanism by which the polycyclic aromatic hydrocarbons ( P A H ) inhibit the formation of hepatic tumors is based on their capability to increase the activity of metabolizing enzymes which degrade the azo dye into non-carcinogenic derivatives. P A H s (e.g. 3-methylcholanthrene) have a carcinogenic action on the skin and subcutaneous tissues, but there is little or no effect on the liver. This was the first experimental evidence for the inducibility of a N A D P H and 0 2 -dependent enzyme in liver microsomes. Extending these findings, a correlation between the inductive effect of certain P A H s on oxidative demethylation and its inhibitory effect on the hepatocarcinogenicity of 3'methyl-4-dimethylaminoazobenzene has been demonstrated by CONNEY et al. (1956).

Many similar observations following the above experimental approach are all based on the same cellular control mechanism, i. e. the capability of the cell to respond to certain substances (inducers) with the biosynthesis of specific enzymes. Due to this adaptive process either the length and intensity of drug action or the extent of toxic effects originating from biotransformation of xenobiotics are altered.

4.2.

Classification of inducers and mechanisms of induction

Up to now 3 classes of cytochrome P-450 inducers are known which induce different families of isozymes with various substrate specificities (WHITLOOK, 1986). (1) the phenobarbital type (including most of inducing drugs) (2) the methylcholanthrene type ( P A H s with different efficiency, T C D D ) (3) the steroid type (most potent (PCN)) (GONZALES et al., 1985).

inducer:

Regulation Mechanisms of Cytochrome P-450

pregnenolone-lfijt-carbonitrile

49

D u e to recent findings, however, this classification has to be extended. J u v o NEN et al. (1985) found a pyrazole inducible form in mice different f r o m other so far known monooxygenases. As investigations on this subject are still in progress no final conclusions can be drawn as yet (GUENGERICH, this volume). The molecular mechanisms of induction are far from being understood in full detail. Available d a t a suggest a transcriptionally controlled mechanism irrespective of the inducer type. Due to t h e toxic consequences, t h e induction of the P A H converting isozymes has been particularly extensively studied and has led to an evidenced sequence of partial reactions during t h e induction process. As p r o t o t y p e of this kind of inducers TCDD has been used. After passage through t h e cell membrane TCDD forms a complex with a cytosolic receptor in t h e liver cell. Subsequently t h e TCDD receptor complex is translocated from the cytosol to the nucleus where it binds site-specifically near the cytochrome P-450c gene. The binding stimulates t h e transcription of specific genes including cytochrome P-450c. The transport of the polvA-m IIXA from the nucleus into t h e cytosol is suggested to represent the rate-limiting step of biosynthesis (IVERSEN et al., 1986). The subsequent translation finally leads to selective biosynthesis of the specific isozyme into which mitochondrial derived heme is inserted to form the holoenzyme. The increased a m o u n t of t h e cytochrome P-450 isozyme results in an enhanced biotransformation of respective substrates with carcinogenic or mutagenic effects (NEBERT et al., 1 9 7 5 ; POLAND e t a l . , 1 9 7 6 ; N E B E R T , 1 9 7 9 ) .

The structural gene for h u m a n cytochrome P 1 -450 which is closely associated with aryl hydrocarbon hydroxylase (AHH) activity has been assigned to chromosome 15 q22—q24 using mitogen stimulated lymphocytes (AMSBAUGH et al., 1986). This finding is in agreement with another recent report using somatic cell hybrids where t h e h u m a n TCDD-inducible cytochrome P ^ S O gene has been assigned to chromosome 15 (HILDEBRANDT et al., 1985). The regulatory gene(s) which previously have been a t t r i b u t e d to h u m a n chromosome 2 required for the expression of A H H activity is not necessarily located at the same chromosome as the structural gene (BROWN et al., 1976). I n contrast to the detailed induction mechanism initiated by TCDD far less information on the mechanism(s) of phenobarbital and PCN induction is available. Recently PIKE et al. (1985) proved a transcriptionally controlled P B induction by showing the same extent of induced m R N A in the nuclei as in t h e cytoplasm. These findings exclude changes in t h e rates of processing, t r a n s p o r t or degradation of m R N A as reason for the enhanced formation of the isozyme. SCHUETZ et al. (1984a, b) have shown t h a t PCN and other glucocorticoides (dexamethasone, a-methylprednisolone) increased t h e concentration of cytochrome P-450 PCN by stimulating the synthesis of P-450 PCN directed m R N A . The induction process not only enhances the overall concentration of cytochrome P-450 b u t considerably alters the p a t t e r n of isozymes. Phenobarbital for example causes a 2.6-fold increase in t h e overall concentration of cytochrome P-450 in t h e liver of male rats, whereas the percentage of t h e specifically in-

50

K. RUCKPAUL; H. REIN

ducible isozyme related to t o t a l cytochrome P-450 is increased f r o m 5 % t o as much as 86% (PHILLIPS et al., 1983). This means t h a t a t t h e same t i m e t h e content of all other isozymes decrease from 85% to 14%. I t is still unclear t o d a t e whether the distribution p a t t e r n of t h e isozymes which are not induced b y phenobarbital is maintained or whether these isozymes are altered individually t h u s changing their m u t u a l ratio and p a t t e r n . Such an assumption is t e m p t i n g because the induction b y /9-naphthoflavone (BNF) does not only lead to a 1.4fold increase in t h e t o t a l content of cytochrome P-450 in t h e microsomal membrane b u t concomitantly causes a repression of t h e translatable m R N A responsible for t h e phenobarbital specific isozyme (PHILLIPS et al., 1983). H A R A D A and OMURA (1983) found somewhat lower values b u t in t h e same range. These finding are remarkable in so far as they provide t h e biochemical basis to u n d e r s t a n d t h e functional complexity of t h e monooxygenase system with respect to pharmacological and toxicological consequences b y its multiplicity and t h u s m a y explain t h e linkage between the activity of t h e cytochrome P-450 system and chemical carcinogenesis.

4.3. Relations between induction and chemical carcinogenesis Studies b y N E B E R T and coworkers h a v e provided compelling evidence for t h e correlation between t h e inducibility of cytochrome P - 4 5 0 ( N E B E R T and J E N S E N , 1979) and t h e occurrence of chemical carcinogenesis. T h e y used mouse strains, which contrary t o normal mice (responsive), are insensitive to induction of cytochrome P X -450 b y P A H (nonresponsive). Relating t h e A H H a c t i v i t y of t h e liver t o 3-methylcholanthrene induced fibrosarcomas revealed a significant lower frequency of t u m o r s in nonresponsive mice as compared to responsive animals. Similar results were f o u n d with lung t u m o r s a f t e r intra-tracheal administration of 3-MC. E x t e n d i n g these results N E B E R T ' S group ( N E B E R T et al., 1 9 7 5 ; POLAND et al., 1 9 7 6 ) have shown t h a t t h e cytosolic T C D D binding receptor is lacking in nonresponsive mice (THORGEIRSSON a n d N E B E R T , 1 9 7 7 ) , t h u s rationalizing t h e observed lower t u m o r frequency in nonresponsive mice. Despite of possible additional influences (e.g. D N A repair mechanisms, immunological status, sensitivity t o viral infections), t h e above results indicate t h a t t h e occurrence of chemically induced t u m o r s is closely associated with t h e inducible A H H activity. I n addition to t h e direct xenobiotic-initiated, transcriptionally controlled regulation of induction, another indirect linkage between chemical carcinogenesis and induction has been described including a u g m e n t a t i o n of transcription and promotor release. T u m o r promoting effects of p h e n o b a r b i t a l were already observed b y PERAINO et al. ( 1 9 7 1 ) . Feeding of r a t s with p h e n o b a r b i t a l a f t e r feeding with 2-acetylaminofluorene (AAF) resulted in a significant increase in t h e incidence of h e p a t o m a s whereas t h e simultaneous administration of both compounds reduced t h e hepatocarcinogenic effects of AAF.

Regulation Mechanisms of Cytochrome P-450

51

BUCHMAN et al. (1986) have studied the influence of 3-MC type induction (treatment with 3,3', 4,4'-tetrachlorobiphenyl) and P B type induction (treatment with 2,2', 4,4', 5,5'-hexachlorobiphenyl) on diethylnitrosamine (DEN) initiated carcinogenesis in rats. Treatment with both types of inducers subsequent to D E N administration extensively enhanced the number of preneoplastic islets in liver as compared to D E N treatment only. Obviously both types of PCBs are potent promoters of DEN-induced preneoplastic foci in rat liver thus confirming earlier findings at the tumor promoting effects of PBBs

(JENSEN e t al., 1983). J O N E S et al. ( 1 9 8 6 ) have shown that the process of induction may be connected with an enhancer function, releasing a promotor. Studying a dioxin responsive genomic element which is flanking the 5'-end of the cytochrome P-450 gene in mouse hepatoma cells the authors found this element to regulate the mouse mammary tumor virus promotor. The TCDD/receptor complex is required to initiate the biosynthesis of this element. The responsiveness of the element towards TODD is maintained at transfection into cells from either a heterologous mouse tissue or a heterologous species (human). The dioxinresponsive genetic element together with TCDD receptors constitute a TCDDregulated enhancer system. The authors conclude that the system augments gene expression by increasing the frequency of transcription initiation. With respect to this category of indirect influences of induction, a related observation was made by FARIS and CAMPBELL ( 1 9 8 1 , 1 9 8 3 ) on the long-term effects of induction. These authors found that neonatal exposure to phénobarbital may irreversibly alter the pattern of cytochrome P-450 catalyzed biotransformations in adult male rats. The mechanism by which this apparent imprinting proceeds remains to be elucidated.

4.4.

Polymorphism of induction

A correlation between chemical carcinogenesis and the cytochrome P-450 dependent activation of certain xenobiotics has been revealed in investigations on laboratory animals. I t was therefore of interest to establish whether these results could be extrapolated to humans, and what role the genetically determined inducibility plays as a risk factor with regard to biotransformation of xenobiotics. KELLERMANN et al. (1973a, b) have provided evidence for the existence of a correlation between A H H activity and tumor incidence in man. Analysing human lymphocytes they have shown a trimodal distribution of A H H inducibility to exist for the Caucasian population of the USA. Patients with bronchial carcinoma predominantly belong to those groups with mean or high A H H inducibility. Numerous investigators have a t t e m p t e d to reproduce the results, with varying degrees of success (cf. P F E I L et al., 1983). The main reasons for the poor reproducibility include seasonal fluctuations (PAIGEN et al., 1981) drug-related and alimentary influences, and effects from secondary

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K . RUCKPAUL; H . REIN

environmental stimuli (e.g. smoking). PELKONEN etal. (1980) have compiled these results from various authors, and concluded that in 7 0 % of the cases, there is a correlation between AHH activity and the occurrence of lung cancer. Unequivocal evidence for the genetic determination of AHH activity in man has been obtained. A study involving 16 pairs of twins has yielded a considerable hereditary component for AHH inducibility in human mitogen stimulated l y m p h o c y t e c u l t u r e s (ATLAS et al., 1 9 7 6 ; OKUDA e t al., 1977). AMSBAUGH et al.

(1986) found that the benzanthracene-induced AHH activity in lymphocytes corresponds with the amount of cytochrome P t - 4 5 0 mRNA. This finding indicates a causal structure/function relationship and more or less excludes any contribution from other non-cytochrome P r 4 5 0 enzymes to the individual differences. This tends to suggest genetic polymorphism as being responsible for the different biotransformation activities. Indeed, anomalous patterns of cytochrome P-450 isozymes can produce defects in biotransformation. Due to its enzymatically different attack, three types of genetic polymorphism in cytochrome P-450 have been differentiated: C-, S- and N-hydroxylations. To the most frequently occurring first group debrisoquine, spartein and bufuralol could be attributed. Recently a connection between bronchocarcinoma and debrisoquine m e t a b o l i s m has b e e n suggested b y AYESH e t al. ( 1 9 8 4 ,

1985).

Persons with bronchocarcinoma appear all as extensive metabolizers and perhaps predominantly homozygotes for extensive metabolism (cf. KALOW, 1987). PLUMMER et al. (1986), however, have shown that debrisoquine hydroxylase does not metabolize or activate any of the known precarcinogens, so that the connection being seen cannot be a major cause but rather should be regarded as a risk factor contributing to bronchocarcinoma. But despite of this the knowledge of each more risk factor will improve our predictions of the causes of cancer.

5.

Concluding remarks and outlook

Regulation of the monooxygenase system at the molecular level proceeds predominantly through interaction of cytochrome P-450 with substrates. The target for the substrate controlled regulation is provided by an equilibrium of two conformers having different functional properties. Substrates shift the spin equilibrium towards the high-spin conformation. This reaction is connected with a redox potential shift to a more positive value resulting in a greater electromotive force for a subsequent electron transfer from the reductase to the monooxygenase. A correlation between spin and redox state and the reduction rate, as well, could be evidenced and indicates the regulatory significance of the substrate dependent spin equilibrium. This regulatory function could be shown not to be limited to the first electron transfer but extended through the whole reaction cycle resulting in an additional correlation with

Regulation Mechanisms of Cytochrome P-450

53

the substrate conversion by use of a homologous series of substrates. Based on thermodynamic data the spin-redox coupling could be rationalized in a corresponding model. I n the polysubstrate cytochrome P-450 LM2, changes of both the spin and the redox equilibrium are mainly entropy driven processes. Conformational changes have been evidenced to be rather small. The regulation of the P-450 system at the membrane level depends on the specificities of the membrane proteins and of the membrane constituting lipids, as well, which offer the structural matrix for the catalytic reaction. The membrane bilaver provides the "volume" for the incorporated proteins, determines their translational and rotational mobility, and may exert diffusional limitations towards protein/protein interactions. The functional properties of the proteins may be further modified by immersion depth and orientation, as well. A heterogenously composed lipid matrix provides the structural basis for protein eompartmentation, hetero-distribution and association, thus generating functional specificities of protein/protein interaction. The electron supply towards P-450 is obviously rate limiting in enzymatic activity. The physiologically relevant NADPH-dependent electron transfer proceeds within functionally active associates of P-450 and reductase (cluster). The electron transfer is controlled by 1:1 complex formation between the exchange proteins. The head group of the lipid exerts a control function in t h a t negatively charged species additionally favour cluster and complex formation. The NADH-dependent cytochrome b 3 system is capable of interaction with the monooxygenase system. The main function of the b 5 system is a synergistic action with respect to the substrate conversion, in t h a t it delivers the second electron in the oxygen activation process, dependent on the P-450 species and the substrate. Besides the redox coupling of both systems, b 3 exerts an effector function towards P-450. Furthermore the b 5 system controls the reaction pathways of the P-450 system. Induction of cytochrome P-450 as cellular regulation mechanism has proved to be physiologically most important due to its accessibility for external influences. Different classes of inducers are capable of changing the isozymic pattern with regard to concentration and distribution. By the induction intensity or length of drug effects can be modified and the conversion of xenobiotics to toxic or carcinogenic compounds can be enhanced or accelerated as well. The extent of these effects is genetically determined and has been shown to depend on polymorphism. A correlation between the individual cytochrome P-450 inducibility and the carcinogenic risk has been evidenced in both laboratory animals and in humans. The determination of the inducibility by means of in vitro assays (e.g. A H H activity assay in human lymphocytes; biotransformation activity of debrisoquine) proves the possibility of prophylactic diagnosis and other biomonitoring applications. Besides utilization for medicinal purposes an application of cytochrome P-450 systems for biocatalytic processes (e.g. conversion to useful compounds, biomonitoring, decontamination) seems possible on the basis of present knowledge. Investigations of monooxygenase systems over about three decades have

54

K . RUCKPAUL; H . R E I N

provided a huge amount of experimental data. Some of them have been selected in this article and may open a window for a comprehensive view. Other data offer a fascinating challenge to stimulate further efforts to open more windows. Unsolved problems identified in this article will provide leads for further research which the reader is invited to take part in. The regulation at the molecular level by a spin/redox coupling mechanism has been evidenced for P-450 LM2. But what do we know about the regulation of high-spin P-450s? Is the catalytic activity of a P-450 species rather determined by a substrate specific conformational state or the respective redox potential and what coupling relations exist between both properties? Alternate pathways of monooxygenases have been described. By which properties of the substrate are they controlled and what determines the isozyme specificity of the different pathways is unclear at present. What properties of the substrate are decisive in the susceptibility towards hydroxylation and what conclusions can be drawn for drug design strategies? The organization of the P-450 system within the membrane is described by different models, but no details exist concerning the composition of proposed clusters. Are they homogeneously composed or mixed up from different isozymes and is the reductase included? Another as yet undescribed area is the isozyme specificity of the P-450/reductase interaction. The molecular basis of the induction mechanism has just begun to be understood for TCDD but what mechanisms do exist for other types of inducers has to be clarified. I t is not clear if inducibility, genetic polymorphism and promotor susceptibility are interrelated and at what level are they regulated. Which structural and chemical peculiarities determine whether a compound will act as substrate or as an inducer is unknown, as well as the question of regulation mechanisms of P-450 dependent transformations of endogenous substrates. In particular, we are only just in the beginning to understand the multiple regulation mechanisms originating from the structural multiplicity and the functional versatility of the system under study and their physiological implications. Many efforts are still necessary to tackle this challenge.

6.

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MATSUBARA, T . , J . BARON, L . L . P E T E R S O N , a n d J . A . P E T E R S O N , ( 1 9 7 6 ) , A r c h .

77, Bio-

chem. Biophys. 172, 4 6 3 - 4 6 9 . MATSUURA, S . , Y . F U J I I - K U R I Y A M A , a n d Y . TASHIRO, ( 1 9 7 8 ) , J . C e l l . B i o l . 7 8 , 5 0 3 - 5 1 9 . MCINTOSH, P . R . , S . KAWATO, R . B . F R E E D M A N , a n d R . J . C H E R R Y , ( 1 9 8 0 ) , F E B S L e t t . 122, 5 4 - 5 8 .

MIKI, X., T. SUGIYAMA, a n d T. YAMANO, (1980), J . Biochem. 88, 3 0 7 - 3 1 6 . MILLER, E. C., J . A. MILLER, a n d R. R. BROWN, (1952), Cancer Res. 12, 2 8 2 - 2 8 3 . MISSELWITZ, R . , G . R . J A N I G , H . R E I N , E . B U D E R , D . Z I R W E R , a n d

K.

RUCKPAUL,

(1976), Acta Biol. Med. Germ. 35, K 1 9 - K 2 5 . MISSELWITZ, R . , G . R . J A N I G , H .

REIN, E.

BUDER, D . ZIRWER, a n d

K . RUCKPAUL,

(1977), Acta Biol. Med. Germ. 36, K 3 5 - K 4 1 . MITANI, F. a n d S. HORIE, (1969a), J . Biochem. 65, 2 6 9 - 2 8 0 . MITANI, F. and S. HORIE, (1969b), J . Biochem. 66, 1 3 9 - 1 4 9 . MIWA, G . T . , S. B . W E S T , M. TUANHUANG, a n d A . Y . H . L u , (1979), J . B i o l . C h e m . 2 5 4 , 5695-5700.

MIWA, G. T. a n d A. Y. H . Lu, (1981), Arch. Biochem. Biophys. 211, 4 5 4 - 4 5 8 . MIWA, G. T. a n d A. Y. H . Lu, (1984), Arch. Biochem. Biophys. 234, 1 6 1 - 1 6 6 . M I Y A K E , Y . , Y . NAKAMURA, X . TAKAYAMA, a n d K . H O R I K E , ( 1 9 7 5 ) , J .

Biochem.

78,

773-783. MORGAN, E. T., D. R. KOOP, a n d M. J . COON, (1982), J . Biol. C h e m . 2 5 7 , 1 3 9 5 1 - 1 3 9 5 7 . MORIMOTO, T . , S . MATSUURA, S . SASAKI, Y . TASHIRO, a n d T . OMURA, ( 1 9 7 6 ) , J .

Cell.

Biol. 68, 1 8 9 - 2 0 1 . M U L L E R - E N O C H , D . , P . CHURCHILL, S . F L E I S C H E R , a n d F . P . G U E N G E R I C H , ( 1 9 8 4 ) ,

J.

Biol. Chem. 259, 8 1 7 4 - 8 1 8 2 . MURAKAMI, K . a n d H . S. MASON, ( 1 9 6 7 ) , J . B i o l . C h e m . 2 4 2 , 1 1 0 2 - 1 1 1 0 . XARASIMHULU, S . , D . Y . COOPER, a n d 0 . R O S E N T H A L , ( 1 9 6 5 ) , L i f e S c i . 4 , 2 1 0 1 — 2 1 0 7 . X A R H I , L . O . a n d A . J . FULCO, ( 1 9 8 6 ) , J . B i o l . C h e m . 2 6 1 , 7 1 6 0 - 7 1 6 9 . N E B E R T , D . W . , J . R . ROBINSON, A . N I W A , K . K U M A K I , a n d A . P . P O L A N D , ( 1 9 7 5 ) , J .

Cell. Physiol. 85, 3 9 3 - 4 1 4 . NEBERT, D. W. (1979), Mol. Cell. Biochem. 27, 2 7 - 4 6 . NEBERT, D . W . a n d N . M. JENSEN, (1979), Critical R e v . B i o c h e m . 6, 4 0 1 - 4 3 7 . NELSON, D . R . a n d H . W . STROBEL, ( 1 9 8 8 ) , J . B i o l . C h e m . 2 3 6 , 6 0 3 8 - 6 0 5 0 . XILSSON, A . a n d B . C . J O H N S O N , ( 1 9 6 3 ) , A r c h . B i o c h e m . B i o p h y s . 1 0 1 , 4 9 4 — 4 9 8 . XORDBLOM, G . D . a n d M . J . COON, ( 1 9 7 7 ) , A r c h . B i o c h e m . B i o p h y s . 1 8 0 , 3 4 3 — 3 4 7 . XOSHIRO, M . , X . H A R A D A , a n d T . OMURA, ( 1 9 7 9 ) , B i o c h e m . B i o p h y s . R e s . C o m m u n . 9 1 , 207-213.

XOSHIRO, M., H. H . RUF, a n d V. ULLRICH, (1980), i n : Biochemistry, Biophysics a n d R e g u l a t i o n of C y t o c h r o m e P - 4 5 0 , ( J . A . GUSTAFSSON, J . C A R L S T E D T - D U K E , A . M O D E ,

J . RAFTER, eds.), Elsevier/Xorth Holland Biomedical Press, A m s t e r d a m , 351 — 35 ROH + H 2 0 These two electrons coming from NADPH are transferred to the cytochrome P-450-heme via the two flavin cofactors of cytochrome P-450 reductase in microsomal monooxygenases. In fact, in the microsomal system, cytochrome P-450 can also receive electrons from NADH via another flavoprotein, cytochrome b 5 reductase, and cytochrome b 5 ( P E T E R S O N and P R O U G H , 1986). In its resting state, two forms of cytochrome P-450 are in equilibrium: a hexacoordinate low-spin iron(III) complex, having a cysteinate and presumably an OH-containing entity as axial ligands, and a pentacoordinate high-spin iron(III) complex with the cysteinate as the only axial ligand. The position of this P-450-Fe r a

+ ip~

ROH

AO=PhIO, H202 ROOH, RC03H. .

70

Fig. 1. Catalytic cycle of cytochrome P-450.

D . MANSUY ; P . BATTIONI

equilibrium is very much dependent on the nature of the cytochrome P-450 isoenzyme considered but also on several factors such as the temperature and the ionic strength. In general, the binding of a substrate to cytochrome P-450 which occurs on a protein hydrophobic active site close to the heme leads to a shift of this equilibrium towards the high-spin pentacoordinate state. The iron(III) coordinated to only one axial ligand in this enzyme-substrate complex is more easily reduced by one electron coming from NADPH via the reductase. This leads to the high-spin pentacoordinate cytochrome P-450-iron(II) complex, a stable intermediate in the catalytic cycle which has been studied by various spectroscopic methods. This ferrous complex has a vacant coordination position and readily binds many ligands such as CO, isocyanides, nitrogenous bases, amines, phosphines and 0 2 . The Fe(II)-0 2 intermediate derived from 0 2 binding was studied by Mossbauer (SHARROCK etal., 1 9 7 6 ) and E X A F S (DAWSON et al., 1986) spectroscopy in the case of the bacterial P-450 c a m . I t appears as a lowspin hexacoordinate complex with a formal Fe(III)0 2 '~ structure analogous to that of oxyhemoglobin. The greater instability of this intermediate in microsomal cytochromes P-450 has so far precluded such spectroscopic studies. A one-electron reduction of the ferrous-dioxygen intermediate leads to the socalled active oxygen complex, the real oxidizing species. However, this key intermediate of the catalytic cycle has a lifetime so short that it could not be observed by any spectroscopic technique. Thus, contrary to those of the other intermediates of the catalytic cycle, the nature of the active oxygen complex is not yet known. Our knowledge of its possible structure is based only on studies of reactions of cytochrome P-450 with single oxygen atom donors (see above) and on comparisons with better known active oxygen complexes derived from other hemoproteins or from iron-porphyrins. From all these data, the most likely mechanism for dioxygen activation by cytochrome P-450 involves (i) a heterolytic cleavage of the O — O bond of a possible Fe(III) — 0 — O H intermediate formed by one-electron reduction of the ferrous-dioxygen complex and protonation of the terminal oxygen atom, (ii) the formation of a high-valent iron-oxo complex derived formally from the ferric state by removal of two electrons, (iii) the transfer of the oxygen atom of this iron-oxo complex into the substrate. Different structures are possible for this iron-oxo complex: a Fe(V) = 0 structure which has never yet been observed in hemoproteins or iron-porphyrins, a (porphyrin + ') — F e ( I V ) = 0 structure which is found in compound I of horseradish peroxidase ( R O B E R T S et al., 1 9 8 1 ) or in iron-porphyrin model complexes (see above), and an Fe(IV) = 0 structure with a protein-centered radical as in compound I of cytochrome c peroxidase (HOFFMAN et al., 1 9 8 1 ) (Fig. 2 ) . All these

+ H+ (P)Fem-0-0H

(P) Fe v = 0

(P)FeIV-0"

(P + ') Fe I V =0

[(P)Fe l v =0 + A + "]

> "H2°

(P = Porphyrin ; A=Amino acid from the protein) P i g . 2 . Possible s t r u c t u r e s for t h e a c t i v e o x y g e n c o m p l e x of c y t o c h r o m e P - 4 5 0 .

Model S y s t e m s for C y t o c h r o m e P - 4 5 0

71

structures derive formally from a two-electron oxidation of the starting ferric state and the binding of an oxygen atom from 0 2 to the iron. It is not known presently whether the cysteinate axial ligand of the iron is still bound in the active oxygen complex, but its presence should greatly influence the nature and intrinsic reactivity of this complex. Th is point has been discussed by several authors (see for instance, O R T I Z D E M O N T E L L A N O , 1 9 8 6 a), but as no high-valent porphyrin (or hemoprotein)-iron-oxo complex bearing a thiolate ligand has been so far obtained, the question of the possible involvement of this ligand remains unanswered. Concerning the last step in the catalytic cycle, the transfer of the oxygen atom from the FeO species into the substrate, which leads to C—H bond hydroxylation, alkene epoxidation, aromatic ring hydroxylation or heteroatom oxidation, concerted and non-concerted mechanisms are theoretically possible. However, a great deal of indirect evidence suggests that nonconcerted mechanisms are most often found (for a review, see O R T I Z D E M O N T E L L A N O , 1 9 8 6 a). Most experimental results can be rationalized if one admits that the active oxygen complex has a free radical nature and reactivity. By considering its possible mesomeric form Fe(IV) — 0", it is easily understandable that it could abstract hydrogen atoms from C—H bonds or add to double bonds of alkenes or aromatic rings. These mechanisms will be discussed in more detail in the following sections by comparison to those of model systems.

2.2.

Oxidations catalyzed by cytochrome P-450 using oxygen atom donors diiierent from dioxygen

During the last ten years, it has been shown that cytochrome P-450-dependent oxidations can be performed by using oxygen atom-donors AO instead of 0 2 and NADPH. With such oxidants, the catalytic cycle of substrate oxidation is considerably simplified, as it consists of only two steps, the transfer of the oxygen atom from AO to the iron and the transfer of this oxygen atom from the FeO species to the substrate (Fig. 1). Single oxygen atom donors such as N a I 0 4 and NaC10 2 ( H R Y C A Y et al., 1975; D A N I E L S S O N and W I K V A L L , 1976; G U S T A F S S O N and B E R G M A N , 1976 a) and later iodosylbenzene ( L I C H T E N B E R G E R et al., 1976; G U S T A F S S O N et al., 1976, 1979) and aryldimethylamine N-oxides ( H E I M B R O O K et al., 1984) have been successfully used for the cytochrome P-450-dependent oxidation of various substrates. Potential oxygen-atom donors involving an 0 — 0 bond such as alkylhydroperoxides or H 2 0 2 have also been used for similar purposes (KADLTTBAR et al., 1973; RAHIMTITLA and O ' B R I E N , 1974; N O R D B L O M et al., 1976). However, with these oxidants, a further problem has been encountered because of the two possible modes of cleavage of their 0 — 0 bond. A heterolytic cleavage of the 0 — 0 bond is required for the formation of the high-valent Fe(V) = 0 species (Fig. 3), whereas a homolytic cleavage leads to an alkoxy radical and an Fe(IV) species. The relative importance of these two modes of cleavage of the 0 — 0 bond in cyto-

72

D . M A N S U Y ; P. BATTIONI

chrome P-450-catalyzed oxidation of substrates by alkylhydroperoxides, peracids or H 2 0 2 has been discussed in a recent review (ORTIZ D E M O N T E L L A N O , 1986 a). ROOH

ROOH

2.3.

(P)Fev=0

•>

(P)FeIV-OH

+ ROH

+ RO"

F i g . 3. T h e t w o possible m o d e s of cleavage of o x i d a n t s c o n t a i n i n g a 0 — 0 b o n d b y c y t o c h r o m e P-450.

How to build up catalytically active models for cytochrome P-450?

I n the following, all the model systems described will be based on a metalloporphyrin catalyst which mimics the cytochrome P-450 heme. An ideal model system would associate with the metalloporphyrin (preferably an iron-porphyrin) a thiolate ligand to mimic the axial cysteinate ligand of cytochrome P-450, a reducing agent and a proton donor necessary for a monooxygenation reaction (see section 1.1), and 0 2 itself as the oxygen atom donor. However, because of the high oxidizing activity of the active oxygen metal-oxo intermediate in such systems, the direct use of thiolate ligands in chemical models seems very difficult: These thiolate ligands are very sensitive towards oxidizing agents. The cysteinate ligand of cytochrome P-450 is not too rapidly oxidized presumably because it is held by the protein far from the oxidizing oxygen atom of the active oxygen species. I n fact, it is separated from this oxygen atom by the heme plane. I t is still difficult to achieve such a clear separation in chemical systems and, so far, most model systems described which were catalytically active for alkane hydroxylation have not involved thiolate ligands. The long catalytic cycle of substrate oxidation by cytochrome P-450, which uses 0 2 as oxygen atom donor and N A D P H , appears difficult to mimic for two main reasons. The multienzyme monooxygenase system allows a complete separation of the iron-oxo hydroxylating species from the reducing agent (NADPH) in excess, since the electrons from N A D P H are transferred to the heme via an electron transfer chain. This separation prevents the direct reaction of the FeO species with NADPH. In chemical model systems, it seems difficult to separate the metal-oxo active species from the reducing agent in excess, explaining the very low yields of substrate oxidation based on the reducing agent observed so far with model systems using 0 2 itself (see following sections). The other role of the protein in the cytochrome P-450 catalytic cycle is to regulate the sequence of the different steps. With a simple chemical system, it appears far easier to mimic the shortened catalytic cycle of cytochrome P-450 which uses single oxygen atom donors. However, the level of difficulty of generating a high-valent iron-oxo complex upon reaction of an iron-porphyrin with an oxygen atom donor depends on the nature of this oxidant. I n t h a t regard, it is very likely that oxidants containing only one transferable oxygen atom linked to a good leaving group, such as iodosylbenzene

Model Systems for Cytochrome P-450

73

PhIO, hypochlorite CIO - or tertiary amine oxides, would transfer their oxygen atom to metalloporphyrins more easily than oxidants containing an 0 — 0 bond. For the latter oxidants, such as alkyhydroperoxides and H 2 0 2 , there are, as mentioned in section 2.2., two possible modes of cleavage of the 0 — 0 bond after reaction with a metallic redox center and only the heterolytic cleavage will lead to the expected high-valent metal-oxo complex (Fig. 3). These considerations explain the order of presentation of the model systems in the following sections. It corresponds to the increasing level of complexity of these model systems as a function of the nature of the used oxygen atom donor (Fig. 4). Fe(III) + AO

**A

»

Fe(V) = 0

Fe(III) + R ' O O H

>

+ RH

Fe(V)=0

>

ROH

+

+ R'OH

Fe(III) + RH

»

ROH

+

Fe(III)

or F e ( I V ) - O H + R ' O ' Fe(III)

+ 02

+

2e"

+

2H+

—»

Fe(III)-00H

+ H+ »

Fe(Y)=0

+ H20

or Fe(IV)=0 t

RH

ROH + Fe(III)

HO'

Fig. 4. Pathways possibly involved in the three kinds of model systems using an ironporphyrin and different oxygen atom donors.

3.

Model systems using oxidants containing a single oxygen atom

3.1.

Iodosylbenzene dependent systems

Since the first report by G R O V E S et al. (1979 a) showing that iodosylbenzene (PhIO) in the presence of catalytic amounts of iron-meso-tetraarylporphyrins was able to perform two important reactions catalyzed by cytochrome P-450, the hydroxylation of alkanes and the epoxidation of alkenes, a great deal of work has been devoted to the study of systems involving PhIO and various metalloporphyrin catalysts.

3.1.1. The various metalloporphyrins used as catalysts The efficacy of a model PhlO-metalloporphyrin system can be evaluated by different parameters such as the catalytic activity of the metalloporphyrin, in moles of product formed per mole of catalyst (turnovers) per min for instance, and the selectivity of the oxidation reaction. But another important characteristic is the ability of the catalyst to remain intact in such a highly oxidizing medium. In fact, metalloporphyrins, like other macrocycles, tend to be oxidatively destroyed by the highly active oxygen species generated in model systems with not only PhIO but also the other oxidants that have been

74

D. M A N S U Y ; P. BATTIONI

used so far. In order to evaluate the robustness of the catalysts in the oxidizing medium, a possible parameter is the total number of turnovers of the catalyst before its complete oxidative destruction. Natural iron-porphyrins such as iron protoporphyrin-IX are very sensitive towards oxidative degradation and have only rarely been used as catalysts in model systems (TATSUNO et al., 1986). Iron-porphyrins with arvl substituents on the four meso positions are more resistant and have been extensively used (Fig. 5). Iron-mesotetraphenyl-

R, =R;=RJ=RT=RS = H TPP f R. = R =R =CH, ^R R ™P R1 = R,=B3=R1=RS=F I R, = RS = CI {

l Rï = Rj=Ri*=H

THPYP

TPFPP .TDCPP

Fig. 5. Structure of different iron-porphyrins used as catalysts in model systems (abbreviations used in the text for the porphyrins are indicated).

porphyrin-chloride (Fe(TPP)(Cl)), which can be easily obtained from pyrrole and benzaldehyde, gives satisfactory results and has been used by most authors. Replacement of the chloride ligand of Fe(TPP)(Cl) by other axial ligands or counterions leads to qualitatively similar results (NAPPA and TOLMAN, 1985). The use of iron-meso-tetramesityl-porphyrin, (Fe(TMP), which contains bulky methyl substituents in all the ortho positions of the mesophenyl rings led to an important stabilization of the intermediate iron-oxo complexes and allowed their characterization by various spectroscopic techniques (GROVES e t al., 1 9 8 1 ; B o s o e t al., 1 9 8 3 ; PENNER-HAHN e t al., 1 9 8 3 , 1 9 8 6 ) .

Major improvements in the catalytic activities and of the catalyst halftimes were obtained thanks to the use of tetraaryl-porphyrins containing halogensubstituted aryl groups such as pentafluorophenyl (CHANG and EBINA, 1981; NAPPA a n d TOLMAN, 1 9 8 5 ) or 2 , 6 - d i c h l o r o p h e n y l (TRAYLOR e t al.,

1984a)

groups. With the iron-porphyrin bearing these electron-withdrawing chlorosubstituents that also provide steric protection, Fe(TDCPP)(Cl) (see fig. 5), as a catalyst, and C 6 F 5 IO in a CH2C12: CH 3 OH: H 2 0 mixture, more than 10,000 epoxide moles were obtained per mole of catalyst, with an initial rate as high as 300 turnovers per second (TRAYLOR et al., 1985). In a more general manner, the use of metal complexes of this porphyrin as catalysts associated with many

Model Systems for Cytochrome P-450

75

other oxidants than PhIO has led to higher catalytic activities and lunger lifetimes of the catalyst (see following sections). Several other porphyrins bearing more or less bulky substituents on the meso-arvl groups have been used, particularly in order to achieve regioselective oxidation. They will be described in details in section 6. All these metalloporphyrins are soluble in hydrophobic solvents and have been used in general in CH2CI2, Cgtly or C H3CN\ However a polar tetra-cationic iron-porphyrin, Fe(tetra-4N-methylpyridylporphyrin=TMPyP) Cl5, was found to catalyze alkene epoxidation by PhIO in CH3OH (LINDSAY-SMITH and MORTIMER, 1985b). The catalytic activity of porphyrin complexes of different metal ions has been evaluated. Fe(III)- and Mn(III)-porphyrins have been used most often and appear to make the best catalysts. However, chromium- (GROVES and K R U P E R , 1 9 7 9 ) , ruthenium- (DOLPHIN et al., 1 9 8 3 ; LEUNG et al., 1 9 8 3 and osmium- (CHE and CHUNG, 1 9 8 6 ) porphyrins were also found to catalyze olefin epoxidation and alkane hydroxylation with lower activities.

3.1.2. Nature of the active oxygen complexes involved in Fe- or Mn-porphyrin-PhIO systems One of the major interests of the study of such systems was the isolation of the first clearly defined high-valent porphyrin-iron-oxo complexes. A porphyrinFe(IV) = 0 complex has been prepared by reaction of 0 2 with Fe(II)(TMP) at low temperature and homolytic cleavage of the 0 — O bond of a (TMP)Fe(III) — 0 — 0 — F e ( I I I ) ( T M P ) intermediate dimer in the presence of 1-methyl-imidazole (BALCH et al., 1 9 8 4 ) (Fig. 6 ) . This complex exhibits spectroscopic properties analogous to those of compounds I I of peroxidases (ENGLISH et al., 1 9 8 3 ; and is a poor oxidant as it is only able to transfer its oxygen atom to phosphines.

2(TMP)Fe"

+

02

—(TMP)Fe

-O-O-Fe

(TMP)

1-CH3-Im

>

2

, .. (Fe'Vy

"MP

N

CH(TMP)Fe 111 — CI

+

CO3H

H20*^

(P+')FeIV=0*

CI (TMP)Fe 1 1 1 - C I

+

PhIO

'

+CH3COOH

+

(P)Fen

F i g . 6 . F o r m a t i o n of high-valent iron-oxo complexes upon reaction of iron-porphyrins with o x y g e n - a t o m donors.

76

D. MANSUY ; P . BATTIONI

On the other hand, the complex derived from its one-electron oxidation is a powerful oxidizing agent. Such a complex having a formal Fe(V) = 0 structure has been also prepared by reaction of PhIO withFe(TMP)(Cl) and treatment by CH3COOH or by direct reaction of meta-chloroperbenzoic acid withFe (TMP)(C1) (GROVES et al., 1981). This green complex is stable below — 4 0 ° C and was studied by iH-NMR, E P R , Môssbauer (Boso et al., 1983) and E X A F S ( P E N N E R HAHX et al., 1983; 1986) spectroscopy. All its characteristics are compatible with a (porphyrin-radical cation) Fe(IV) = 0 structure and similar to those of horseradish peroxidase compound I. It transfers its oxygen atom to alkenes almost quantitatively and exchanges its oxygen atom with water (GROVES et al., 1981) (Fig. 6). Recently, such (TMP + ")Fe(IV) = 0 complexes have been prepared by chemical (BALCH et al., 1985) and electrochemical ( L E E et al., 1985a; CALDERWOOD and BRUICE, 1986; YUAN et al., 1985a; GROVES and G I L BERT, 1986) oxidation of (TMP)Fe(IV) = 0 . Porphyrin-Fe(IV) = 0 complexes have been also prepared by electrochemical oxidation of porphyrin-Fe(III)OH complexes or by reduction of porphyrin-Fe(II)-0 2 complexes (SCHAPPACHER et al., 1985).

(TPP)Mn 111 - X + PhIO

=>

ROH

Fig. 7. Complexes isolated upon reaction of Mn(III)-porphyrins with P h I O a n d their reaction with hydrocarbons.

Reaction of Mn(III)-porphyrins with PhIO leads to various complexes the structure of which varies as a function of the nature of the solvent and of the counterion. The complexes characterized so far by X-ray analysis exhibit no M n = 0 bond. They are either 11-oxo Mn(IV)-0-Mn(IV) dimers or Mn(IV) monomers as shown in Fig. 7 (SCHARDT et al., 1981, 1982; CAMENZIND et al., 1982; SMEGAL et al., 1983; SMEGAL and H I L L , 1983a, 1983b). These Mn(IV) complexes are believed to be in equilibrium with a very reactive Mn(V) = 0 complex which could be responsible for their reactions with alkanes and alkenes (Fig. 7) (SMEGAL a n d H I L L , 1 9 8 3 a ) .

Model Systems for Cytochrome P-450

77

3.1.3. Different reactions performed by model metalloporphyrin-PhIO systems I t is now clear t h a t these model systems are able to perform all the kinds of oxidation catalyzed by cytochromes P-450 under similar mild conditions, i. e. the hydroxylation of alkanes, the epoxidation of alkenes, the hydroxylation of aromatic rings and the oxidation of molecules containing a heteroatom (0, N or S). The characteristics of these different reactions will be considered by comparison with those of the corresponding cytochrome P-450-dependent reactions. 3.1.3.1. Alkane hydroxylation The hydroxylation of several alkanes such as cyclohexane, adamantane, decalin and n-heptane, is performed with satisfactory yields by using P h I O in the presence of Fe(III)- or Mn(III)-porphyrins. Alcohols are the major products and, in general, only minor amounts of the corresponding ketones are observed. Important characteristics of the hydroxylation of alkanes by these model systems which are similar to those found in the case of cytochrome P-450 are

Ooh
—OH +

o°

(15 = 1 )

k H / k D - 13 OH Fe(TPP)(Cl)

+

OH ( 9 0 MO)

PhIO

OH OH

+

(48M)

Fig. 8. Some characteristics of a l k a n e h y d r o x y l a t i o n b y P h I O catalyzed by iron-porphyrins.

illustrated in Fig. 8: (i) a very high isotopic effect k H /k D of 12.9 ^ 1 for cyclohexane hydroxylation (GROVES et a!., 1983c), (ii) a high retention of configuration in the hydroxylation of cis-decalin into 9-decalol (GEOVES and NEMO, 1983b; LINDSAY-SMITH and SLEATH, 1983b), (iii) the preferential hydroxylation of tertiary C—H bonds in alkanes containing CH and CH 2 groups (relative reactivity of tertiary versus secondary C—H bonds in adamantane of about

78

D. MANSUY; P. BATTIONT

50) ( G R O V E S and N E M O , 1983b). It is noteworthy that the Mn(TPP)(Cl)—PhIO system leads very often to higher yields of hydroxylated products of alkanes than theFe(TPP)(Cl)—PhIO system, but also to higher ketone: alcohol ratios (GROVES e t al., 1 9 8 0 ; H I L L a n d SCHARDT, 1 9 8 0 ; FONTECAVE a n d

MANSUY,

1984a). Alkane activation by the Mn(TPP)(X) —PhIO system leads, in addition to the expected alcohols, to products deriving from the substitution of a hydrogen atom by a halogen X , coming from the catalyst or from the solvent, or by N 3 in the presence of NaN 3 in excess ( G R O V E S et al., 1980; H I L L and S C H A R D T , 1980; H I L L and S M E G A L , 1982; H I L L et al., 1983). These results have been interpreted by the involvement of free radicals derived from the alkane which can either abstract a halogen atom from the solvent or be oxidized by the Mn(IV)—X intermediate with transfer of a halogen or N 3 ligand. Intermediate formation of an alkane-derived free radical in these reactions was confirmed by a study of the oxidation of norcarane, in which products derived from an isomerisation of a norcaranyl radical were observed ( G R O V E S et al., 1980) (Fig. 9).

Mn(TPP)(Cl) + PhIO

Fig. 9. Some characteristics of alkane oxidation by PhIO catalyzed by Mn-porphyrins.

3.1.3.2. Alkene oxidation

The four reactions observed upon cytochrome P-450-dependent oxidation of alkenes (i. e. the epoxidation of the double bond, hydroxylation of allylic C—H bonds, minor formation of aldehydes RCH 2 CHO from R C H = C H 2 and the slow transformation of the iron-porphyrin catalyst into an N-alkyl porphyrin) have been easily reproduced by the iron-porphyrin—PhIO system. This system epoxidizes olefins with high yields based on the starting oxidant especially when Fe(TPFPP)(Cl) ( C H A N G and E B I N A , 1981) or Fe(TDCPP)(Cl) ( T R A Y L O R et al., 1984) (Fig. 5) are used as catalysts. This reaction is totally stereospecific and corresponds to a syn addition of the oxygen atom to the double bond ( G R O V E S and N E M O , 1983a; L I N D S A Y - S M I T H and S L E A T H , 1982). It is noteworthy that the ci.s-1,2-disubstituted olefins are much more reactive than their ¿raws-isomers ( G R O V E S et al., 1983a). This has been explained by an approach of the double bond plane of the olefin almost perpendicular to the

Model Systems for Cytochrome P-450

79

porphyrin plane. Theoretical calculations have indicated an approach with the double bond plane making a 4 5 ° angle with that of the porphyrin ( S E V I N and F O N T E C A V E , 1986). Kinetic analyses of these epoxidation reactions led to important advances in our understanding of their mechanisms. In fact, two different results were obtained depending upon the conditions used. With the Fe(TDCPP)(Cl)-C 6 F 5 IO system in CH 2 C1 2 :CH 3 0H:H 2 0 (80:18:2), the reaction order is one for the oxidant and for the catalyst and the reaction rate is not dependent on the alkene concentration ( T R A Y L O R et al., 1985). This indicates that the formation of the reactive iron-oxo complex is the rate-limiting step under these conditions. With Fe(TPFPP)(Cl) and C 6 F 5 IO in CH2C12, the epoxidation rate was independent of the olefin concentration but different alkenes were epoxidized at different rates and one alkene acted as a competitive inhibitor for the epoxidation of another (COLLMAN et al., 1985b). This result was interpreted by the kinetic scheme shown in Fig. 10, where the rate-determining step is the transfer of the oxygen atom from the iron-oxo complex to the alkene and where a preequilibrium between the iron-oxo complex, the alkene and an intermediate complex containing the elements of the iron-oxo complex and the alkene, precedes the oxygen atom transfer.

(P)Fe m

^ ^ -Phi

(P)Fe v =0

+

^

R

^

Intermediate Complex step

0

K

R

'

Fig. 10. Kinetic scheme proposed for alkene epoxidation by PhIO catalyzed by (FeTPP) (CI).

During the epoxidation of monosubstituted alkenes by Fe(TPP)(Cl) (MANet al., 1 9 8 5 ) or Fe(TDCPP)(Cl) ( M A S H I K O et al., 1 9 8 5 ; COLLMAN et al., 1986 b) and PhIO, a slow transformation of the catalyst occurs with formation of a green pigment. Acidic demetallation of the pigments formed upon oxidation of R C H = C H 2 alkenes catalyzed by Fe(TDCPP)(Cl) leads to Ar-alkylporphyrins which exhibit an X—CH 2 CHOHR structure. An identical structure has been found for the green pigments derived from cytochrome P - 4 5 0 which accumulate in the liver after oxidative metabolism of such R C H = C H 2 alkenes ( O R T I Z D E M O N T E L L A N O and R E I C H , 1 9 8 6 ) . In the model system as in cytochrome P - 4 5 0 , the stereochemistry found for the final Af-alkylporphyrin corresponds to a syn addition of t he oxygen atom and of a pyrrole nitrogen atom on the alkene double bond ( C O L L M A N et al., 1986b) (Fig. 11). The iV-alkylporphySUY

Fc (TDCPP) (CI) + PhIO

> [N-Fev-N] — \ C ^N II +• ? " 'I 0 H^i HO L-'D I H T-'H Ph

Ph

Fig. 11. Stereochemistry of the formaton of N-alkylporphyrins upon alkene oxidation by PhIO catalyzed by Fe(TDCPP) (CI).

80

D . MANSUY; P . BATTIONI

rins with an N—CH 2 CHOHR structure, which have been isolated from oxidation of R C H = C H 2 alkenes either by Fe(TDCPP)(Cl) and P h I O or b y cytochrome P-450, derive formally from addition of t h e pyrrole nitrogen a t o m to the less substituted carbon of the alkene double bond. 2V-alkylporphyrins deriving from addition of the pyrrole nitrogen atom to the more substituted carbon atom of the alkene double bond have recently been isolated from the oxidation of alkenes R C H = C H 2 b y P h I O in the presence of less hindered ironporphyrins such as Fe(TPP)(Cl) (AETATTD et al., 1987) (see Fig. 12). RCH2CHO y N —Fe-N

— *

N—Fe'"-N X

X

0 S

r N —Fe-N X B

—

*

N-Fe m -N _

X B'

Fig. 12. Possible mechanisms involved in t h e oxidation of alkene double b o n d s b y P h I O a n d iron-porphyrins.

Besides the epoxides which are always the m a j o r products of alkene oxidation by these model systems, and the JV-alkylporphyrins, another class of products deriving from the oxidation of the double bond of some R C H = C H 2 alkenes, the aldehydes R C H 2 C H O , have been detected in minor a m o u n t s (MANSUY et al., 1984d; GROVES and NEMO, 1983a; COLLMAN et al., 1986a). This aldehyde formation, which was also observed in cytochrome P-450dependent reactions (MANSUY et al., 1984d), is particularly i m p o r t a n t in oxidation of styrenes. The aldehydes are not derived from a n isomerisation of the corresponding epoxides b u t from the migration of a hydrogen a t o m during the reaction. Figure 12 gives a possible mechanism which allows one to explain all the aforementioned results concerning the oxidation of alkene double bonds b y iodosoarenes in the presence of ironporphyrins. I n this mechanism, t h e intermediate formed by reaction of the iron-oxo species with the alkene, for which kinetic (COLLMAN et al., 1985b) and spectroscopic evidence (GROVES and WATANABE, 1986 a) has been obtained, is formulated as a four-membered metallocycle as suggested by several authors (MANSUY et al., 1984a; COLLMAN et al., 1985b; GROVES and WATANABE, 1986a). There is still no direct evidence

Model Systems for Cytochrome P-450

81

for it, and other possible structures have been proposed ( G B O V E S and W A T A N A B E , 1986a; T R A Y L O R et al., 1986a, b). However, the various final observed products can easily be explained by its different possible reactions. In fact, depending upon the sense of addition of the Fe = 0 species to the alkene double bond, two four-membered metallocycles may be formed. Both of them may undergo reductive elimination of their oxygen and cr-alkyl cis ligands of the iron to give the epoxide. Metallocycle A has another possible fate : a rearrangement with a 1,2-shift of a hydrogen atom in « position relative to the oxygen which leads to the observed aldehyde RCH 2 CHO. Both metallocycles may also undergo a migration of their c-alkyl ligand from the iron to a pyrrole nitrogen atom. Such Fe - » N migrations have been previously observed for high-valent (r-alkyl-iron-porphyrin complexes formed as intermediates in the oxidation of u-alkyl Fe(III) — R complexes ( M A N S U Y et al., 1982a; L A N Ç O N et al., 1984; B A L C H and R E N N E R , 1986). This isomerisation of metallocycles A I 1 and B leads to five-membered Fe—0—C—C—N metallocycles which, upon acidic demetallation, give the isolated iV-alkylporphyrins. From the available data, it seems t h a t the structure of the final iV-alkylporphyrins depends greatly on the structure of the starting iron-porphyrin catalyst. With Fe(TDCPP)(Cl), N — C H J C H O H R structures have been found ( M A S H I K O et al., 1985; COLLMAN et al., 1986b) whereas with Fe(TPP)(Cl) or Fe(TpClPP)(Cl), iV-alkylporphvrins with an N — C H ( C O O H ) R structure have been isolated ( A R T A U D et al., 1987). The latter derive presumably from a further oxidation of metallocycle A' in the reaction medium (Fig. 12). During olefin oxidation by the model iron-porphyrin-ArlO systems, allylic alcohols are formed as well as epoxides ( G R O V E S and S U B R A M A N I A N , 1984; M A N S U Y et al., 1984a). Allylic alcohols coming from deuterated cyclohexene are mainly derived from the insertion of an oxygen atom into an allylic C—H bond but also from an allylic rearrangement of a possible free radical intermediate ( G R O V E S and S U B R A M A N I A N , 1984). The allylic alcohol:epoxide ratio depends greatly on the nature of the porphyrin substituents and on the nature of the metal ( M A N S U Y et al., 1984a).

3.1.3.3. Aromatic hydrocarbon oxidations Much less work has been devoted to the study of the oxidation of aromatic hydrocarbons by the metalloporphyrin-PhIO systems. In fact, yields of phenols in such reactions are generally considerably lower than the yields of alkane hydroxylation and alkene epoxidation. For instance, with the very efficient Fe(TPFPP)(Cl) catalyst and PhIO, cyclohexane is hydroxylated with a 71% yield and cyclohexene is epoxidized with a 95% yield whereas anisole is hydroxylated at the ortho and para positions with a total yield of only 11% ( C H A N G and E B I N A , 1 9 8 1 ) . Hydroxylation of para-deutero-anisole by this system is performed with 72% retention of deuterium in the product ^ara-methoxyphenol. Such a shift of a deuterium atom from the carbon to be hydroxylated

82

D . M A N S U Y ; P . BATTIONI

to the adjacent one is classical in cytochrome P-450-catalyzed hydroxylation of aromatic rings ( J E R I N A and D A L Y , 1974). The level of deuterium retention in the 2>ara-hydroxylation of para-deutero-anisole by PhIO catalyzed by different metalloporphyrins depends on the nature of the catalyst: 7 2 % for F e ( T P F P P ) (CI), 6 0 % for Fe(TPP)(Cl) and Mn(TPFPP)(Cl) and only 2 6 % for Mn(TPP)(Cl) (CHANG and E B I N A , 1981). 1-Naphthol is also formed in low yields with 6 8 % deuterium retention from the oxidation of labelled naphthalene by Fe(TPP)(Cl) and PhIO ( L I N D S A Y - S M I T H and S L E A T H , 1982b) (Fig. 13).

OH,

€

H

»

FelTPFPPKCI)"'

» 0 - O - 0 »

-

(3%)

C

H

"

(0,8°/.)

Fe(TPP)(Cl) F i g . 1 3 . S o m e e x a m p l e s of a r o m a t i c c o m p o u n d h y d r o x y l a t i o n b y P h I O a n d iron-porphyrins

3.1.3.4. Oxidation of compounds containing a heteroatom Ethers containing an OCH 3 group are O-demethylated with low yields by the model Fe(TPP)(Cl)-PhIO system. An isotope effect ku :kD of 9 is reported for the O-demethylation of anisole by this system ( L I N D S A Y - S M I T H et al., 1982; L I N D S A Y - S M I T H and S L E A T H , 1983 a). This large isotope effect is close to that found for anisole-O-demethylation by rat liver microsomal cytochrome P-450 ( L I N D S A Y - S M I T H and S L E A T H , 1983a). Tertiary amines are also iV-dealkylated by these model systems but with an isotope effect ku :kD of 1.3, this oxidation being initiated by a one-electron transfer from the amine to the iron-oxo complex and not by the abstraction of a hydrogen atom in oc position to the nitrogen ( L I N D S A Y - S M I T H and M O R T I M E R , 1985 a). Thioethers are oxidized with good yields by PhlO-metalloporphyrin systems. Sulfoxides are formed in a first step but a further oxidation leads to sulfones (ANDO et al., 1982). As the Fe- or Mn-porphyrin-PhIO systems were shown to reproduce quite well all the kinds of reaction catalyzed by cytochrome P-450, they have been used to mimic some oxidations of compounds which have important implications in pharmacology and toxicology. For instance, the oxidations of iV-nitrosoamines ( L I N D S A Y - S M I T H et al., 1984) and of cyclopropylamines (GTTENGERICH et al., 1984) by model systems have been found similar to the enzymatic oxidations.

M o d e l S y s t e m s for C y t o c h r o m e P - 4 5 0

83

3.1.4. The use of nitrogen and carbon analogues of iodosylbenzene In the presence of Fe- or Mn-porphyrins, P h I = N - t o s y l (a nitrogen analogue of PhIO) is able to transfer its iV-tosyl-nitrenc moiety to alkenes and alkanes, leading respectively to ¿V-tosyl-a/.iridines and iV-tosylamines ( B R E S L O W and G E L L M A N , 1982, 1983; M A N S U Y et al., 1984b; M A N S U Y and B A T T I O N I , 1986). This subject is beyond the scope of this article and will not be discussed in detail (for a recent review, see M A N S U Y and B A T T I O N I , 1986). I t is only mentioned to show that a chemistry of transfer of nitrogen into hydrocarbons exists as well as the oxygen transfer into the same substrates (Fig. 14). Carbon analogues of PhIO (the iodonium ylids P h I = C R R ' ) also easily react with iron porphyrins with transfer of their carbene moiety to form stable metallocyclic iron complexes (Fig. 15) ( B A T T I O N I et al., 1986a). The structures of these complexes are similar to those found in alkene oxidation by PhIO catalyzed by iron porphyrins (Fig. 12 and 15).

o

NHTs

Phi = NTs

Fe (P) (CI) N

I

Ts

P = porphyrin Fe(TPPXX) +

3.2.

)

N

N

Fig. 14. Insertion of the Xtosyl moiety of PhlNTs into hydrocarbons catalyzed by iron-porphyrins

Fig. 15. Formation of I 1 Fe —O —C—C —N metallocycles upon reaction of a carbon equivalent of PhIO with ferric porphyrins.

Hypochlorite-dependent systems

The use of diluted solutions of NaOCl in water at pH around 12, in the presence of phase transfer agents, allows the oxidation of substrates dissolved in organic solvents in the presence of an Mn-porphyrin catalyst ( T A B U S H I and K O G A , 1979a; G U I L M E T and M E U N I E R , 1980). With Mn(TPP) (CI) as catalyst, benzyl alcohol is oxidized to benzaldehyde and alkanes are oxidized into the corresponding alkyl chlorides ( T A B U S H I and K O G A , 1979 a). Styrene is epoxidized in good yield by such systems using NaOCl and Mn(TPP)(Cl) but alkenes containing

84

D . MANSUY; P . BATTIONI

allylic hydrogens lead to only low yields of epoxidation (GUILMET and MEUNIER, 1980). The system is considerably improved by the addition of nitrogen bases that can act as axial ligands to the catalyst, such as pyridine (GUILMET and MEUNIER, 1982) or imidazoles substituted by an aryl group (COLLMAN et al., 1983). With such ligands, the initial rate of styrene epoxidation can reach 11 turnovers per second (COLLMAN et al., 1985a) and the system becomes applicable to a large series of alkenes (MEUNIER et al., 1984). The use of pyridine leads also to an increase in the stereoselectivity of the epoxidation of stilbenes (GUILMET and MEUNIER, 1982). As with iodosoarenes, the use of Mn-tetraarylporphyrins containing bulky substituents in ortho position of the meso-phenyl rings leads to an increase in the epoxidation rates (BORTOLINI et al., 1983; COLLMAN et al., 1985a) and in the stereoselectivity of the epoxidation (BORTOLINI and MEUNIER, 1984). Moreover, the use of Mn-tetraarylporphyrins bearing halogen substituents at the ortho position of the meso-phenyl groups even leads to an efficient epoxidation of poorly reactive monosubstituted alkenes such as oct-l-ene (60—80% yield) ( D E POORTER and MEUNIER, 1984). Olefin epoxidations can also be performed with increased rates by using systems in which the Mn-porphyrin is bound to a polymer ( V A N DER MADE et al., 1983) or where the pH value of the NaOCl solution is decreased to 9.5 (MONTANARI et al., 1985). Kinetic and spectroscopic studies indicate that epoxidations by the hypochlorite-Mn-porphyrin systems have mechanisms very similar to those of the PhIO-Mn-porphyrin sytems. They involve a high-valent Mn-oxo complex (BORTOLINI et al., 1986) as the epoxidizing species and the formation of an intermediate complex between this Mn-oxo species and the alkene which could be a four-membered metallocycle (COLLMAN et al., 1984, 1985a; NOLTE et al., 1986) analogous to that described in the preceding section in PhiO-dependent systems (Fig. 16). NaOCt

+ (P)Mn'"-X

+

M n ' " -Porphyrin R4N+Cr in C H 2 C l 2 : H 2 0

0

+

NaCl

-NaCl

(P)Mnv=0

I

X

0 (P) M n ' X

'Q R

F i g . 16. A l k e n e e p o x i d a t i o n s using N a O C l a n d M n - p o r p h y r i n s . A possible m e c h a n i s m .

3.3.

Systems dependent on other single oxygen atom donors

3.3.1.

Amine-oxides

Dimethylaniline-2V-oxides can act as oxygen atom donors for Fe- and Mnporphyrins (SHANNON and BRUICE, 1 9 8 1 ; N E E and BRUICE, 1 9 8 2 ) as well as for cytochromes P - 4 5 0 (HEIMBROOK et al., 1 9 8 4 ) . They have been used for alkene

Model S y s t e m s f o r C y t o c h r o m e P - 4 5 0

85

epoxidation. However, yields of alkene epoxidation and especially of alkane hydroxylation remain low when compared to those obtained with P h I O - or CIO -dependent systems. This is due to competition between the substrate and dimethylaniline formed after transfer of the oxygen atom, for reaction with the metal-oxo species. A very detailed kinetic study of the different steps involved in the reactions performed by these TV-oxides in the presence of Fe( D I C K E N et al., 1985) or Mn- ( P O W E L L et al., 1984) porphyrins has been reported. An oxaziridine has also been used as an oxygen atom donor for the epoxidation of alkenes in the presence of Mn-porphvrins (YUAN and BRUICE, 1985a). 3.3.2. Potassium hydrogen persulfate This water-soluble oxidant, K H S 0 5 , has been used in a biphasic system (CH 2 Cl 2 -buffer, pH8) in the presence of catalytic amounts of Mn(III)-porphyrins for alkene epoxidation and alkane hydroxylation (DEPOORTER et al., 1985; D E P O O R T E R and M E U N I E R , 1 9 8 5 a, b). Its efficacy in olefin epoxidation is considerably increased by the presence of pyridine, like the previously described hypochlorite system. I t is much superior to the latter system for alkane hydroxylation as it leads to good conversion of cyclohexane and adamantane in mild conditions. 3.3.3. Periodates A periodate soluble in organic solvents, N B u 4 I 0 4 , has been used as oxygen donor in the presence of catalytic amounts of an iron-porphyrin for the oxidation of thioethers into sulfoxides (TAKATA and ANDO, 1983). This system leads also to alkene epoxidation but less efficiently than P h I O or CIO - .

4.

Model systems using hydroperoxides as oxidants

4.1.

Alky Hydroperoxides as oxidants

Alkylhydroperoxides R O O H (R = cumene or t-butyl), in the presence of catalytic amounts of Fe(TPP)(Cl), oxidize alkanes to the corresponding alcohols and ketones with good yields (80% for cyclohexane) (MANSUY et al., 1980). However, the Fe(TPP)(Cl) —ROOH system is completely unable to epoxidize alkenes (MANSUY et al., 1982b). A comparison of the properties of this system and of the Fe(TPP)(Cl)—PhIO system shows that different active oxygen species are formed. Reaction of Fe(TPP)(Cl) with cumenehydroperoxide fails to lead to the Fe(V) = 0 species characteristic of the PhIO system. In fact, this reaction leads presumably to a homolytic cleavage of the alkylhydroperoxide 0 — 0 bond with formation of an alkoxy radical as reactive species (MANSUY et al., 1982b) (Fig. 17). This radical can abstract hydrogen from alkanes, explaining why alkanes are hydroxylated eventually, while it cannot perform

86

D . MANSUY ; P . BATTIONI

alkene epoxidation. However, cytochrome P-450 catalyzes alkene epoxidation by cumenehydroperoxide. This suggests that the strong electron-donating cvsteinate ligand of cytochrome P-450-iron could play a key role in allowing heterolytic cleavage of the alkylhydroperoxide 0 — 0 bond ( M A N S U Y and BATTIONI, 1986). Imidazole seems to play a similar role since its addition to the Fe(TPP)(Cl) — ROOH or Mn(TPP)(Cl) — ROOH (R = cumene or t-butyl) systems allows them to epoxidize alkenes such as cyclohexene, cis-stilbene and 2-methylhept-2-ene with yields between 20 and 50% (MANSUY et al., 1984c) (Fig. 17). + Fe(TPP)(Cl)

ROOH

+ P-450

Fe(TPP)(Cl) +

>

>

>

[CI(TPP)FeIV-0H]

+ RO" — >

„ n [ P - 4 5 0 Fe — O J

r

j-cl(Tpp)FeV_0j

I m

-ROH

i im

A l k a n e hydroxylation no alkene epoxidation

> Alkane hydroxylation alkene epoxidation —

>

A l k a n e hydroxylation alkene epoxidation ( 2 0 - 5 0 % )

Fig. 17. Reactions of cumenehydroperoxide with P-450 or with iron porphyrins with or without imidazole. Possible active intermediates.

Kinetic studies on the heterolytic cleavage of the 0 — 0 bond of various peroxide compounds including peracids and alkylhydroperoxides by Fe(III)- and Mn(III)-porphyrins show that this type of cleavage and the formation of a highvalent metal-oxo species occurs more easily with acidic ROOH compounds and is facilitated by the presence of nitrogenous bases such as imidazole (TRAYLOR et al., 1984b; Y U A N and B R U I C E , 1985b, 1986; L E E and B R U I C E , 1985).

4.2.

Hydrogen peroxide as oxidant

This oxidant appears especially interesting for two main reasons: (i) its reaction with Fe(III)-porphyrins should lead to an intermediate very similar to that formed in cytochrome P-450 reactions after a two-electron reduction of dioxygen, (ii) when diluted in water, it is a cheap and simple oxidant especially attractive for preparative oxygenation of substrates as the secondary product is water. I t has recently been reported that H 2 0 2 can be used in the presence of Mn(III)-porphyrins and imidazole for very efficient epoxidation of alkenes ( R E N A U D et al., 1985) and very efficient hydroxylation of alkanes ( B A T T I O N I et al., 1986b). For both reactions, imidazole is absolutely required. When Mn(TDCPP)(Cl) is used as a catalyst, quantitative conversions of various alkenes, including the poorly reactive non-l-ene, are obtained with very high yields (90—100%) within one hour at 20°C. Epoxidation of 1,2-dialkyl (or diaryl)-alkenes is totally stereospecific and corresponds to a syn addition of the oxygen atom to the double bond. The Mn(TDCPP)(Cl)-imidazole-H 2 0 2 system

Model Systems for Cytochrome P-450

87

h y d r o x y l a t e s alkanes such as cyclohexane, cyclooctane, a d a m a n t a n a n d ethylbenzene with yields between 40 a n d 8 5 % . Alcohols a n d ketones are f o r m e d in a 1 : 1 ratio, except for a d a m a n t a n which leads to a d a m a n t a n - l - o l as a m a j o r p r o d u c t (Fig. 18) (BATTIONI et al., 1986b).

Vs (90%)

(70%) no trans Isomer

" UM

+ol-2(19%)

(63%)

+ o n e - 2 ( 3%)

Fig. 18. Oxidations by H 2 0 2 catalyzed by Mn-porphyrins and imidazole (yields based on the starting hydrocarbon).

I n t h e presence of t h e water-soluble p o r p h y r i n Fe(TMPyP)(Cl) 5 , H 2 0 2 h y d r o x y l a t e s phenylalanine in tyrosine a n d hydroxytyrosine (SHIMIDZU et al., 1981). These a r o m a t i c hydroxylations occur with a 7 0 % yield based on phenylalanine b u t a t a low r a t e (7 turnovers per hour). Oxidation of thioethers i n t o sulfoxides is also p e r f o r m e d b y H 2 0 2 in t h e presence of Fe(TPP)(Cl). Also in this case, t h e addition of imidazole improves t h e rates a n d yields (OAE et al., 1982).

5.

Model systems using 0 2 in the presence of a reducing agent

5.1.

Different systems reported

Borohydrides h a v e been used as reducing agents in 0 2 - d e p e n d e n t oxidations of h y d r o c a r b o n s catalyzed b y Fe- or Mn-porphyrins. W i t h N a B H 4 a n d Mn(TPP)(Cl), cyclohexene is oxidized to cyclohexanol as a m a j o r p r o d u c t with cyclohexen-3-ol as a minor p r o d u c t (TABTJSHI a n d KOGA, 1979b). Cyclohexanol is believed t o derive f r o m t h e reduction of cyclohexene-oxide b y N a B H 4 in excess, whereas cyclohexen-3-ol comes f r o m allylic h y d r o x y l a t i o n of cyclohexene. Similar systems using N a B H 4 in benzene-methanol m i x t u r e s a n d ironp o r p h y r i n catalysts either soluble in t h e m e d i u m (SANTA et al., 1985) or supp o r t e d on polypeptides (MORI et al., 1985) have been also reported. B y using tetra-JV-butyl-ammonium borohydride, which is soluble in CH2C12, a n d Mn(TPP)(Cl), several terminal olefins such as styrene are oxidized t o a m i x t u r e

88

D. MANSUY; P . BATTIONI

of the corresponding methyl ketone and of the alcohol derived from its reduction by the borohydride in excess (PERREE-FATTVET and GAUDEMER, 1981) (Fig. 19). Sodium ascorbate can also be used as a reducing agent in a biphasic system where the Mn(TPP)(Cl) catalyst and the hydrocarbon substrate are present in benzene whereas the reducing agent is present in an aqueous phase buffered at p H 8.5 (Fig. 20). If a catalytic amount of a phase transfer agent is added to that system, it becomes able to oxidize alkenes very selectively into epoxides, OH

O

Mn(TPP)(Cl )

0 2 -NaBH 4

5

0 Ph-^

o

Mn(TPP)(a)^ 02-NBU4BH4

• 0

P h

OH X

Ph

Fig. 19. Examples of alkene oxidations by 0 2 in the presence of borohydrides catalyzed by Mnporphyrins.

Mn(TPP)(Cl) in C 6 H 6 Ascorbate in H 2 0 pH 8.5

02

/ = V -OH

a0 "w CHo

CH3 0 H H

Fig. 20. Some reactions performed by the biphasic M n ( T P P ) (Cl)-ascorbate-0 2 system.

and alkanes into alcohols and ketones (MANSTTY et al., 1983). 1,2-dialkylethylenes are epoxidized by this system in a completely stereospecific manner whereas stilbenes are not. In fact, cis-stilbene leads to a 37:63 mixture of cis and trans epoxides (FONTECAVE and MANSUY, 1984a). This proportion is very similar to those reported for other systems using Mn(TPP)(Cl) as a catalyst and other oxidants such as P h I O or NaOCl. However, when compared to the M n ( T P P ) (CI)—PhIO system, the ascorbate—0 2 —Mn(TPP) (CI) system exhibits two major different characteristics: (i) it oxidizes cyclohexene exclusively into cyclohexene-oxide with no formation of allylic alcohol, (ii) it oxidizes alkanes with major formation of the corresponding ketones, whereas the M n ( T P P ) (CI)—PhIO system leads predominantly to the alcohols. This suggests that different active oxygen intermediates are involved in these two systems (FONTECAVE and MANSUY, 1984 a).

Hydrogen itself has been used successfully as a reducing agent for monooxygenations by 0 2 catalyzed by metalloporphyrins. In the presence of colloidal

Model Systems for Cytochrome P-450

89

platinum and Mn(TPP)(Cl) as catalysts, cyclohexene is oxidized by 0 2 and H 2 into the expected epoxide and minor amounts of cyclohexen-3-one ( T A B U S H I and Y A Z A K I , 1 9 8 1 ) . This M n ( T P P ) ( C l ) - 0 2 - H 2 - P t system also epoxidizes several other olefins with an order of reactivity of these olefins very similar to that found for the Mn(TPP)(Cl)—PhIO system. Addition of imidazole improved the epoxidation rates. The system also hydroxylates adamantan with predominant formation of the tertiary alcohol. TheMn(TPP)(OCOCH 3 )—0 2 —H 2 -Pt system was also used for the oxidation of more complex molecules such as geranyl acetate, and was found to epoxidize the double bond not carrying the

90

10

Fig. 21. Epoxidation of geranyl acetate by the Mn(TPP)(OAc) —0 2 —H 2 —Pt system.

RCOO"

Fig. 22. The different steps involved in hydrocarbon oxidations by the Fe(TpivPP) (CI) —0 2 —H 2 —Pt—(PhC0) 2 0 system.

function in a regioselective manner ( T A B U S H I and MIEIMITSTJ, 1984) (Fig. 21). Iron-porphyrins act also as catalysts in 0 2 -dependent monooxygenation of hydrocarbons using H 2 and colloidal platinum. With the iron"picket-fence"-porphyrin, Fe(TpivPP)(01), as a catalyst, the various steps of alkene epoxidation by such an 0 2 — H 2 — P t system have been studied ( T A B T J S H I et al., 1985). A crucial step is the heterolytic cleavage of the O—O bond of an Fe(III)—0 —0~ intermediate (Fig. 22). The presence of protons, but also more importantly of an acid anhydride, greatly facilitates this step. This easy heterolytic cleavage of 0 — 0 bonds in porphyrin—Fe(III) — 0 — 0 —CO—R complexes has recently been demonstrated ( G R O V E S and W A T A N A B E , 1 9 8 6 b). CH2OCOCH3

90

D . MANSUY; P . BATTIONI

I t involves an extraordinarily low enthalpy of activation of 16.7 k j per mol and is acid-catalyzed. I n fact, this easier cleavage of the 0 — 0 bond of Fe(or Mn)superoxo intermediates after acylation of the terminal oxygen atom b y acid chlorides or acid anhydrides added to the medium was successfully used in systems based on Mn-porphyrins (GROVES et al., 1983) or Fe-porphyrins (KHENKIN and SHTEINMAN, 1984) and 0 2 ' . This heterolytic cleavage leads to the expected high-valent iron-oxo complex which can epoxidize alkenes and hydroxylate alkanes. Accordingly, the F e ( T p i v P P ) ( C l ) — 0 2 — H 2 — P t — ( P h C 0 ) 2 0 system epoxidizes cvclohexene at a rate of 36 turnovers per hour ( T A B U S H I e t al., 1985). I n t h e s y s t e m d e s c r i b e d b y K H E N K I N a n d S H T E I N M A X ,

the electrons necessary for the monooxygenations come f r o m an electrode, while F e ( T P P ) ( C l ) or F e ( T M P ) ( C l ) is used as catalyst and benzoyl chloride as a stoichiometric acylating agent. This system demethylates anisole with a kH :lcD isotope effect of 7. All these 0 2 -dependent systems are able to reproduce qualitatively the main reactions of cytochrome P-450 and particularly the hydroxylation of inert C — H bonds of alkanes under mild conditions. However, their efficacy remains inferior to that of the enzymatic system especially in terms of catalytic a c t i v i t y (number of turnovers per min) and of yields based on the reducing agent. I n the F e ( T p i v P P ) ( C l ) — 0 2 — H 2 — P t system, it was shown that the rate-limiting step is the arrival of the second electron in the catalytic cycle (TABUSHI et al., 1985). The low yields based on the reducing agent obtained with these model systems (between 0.1 and 5 % for the borohydride-, ascorbate- and H 2 — P t dependent systems as a function of the nature of the substrate) are due to competition at the level of the high-valent metal-oxo intermediate between the substrate and the reducing agent in excess (Fig. 22). As there is no separation between the active oxygen species and the reducing agent in excess in the model systems, unlike in the enzymatic system, the reduction of the metal-oxo species b y the reducing agent is faster than its reaction with the hydrocarbons (FONTECAVE a n d M A N S U Y , 1 9 8 4 a ; TABUSHI et al., 1985). T h e b e s t y i e l d b a s e d

on the reducing agent reported so far, 56%, was obtained for cyclooctene epoxidation b y an electrochemical system using M n ( T P P ) ( C l ) as a catalyst and ( P h C 0 ) 2 0 as a stoichiometric acylating agent (CREAGER et al., 1986). However, because of the slow arrival of the electrons in that system, the rate remained low (about 2 turnovers per hour). V e r y recently, t w o systems were reported to give good yields based on the reducing agent (up to 5 0 % ) and higher rates (up to 3 — 9 turnovers per min) which are not too far f r o m those of cytochrome P-450. T h e first one employs a dihydropyridine as a reducing agent in the presence of a flavin mononucleotide, 0 2 as the o x y g e n atom donor, and a water-soluble anionic Mn-porphyrin and iV-methyl-imidazole as catalysts. Nerol and cyclohexene are epoxidized b y this system with turnover frequencies of 9 and 3.6 moles of epoxide formed per mole of catalyst per min, and yields based on the reducing agent up to 3 3 % (TABUSHI and KODERA, 1986). T h e second system uses Zn powder as a reducing agent, 0 2 as the o x y g e n atom donor, acetic acid as a source of protons and M n ( T P P ) ( C l ) and iV-methvl-

M o d e l Systems f o r C y t o c h r o m e P-450

91

imidazole as catalysts ( B A T T I O N I et al., 1 9 8 7 ) (Fig. 2 3 ) . Cyclooctene is epoxidized by this system with a turnover frequency of 3.3 moles of products formed per mole of catalyst per min and with yields based on the reducing agent of 50%. Other alkenes including cyclohexene and non-l-ene are epoxidized with

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turnover frequencies between 0.4 and 3 min The system hydroxylates alkane) such as cyclooctane and adamantan with rates (28 and 32 turnovers per hours and yields based on the reducing agent (15 and 12%) which remain satisfactory. Interestingly, this system epoxidizes cyclooctene with a 95% conversion and 90% yield of cyclooctene-oxide within 1 h if a three-fold excess of Zn relative to cyclooctene is used. I t is noteworthy that either iV-methylimidazole or imidazole is absolutely required for monooxygenation to occur. 5.2.

The possible roles of the porphyrin and axial ligand of iron in cytochrome P - 4 5 0 and model systems

Very few studies have been devoted to comparisons between porphyrin- and non-porphyrin-Fe (or Mn) complexes under identical conditions. A comparison of the characteristics of the oxidation of hydrocarbons by PhIO catalyzed by either Fe(TPP)(Cl) or non-porphyrin-Fe(III) salts such as FeCl 3 or Fe(acetylacetonate) 3 showed that the porphyrin ligand played at least two roles: (i) to control more efficiently the substrate-derived intermediate free radicals by the iron complexes, and (ii) to trigger the chemoselectivity of the active oxygen iron complexes ( F O N T E C A V E and M A N S U Y , 1984b). As far as the role of the axial iron ligand is concerned, the results obtained with the PhlO-iron-porphyrin

92

D . MANSUY; P . BATTIONI

system, which does not involve any axial thiolate ligand and which reproduces quite well the reactions of cytochrome P-450, indicated that the cysteinate ligand of iron in cytochrome P-450 is not necessary for the transfer of the oxygen atom of PhIO into the substrate. In systems using oxidants containing an O — O bond such as alkylhydroperoxides or H 2 0 2 , monooxygenation of hydrocarbons is observed only in the presence of an imidazole axial ligand of the Fe- or Mn-porphyrins (section 4.). I t seems that the presence of this good electron-donor ligand allows the heterolytic cleavage of the 0 — 0 bond of the oxidant which is necessary for the formation of a formal Fe(V) = 0 active intermediate. Thus, it is tempting to speculate that a possible role of the good electron-donor cysteinate ligand in cytochrome P-450 is to permit also the heterolytic cleavage of the 0 — 0 bond of hydroperoxides. In oxidations by 0 2 itself, a key or beneficial role of imidazole was most often observed in model systems (see above). This suggests that a good electron-donating axial ligand of the iron (imidazole in model systems or cysteinate in cytochrome P-450) greatly facilitates the heterolytic cleavage of the 0 — 0 bond of the possible Fe(III)- (or Mn(III)) — OOH intermediate derived from a two-electron reduction of 0 2 - Besides this very important role in the 0 — O bond cleavage, the axial iron ligands should modulate the chemoselectivity and regioselectivity of the reactive metal-oxo intermediate.

6.

Metalloporphyrin catalysts for regioselective and asymmetric oxidations

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Multiple Activities of Cytochrome P-450

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These reactions hamper measurement of the stoichiometry of oxidase reactions. Thus, to determine the amount of H 2 0 2 formed via direct two-electron oxygen reduction and that of 0,f, one must exclude reactions 4 and 6. While this can be easily done with reaction 6 by adding specific catalase inhibitors, such as azide, hydroxylamine, etc., no means of inhibiting reaction 4 is available. Therefore, straightforward determination of 0 2 and directly formed H 2 0 2 is impossible. The OjT formation rate can be calculated by extrapolating the experimentally obtained values to infinite concentration of the indicator, and the amount of direct H 2 0 2 formation can then be determined by subtracting half the OjT from the net H 2 0 2 formation rate.

2.2.

Superoxide anion formation

CKT formation has been demonstrated repeatedly using various approachesboth in microsomes ( A U S T et al., 1972; D E B E Y and B A L N Y , 1973; S A S A M E et al., 1 9 7 5 ; N E L S O N et al., 1 9 7 6 ; BARTOLI e t al., 1 9 7 7 ; UEMURA e t al., 1 9 7 7 ; AUCLAIR

et al., 1978; P o w i s , 1979; K U T H A N and U L L R I C H , 1982; S O O D A E V A et al., 1982; Z H U K O V and A R C H A K O V , 1982, 1985a) and reconstituted P-450-eontaining systems ( K C T H A N et al., 1978; L \ o E L M A N - S R N I I B E R G and J O H A N S S O N , 1980; S O O D A E V A et al., 1982). The question of the site of O^" formation in microsomes has been discussed for a long time, an important role in Oif generation being ascribed to such components of the oxygenase system as NADPH-dependent reductase and cytochrome b 5 . More recently, however, it has been shown that such commonly used indicators as epinephrine and nitroblue tetrazolium can either catalyze autoxidation of electron-transport proteins or themselves undergo oxidation to yield OjT, leading to considerable overestimation of the reductase and cytochrome b 5 ability to generate 0 7 ( B E R M A N et al., 1976 ; B O R S et al., 1978; S C H E N K M A N et al., 1979). The development of a procedure utilizing the non-autoxidizable cytochrome c subjected to succinvlation in order to reduce its affinity to the reductase ( K U T H A N et al., 1982) has solved this problem. Using this procedure K U T H A N and U L L R I C H have shown microsomal OJT formation to be carbon monoxide sensitive with maximal reversibility by visible light observed at 450 nm. The reactions of O j formation and 7-ethoxvcoumarin deethylation show equal sensitivity to CO indicating that 0 ^ formation is completely due to P-450, the contribution of other microsomal electron carriers being small ( K U T H A N and U L L R I C H , 1982). The same is indicated by sharp enhancement of 0.7 formation on the addition of P-450 to a reconstituted system containing N A D P H - d e p e n d e n t reductase and N A D P H ( S O O D A E V A et al., 1982). In microsomes, antibodies to cytochrome b 5 increase the 0 ; R generation rate ( D Y B I N G et al., 1976), while X A D H inhibits the reaction ( S A S A M E et al., 1975). 0^" generation is also retarded by the addition of cytochrome b 5 to a reconstituted system containing the NADPH-dependent reductase, P-450, N A D P H , and ^-nitroanisole ( I N G E L M A N - S U N D B E R G and J O H A N S S O N , 1980). These data indicate that OiT results from the decay of

158

A . I . ARCHAKOV; A . A . ZHTJKOV

oxvcytochromc P - 4 5 0 whose concentration and, hence, decomposition rate is decreased by cytochrome b.- r mediated reduction. T h e fact t h a t the addition of cytochrome br> to the reconstituted system improves coupling between N A D P H and substrate oxidation without affecting N A D P H oxidation rate (IMAI and SATO, 1977; INGELMAN-SUNDBERG and JOHANSSON, 1980; IMAI, 1981 ; GORSKY and COON, 1986) can also be accounted for by a decreased f r a c tion of N A D P H consumed for O.7 formation via oxycytochrome P - 4 5 0 decay. Isolated cytochrome P - 4 5 0 CAM can also undergo autoxidation to give 0 ^ . T h e oxygenated complex of this hemoprotein is relatively stable and decomposes with a first-order rate constant of 0.008 s _ 1 (SLIGAR et al., 1977). However, no OJT formation can be detected during the autoxidation of the rat liver microsomal P - 4 5 0 isozyme LM 4 and direct formation of H 2 0 2 and water takes place (OPRIAN et al., 1983). Substrates stabilize oxvcytochromc P - 4 5 0 thus saving it for reduction bv a second electron. Camphor binding increases oxycytochrome P - 4 5 0 CAM lmlf-life 12-fold (OPRIAN et al., 1983), and cholesterol binding to cytochrome P - 4 5 0 CAM increases the oxycomplex half-life 15-fold (TUCKEY and KAMIN, 1982).

2.3.

Hydrogen peroxide formation

H 2 0 2 generation during N A D P H oxidation in microsomes was first detected in 1957 (GILLETTE et al., 1957) and repeatedly confirmed later both in the presence and in the absence of P - 4 5 0 substrates (THURMAN et al., 1972 ; BOVERIS et al., 1972; HILDEBRANDT et al., 1973, 1982; STAUDT et al., 1974; NORDBLOM and COON, 1977; ZHUKOV and ARCHAKOV 1982, 1 9 8 5 a ; KUTHAN and ULLRICH, 1982; HILDEBRANDT and ROOTS, 1975; ROESSING et al., 1985; RITTER et al., 1985). As with OJT generation, the majority of investigators agree t h a t H 2 0 2 is produced mainly by cytochrome P-450. This is indicated b y equal activation energies, affinities for N A D P H , and sensitivities to carbon monoxide for the reactions of ethylmorphine deethylation and H 2 0 2 production (ESTABROOK and WERRINGLOER, 1974). Maximum reversibility of CO inhibition by visible light is observed at 450 nm (KUTHAN and ULLRICH, 1982). H 2 0 2 generation is inhibited by the typical P - 4 5 0 inhibitor metyrapone (ROOTS et al., 1980; RITTER et al., 1985). T h e formation of a complex containing an N A D P H and a substrate molecule bound, apparently, to the reductase and P - 4 5 0 , respectively, is required for H 2 0 2 generation to proceed (HILDEBRANDT et al., 1982). P - 4 5 0 substrates influence H 2 0 2 formation differently. T h e effect depends on both the substrate nature and P - 4 5 0 isozyme involved. Aminopyrine, for instance, does not affect the initial rate of H 2 0 2 formation in the microsomes of phenobarbital-induced and untreated tats, but decreases it in the case of induction by 3-methylcholanthrene and increases it with pregnenolone-16:xcarbonitrile induction (HILDEBRANDT et al., 1973). Hexobarbital stimulates H 2 0 2 production in the microsomes of phénobarbital- and pregnenolone-1 fitcarbonitrile-treated animals, while ethylmorphine shows this effect only in t h e

Multiple Activities of Cytochrome P-450

159

latter case and does not affect the rate in the former (ESTABROOK and WERRINGLOER, 1974). Benzphetamine enhances H 2 0 2 production in the microsomes of phénobarbital- but not 3-methylcholanthrene-induced and untreated rats (MANNERING, 1981). H 2 0 2 production can be stimulated by the product of substrate oxidation on cytochrome P-450 rather than by substrate itself as shown, for instance, for propranolol (HILDEBRANDT et al., 1982). The reaction in this case is characterized by a distinct lag-period. The same action is exhibited by the benzphetamine demethylation product, benzylamphetamine (NORDBLOM and COON, 1977). Stimulation of H 2 0 2 formation may underlie the inhibitory action of certain compounds on oxygenase reactions. Thus, low concentrations of the naturally occurring flavonoid quercetin inhibit the oxidation of ethoxyresorufin, y-nitroanisole and benzopyrene in liver microsomes of rats treated with /? - n a p h t h of 1 a v o ne. The substance does not affect the rates of N A D P H and oxygen consumption but doubles the rate of H 2 0 2 formation (SousA and MARLETTA, 1985). Some authors considered the observed uncoupling in general and H 2 0 2 production in particular as an experimental artefact arising from disintegration of endoplasmic reticulum during isolation of the microsomal fraction. I t has been recently shown, however, that aminopyrine administration to rats stimulates H 2 0 2 production in vivo, especially against the background of phénobarbital pretreatment. With guinea pigs, phénobarbital induction alone stimulated H 2 0 2 production without administration of substrates (PREMEREUR et al., 1985).

As pointed out above, two routes of P-450-dependent H 2 0 2 formation are theoretically feasible, namely protonation and dissociation of the peroxycomplex (direct route) and dismutation of preformed superoxide anions (indirect route). The choice between these two alternatives may be made by measuring the stoichiometry of 0 7 and H 2 0 2 generation. In the case of indirect H 2 0 2 formation, the ratio of the 0 7 to H 2 0 2 formation rate should equal 2 according to the stoichiometry of reaction 4, while if H 2 0 2 is in part formed by the direct route the ratio will be less than 2 and the closer to zero the greater the fraction of directly formed H 2 0 2 . The stoichiometry of 07 and H 2 0 2 formation was first studied by KUTHAN et al., (1978), who used succinvlated cytochrome c as an trap. The 10^ : ZlH 2 0 2 ratios measured in reconstituted systems containing NADPH-dependent reductase, P-450, and 7-ethoxycoumarin in different combinations were considerably greater than two, which apparently points to the incorrectness of the procedure used for calculating the 0 7 generation rate. Later, such a procedure was developed in detail in the same laboratory and ratios close to unity were obtained both in the absence and in the presence of substrates (KUTHAN et al., 1982). The authors, failed however, to demonstrate a linear dependence between the 0 ^ formation rate and concentration of microsomal protein, and the results were extrapolated to zero concentration, where the ratio of 2 indicative of the indirect reaction was found. Since the validity of such extrapolation is not obvious, a somewhat different calculation procedure

160

A. I. ARCHAKOV; A. A. ZHUKOV

was used in our laboratory, and the dependence obtained was linear over the whole range of protein concentrations tested (ZHUKOV and ARCHAKOV, 1985 a). Using this procedure and liver microsomes of phenobarbital-induced rabbits, we found that the / J 0 ^ : z l H 2 0 2 ratio was essentially smaller t h a n 2, which points to direct H 2 0 2 formation as proceeding in parallel with the indirect reaction. The contribution of the latter increases with p H (along with an increase in the oxv-P-450 steady-state level), ionic strength and on the addition of M g 2 + ions (ZHUKOV a n d ARCHAKOV, 1 9 8 5 a, b).

One of the objections to the possibility of direct H 2 0 2 formation via peroxy-complex decomposition was the lack of synergism of N A D P H and N A D H (ESTABROOK a n d WERRINGLOER, 1 9 7 4 ; ESTABROOK e t al., 1 9 7 9 ; KUTHAN

and

observed normally in the oxidation of the m a j o r i t y of P - 4 5 0 substrates proceeding through the intermediate formation of the peroxvcomplex. I n our opinion, however, the lack of synergism does not point to o x y - P - 4 5 0 autoxidation as the only route of H 2 0 2 formation. I f this were the case, N A D H , by accelerating oxy-P-450 reduction and thus decreasing its concentration, would decrease the rate of indirect H 2 0 2 formation, and, hence, antagonism rather than an additive effect would be observed. I t has been shown in our laboratory t h a t antagonism is indeed observed at high p H when, as judged from the stoichiometry, H 2 0 2 results mainly from OjT dismutation. B u t at low pH, when the direct route of H 2 0 2 formation is the m a j o r one, N A D P H and N A D H act as antagonists ( Z H U K O V and A R C H A K O V , 1 9 8 5 A , b). T h e additive effect observed at neutral p H ( E S T A B R O O K and W E R R I N G L O E R , 1 9 7 4 ; E S T A B R O O K et al., 1 9 7 9 ; K U T H A N and U L L R I C H , 1 9 8 2 ) , when the fractions of directly and indirectly formed H 2 0 2 are approximately equal, is apparently due to the decrease in the o x y - P - 4 5 0 level being balanced b y the enhancement of direct reaction via peroxy-complex decomposition. ULLRICH, 1 9 8 2 )

When considering the direct route of H 2 0 2 formation, the second electron is normally assumed to be supplied by cytochrome b 5 or N A D P H - d e p e n d e n t reductase. However, an alternative viewpoint exists ascribing t h e role of an electron donor to substrate. Heinemeyer et al. (1980), in their study of the stoichiometry of hexobarbital oxidation, have demonstrated t h a t the sum of H 2 0 2 formed and hexobarbital consumed was not accounted for by N A D P H oxidized. They therefore assumed t h a t the substrate was consumed for the reduction of oxy-P-450, and the peroxy-complex thus formed decomposed to give H 2 0 2 . E S T A B R O O K believes t h a t this mechanism, which he terms " c a r b o n activation", is operating in H 2 0 2 formation during the oxidation of arachidonic acid by P - 4 5 0 ( E S T A B R O O K , 1982; E S T A B R O O K et al., 1982). Generally speaking, the relative probability of o x y - P - 4 5 0 being reduced by substrate or the reductase (cytochrome b 5 ) is considered to depend on the substrate's ability to a c t as an electron donor (ESTABROOK, 1982).

2.4.

Water formation

I t has been demonstrated in a number of investigations dealing with the stoichiometry of P-450-catalyzed reactions that a fraction of N A D P H oxidized

Multiple Activities of Cytochrome P-450

161

and oxygen consumed may remain unaccounted for by substrate oxidation and H202

formation

(ESTABROOK a n d WERRINGLOER,

1974;

ESTABROOK e t

al.,

1 9 7 9 ; H E I N E M E Y E R e t a l . , 1 9 8 0 ; MORGAN e t a l . , 1 9 8 2 ; ZHUKOV a n d ARCHAKOV,

1982). Only half of the NADPH oxidized in microsomes in the absence of substrate corresponds to H 2 0 2 formation (ESTABROOK and WERRINGLOER, 1974; ZHUKOV a n d ARCHAKOV, 1 9 8 2 ; HEINEMEYER e t a l . , 1 9 8 0 ) , a n d in t h e p r e s e n c e

of benzphetamine formaldehyde and H 2 0 2 formed account for about 4 0 % of NADPH consumed (ESTABROOK et al., 1979). Since both substrate oxidation and H 2 0 2 formation require equimolar consumption of both NADPH and oxygen, one can conclude that the cosubstrates participate in a third reaction. This may be either the monooxygenation of a certain endogenous substrate or the direct four-electron reduction of the dioxvgen molecule to yield two molecules of water. The alternatives can be distinguished by calculating the stoichiometry of NADPH and oxygen consumption unaccounted for by substrate oxidation and H 2 0 2 formation. The oxidase mechanism (equation 7) requires the NADPH to 0 2 ratio to equal 2, while it should be unity for the monooxygenase reaction (equation 1). The ratio of 2 has been obtained by STAUDT et al. in the presence of the pseudosubstrate perfluorohexane (STAUDT et al., 1974). The conclusion about direct water formation was, however, not valid since no measures were taken to block microsomal catalase, in the presence of which the obtained stoichiometry could also be observed with indirect reaction involving the intermediate formation of free hydrogen peroxide (see above). With sodium azide added to block catalase, the present authors have demonstrated that direct water formation through complete four-electron reduction of the dioxygen molecule is indeed catalyzed by liver microsomes of phenobarbital-induced rabbits both under conditions of NADPH-free oxidation and in the presence of various cytochrome P-450 substrates and pseudosubstrates (ZHUKOV and ARCHAKOV, 1982, 1985a, b). Similar results have been obtained by GORSKY et al. with reconstituted systems containing NADPH-dependent reductase, phospholipid, and different isozymes of rabbit liver cytochrome P-450. Water formation was not demonstrated to involve reversible dissociation of peroxy-P-450 to yield H 2 0 2 (GORSKY et al., 1984). Perfluorinated hydro-

carbons are distinguished from true substrates by their ability to sharply stimulate the oxidase reaction of water formation both in microsomes and the reconstituted system. The reaction accounts for more than 8 0 % of NADPH, consumed compared to 5 0 % under the conditions of free oxidation (GORSKY e t a l . , 1 9 8 4 ; ZHUKOV a n d ARCHAKOV, 1 9 8 5 a ) .

Interestingly, with some substrates, water formation is observed only in microsomes, while in the reconstituted system NADPH consumption is entirely accounted for by substrate oxidation and H 2 0 2 formation. This was shown, for instance, for benzphetamine and dimethylaniline in the case of their oxidation in rabbit liver microsomes (ZHUKOV and ARCHAKOV, 1985 b) and in a reconstituted system containing P - 4 5 0 L M 2 (NORDBLOM and COON, 1 9 7 7 ;

GORSKY et al., 1984). Water formation in the presence of these substrates is probably catalyzed by P-450 isozymes other than P-450 LM2.

162

A . 1. ARCHAKOV; A . A .

ZHUKOV

As to the precise mechanism of water formation, the first molecule is apparently released during peroxy-P-450 decay to yield the oxenoid species [ F e O ] 3 + . The further fate of the oxenoid oxygen, which can be either inserted into substrate or reduced to yield the second water molecule, appears to depend both on the substrate reactivity and on the specificity of the P-450 isozyme to this substrate. With such inert compounds as perfluorinated hydrocarbons, insertion is impossible and further reduction takes place, while with more reactive substrates, both reactions may run in parallel. Good experimental support for this interpretation is provided by recent findings of M I W A et al. (1985). They studied 7-ethoxycoumarin oxidation in liver microsomes of phenobarbitalinduced rabbits and showed that deuteration of Ci in the ethyl group decreases 5-fold the rate of 7-deethylation, but does not affect those of 6-hydroxylation, N A D P H oxidation, oxygen consumption, and H 2 0 2 formation. The authors conclude that a pathway for [FeO] 3 + decay exists in addition to oxygen insertion into substrate, the reaction taking this way when the insertion is hampered ( M I W A et al., 1985). This pathway is likely to represent oxygen reduction to water. The model proposed allows one to account, without rejecting the feasibility of direct H 2 0 2 formation, for the fact that perfluorinated hydrocarbons fail to increase the rate of H 2 0 2 formation over that observed in the presence of their non-fluorinatecl analogues ( K U T H A N and U L L R I C H , 1 9 8 2 ) . Indeed, if peroxyP-450 decay is assumed to begin from the release of a water molecule and the formation of oxenoid species [FeO] 3 + , then in the case of the coupled reaction the second oxygen atom will be inserted into substrate, while uncoupling will result in the formation of the second water molecule rather than hydrogen peroxide. The sequence and number of steps leading to the release of the second water molecule cannot be unequivocally elucidated on the basis of the data available, and Fig. 1 therefore shows only one of the alternatives. The third and fourth electrons can be supplied, for instance, by cytochrome b 5 . This is consistent with the fact that the greater the uncoupling, the lower the level of reduced b 3 in microsomes and the higher the rate of its oxidation ( S T A U D T et al., 1 9 7 4 ) .

3.

Peroxidase activity

et al. (1973) have first found that liver microsomes can catalyze oxidative dealkylation of amines in the presence of organic hydroperoxides. I n later studies summarized in several reviews ( O ' B R I E N , 1978; W H I T E and COON, 1980; M E T E L I T Z A , 1984; S L I G A R et al., 1984), it has been shown that the KADLUBAE

r o l e o f c o s u b s t r a t e m a y b e p l a y e d b y p e r a c i d s ( B L A K E a n d COON, 1 9 8 0 )

and

hydrogen peroxide ( N O R D B L O M et al., 1976; W E R R I N G L O E R and K A W A N O , 1980; K A R U Z I N A et al., 1983) as well as by compounds containing a single oxygen atom, such as sodium periodate (HRYCAY et al., 1975a), sodium chlorite

(NORDBLOM

et

al.,

1976),

M u l t i p l e A c t i v i t i e s of C y t o c h r o m e P - 4 5 0

iodosobenzene

(LICHTENBERGER

et

al.,

163

1976), iodosobenzene diacetate (GUSTAFSSON et al., 1979), pyridine iV-oxide et al., 1980). All the reactions catalyzed by P-450 in the presence of N A D P H and oxygen may also proceed in the presence of the above oxidants. These are aromatic hvdroxvlation ( R A H I M T U L A and O ' B R I E N , 1974; M E T E L I T Z A et al., 1979), 0 - and A T -dealkylation ( K A D L U B A R et al., 1973; R A H I M T U L A and O ' B R I E N , 1975; N O R D B L O M et al., 1976; S C H E L L E R et al., 1976), epoxidation ( A K H R E M et al., 1977), oxidation of saturated and heterocyclic compounds ( E L L I N and O R R E N I U S , 1975; G U S T A F S S O N and B E R G M A N , 1976) and alcohols ( R A H I M T U L A and O ' B R I E N , 1977a). (SLIGAE

The main question concerning the hydroperoxide-dependent reactions of P-450 as compared to classical peroxidases is that of tire mechanism of the O — 0 bond scission after the formation of cytochrome-hydroperoxide complex and of the further fate of the active iron-oxo species so formed. Cytochromes P-450 are well known to differ from peroxidases in that they contain cysteine thiolate as the fifth axial ligand of heme iron instead of histidine (BRUTCE, 1986). The mechanism of peroxidase reactions has been studied in detail. At the first step of the reaction the iron-protoporphyrin moiety F e ( I I I ) P r I X undergoes two-electron oxidation by a peroxide to form the oxo-liganded ji-cation radical O F e ( l V ) P r I X + , termed "Compound I " , the second oxygen atom from the peroxide being released as water (or an alcohol). Compound I then oxidizes substrate by electron or hydrogen abstraction to give substrate radical and ferro-oxo species O F e ( I V ) P r I X termed "Compound I I " . Compound I I abstracts another electron from substrate to give water, oxidized substrate, and the enzyme in the initial state. Thus the classical peroxidase reaction necessarily involves the step of one-electron substrate oxidation to give a frea radical. With P-450, the formation of a compound I-like species is also beyond doubt, but due to the presence of a thiolate-anion in the P-450 active site instead of a histidine, the enzyme is capable of oxygen insertion into the substrate C—H bond in addition to electron abstraction. The reaction can also involve intermediate formation of a substrate radical which then reacts with the iron-bound hvdroxyl radical ("oxygen rebound" mechanism) (GROVES and MCCLUSKY, 1 9 7 6 ; GROVES,

OFe(IV)S° + AH

1986)

HOFe(IV)S~ + A"

F e ( l I I ) S - + AOH.

The difference between peroxidases and cytochrome P-450 is that with the former, hydrogen is abstracted from substrate by porphyrin cation radical, while with the latter it is the oxenoitl species that attacks substrate. However, the mechanism of 0 — 0 bond cleavage by P-450 has not yet been definitely established. A considerable body of evidence favours a heterolytic mechanism with the formation of a compound I-like species. Thus, when added to microsomes or isolated P-450 LM2, hydroperoxides produce difference spectra identical to those of compound I of certain peroxidases (such as cytochrome c peroxidase) ( Y O N E T A N I and R A Y , 1 9 6 5 ) . Mixing microsomes with

164

A . I . ARCHAKOV; A . A . ZHUKOV

c u m e n e hydroperoxide ( C H P ) gives rise to a signal in t h e E S R s p e c t r u m with g = 2 t h a t is, a radical species similar t o c o m p o u n d I is f o r m e d (RAHIMTULA et al., 1974). [ 4 - 3 H ] A c e t a n i l i d e o x i d a t i o n in t h e presence of h y d r o p e r o x i d e s a n d in t h e c o m p l e t e s y s t e m is c h a r a c t e r i z e d b y e q u a l values of N I H - s h i f t , which p o i n t s t o a c o m m o n m e c h a n i s m of o x y g e n insertion i n t o s u b s t r a t e (RAHIMTULA e t al., 1978). O x y g e n a t i o n of a v a r i e t y of s u b s t r a t e s in t h e a b o v e t w o s y s t e m s shows close values of t h e kinetic a n d t h e r m o d y n a m i c p a r a m e t e r s (AKHREM et al., 1977). T h e f o r m a t i o n of a c o m p o u n d I - l i k e species is f a v o u r e d b y t h e f a c t t h a t o x y g e n a t i o n c a n b e carried o u t b y s i n g l e - o x y g e n donors such as iodosobenzene in which h o m o l y t i c I — 0 bond c l e a v a g e is difficult t o believe. T h a t h e t e r o l y t i c iodosobenzene b r e a k d o w n does indeed o c c u r was shown in E S R studies witli c h r o m i u m - a n d m a n g a n e s e - s u b s t i t u t e d p o r p h y r i n s (GROVES et al., 1 9 8 0 ; GROVES a n d HAUSHALTER, 1981). A c o m p l e x with similar s p e c t r a l p r o p e r t i e s is f o r m e d on iodosobenzene i n t e r a c t i o n w i t h P - 4 5 0 CAM c o n t a i n i n g m a n g a n e s e - s u b s t i t u t e d p o r p h y r i n (GELB et al., 1 9 8 2 b ) . I o d o s o b e n z e n e dependent c a m p h o r o x y g e n a t i o n b y P - 4 5 0 CAM b e i n g i n h i b i t e d b y e x c e s s s u b s t r a t e (HEIMBROOK a n d WHITCOMBE, 1981) is i n d i c a t i v e of t h e p i n g - p o n g k i n e t i c m e c h a n i s m , which m e a n s t h a t t h e iodobenzene s k e l e t o n c a n n o t d i r e c t l y p a r t i c i p a t e in s u b s t r a t e o x y g e n a t i o n since it c a n n o t b e p r e s e n t in t h e a c t i v e site t o g e t h e r with s u b s t r a t e . E P R a n d optical s p e c t r o s c o p y suggest a c o m m o n i n t e r m e d i a t e for iodosobenzene-, hydroperoxide-, a n d p e r a c i d - d r i v e n r e a c t i o n s (ULLRICH, 1 9 7 7 ; LICHTENBERGER a n d ULLRICH, 1 9 7 7 ; GROVES e t a l . ,

1981).

T h e possibility of a h o m o l y t i c 0 — 0 b o n d c l e a v a g e is inferred f r o m e x p e r i m e n t s in which t h e r a t e of C H P - d e p e n d e n t o x y g e n a t i o n has b e e n s h o w n t o depend on t h e n a t u r e of s u b s t i t u e n t s in t h e m o l e c u l e of b o t h s u b s t r a t e a n d c o s u b s t r a t e (BLAKE a n d COON, 1981). T h i s c a n b e a c c o u n t e d for b y t h e p a r t i c i p a t i o n in t h e r a t e - l i m i t i n g step of c u m y l o x y - r a d i c a l f o r m e d t h r o u g h t h e C H P 0 — 0 b o n d homolysis. B e n z o [ a ] p y r e n e conversion in t h e N A D P H - a n d C H P s u p p o r t e d s y s t e m s proceeds b y different ways, p h e n o l s being t h e m a j o r prod u c t s in t h e f o r m e r a n d quinones in t h e l a t t e r case (CAPDEVILA e t al., 1 9 8 0 ) , which m a y also b e due t o different m e c h a n i s m s . I n our l a b o r a t o r y , C H P - d e p e n d e n t r e a c t i o n s were d e m o n s t r a t e d t o subs t a n t i a l l y differ f r o m N A D P H - d e p e n d e n t ones in t h e i r s e n s i t i v i t y t o inhib i t o r s , mucli g r e a t e r s i m i l a r i t y being observed b e t w e e n t h e N A D P H - a n d H 2 0 2 - s u p p o r t e d s y s t e m s (KARUZINA et al., 1983). P e r a c i d homolysis will give a c a r b o x y - r a d i c a l w h i c h shows a t e n d e n c y t o d e c a r b o x y l a t i o n . W i t h p e r o x y p h e n y l a c e t i c acid used b y WHITE et al., decarb o x y l a t i o n will give a b e n z y l radical which, upon r e c o m b i n a t i o n w i t h t h e ironoxo c o m p l e x , will give b e n z y l alcohol. T h e s e a u t h o r s d e m o n s t r a t e d t h a t b e n z y l alcohol is indeed f o r m e d during t h e o x y g e n a t i o n of v a r i o u s s u b s t r a t e s b y pero x y p h e n y l a c e t i c acid in b o t h h e p a t i c a n d b a c t e r i a l P - 4 5 0 - d e p e n d e n t s y s t e m s (WHITE et al., 1980). I n t e r e s t i n g l y , t h e a b i l i t y t o c a t a l y z e p e r a c i d d e c a r b o x y l a t i o n is a u n i q u e p r o p e r t y of P - 4 5 0 n o t f o u n d in o t h e r h e m o p r o t e i n s (horseradish p e r o x i d a s e , chloroperoxidase, c a t a l a s e , m y o g l o b i n ) (MCCARTHY a n d WHITE, 1 9 8 3 a). L a t e r , however, t h e s a m e a u t h o r s showed b y k i n e t i c a n d in-

Multiple Activities of Cytochrome P-450

165

hibitorv analysis that peroxyacetic acid decarboxylation and substrate oxygenation by cytochrome P-450 proceed via entirely independent routes without formation of any common intermediate (MCCARTHY and WHITE, 1983b). Therefore, the mechanism of 0 — 0 bond cleavage during peracidsupported hydroxylation remains unclear. Thus, no definite conclusion can at present be drawn concerning the mechanism of 0 — 0 bond cleavage in the peroxidase reactions of P-450. The majority of results favours heterolysis. At the same time, the data of BLAKE and COON conclusively indicate that homolytic mechanism is operating with CHP (BLAKE and COON, 1981). The mechanism could depend on the oxidant properties. Thus, it has been shown in the studies of interaction between hydroperoxides and iron(III)porphyrins that the second-order rate constant for oxygen transfer to the porphyrin depends on the p K of the leaving group (ROH). The dependence shows break at p K 11, which might result from a heterolytic mechanism being operational at pK < 1 1 (peracids) and a homolytic one at p K > 11 (hydroperoxides) (LEE and BRUICE, 1985). In view of the above data, one could expect the H 2 0 2 -dependent system to model the NAI)PH/0 2 -dependent one best. However, different product patterns and responses to methylcholanthrene treatment were observed with NADPH- and H 2 0 2 -dependent oxygenation of prostaglandins P C E , and P G E 2 in rat liver microsomes (HOLM et al., 1985). Care should, however, be taken in treating results obtained with microsomes since differences in regioselectivity observed may well arise from different P-450 isozymes being involved in NADPH- and hydroperoxide-dependent reactions. Thus, the fact that induction of rats by phénobarbital, methylcholanthrene, and Arochlor 1254 affects NADPH- but not CHP-dependent benzo[a]pyrene oxidation might be due to the involvement of constitutive isozymes in the latter case (WONG et al., 1986). As pointed out above, cytochrome P-450 differs from peroxidases in its ability to insert oxygen into the substrate molecule, while with peroxidases the pathway involving two subsequent one-electron reductions of the oxenoid species is more common. With P-450, however, the reaction can also take the latter pathway. Thus, P-450 catalyzes cytochrome b 5 oxidation in the presence of C H P (RAHIMTULA and O'BRIEN, 1977 b). N A D H is also oxidized b y hydro-

peroxides in the presence of microsomes (HRYCAY and O'BRIEN, 1974). In the experiments with the reconstituted system, this reaction has been shown to require cytochrome b 5 and NADH-dependent reductase for maximal activity (HRYCAY et al., 1975b). NADPH is also oxidized under these conditions (HRYCAY et al., 1975b). These results indicate that oxenoid species can be reduced by electrons supplied from cytochrome b 5 or NADPH-dependent reductase, which appears to occur not only in the presence of hydroperoxides, but also during the P-450-catalyzed four-electron oxidase reaction (see Fig. 1). The role of hydrogen donor in P-450-dependent peroxidase reactions may be played by such compounds as tetra- and dimethylphenylenediamine (HRYCAY and O'BRIEN, 1971), pvrogallol (MCCARTHY and WHITE, 1983a), the 1,4-di-

166

A. 1. ARCHAKOV; A. A. ZHUKOV

hydropyridine derivative felodipine (BAARNHIELM et al., 1984). In the latter two cases the reaction may also proceed in the presence of NADPH and oxygen and, as shown for felodipine, is inhibited by carbon monoxide (Baarnhielm et al., 1984). While the mechanisms of substrate oxidation in hydroperoxideand NADPH/0 2 -dependent systems may be identical, in the latter case the reaction should be treated as a four-electron oxidase rather than peroxidase one since the dioxygen molecule appears to undergo four-electron reduction to yield two molecules of water. Though P-450 ability to utilize hydrogen peroxide and organic hydroperoxides as cosubstrates undoubtedly allows the enzyme to be formally considered a peroxidase, the question still remains as to the degree of similarity between P-450 and "classical peroxidases" such as horseradish peroxidase, catalase, and cytochrome c peroxidase. This point has been recently discussed in a review by MARNETT et al., (1986) where the authors point to considerable differences between the above classical peroxidases on the one hand and such hemoproteins as cytochrome P-450 and metmyoglobin on the other. Thus, turnover numbers of P-450 and metmyoglobin are close and several orders of magnitude lower than those of peroxidases. This appears to result from the fact that, contrary to peroxidases, P-450 reacts slowly with hydroperoxides and the formation of the higher oxidation state rather than its reduction is ratelimiting. The reason for such different reactivities might be the availability in peroxidases of distal histidine participating in general base-general acid catalysis as well as of an arginine capable of stabilizing the negative charge formed in the transition state for hydroperoxide heterolysis (POULOS and KRAUT, 1980), while such residues are lacking in P-450 and its heme environment is nonpolar. Certain other peculiarities, such as different products formed from some hydroperoxides on P-450 and peroxidases also distinguishes the former from classical peroxidases (MARNETT et al., 1986).

4.

Dioxygenase activity

CHEN and LIN (1969) have demonstrated that fluorene hydroxylation in rat liver microsomes proceeds in two steps. First, 9-hydroperoxyfluorene is formed, which is then reduced to the 9-hydroxy derivative. Tetralin oxidation proceeds by the same way (CHEN and LIN, 1968). With dimethylbenzanthracene, endoperoxide is formed at the first step which is then reduced to a (liol (CHEN and TIT, 1977). Later, the same reaction sequence was demonstrated for 9-methylfluorene whose hydroperoxide intermediate is more stable. Both steps required P-450 and N A D P H

(CHEN e t a l . , 1 9 8 5 ) . 9 - H y d r o p e r o x y m e t h y l f l u o r e n e

a l s o f o r m e d in t h e p r e s e n c e of C H P (CHEN a n d GITRKA, 1 9 8 5 ) .

was

ESTABROOK

et al. (1982) believe that one of the pathways for P-450-dependent oxidation of arachidonic acid also involves intermediate hydroperoxide formation. I t is quite possible that P-450 also participates in the biosynthesis of prostaglandins at the s t e p of a r a c h i d o n a t e c y c l i z a t i o n t o y i e l d P G G 2 (GRAE a n d ULLRICH, 1 9 8 2 ) .

Thus, in the above cases P-450 can activate substrate for dioxygen addition, exhibiting dioxygenase activity. The mechanism of such activation remains, however, unclear. ESTABROOK et al., (1982) believe that activation involves hydrogen abstraction from arachidonic acid by oxy-P-450 to yield substrate radical and hydrogen peroxide. On the other hand, the possibility of NADPH and 0 2 substitution for CHP in the formation of 9-hydroperoxy-9-niethylfluorene (CHEN and GURKA, 1985) indicates that another species, such as the oxenoid one, may activate substrate. The further fate of the incorporated dioxygen depends on the chemical nature of the substrate. Hydroperoxides may be stable and exist in the cell as such. Otherwise, they can undergo further catalytic or spontaneous conversion with fixation of both oxygen atoms (formation of diols and carboxyls, cvclization) or decompose to release one or both oxygen atoms as water or singlet o x y g e n (PORTER e t a l . , 1 9 8 4 ; ULLRICH, 1 9 8 4 ) .

5.

Reductase activity

Not only oxygen can serve as an electron acceptor for P-450. Rat liver microsomes catalyze the reduction of azo dyes, the reaction being partly inhibited by carbon monoxide and stimulated by plienobarbital and 3-methvlcholanthrene treatment (HERNANDEZ et al., 1967). Similar sensitivity to CO and induction of P-450 is observed with the reduction of nitro compounds to amines in liver microsomes of mice, rats, and rabbits (GILLETTE et al., 1968). i\T-Ox ides can also serve as substrates for P-450-catalyzed reduction (SUGIURA et al., 1974, 1976; IWASAKI et al., 1977). Unlike azo- and nitroreduction, only P-450 but not NADPH-dependent reductase or cytochrome b 5 can catalyze this reaction (SUGIURA et al., 1976; IWASAKI et al., 1977). iV-Oxides form complexes with P-450 apparently through oxygen coordination with the heme iron and undergo two-electron reduction to give water and an amine. A stoichiometry of 1 : 1 exists between NADPH oxidation and A—oxide reduction (SUGIURA et al., 1976): Finally, P-450 catalyzes reductive dehalogenation of a variety of haloalkanes, such as halothane (2-bromo-2-chloro-l,l,l-trifluoroethane) (FUJII et al., 1981, 1982; AHR et al., 1982), carbon tetrachloride (AHR et al., 1980; NASTAINCZYK et al., 1982), liexa- and pentachloroethane (NASTAINCZYK e t a l . , 1 9 8 2 ) , d i b r o m o e t h a n e (TOMASI e t a l . , 1 9 8 3 ) .

ULLRICH et al., have proposed a scheme for P-450-dependent metabolism of haloalkanes (AHR et al., 1982; NASTAINCZYK et al., 1982). The scheme implies that upon the formation of the enzyme-substrate complex it is reduced by an electron supplied by the NADPH-dependent reductase to yield a substrate free radical and release a halogenide anion. Either the radical may abstract hydrogen from other compounds or the enzyme molecule, which will result in lipid peroxidation, mutagenesis and enzyme inactivation or undergo a second reduction, preferably via cytochrome b5. In the latter case carbanion is formed tending to /S-elimination. The ratio of two- to one-electron reduction products

168

A. I. AECHAKOV; A. A. ZHUKOV

depends mainly on substrate. Carbon tetrachloride is metabolized entirely via t h e one-electron p a t h w a y (AHR et al., 1980), with halothane t h e ratio is close to two (AHR et al., 1982), while with liexa- and pentachloroethane t h e contribution of two-electron reduction is 99.5 and 9 6 % , respectively (NASTAINCZYK et al., 1982). A stoichiometry of 1 : 1 is observed in t h e latter case between X A D P H consumed and p r o d u c t formed. N A D P H and N A D H exhibit synergism as is t h e case with monooxygenase reactions (NASTAINCZYK et al., 1982). The property shared by t h e m a j o r i t y of P-450-dependent reductase reactions in vitro is t h a t t h e y do not proceed under aerobic conditions since oxygen appears to compete efficiently for electrons supplied to t h e hemoprotein. With haloalkanes, metabolism in t h e presence of oxygen proceeds via t h e monooxygenase p a t h w a y , and halothane, for instance, is converted to trifluoroacetic acid (KARASHIMA et al., 1977), though t h e formation of triehloromethyl radical from CC14 is still possible under aerobic conditions. However, t h e results obtained in vitro cannot be extended to physiological conditions since oxygen tension in tissues is considerably lower (about 4 % ) (KRATZ and STAUDINGER, 1965). P-450-dependent hexachloroethane reduction a t this tension is inhibited b y less t h a n 5 0 % (NASTAINCZYK et al., 1 9 8 2 ) . Other d a t a also indicate t h a t monooxygenation and reduction of haloalkanes can proceed in parallel. Thus, t h e products of halothane covalent binding retained b o t h labelled chlorine and 3 H , which points to reductive elimination of bromine ( V A N D Y K E and GANDOLFI, 1 9 7 6 ; GANDOLFI et al., 1 9 8 0 ) . Deuteration of this compound did not affect toxicity, but decreased t h e rate of oxygenation (Sipes et al., 1980).

6.

Oxygen transport

et al. ( 1 9 7 1 ) in their studies on respiration of tissue slices h a v e found t h a t with liver slices, unlike brain ones, t h e dependence of respiration r a t e on t h e partial pressure of oxygen is inconsistent with t h e model suggesting passive oxygen diffusion in tissues. The d a t a obtained could be accounted for if a n oxygen carrier providing facilitated diffusion was assumed t o exist in hepatocytes. Since t h e liver is distinguished from t h e brain b y much higher P-450 content and since oxygen diffusion in tissues proceeds mainly along t h e membranes of endoplasmic reticulum (LONGMUIR, 1981), t h e authors h a v e suggested t h a t t h e role of reversible oxygen binder might be played b y ferrous P-450. I n t h e experiment when oxygen was added to a dithionite-reduced microsomal suspension, subsequent addition of CO resulted in oxygen evolution, t h e a m o u n t of oxygen released being closely correlated with t h a t of P-450 in t h e suspension. This was considered a result of oxygen displacement b y CO f r o m t h e complex with ferrous P - 4 5 0 (LONGMUIR et al., 1 9 7 3 ) . These results cannot be t r e a t e d as t h e definite proof for P-450 participation in facilitated oxygen diffusion in t h e cell. However, taking into account t h a t t h e rate c o n s t a n t of oxy-P-450 dissociation without charge transfer is sufficiently high (about 1 s^ 1 (PEDERSON a n d LONGMUIR

M u l t i p l e A c t i v i t i e s of C y t o c h r o m e P - 4 5 0

169

and t h a t about 3 0 % of total P - 4 5 0 amount is found as oxycomplex in the steady state (WERRINGLOER, 1982), such a possibility cannot be rejected. GUNSALUS, 1 9 7 7 ) )

7.

Oxene transferase activity and singlet oxygen formation

et al. (ULLRICH et al., 1982; GRAF et al., 1983; ULLRICH, 1984) have demonstrated t h a t , when catalyzing the formation of hydroperoxides from PGH 2 , cytochromes P-450 termed thromboxane A2 synthase and prostacyclin synthase act as the carriers of active oxygen atoms, using endoperoxides as substrates. Thromboxane synthase isolated from pig aorta exhibited all properties of P-450 except t h a t it could not bind typical substrates or be reduced by N A D P H in the microsomal fraction (ULLRICH et al., 1981; GRAF and ULLRICH, 1982). The reaction is likely to involve intermediate formation of an oxenoid species [FeO] 3+ and similar to the iodosobenzene supported peroxidase reaction. The heme iron does not undergo reduction in this reaction, and CO shows no inhibition. As a matter of fact, the reaction represents P G H 2 isomerisation to prostacyclin or thromboxane A2 (ULLRICH et al., 1985). The formation of an oxenoid species in the course of PGG 2 oxidation is also suggested by the possibility of cooxygenation of such a typical P-450 substrate as cyclohexane in this reaction. When added to the mixture containing prostaglandin endoperoxide synthase and PGG 2 , cyclohexane was converted to cycloliexanol, the isotope effect being the same as t h a t observed with iodosobenzene (GROSS et al., 1984). A characteristic feature of both reactions is the formation of singlet oxygen as follows (CADENAS et al., 1983; ULLRICH, 1984; NASTAINCULLRICH

ZYK a n d ULLRICH, 1 9 8 4 ) ,

ROOH, PhIO Fe(III)

^ ROH, P hi

ROOH, PhIO -* [FeO]

3+

• ^

+ Fe(III)

ROH, P h i

This appears to be the only enzyme reaction conclusively demonstrated as accompanied by the formation of singlet oxygen in the enzyme active center. Thus, in prostaglandin biosynthesis P-450 acts not only as a dioxygenase at the step of PGG 2 formation from arachidonic acid (cyclooxygenase) and an oxene transferase in the formation of thromboxane A2 and prostacyclin from P G H 2 (thromboxane A2 synthase and prostacyclin synthase), but also as an enzyme capable of generating singlet oxygen as, apparently, a side product.

8.

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Multiple Activities of Cytochrome P-450

175

Chapter 5 The Hormonal and Molecular Basis of Sexually Differentiated Hepatic Drug and Steroid Metabolism in the Rat E . T . MORGAN a n d J . - A .

GUSTAFSSON

1.

Introduction. Sex-dependent expression of P-450 isozymes

2. 2.1. 2.2. 2.3. 2.4. 2.5.

Properties of sex-specific isozymes P-450 16* P-450 15/9 P-450 DEa P-450 g P-450 PCX

177 177 179 180 182 182

3. 3.1. 3.2. 3.3.

Some sex-specific P-450 isozymes are structurally related Immunochemical evidence Protein sequencing and peptide mapping C.DXA sequencing

184 184 186 187

4. 4.1. 4.2.

Hormonal regulation of sex-specific P-450 isozymes P-450-dependent enzyme activities P-450 isozyme levels

188 188 189

5.

Growth hormone regulates P-450 16« and P-450 15(i at a pretranslational level 190

6.

Unanswered Questions

191

7.

References

192

176

. . . .

177

1.

Introduction.

Sex-dependent expression of cytochrome P - 4 5 0 isozymes I n the late 1970s, the concept of sexually differentiated isozymes of hepatic cytochrome P - 4 5 0 catalyzing sex-specific drug and steroid hvdroxylations in the rat received increasing attention due to the following observations: (a) cytochrome P - 4 5 0 is a family of structurally and catalytically distinguishable isozymes which display different responses to different xenobiotic inducers (Lu and WEST, 1980); (b) most drug and many steroid hydroxylations in the liver are P - 4 5 0 1 catalyzed (KUXTZMAN et al., 1964; CONNEY, 1967; L u and WEST, 1980) and (c) many rat hepatic drug- and steroid-metabolizing activities show significant sex differences. I t should be noted that the terms " s e x specific" and "sexually differentiated" used here are usually not intended to mean a total lack of an isozyme in one sex, but rather a significant quantitative difference in the level of expression. I n 1980, we reported the partial purification of a P - 4 5 0 fraction from female rat liver which possessed the female-specific 5»-androstane-3(X,17/?diol disulfate 15/?-hydroxylase activity ( G U S T A F S S O N et al., 1980; L E F E V R E et al., 1980). The major protein-staining band in the electrophoretic analysis of this fraction was absent from the equivalent fraction purified from male rats ( G U S T A F S S O N et al., 1980; L E F E V R E et al., 1980). At the same time, C H U N G and CHAO (1980) reported t h a t the male-specific testosterone 16«-hydroxylase activity was present in a partially purified P - 4 5 0 fraction from male rat liver, but not in the same fraction from females. These early studies strongly indicated that at least two sexually differentiated hepatic microsomal enzyme activities were attributable to distinct, sex-specific isozymes of cytochrome P-450. At least four isozymes of P - 4 5 0 are now known to be expressed in a sexually differentiated manner in the rat, and as discussed below there is evidence for a fifth, namely P - 4 5 0 D E a . These isozymes and their nomenclatures in various laboratories are shown in Table 1, together with some preferred substrates of each isozyme. Only those isozymes showing well-characterized, several-fold sex differences are included, although smaller sex differences in the expression of other isozymes may occur.

2.

Properties of sex-specific isozymes

2.1. P-450 16« and S C H E N K M A N ( 1 9 8 2 ) were the first to purify a sex-specific P - 4 5 0 isozyme to electrophoretic homogeneity, but were unaware a t t h a t time t h a t CHENG

1 The abbreviations used are: P-450, cytochrome P - 4 5 0 ; GH, growth hormone; G H S P , growth hormone secretory pattern; Hx, hypophysectomized

177

Table 1. Sex specific isozymes of cytochrome P-450 in r a t liver

Isozyme

Alternative nomenclature

Dominant gender

Preferred substrates

P-450 g 1 P-450 16a 2

RLM33 h 1 , RLM 5 3 , UT-A 4 , 2c 5 , male 6

male male

benzphetamine steroids (2-, 16a-), 7 - p r o p o x y c o u m a r i n , aminopyrine, b e n z p h e t a m i n e , ethylmorphine, p-tolethylsulfide

P-450 15/S7

i 1 , 2d 5 , UT-I 8 , female 6

female

steroid sulfates (15/?-), a m i n o p y r i n e , ethylmorphine

P-450 PCN 9

PCN-E4, PB-2a 8 , p 1 0

male

e t h y l m o r p h i n e , estradiol (2-, 4-), androstenedione (6/?-)

female

ethylmorphine

P-450 D E a '

-

et al. (1984); 2 M O R G A N e t a l . (1985b); 3 C H E N G and S C H E N K M A N (1982); j G U E X et al. (1982); 5 W A X M A N (1984); 6 K A S I A T A K I et al. (1983); 7 M A C G E O C H et al. (1984); 8 W A X M A N et al. (1985); 9 E L S H O U R B A G Y and G U Z E L I A N (1980); 10 W R I G H T O N et al. (1985). I t should be noted t h a t t h e isozymes e q u a t e d with P-450 PCN here m a y be different homologs a n d all are n o t necessarily sexually differentiated. 1

RYAN

GERICII

P-450 R L M 5 and R L M 3 were male-specific proteins. Using a modification of their protocol, Ave purified P-450 R L M 5 from male rat liver and showed it to be male-specific using an immunoadsorbed polyclonal antibody preparation which specifically recognizes R L M 5 on Western blots (MORGAN et al., 1985a). These antibodies also inhibited 60% of the testosterone 16a- and 2a-hydroxylase activity of male rat liver microsomes, but had no effect on the much lower activities in female microsomes or on testosterone 6/?-hydroxylase activity of microsomes from either sex (MORGAN et al., 1985a). Our results were in agreement with those of W a x m a n (1984) for P-450 2c. W e concluded t h a t P-450 RLM 3 /2c was the male-specific testosterone and 4-androstene-3,17-dione 16a-hydroxylase, and renamed it P-450 16a (MORGAN et al., 1985a, b). As indicated in Table 1, P-450 16a has also been named UT-A, h and male in various laboratories. P-450 16« catalyzes the 16a-hydroxylation of testosterone,

androstene-

d i o n e a n d p r o g e s t e r o n e (CHENG a n d SCHENKMAN, 1 9 8 2 ; WAXMAN, 1 9 8 4 ; MORGAN et al., 1985a), as well as the 2a-hydroxylation of testosterone and

progesterone (WAXMAN, 1984), characterized sex-differences in MORGAN et al., 1985a). CHENG P-450 16a effectively catalyzes

178

and appears to be responsible for the wellthese microsomal activities (WAXMAN, 1984; and SCHENKMAN (1982) also reported t h a t the 2-hydroxylation of 17/9-estradiol, a fact

E . T. MORGAN; J . - À .

GUSTAFSSON

confirmed by R Y A N et al. (1984) and D A N N A N et al. ( 1 9 8 6 b ) . T h e latter authors also showed that P - 4 5 0 16» contributes significantly to the sex-specific microsomal estrogen 2-hydroxylase activity of rat liver. This conclusion is supported by the observation t h a t the microsomal estrogen 2-hydroxylase activity is subject to growtli hormone regulation (JELLINCK et al., 1985), as is P - 4 5 0 1 6 * expression (MORGAN et al., 1985b). Recently, A N D E R S S O N and J O R N V A L L (1986) reported t h a t P - 4 5 0 1 6 * catalyzes the 25-hydroxylations of vitamin D 3 and 5/?-cholestane-3«,7*,12«triol, and showed t h a t this isozyme is responsible for these microsomal activities in male rat liver. The lower but significant activities in female liver microsomes were attributed to other isozymes (ANDERSSON and JORNVALL, 1986). P - 4 5 0 1 6 * contributes significantly to the masculine microsomal metabolism of 7-propoxycoumarin, benzo[a]pyrene and aminopvrine, but other isozymes are also significant in these reactions (KATO et al., 1986). I n addition, purified P - 4 5 0 16« metabolizes ^j-tolethylsulfide, toluene and dimethylaniline (WAXMAN, 1984), benzphetamine, aminopvrine and ethvlmorphine (MORGAN et al., 1985a), R - and ^-warfarin (GUENGERICH et al., 1982), hexobarbital, zoxazolamine and p-nitroanisole (RYAN et al., 1984) with moderate to high efficiency. Such a versatile catalyst could conceivably account for many of the known male-specific drug-metabolizing activities in rat liver, and for others not yet documented. However, in order to unequivocally ascribe any given sex-dependent activity to P - 4 5 0 16« or any other isozyme, detailed antibody inhibition studies will be required. I n Sprague-Dawley rats, P - 4 5 0 1 6 * is present as two allelic variants, distinguishable by two-dimensional gel electrophoresis (RAMPERSAITD et al., 1985). Some populations of this strain express both allozymes and some express only one. Several inbred strains of rat were each found to express only a single variant, and in some strains there was a third form not found in SpragueDawley rats (RAMPERSATJD et al., 1985). As the authors noted, it will be interesting to learn whether there are any differences in the function of these allozymes, or in the developmental and hormonal regulation of their expression. N E G I S H I and co-workers have purified a male-specific testosterone 16«hydroxylase from 1 2 9 / J mouse liver (HARADA and NEGISHI, 1985). This isozyme is 10 times more active in testosterone 16«-hydroxylation than is rat P - 4 5 0 16«, and is responsible for > 9 0 % of the microsomal activity in mice of either sex. I t s structural and genetic relationship to rat P - 4 5 0 16« has not yet been reported.

2.2.

P - 4 5 0 15ii

Although we had obtained a partially purified female P - 4 5 0 fraction which catalyzed the 15/S-hydroxylation of 5*-androstane-3«,17/?-diol 3,17-disulfate as early as 1980 ( G U S T A F S S O N et al., 1980; L E F E V R E et al., 1980), we were unable to further purify the enzyme to homogeneity from this fraction, due to the persistence of a high-Mr contaminant.

Hormonal Regulation of Sex-Specific P-450s

179

During the purification of P - 4 5 0 16a, we noticed t h a t the contaminant seen in our 15/?-hydroxylase preparations was removed at an early stage in the purification procedure. Accordingly, we tried to use the same protocol for the purification of the 15/?-hydroxylase (MACGEOCH et al., 1984). Using a minor modification of this method, we obtained an electrophoreticallv homogeneous P - 4 5 0 isozyme which catalyzed the 15/?-hydroxylation of 5*-androstane3«,17/?-diol 3,17-disulfate at a rate which was five times faster t h a n t h a t of female rat liver microsomes (MACGEOCH et al., 1984). Immunoabsorbed antibodies to this isozyme were used to demonstrate its very low abundance in male rat liver and its regulation by growth hormone (MACGEOCH et al., 1984; 1985). W e concluded that this enzyme is the female specific steroid sulfate 15/?-hydroxylase of rat liver, and named it P - 4 5 0 15/?. Although polyclonal antibodies to P - 4 5 0 15/3 inhibited neither the 15/?-hvdroxylase activities of microsomes nor the purified enzyme (MACGEOCH et al., 1984), our conclusion was supported by our later observation t h a t monoclonal antibodies to P - 4 5 0 15/3 inhibit the 15/9-hydroxylase activity of female rat liver microsomes by 7 0 % ( M O K G A N et al., 1987). P - 4 5 0 15/? has also been purified in other laboratories and named P - 4 5 0 female, 2d or i (Table 1). P - 4 5 0 15/? also catalyzes the metabolism of some drug substrates in a reconstituted system containing the purified isozyme. These include aminopyrine, ethylmorphine and benzphetamine ( M A C G E O C H et al., 1 9 8 4 ; K A M A T A K I et al., 1 9 8 3 ; W A X M A N , 1 9 8 4 ) . However, none of these activities is female-specific. Indeed, their microsomal metabolism is more efficient in males. Thus, 15/?hydroxylation of steroid sulfates is the only female-specific activity which has been attributed to P - 4 5 0 15/?.

2.3. P-450 DEa During purification of the female-specific P - 4 5 0 15/?, another P - 4 5 0 isozyme was isolated from female rat liver, namely P - 4 5 0 D E a (MACGEOCH et al., 1984). Figure 1 shows the relative mobilities of purified P - 4 5 0 isozymes 16«, 15/? and

%K67 K -

43KFig. 1. SDS-polyacrylamide gel electrophoresis of purified P-450 isozymes. 1.0 [j.g of each isozyme was electrophoresed on a 7 . 5 % Polyacrylamide gel containing 0 . 1 % SDS.

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E . T . MOEGAN; J . - A . GUSTAFSSON

D E a on SDS-polyacrylamide gel electrophoresis. P - 4 5 0 15/3 has an Mr of 50,000, while purified D E a and 16« both have Mr values of 52,000 in this system. P - 4 5 0 D E a has been named arbitrarily by us on the basis of its elution profile relative to P - 4 5 0 15/? on a D E A E - S e p h a r o s e column. Although we have purified it from female rat liver, we have not yet prepared specific antibodies to P - 4 5 0 D E a . Consequently, we have been unable to determine whether it is sex-specific or not.

Pig. 2. Western blot showing apparent sex-specificity of P-450 DEa. Microsomes from the indicated sources were electrophoresed on a 7 % polyacrylamide gel containing SDS. The proteins were blotted onto a nitrocellulose filter and probed with a polyclonal antibody to P-450 16a. The antibody-antigen complex was visualized by incubation with goat anti-rabbit IgG followed by a horseradish peroxidase-rabbit antiperoxidase complex. The microsomal samples in each lane were as follows: 1) 2 fig male + 2 fj.g female, 2) 2 y.g male, 3) 2 fig female, 4) 5 fig male + 5 [ig female, 5) 5 fig male, 6) 5 fxg female.

Purified P - 4 5 0 D E a is electrophoretically indistinguishable from P - 4 5 0 1 6 * and is structurally related to this isozyme as observed by immunological crossreactivity and peptide mapping (MORGAN et al., 1985a). However, P - 4 5 0 16% and D E a can also be discriminated b y the latter two criteria. R Y A N et al., (1984) have purified a P - 4 5 0 isozyme, P - 4 5 0 f, which is also electrophoretically, immunologically and structurally similar to P - 4 5 0 16«, and it is possible t h a t P - 4 5 0 f and P - 4 5 0 D E a are the same enzyme. A c D N A for P - 4 5 0 f has been cloned, and used to show t h a t P - 4 5 0 f expression is about twice as high in female rat liver as in males (GONZALEZ et al., 1986a). A similar sex difference in the microsomal P - 4 5 0 f apoprotein levels has beenreported ( B A N D I E R A et al., 1986). However, P - 4 5 0 D E a purified in our laboratory is a low-spin hemoprotein ( M A C G E O C H et al., 1984), whereas P - 4 5 0 f isolated by R Y A N et al. (1984) is in the high-spin state. This suggests t h a t the two isozymes may be different, although the difference in spin state could be explained by the presence or absence of a ligand bound in vivo, possibly originating from the diet or environment. Figure 2 shows the result of a recent experiment designed to answer t h e question of whether P - 4 5 0 D E a is a sex-specific isozyme. W e have consistently noted t h a t although purified P - 4 5 0 16« and D E a are difficult to resolve by SDS-polyacrylamide gel electrophoresis, the two proteins can be resolved in

Hormonal Regulation of Sex-Specific P-450s

181

partially purified or microsomal preparations containing either isozyme. Figure 2 is a Western blot of male and female rat liver microsomes run separately or together on a 7 % SDS-polyacrylamide gel, blotted on nitrocellulose and probed with a polyclonal antibody to P-450 16a. This antibody crossreacts with P-450 DEa, P-450 15/?, and a male-specific protein presumed to be P-450 g. The upper immunologicallv-stained bands in each lane correspond to P-450 16« and P-450 DEa in liver microsomes from male and female rats, respectively (Lanes 2, 3, 4 and 6). A distinctly higher mobility of P-450 16* compared to P-450 DEa is noted. Lane 1 shows that in a mixture of male and female rat liver microsomes, the upper band is a doublet. Thus, microsomal P-450 16a and DEa can be resolved in this system. The presence of only a single upper band in male rat liver microsomes, corresponding to P-450 IQx, indicates that P-450 DEa is absent from, or expressed at low levels in male rat liver (compare lanes 1 and 2). This experiment does not conclusively demonstrate that P-450 DEa is female-specific, but strongly indicates that further investigation with a specific P-450 DEa antibody is needed. 2.4.

P-450 g

P - 4 5 0 g is a male-specific rat liver isozyme isolated by RYAN et al. (1984) and is a p p a r e n t l y e q u i v a l e n t t o t h e R L M 3 of CHENG a n d SCHEXKMAN ( 1 9 8 2 ) .

It

catalyzes the 6/?-hydroxylation of testosterone, as does the male-specific isozyme P-450 PCN (below). P-450 g differs from the latter isozyme, however, in both catalytic and structural properties (RYAN et al., 1984; HANIU et al., 1984). While the sex-specificitv of P-450 g, and its developmental induction in postpubertal males have been clearly demonstrated (BANDIERA et al., 1986), its contributions to any sexually differentiated microsomal drug- or steroidmetabolizing activities have yet to be shown. Interestingly, BANDIERA et al. (1986) have found that adult male outbred rats can be divided into two populations expressing P-450 g at different levels. The levels of P-450 g in the livers of these animals are not dependent on their serum testosterone concentrations. Genetic studies using inbred strains of rats expressing high and low levels of P-450 g in the males have shown that the gene regulating this trait is cis acting, autosomal, and inherited additivelv (RAMPERSATTD e t a l . , 1 9 8 7 ) . I n a d d i t i o n , MCLELLAN-GREEN e t al. ( 1 9 8 7 ) show-

ed that while female Sprague-Dawley and male Fischer rats both have negligible levels of translatable hepatic P-450 g mRXA, male Sprague-Dawley rats expressing high or low levels of the isozyme have similar levels of themRNA. This indicates a translational or postradiational regulation of P-450 g expression in Sprague-Dawley rats, that differs between the two populations. 2.5.

P - 4 5 0 PCN

P-450 PCN is an isozyme first isolated from the livers of rats treated with pregnenolone-16«-carbonitrile, and is induced by that compound (ELSHOURBAGY and GTJZELIAN, 1980). Isozymes with highly similar physical and immuno-

182

E . T . MORGAN; J . - A .

GUSTAFSSON

Table 2 . M o n o c l o n a l a n t i b o d i e s t o P - 4 5 0 1 6 a , 15ß a n d D E a "

Antigen

N u m b e r oí g r o w i n g clones

N u m b e r of positive wells

16«

1224

161

456

60

15ß +

0

b

c

DEa

N u m b e r of specific c l o n e s 6

1 5C

F e m a l e B A L B / c m i c e were i m m u n i z e d w i t h e i t h e r pure P - 4 5 0 1 6 a or a p a r t i a l l y purified f e m a l e P - 4 5 0 f r a c t i o n c o n t a i n i n g P - 4 5 0 15/3 a n d D E a as t h e m a j o r c o m p o n e n t s . A f t e r fusion, growing h y b r i d o m a s were screened b y E L I S A for a n t i b o d i e s t o t h e i n j e c t e d a n t i g e n . P o s i t i v e s were rescreened i m m e d i a t e l y for c r o s s - r e a c t i v i t y t o the antigen from the other sex. N u m b e r of clones p r o d u c i n g a n t i b o d i e s w h i c h did n o t c r o s s - r e a c t in t h e s e c o n d E L I S A screen. All of t h e s e clones p r o d u c e d a n t i b o d i e s specific for P - 4 5 0 15/?.

logical properties to P-450 PCN have been isolated in various laboratories, and have been designated P-450 P B - 2 a (WAXMAX et al., 1985), P B / P C N - E or P C N - E (GUENGERICH et al., 1982; W A X M A N et al., 1985), or P-450 p (WRIGHTON et al., 1985). P-450 PB-2a/PCN is inducible by phenobarbital as well as by pregnenolone-16(x-carbonitrile, while P-450 p has been shown to be induced by glucocorticoids and macrolide antibiotics (SCHUETZ et al., 1984; W R I G H T O N et al., 1985). P-450 PB-2a/PCN is found at much higher levels in hepatic microsomes from male rats than from female rats (WAXMAN et al., 1985), and is neonatally imprinted and androgen-regulated (DANNAN et al., 1986b). Based on these observations and the fact that antibodies to this isozyme effect an 8 7 % inhibition of male rat liver microsomal androstenedione 6/3-hydroxvlase activity (WAXMAN et al., 1985), these authors have proposed that P-450 P B - 2 a / P C X is the isozyme responsible for this sexually differentiated activity in male rat liver. The purified isozyme is not an effective catalyst of 6/3-hvdroxylation, but this is explained by the isozyme's propensity to lose catalytic activity during purification (ELSHOURBAGY and GUZELIAN, 1980). Similarly, P - 4 5 0 P C N / P B 2a/PCN-E can be shown by antibody inhibition to contribute significantly to the male-specific microsomal ethvlmorphine demethvlase (ELSHOURBAGY and GUZELIAN, 1980) and 17/3-estradiol 2- and 4-hydroxylase activities (DANNAN et al., 1986a) despite relatively low activities of the purified enzyme towards these substrates. Recently, GONZALEZ et al. (1986b) have found two P-450 PCN-related m R N A s to be expressed in rat liver, one of which is PCN-inducible and one of which is constitutive. The constitutive mRNA is sexually differentiated and follows the same developmental pattern as P-450 P B - 2 a / P C N - E . However,

H o r m o n a l R e g u l a t i o n of S e x - S p e c i f i c P - 4 5 0 s

183

the NHj-termini of the cloned mRNAs are distinct from those of P-450 PCN-E and P-450 p (GONZALEZ et al., 1986b). Thus, it is possible that P-450 PCN, p, P B - 2 a and PCN-E isozymes are not identical, and/or contain two or more highly homologous forms of the isozyme which might be under different hormonal and xenobiotic regulation. I t is still apparent, however, that one or more members of the P-450 PCN gene family are responsible for the male-specific 6/9-hydroxvlase activity.

3.

Some sex-specific P - 4 5 0 isozymes are structurally related

Cytochrome P-450 isozymes are the products of a multigene superfamily (WHITLOCK, 1986), which can be classified into different families on the basis of protein and DNA sequence homology. Members of a gene family have greater than 5 0 % amino acid sequence homology, and members of a subfamily are at least 7 0 % homologous (J)AYHOFF, 1976). Peptide mapping, NH 2 -terminal amino acid sequencing and immunological cross-reactivities indicate that some sexually differentiated P-450 isozymes have greater structural similarities to each other than to other known P-450 isozymes, indicating that they may belong to a new gene subfamily. cDNA cloning and sequencing have demonstrated that P-450 16« belongs to the same subfamily as P-450 P B - i and f, part of the P-450 b/e gene family.

3.1.

Immunochemical evidence

Four independent laboratories have reported cross-reactivity of polyclonal antibodies to isozymes from the group P-450 16«, P-450 15/3, P-450 f, P-450 g and P-450 D E a with one or more other members of the group. Using a Western blot technique, we showed that rabbit antibodies to P-450 16» cross-react with P-450 15(9 and D E a (MORGAN et al., 1985a), and that antibodies raised to P-450 15/9 cross-react with P-450 16« and D E a (MACGEOCH et al., 1984). This followed a report by KAMATAKI et al. (1983) that antibodies to P-450 16« crossreact with P-450 15/9 in Ouchterlony immunodiffusion analysis, although these authors saw no cross-reactivity of anti-P-450 15/9 serum with P-450 16». Both laboratories reported that cross-reactivity could be removed from the antibody preparations by immunoadsorption with a heterologous crude antigen i. e. p a r t i a l l y p u r i f i e d P - 4 5 0 (MACGEOCH e t a l . , 1 9 8 4 ; MORGAN e t a l . , 1 9 8 5 a )

or

microsomes (KAMATAKI et al., 1983) from rats of the opposite sex. In contrast, WAXMAN (1984) observed residual cross-reactivity of anti-P-450 16« with P-450 15/9, measured by an E L I S A, even after immunoadsorption. He also reported that antibodies to isozyme PB-1, a protein moderately induced by phénobarbital, cross-reacted significantly with P-450 15/3 (WAXMAN, 1984). BANDIERA et al. (1985) also found significant immunochemical similarities among sex-specific P-450 isozymes of rat liver. They raised antibodies to P-450 f and the male-specific P-450 g. Not only did antibodies to these two iso-

184

E . T . MORGAN; J . - A . GUSTAFSSON

z y m e s cross-react w i t h e a c h o t h e r , b u t t h e y also r e c o g n i z e d P - 4 5 0 1 6 « a n d P - 4 5 0 15/? in b o t h O u c h t e r l o n y i m m u n o d i f f u s i o n , E L I S A a n d W e s t e r n b l o t assays (BANDIERA et al., 1985). A n t i - P - 4 5 0 f also showed w e a k c r o s s - r e a c t i v i t y w i t h t h e p h e n o b a r b i t a l - i n d u c i b l e isozymes P - 4 5 0 b a n d e, c o m p l e m e n t i n g a n earlier o b s e r v a t i o n t h a t a n t i - P - 4 5 0 b recognizes P - 4 5 0 f (RYAN e t al., 1 9 8 4 ) . T h e same a u t h o r s were able t o render t h e a n t i - P - 4 5 0 f a n d g a n t i b o d i e s specific for their r e s p e c t i v e a n t i g e n s b y i m m u n o a d s o r b i n g t h e m w i t h t h e c r o s s - r e a c t i v e a n t i g e n s in p a r t i a l l y purified f o r m ( BANDIERA et al., 1 9 8 6 ) . T h e a b o v e o b s e r v a t i o n s i n d i c a t e t h a t t h e r e m a y b e e x t e n s i v e a m i n o acid s e q u e n c e h o m o l o g y a m o n g various m e m b e r s of t h e P - 4 5 0 i s o z y m e g r o u p 16 a , 15/5, f, g a n d D E a , a n d also i n d i c a t e h o m o l o g y of a t l e a s t P - 4 5 0 f a n d P - 4 5 0 15/3 w i t h P - 4 5 0 b and P B - 1 , t h e p h e n o b a r b i t a l - i n d u c i b l e isozymes. BANDIERA e t al. (1985) h a v e noted t h a t o t h e r e x a m p l e s of i m m u n o l o g i c a l c r o s s - r e a c t i v i t y a m o n g P - 4 5 0 isozymes or o t h e r globular p r o t e i n s are a c c o m p a n i e d b y a > 6 0 % a m i n o acid s e q u e n c e h o m o l o g y . I t is c o n c e i v a b l e t h a t some or all of t h e c r o s s - r e a c t i v i t i e s discussed a b o v e could be due to t h e p r e s e n c e of t h e c r o s s - r e a c t i n g a n t i g e n s in t h e purified p r o t e i n s used for i m m u n i z a t i o n . BANDIERA et al. ( 1 9 8 5 ) showed t h a t t h e a n t i P - 4 5 0 f a n d g a n t i b o d i e s did n o t recognize a n y c o n t a m i n a t i n g p r o t e i n s in t h e purified a n t i g e n p r e p a r a t i o n s , arguing against t h i s possibility. H o w e v e r , t h e level of c o n t a m i n a t i o n required t o p r o d u c e t h e c r o s s - r e a c t i n g a n t i b o d i e s would, n o t necessarily be t h e s a m e as t h a t required for d e t e c t i o n of t h e c o n t a m i n a n t s b y Western blotting. W e have produced monoclonal antibodies to P - 4 5 0 16a and 15/5 which s i g n i f i c a n t l y c r o s s - r e a c t with t h e heterologous a n t i g e n a n d / o r P - 4 5 0 D E a , proving t h a t t h e o b s e r v e d i m m u n o l o g i c a l c r o s s - r e a c t i v i t i e s are indeed due to c o m m o n a n t i g e n i c d e t e r m i n a n t s a m o n g t h e a n t i g e n s . T h e s e studies are described below (MORGAN e t al., 1987). P u r i f i e d P - 4 5 0 1 6 * was used t o i m m u n i z e f e m a l e B A L B / c m i c e . S p l e e n cells were p r e p a r e d f r o m m i c e e x h i b i t i n g a high serum t i t e r t o P - 4 5 0 1 6 » a n d f u s e d t o S P 2 / 0 m y e l o m a cells. Cells were p l a t e d in m i c r o l i t e r wells, a n d h y b r i d o m a s selected in m e d i u m c o n t a i n i n g h y p o x a n t h i n e , a m i n o p t e r i n a n d t h y m i d i n e . Growing h y b r i d o m a s were screened b y E L I S A f o r p r o d u c t i o n of a n t i b o d i e s t o P - 4 5 0 1 6 » . Clones giving a p o s i t i v e signal in this a s s a y were t h e n s c r e e n e d b y E L I S A for c r o s s - r e a c t i v i t y w i t h a m i x t u r e of f e m a l e r a t liver p r o t e i n s c o n t a i n ing P - 4 5 0 15/9 a n d D E a as t h e m a j o r c o m p o n e n t s . T a b l e 2 shows t h a t one in eight growing h y b r i d o m a s p r o d u c e d a P - 4 5 0 1 6 « a n t i b o d y , b u t o n l y one of t h e s e a n t i b o d i e s ( < 1 % ) did n o t also recognize t h e P - 4 5 0 15/3/DEa m i x t u r e . S i m i l a r l y , when m i c e were i m m u n i z e d with t h e P - 4 5 0 15/S/DEa m i x t u r e , one in eight clones produced a n a n t i b o d y t o this a n t i g e n . O n l y five of t h e s e ( 8 % ) did n o t recognize P - 4 5 0 1 6 « , a n d all five were specific for P - 4 5 0 15/3 ( T a b l e 2). ANDERSSON a n d JORNVALL (1986) h a v e also r e p o r t e d a low f r e q u e n c v of specific m o n o c l o n a l a n t i b o d i e s t o P - 4 5 0 1 6 « . O n l y t w o of 2 7 p o s i t i v e h y b r i d o m a s produced a n t i b o d i e s t h a t were monospecific f o r t h e i s o z y m e . W e f o u n d t h a t even a m o n g t h o s e m o n o c l o n a l a n t i b o d i e s which specifically recognized P - 4 5 0 15/5 or P - 4 5 0 1 6 « in t h e initial E L I S A screens, s i g n i f i c a n t cross-

Hormonal Regulation of Sex-Specific P-45()s

185

reactivity could be observed depending on the assay system used. Anti-P-450 15/3 monoclonal antibodies F22 and F23 were specific for P-450 15/3 in ELISA, Western blotting and Sepharose-coupled immunoadsorption assays, whereas antibodies F3, F1 and F20 each showed significant cross-reactivity with P-450 16* and/or P-450 D E a in at least one of these assays. I t is of interest to note t h a t all five of the P-450 15/3-specific monoclonal antibodies were able to compete for each other's binding to P-450 15/3, and all inhibited the catalytic activity of microsomal P-450 15/? by up to 70%. These observations indicate (a) t h a t those antigenic sites on P-450 15/3 which are not shared by P-450 16a and P-450 D E a are confined to one part of the molecule and (b) t h a t this part of the molecule is near the P-450 15/3 active site. The observed inhibition of microsomal 15/?-hydroxylase provides the proof that P-450 15/? is indeed the isozyme responsible for this activity. The significant cross-reactivities observed with F l , F3 and F20 antibodies in some systems, but not in others, argues that even those epitopes which distinguish P-450 15/3 from P-450 16* and D E a bear some structural resemblance to those on their counterparts. Even the apparently specific F22 and F23 antibodies showed some affinity for P-450 16:%, since they effectively inhibited the testosterone 16*- and 2*-hydroxylase activities of male rat liver microsomes. We propose t h a t F22 and F23 recognize epitopes at the active sites of both P-450 15/? and P-450 16«, but with a significant difference in affinity. Thus, under conditions favoring high-affinity binding, i. e. procedures with many washing steps such as Western blotting, ELISA and immunoaffinity adsorption, P-450 15/? binds the antibodies with much greater efficiency. However, under conditions involving constant incubation of the antibodies with the enzymes, lower affinity interactions are detected, and thus inhibition of P-450 16* catalytic activity is observed.

3.2.

Protein sequencing and peptide mapping

Several laboratories have studied the structural relationship among two or more of the sex-specific P-450 isozymes by electrophoretic analysis of the peptides formed by limited proteolysis of the isozymes by different peptidases ("peptide mapping"). We compared the peptide maps of P-450 16% and D E a formed by *-chymotrypsin and S. aureus V 8 protease (MORGAN et al., 1985a). Although significant differences were observed in the sizes of some of the peptides generated, many similarities could be seen in the peptide patterns, indicating structural similarities between these isozymes. W A X M A X (1984) also found similarities in the peptide maps of P-450 16* and 15/3 generated by *-chymotrypsin. I n a comparison by R Y A N et al. (1984) of the peptide maps of P-450 f, g and 16* with those of five other isozymes, similarities between the patterns for P-450 g and 16* can be discerned, while those of P-450 f appear to be different. The NH 2 -terminal sequences of P-450 f, g, 16* and 15/? have been elucidated

186

E . T . MORGAN; J . - A . GUSTAFSSON

(HANTI: et al., 1984). Of the first 15 NHj-terminal amino acid residues, P - 4 5 0 16« shares 1 0 - 1 1 with P - 4 5 0 f, g and 15/3. P - 4 5 0 g also shares 12/15 residues with P - 4 5 0 15/?. However, homology in this region is less marked between P - 4 5 0 f and P - 4 5 0 g (7/15) and P-450 f and P - 4 5 0 15/3 (8/15), suggesting that P - 4 5 0 f may be more distantly related to the sex-specific isozymes. The NHj-terminal sequence of P - 4 5 0 16« also showed high homology to P - 4 5 0 P B - 1 (13/20 residues), the phenobarbital-inducible isozyme.

3.3.

c D N A sequencing

GONZALEZ e t al. ( 1 9 8 6 A ) h a v e c l o n e d f u l l l e n g t h c D N A s f o r P - 4 5 0 f a n d P - 4 5 0

P B - 1 . The deduced amino acid sequences show 7 5 % similarity, placing these two isozymes in the same gene subfamily. Both isozymes are 5 0 % homologous to P - 4 5 0 b, indicating t h a t the new P B - l / f subfamily belongs in the P - 4 5 0 b family. W e recently cloned and sequenced the cDNA for P - 4 5 0 16« (STROM et al., unpublished observations), using polyclonal antibodies to screen a library created in / g t 11. Identification of the cDNA as P-450 16« was based on (a) comparison of the nucleotide sequence with the NH 2 -terminal protein sequence (b) recognition of the expressed cDXA-/?-galactosidase fusion protein by specific polyclonal and monoclonal antibodies and (c) hormonal regulation of the m R X A (see below). The sequence of our cDNA is identical to t h a t recently p u b l i s h e d f o r P - 4 5 0 (M-L) (YOSHIOKA e t a l . , 1 9 8 7 ) . P - 4 5 0 1 6 « is 6 9 a n d

73%

homologous to P - 4 5 0 f and P B - 1 , respectively, placing it in the P B - l / f subf a m i l y . I n t e r e s t i n g l y , RAMPEBSAUD a n d WALZ ( 1 9 8 7 ) m a p p e d P - 4 5 0 b a n d e

to a different chromosome from P - 4 5 0 g and h, suggesting t h a t b/e and g/h represent different P - 4 5 0 gene families. This conclusion is obviously at odds witJi the deduced sequences for P - 4 5 0 b and h, which are 5 2 % similar. There is some question as to the size of the P B - l / f subfamily. GONZALEZ et al. (1986a) favored a maximum of three members, based on genomic blot data. FRIEDBERG et al. (1986) using the same technique, found evidence for a fairly large P B - l / f subfamily. YOSHIOKA et al. (1987) have reported t h a t a cDNA isolated from female rat liver shows high sequence similarity with P - 4 5 0 1 G-v, but the information given does not preclude its identity with P - 4 5 0 f or P - 4 5 0 P B - 1 cDNAs. The immunochemical and physical evidence available indicate that P - 4 5 0 g, 16x and 15/3 share some features of both primary and secondary structure. Immunochemical evidence for structural similarities between P - 4 5 0 15¡3 and P - 4 5 0 P B - 1 has also been presented (WAXMAN, 1984). The familial relationships among these isozymes will be revealed when the cDNA sequences for all of them become available. T o t h a t end, we have cloned a cDNA for P - 4 5 0 15/3 (MACGEOCH et al., 1987) and are currently determining its nucleotide sequence.

Hormonal Regulation of Sex-Specific P-450s

187

4.

Hormonal regulation of sex-specific P-450 isozymes

4.1.

P-450-dependent enzyme activities

During the 1970s and early 80s, the G U S T A F S S O N laboratory established that pituitary growth hormone is the primary hormone involved in maintenance of sexually differentiated steroid and drug metabolism in rat liver (for a review, see G U S T A F S S O N et al., 1983). Growth hormone secretion is in turn regulated by the action of gonadal steroids, establishing a gonadal-hypothalamopituitary-liver axis. Blood GH levels in the rat display a sexually dimorphic, temporal variation ( E D E N , 1 9 7 9 ) . In the male, this is characterized by regular peaks every 3 — 4 h, with troughs of almost zero levels between the peaks. In the female blood GH levels are more constant, with smaller peaks occurring at irregular intervals and higher baseline levels than in the male. These fluctuations in blood GH have been attributed to episodic pituitary GH secretion, and we speak of the sexually differentiated GH secretory pattern (GHSP). I t should be noted, however, t h a t a direct connection between the blood GH pattern and episodic pituitary GH secretion has not been demonstrated formally. The hepatic drug- and steroid-metabolizing activities of a male or hvpophysectomized rat liver can be feminized by continuous infusion of GH, mimicking the female G H S P ( M O D E et al., 1 9 8 1 ) . However, intermittent injection of the same daily dose of GH to H x rats has no such effect. There are no sex differences in Hx rats. These observations clearly demonstrate the major role of the sexually dimorphic GHSP in regulation of these enzyme activities. Sex steroids can also modulate hepatic drug- and steroid-metabolizing activities, but this action is achieved via the hvpothalamo-pituitary axis, since gonadectomv or gonadal hormone treatments have no effect in Hx rats ( G U S T A F S S O N and S T E N B E R G , 1 9 7 6 ) . In the male rat, testicular androgens have a dual role. Androgens secreted in the neonatal period imprint the hypothalamus to direct a masculine G H S P ( J A N S S O N et al., 1 9 8 5 ) , and thus a masculine pattern of hepatic drug and steroid metabolism, from the advent of puberty ( E I N A R S S O N et al., 1 9 7 3 ; G U S T A F S S O N et al., 1 9 8 3 ) . Thus, males castrated after puberty still exhibit an essentially masculine profile of hepatic activities. The second role of androgens is in maintaining the masculine pattern of hepatic drug and steroid metabolism from puberty onwards (EINARSSON et al., 1973; G U S T A F S S O N et al., 1 9 8 3 ) . I t has been clearly demonstrated that androgen treatment of unimprinted, mature rats can produce a normal male G H S P ( J A N S S O N and F R O H M A N , 1 9 8 7 ) and a fully masculine hepatic steroid-metabolizing profile ( J A N S S O N et al., 1 9 8 5 ) , disproving the previous concept that only neonatally imprinted rats can fully respond to androgen administered in the adult phase. The female GHSP, and hence the female pattern of hepatic drug and steroid metabolism, appears to be more dependent on the absence of androgen than on the presence of estrogen, since gonadectomy of female rats has relatively

188

E . T . MORGAN; J . - Á .

GUSTAFSSON

little effect compared to that seen in males ( G U S T A F S S O N et al., 1 9 8 3 ) . However, estrogen administration to intact or castrated male rats does feminize hepatic steroid-metabolizing activities ( B E R G and G U S T A F S S O N , 1 9 7 3 ; G I T S T A F S S O N and INGELMAN-SUNDBEBG,

4.2.

1974).

P - 4 5 0 isozyme levels

The male-specific microsomal steroid 16«-hydroxylase and female-specific steroid sulfate 15/3-hvdroxylase activities of rat liver are two activities regulated by G H and testosterone as described above. With the purification of the P-450 isozymes catalyzing these reactions in untreated adult rats, and development of specific immunoassays for them, we and others have been able to study the hormonal regulation of expression of these isozymes in rat liver. These independent studies have clearly established that developmental and hormonal regulation of hepatic microsomal 16*- and 15/?-hvdroxylase activities is achieved by modulation

o f t h e l e v e l s o f P - 4 5 0 1 6 * a n d P - 4 5 0 15/3 i n t h e

endoplasmic

reticulum, and that the G H S P is the major determining factor of the hepatic P - 4 5 0 16«/15/9 p h e n o t v p e .

Thus,

expression

of

P-450 16«

and

P - 4 5 0 15/3

is

low until puberty, when the surge of sex hormones is responsible for the development of sex-specific expression around age 4 — 5 weeks ( M A C G E O C H et al., 1985: M O R G A N et al., 1985b; M A E D A et al., 1984; W A X M A N et al., 1985). The male phenotvpe is neonatally imprinted by androgen, inducible by androgen in the adult ( M O R G A N et al., 1985a; M A C G E O C H et al., 1985; W A X M A N et al., 1985), and inducible by discontinuous G H administration to H x rats ( M O R G A N et al., 1985b; K A T O et al., 1986). Similarly, the feminine phenotvpe is favored by a lack of neonatal androgen imprinting and is inducible by estrogen in intact male rats or gonadectomized rats of either sex ( M A C G E O C H et al., 1985; M O R G A N et al., 1985b; D A N N A N et al., 1986a). Continuous G H infusion to intact male or Hx rats, mimicking the female G H S P , also feminizes the hepatic P-450 phenotvpe ( M A C G E O C H et al., 1985; M O R G A N et al., 1985b; K A T O et al., 1986). Hx rats exhibit no sex difference, and an intermediate phenotvpe ( K A M A T A K I et al., 1985; M A C G E O C H et al., 1985; M O R G A N et al., 1985b). Sex steroids have no effect in Hx rats, confirming that they act via the hypothalamo-pituitary axis ( K A M A T A K I et al., 1985). The roles of the pituitary gland and of constant blood levels of G H in maintaining the female P-450 16«/15/3 phenotype are shown in Figure 3, where it can be seen that hypophysectomy of male and female rats abolishes the sex differences in P-450 16« and 15/3 expression. Infusion of human G H induces the female phenotype in intact male rats or Hx rats of either sex. Male-specific expression of P-450 PCN is also neonatally imprinted by androgen ( W A X M A N et al., 1 9 8 5 ) but the development of sex-specific expression of P - 4 5 0 P C N

i s d i f f e r e n t f r o m t h a t o f P - 4 5 0 1 6 « a n d P - 4 5 0 15/3. P - 4 5 0

PCN

expression is near adult male levels in immature rats of both sexes, then begins to fall steadily in the female from between weeks 2 and 4 to week 8 ( W A X M A N

H o r m o n a l R e g u l a t i o n of S e x - S p e c i f i c P - 4 5 0 s

189

et al., 1985). P-450 PON is GH-regulated, but is not dependent on the G H S P since it is induced in H x males regardless of whether administration is intermittent or continuous (YAMAZOE et al., 1 9 8 6 ) .

•

o

H

o ffl 9 5 % ) sequences (Table 2). Several published sequences not subjected here to extensive analysis are listed in Table 3 in connection with their closest relatives in t h e 38 sequences in Table 2. Basically we avoided mixing two or more sequences of different origin for t h e

202

O . GOTOH; Y . F U J I I - K U R I Y A M A

Table 3 a. Variant Sequences Present Sequence Species

Form

Variant Sequence Residue

Rat

Ile(53)

Rat

Identical

Human

Phe(381) Ile(462) -

Rat

-

Residue Form Source

Reference

Met

c

cDNA

YABUSAKI e t al.

c

gene

HINES e t al. (1985)

Leu Val

pi pi

cDNA gene

JAISWAL e t al. ( 1 9 8 5 a )

Arg(173) Cys(403) -

His Arg

d

gene

SOGAWA e t a l . (1984)

Lys

d

aa

HANIU e t a l . (1986)

gene

GONZALEZ

P2

cDNA

KIMURA et al. (1986)

3

cDNA

JAISWAL e t al.

b

gene

SUWA e t al. (1985)

(1984)

JAISWAL e t al. (1986 b)

Rat

d

Arg(31) -

Mouse

P3

Identical

Mouse

p3

IIe(384)

• Met

Human

lm4

Ser(79) Leu(51i) • Phe(512) •

Arg Del. Arg

Rat

b

Identical

Rat

e

Met(473) -

Lys

e

aa

YUAN e t al.

c21

Ser(14) Tyr(401) -

Ala His

c21

gene

CHUNG e t al. ( 1 9 8 6 a)

(123/124)Tyr(401) Ser(431) -

Thr His Cys

c21

cDNA

JOHN e t al. (1986)

Bovine Bovine

c21

3

P

P

et al. (1985a)

(1986)

(1983)

Human

c21

Identical

c21

gene

WHITE et al. (1986)

Human

see

Cys(16) - Tyr Leu(274) - Phe Ile(301) - • Met

see

cDNA

CHUNG

Bovine

see

Asn(57) - Asp Asp(106) - Asn Asp(197) - Asn

see

aa

CHASHCHIN

et al. (1986b)

et al. (1986)

s a m e f o r m of P - 4 5 0 . H o w e v e r , r a b b i t P - 4 5 0 l m 4 s e q u e n c e a n a l y z e d h e r e is a m i x t u r e of t w o s o u r c e s ; r e s i d u e s 2 t o 92 w e r e t a k e n f r o m t h e p r o t e i n s e q u e n c e of O Z O L S (1986), w h e r e a s t h e r e s t of t h e s e q u e n c e is d e r i v e d f r o m t h e c D N A s e q u e n c e of O K I N O e t al. (1985). T r a n s l a t i o n e r r o r s w e r e c o r r e c t e d w i t h o u t a n o t i c e w h e n g e n o m i c or c D N A s e q u e n c e is a v a i l a b l e . T h e f o u r t h r e s i d u e in r a t P - 4 5 0 b w a s c o r r e c t e d t o b e Ser ( Y U A N e t al., 1983; S U W A e t al., 1985) i n s t e a d of T h r in t h e original N - t e r m i n a l s e q u e n c e r e p o r t e d b y B O T E L H O e t al., (1979).

Evolution, Structure, Gene Regulation of P-450

203

Table 3 b. Other P - 4 5 0 Sequences Closest Sequence

Sequence

Species

Species

Form

Rat

e(U. C.)

Form

Rat

Rat

Rat

Rat

Rat

Rabbit

lm2

Rabbit

pb24

lm2

Comparable Positions ( % of Total)

No. of Reference Difference ( % Identity)

168-491 (66.0)

(99.1)

281-491 (43.0)

5 (97.6)

KUMER et al.

295-491 (40.1)

37

AFFOLTER e t al.

1—491 (99.6)

17

PHILLIPS e t a l . ( 1 9 8 3 )

HEINEMANN

(96.5)

lm4

Rabbit

lm4

Rabbit

lm3b

Rabbit

pc3

87-490 (82.4)

Rat

f

Rat

tfl

87-490 (82.4)

(97.8)

162-492 (67.7)

13 (96.5) 138 (59.0)

Rat

a

Human

1

(1986)

(81.2)

Rabbit

(68.0)

(1983)

OZOLS

and

(1983)

FUJITA e t a l . ( 1 9 8 4 )

11

(96.9)

LEIGHTON e t al.

(1984)

9 FRIEDBERG et al.

(1986) PHILLIPS e t al.

(1985)

R a b b i t P - 4 5 0 lm2 of HEINEMANN and OZOLS and P - 4 5 0 lm4 of FUJITA e t al. were determined b y protein sequencing methods. Other sequences were deduced from c D N A sequences.

The N-terminal 50 residues of rabbit P-450 lm6 were taken from M I H A R A et al. (1985). The missing part in rabbit P-450pc2 cDNA ( L E I G H T O N et al., 1984) was made up by the corresponding gene sequence ( G O V I N D et al., 1986). Ser(262) of rat P-450d inferred from the cDNA sequence ( K A W A J I R I et al., 1984a) was corrected to be Phe as described previously (SOGAWA et al., 1985). The 2498th and 2499th nucleotides in human P-450c 21b gene reported by H I G A S H I et al. (1986) were found to be inverted, leading to correction from Pro to Arg at the amino acid site 426 as in the sequence of W H I T E et al. (1986). No other change was made, but the initiator Met was added to rabbit P-450 lm4 and rabbit P-450 lm3a to keep consistency in numbering system with those for related sequences. Throughout this chapter, we use a conventional name of each gene or protein usually given by the author(s) who determined the sequence. We omit hyphens and parentheses, and use lower case letters to identify individual

204

O. GOTOH; Y .

FUJII-KURIYAMA

forms of P-450. To facilitate cross references, a universal nomenclature recently proposed by Nebert et al. (1987) is shown together with the conventional names in Table 2. 3.3.

P-450 protein families

The terms protein family and superfamily have been used with different meanings ( D A Y H O F E , 1 9 7 8 ; D O O L I T T L E , 1 9 8 1 ) . According to D O O L I T T L E , any sequences that are demonstrably related to each other belong to the same family, while a superfamily is composed of two or more families not all of whose members are demonstrably homologous with all the other members in each family. According to this definition, all bacterial and eukaryotic P-450s belong to a single family, while P-450, cytochrome b, and some subunits of cytochrome c oxidase may possibly compose a superfamily. On the other hand, more popular notions of protein family and superfamily are those of DAYHOFF (1978). By her definitions, two significantly related sequences belong to a superfamily if they differ at more than 50% positions, belong to a family if they differ at more than 20% but less than 50% positions, and belong to a subfamily if they differ at less than 20% positions. When we apply these definitions to known P-450 sequences, we identify nine families in the unique superfamily. Although the cut-off value discriminating families is more or less arbitrary, we will use this classification of P-450 families until solid criteria based on gene structure or other means become available. The nine family names (Table 2) used in this chapter should be familiar to most readers. 3.4.

Sequence alignment

A collection of related protein sequences provides maximal information when they are properly aligned. Thus it is crucially important to get a good alignment. Optimal alignments of two or three sequences are obtained in a rigorous manner ( N E E D L E M A N and W U N S C H , 1 9 7 0 ; G O T O H , 1 9 8 2 , 1 9 8 6 ) . Here, an optimal alignment means t h a t the distance value associated with the alignment is minimal (or equivalently, similarity is maximal) among all alternative alignments, where a distance (or similarity) is defined as a sum of weights given to individual matches, replacements, deletions and insertions. Unfortunately, no practical method is known for aligning many sequences in a mathematically rigorous manner. Our approach is to recurrently apply the pairwise or three-way alignment method to pre-aligned sets of sequences. Namely, we first constructed a preliminary tree t h a t represents relationships among all the P-450 sequences considered. The preliminary tree was obtained on the basis of distance values for selected pairs of the sequences. Inspecting the tree, we chose two or three sequences that were most closely related and obtained their optimal alignment. The aligned sequences were combined and treated as a single sequence. The process was repeated until all the original

Evolution, Structure, Gene Regulation of P-450

205

sequences were combined into a single alignment. For example, we first combined rat P-450c and mouse P-450 pi sequences. The combined sequence was aligned with human P-450c and rabbit P-450 lm6 by the three-way method. The c-group sequence was aligned with the similarly combined d-group sequence to generate the MC-family sequence. The MC-family sequence was then aligned with the PB-family sequence and the combined C21 — 17a, sequence, and so on. A position in a combined sequence indices a column vector composed of M aligned residues, where M is the number of original sequences included in the combined sequence. A deletion (represented by a dash) is counted as a residue for convention. In calculation of an optimal alignment, the weight given to a matched pair of positions (column vectors) was evaluated by averaging the mutation data matrix elements (DAYHOFF, 1978) corresponding to all combinations of residues in one column and those in the other. A deletion matched with any residue but a deletion was weighted by 6, whereas a null weight was given to a deletion matched with a deletion in another column. Additional weight of 6 was given to a consecutive run of deletions introduced by the matching algorithms (GOTOH, 1 9 8 2 , 1 9 8 6 ) .

The alignment thus obtained satisfied several criteria, such as coincidence of predicted secondary structures and hydrophobic patterns in different groups of P-450s. However, sequences in the central region of about 130 residues are extremely variable, so that we could not establish a solid alignment in this region. I t was most difficult to align the combined animal sequences witli P-450 cam, perhaps because considerable structural rearrangement may have occurred in this variable region since the divergence of prokaryote and eukarvote. From structural considerations (see next section), we imposed a constraint that Arg(187) in P-450 cam should match Lys(256) in rat P-450c and Lys(236) in rat P-450b; without this constraint, Arg(187) in P-450 cam could alternatively match Arg(245) in rat P-450c and Lys(225) in rat P-450b. Figure 1 shows the final output of the alignment procedure. The residues lost in a mature protein are underlined. Every 20 positions are numbered above the sequences. The residue numbers of individual sequences for the left-most and the right-most amino acids in each row are given on the both sides. Hereafter, we will refer to a position number in square brackets while a residue number in parentheses, e. g., Cys[497] = Cys(461) for rat P-450c and = Cys(436) for rat P-450b. Our residue numbers for rabbit P-450 lm4, rabbit P-450 lm3a and P.putida P-450 cam differ by 1 from those in the literature (OZOLS, 1986; KHANI et al., 1987; POULOS et al., 1985), because we regard the initiator Met as residue 1 despite of its absence in purified proteins. 3.5.

Construction of a phylogenetic tree

Based on the alignments in Figure 1, we estimated the number of accepted point mutations per 100 residues (PAM) for every pair of the 38 P-450 sequences. Note that PAM is generally greater than the percent difference between

206

O. GOTOH; Y . FUJII-KURIYAMA

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present-dav sequences, since multiple mutations may have occurred at a locus. DAYHOFF (1978) proposed a table that relates a percent identity to a PAM, Instead of using this table, we adopted a maximum likelihood method (CRAMEK, 1946) to estimate a PAM on the assumption that evolutionary changes of amino acids follow a Markov process determined by the transition matrix presented in Figure 82 in DAYHOFF (1978). Briefly, we first count the individual numbers of the 20 X 20 types of amino-acid changes associated with the conversion from one sequence to the other. The diagonal elements of this table represent the numbers of unchanged residues. The PAM value that maximizes the probability of observing these numbers of amino-acid changes and identities is defined as the maximum likelihood estimate of PAM. We omitted deletions or insertions from the present analysis, since the frequency of evolutionary events that change sequence length is not well understood. Using the 703 ( = 38X37/2) PAM values, we constructed a phvlogenetic tree of P-450 protein sequences (Fig. 2) by UPGMA (unweighted pair group method of averaging) (SOKAL and SNEATH, 1963). Although UPGMA assumes a constant evolutionary change rate, a constant rate is not realized by P-450 sequences as discussed below. However, the tree topology may not be significantly affected even if other tree reconstruction methods are used, since the clusters are well separated from one another. 3.6.

Rates of amino-acid change

The PAM values between orthologous proteins in different mammalian species (Table 4) show that no pair of the four species, human, rat or mouse, rabbit and bovine, is apparently closer than other pairs. This observation is consistent with the well-accepted scheme that various orders of mammals differentiated in a short period around 80 million years (My) ago (DICKERSON, 1972; MCLAUGHLIN and DAYHOFF, 1972). If we accept this scheme, the average rate of amino acid replacement is calculated to be 1.8 ± 0.3 (SD) Pauling, or equivalently the unit evolutionary period (UEP) equals 2.8 ± 0.5 (SD), where one Pauling stands for 10 9 replacements per site per year (KIMURA, 1969) and the UEP is defined as the time in million years for a 1 % change in amino acid sequence to occur (DICKERSON, 1972). As shown in Table 4, the variation in the rates of different lines is significantly greater than that expected from purely statistical variations (approximately i 0.13 Pauling). Functional and/or structural constraints must have operated differently on one line versus another. It was rather unexpected that the P-450s such as C21, 17«, and SCC that catalyze specific metabolic reactions would show relatively high evolutionary rates compared to those with broad substrate specificities (members in PB and MC families). The alcohol inducible forms (rat and human P-450j and rabbit P-450 lm3a) show the slowest rates of 1.3 ~ 1.5 Pauling (UEP = 3.4 ~ 3.8). A similar rate (1.3 Pauling or UEP = 3.9) is observed for the divergence between chicken P-450 pbl and a mammalian group composed of the alcohol inducible forms and the so-called "constitutive" forms.

212

O. GOTOH; Y. FUJII-KURIYAMA

Table 4. Comparison of orthologous P-450 sequences

Gene

Name

Species 1

Species 2

PAM

Match %

Rate 1 Pauling

IA1

c

rat rodent 2 rodent human

mouse human rabbit rabbit

6.15 21.73 30.42 29.09

93.13 78.15 73.66 73.93

1.80 1.36 1.90 1.81

2.76 3.68 2.62 2.75

IA2

d

rat rodent rodent human

mouse human rabbit rabbit

6.03 30.92 30.16 24.84

93.37 73.26 73.94 77.71

1.77 1.93 1.89 1.55

2.82 2.59 2.65 3.22

1IB1

b

rat

rabbit

26.32

77.19

1.65

3.04

IIE1

j

rat rat human

human rabbit rabbit

23.54 20.83 23.54

78.70 81.14 79.11

1.47 1.30 1.47

3.40 3.84 3.40

XXI

c21

mouse mouse human

human bovine bovine

36.18 35.78 24.99

70.77 70.97 78.11

2.26 2.24 1.56

2.21 2.24 3.20

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vestiges of transposon-like sequences t h a t have been inserted into genes, become fixed and then diverged in nucleotide sequence. I n the case of P - 4 5 0 genes, however, we have not yet found J ) N A sequences reminiscent of the insertion of transposon-like sequences in the introns. T h e D N A sequences in the introns m a y have diverged so rapidly after insertion into the preexisting gene t h a t their characteristic features cannot be recognized.

5.3.

Chromosomal localization

Chromosomal localization of P - 4 5 0 genes is now in progress in several laboratories by in situ hybridization techniques and Southern blot analysis of hybrid cell DNAs. So far several P - 4 5 0 genes have been determined for their chromosomal localizations in human and mouse genomes (WOLF, 1986; NEBERT and GONZALEZ, 1987). I n view of the evolutionary distance between these various families of P - 4 5 0 , it may not be surprising t h a t their genes are widely dispersed among chromosomes. I n mouse, PB-inducible P - 4 5 0 (equivalent t o rat P - 4 5 0 b and P-450e) has been reported to locate on chromosome 7 (SIMMONS and KASPER, 1983), while MC-inducible P - 4 5 0 (equivalent to rat P - 4 5 0 c and P - 4 5 0 d ) is found on chromosome 9 (TUKEY et al., 1984). P - 4 5 0 pen and steroidogenic P - 4 5 0 c21 are on chromosome 6 (SIMMONS et al., 1984) and 17 (WHITE et al., 1984), respectively.

6.

Regulation of expression of cytochrome P - 4 5 0 genes

Most of P - 4 5 0 species are known to be regulated for their gene expression b y endogenous or exogenous stimuli and show temporal and tissue-specific expressions. Exogenous substances such as P B , 3-MC, 2,3,7,8-tetrachlorodibenzo/j-dioxin (TCDD), PCN, ethanol, polychlorinated biphenyl (PCB), isosafrole and others are known to be inducers of their specific forms of cytochrome P - 4 5 0 , while ACTH (adrenocorticotropin), F S H (follicle-stimulating hormone), L H (luteinizing growth hormone), and many steroids regulate the synthesis of other forms of P - 4 5 0 s as endogenous stimuli (ADESNIK and ATCHISON, 1986; WHITLOCK, 1986; WATERMAN et al., 1986). I n general, it has been clarified from the studies using translational and transcriptional inhibitors and in vitro translational systems directed by the isolated m R N A t h a t the increased levels of various forms of P - 4 5 0 result from increased rates of de novo synthesis of the P - 4 5 0 apoproteins which can be accounted for b y the increase in the corresponding m R N A within cells treated with inducers. Evidence has been accumulating t h a t the increase in the P - 4 5 0 m R N A is primarily a consequence of transcriptional activation of the corresponding genes. The availability of hybridization probes to measure precisely the m R N A content and transcription rate makes it possible to clarify the level

232

O. GOTOH; Y. FUJII-KURIYAMA

a t which regulation is effected, and facilitate accurate a n d direct m e a s u r e m e n t of t h e changes in levels of gene expression. These observations h a v e established t h e inducible n a t u r e of cytochrome P-450s to open t h e way t o w a r d studies of t h e induction mechanism a t a molecular level a n d h a v e been extensively documented in t h e excellent review articles published recently (ADESNIK a n d A T C H I S O N , 1986; W H I T L O C K , 1986; W A T E R M A N et al., 1986; E I S E N , 1986). Accordingly, we will focus mainly on t h e molecular biological studies using isolated genes and gene transfer m e t h o d s to provide deeper insights into t h e molecular aspects of t h e induction mechanisms in this section. Several genes of t h e P-450 superfamily h a v e been isolated and analysed for their structures as described in t h e previous section, b u t only a few successful examples h a v e been reported in which t h e P-450 genes or their derivative genes transfected into t h e cultured cell lines expressed their products in response t o t h e inducers faithfully mimicking t h e expression of t h e endogenous corresponding genes in their specific tissue. These are genes for drug-metabolizing mouse P-450 p i or t h e r a t equivalent P-450c which is induced in liver b y lipophilic xenobiotics such as 3-MC, TCD1) and P C B and for steroidogenic P-450 c21 whose expression is positively regulated in adrenal cortex b y ACTH.

6.1.

Poly cyclic aromatic hydrocarbon inducible P-450 gene

Several lines of evidence h a v e suggested t h a t t h e induction of this group of cytochrome P-450s b y TCDD or 3-MC is mediated b y t h e receptor for these chemicals which is believed t o be a p r o d u c t of t h e Ah locus (NEBERT e t al., 1972; T H O M A S et al., 1972). The inducers t a k e n up in t h e cells are first recognized b y t h e receptor t o f o r m t h e receptor-inducer complex. I t has been proved t h a t t h e receptor associated with t h e inducer, in t u r n , moves to t h e nucleus t o activate t h e transcription of r a t P-450c gene or mouse equivalent (P-450 p i ) probably b y interacting with a regulatory region of t h e gene (POLAND a n d G L O V E R , 1973; P O L A N D et al., 1974). The presence of t h e cis-acting regulatory sequence in response to T C D D or 3-MC has been reported b y J O N E S et al. (1985, 1986a, 1986b), GONZALEZ and N E B E R T (1985), a n d F U J I S A W A - S E H A R A et al. (1986) in t h e u p s t r e a m region of a high-activity v a r i a n t P-450 p i gene, n o r m a l P-450 p i gene and r a t P-450c gene, respectively. All these groups constructed chimeric genes b y ligating t h e 5' u p s t r e a m regions of their respective genes t o t h e chloramphenicol acetyltransferase (CAT) s t r u c t u r a l gene a n d t h e n introduced t h e m into H e p a - 1 cells for investigating t h e inducibility of t h e CAT activity. The transfection experiments showed t h a t these fusion genes expressed t h e CAT activity in response t o TCDD or 3-MC. T h e t r a n s i e n t expression system of t h e CAT activity was shown to mimic f a i t h f u l l y t h e induction phenomena of P-450c gene in r a t livers ( F U J I S A W A - S E H A R A et al., 1986), b y all criteria tested such as inducer-specificity, cell-specificity, inducibility (ratio of t h e induced to t h e non-induced activity) and correct transcription site determined b y SI nuclease mapping. Essentially similar results h a v e been obtained

Evolution, Structure, Gene Regulation of P-450

233

with transformants of Hepa-1 cells cotransfected with P-450c fusion gene and pSV2-neo. Because of the apparently aberrant construction of the P-450 fusion gene reported by JONES et al., (1985,1986 a), the transcription in their constructs seemed to use a cryptic start site in the middle of the first intron in response to TCDD. The deletion experiments revealed that the upstream regulatory regions up to —6.3 kb contain at least three functional domains; —44 to —200 bp region immediately upstream of the T A T A box functions in the basal level of transcription and the other two which are located in the sequences from —0.8 to —1.1 kb and from —1.1 to —6.3 kb enhance, in combination, the transcription in response to inducers (FUJISAWA-SEHARA et al., 1986; SOGAWA et al., 1986). Besides these functional regions, Jones et al. (1985) have suggested that the sequence from —50 to —350 bp (— 700 to —1000 bp in their numbering system) is an inhibitory domain which presumably interacts with the labile repressor. Further subdivision of the sequence from —0.8 to —1.1 kb which is the most important for the inducibilitv-conferring activity revealed that two homologous core sequences of 15 base pairs are tandemly arranged in the inverted orientation with each other in the sequences from —1007 to —1021 bp and from — 1088 to —1092 bp in rat gene (FUJISAWA-SEHARA et al., 1987). The homologous core sequences are also found in the corresponding positions in the mouse JONES e t al., 1986 a ; GONZALEZ e t al., 1 9 8 5 a ) a n d h u m a n g e n e s ( K A W A J I R I e t

GGCC;

AC

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XRE2

AGCC

human

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• JmlLf S

, CTOTT

CTTAA

MT-llA

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AGAT CAAA

chicken

lysozyme

TGTG

chicken

lysozymr-

MSV

GCTTA H C A A

P i g . 9. D N A sequences of X R E s a n d several G R E s . D N A sequences of h u m a n metallothionein I I A ( M T - I I A ) gene f r o m - 2 6 4 to - 2 3 6 ( K A R I N et al., 1984), M M T V D N A f r o m — 186 to - 1 5 8 ( P A Y V A R et al., 1983; SCHEIDEREIT a n d BEATO, 1984), M S V D N A f r o m — 175 to

— 2 0 3 (MIKSICEK et al., 1986; D E F R A N C O a n d YAMAJIOTO,

1986), a n d the

sequences of chicken lysozyme gene f r o m — 5 0 to — 7 7 a n d f r o m — 1 9 1 to — 1 6 4 ( R E N K A WITZ et al., 1984) were c o m p a r e d with X R E 1 a n d X R E 2 . T h e distal G R E in chicken l y s o z y m e ( m a r k e d b y an asterisk)

is also recognized b y progesterone receptor

(Von

DER A H E et al., 1986). T h i c k l y stippled lanes indicate the positions in w h i c h a single nucleotide occupies 5 or more positions out of 7, a n d lightly stippled lanes indicate the positions in w h i c h either of the t w o alternative nucleotides occupies 6 or more positions.

234

O . GOTOH; Y . F U J I I - K U R I Y A M A

al., 1 9 8 6 ) . These two core sequences express the CAT activity in response to 3-MC, even when they are placed separately on the heterologous promoter of S V 4 0 early gene without the enhancer sequence which is fused to the CAT gene (SOGAWA et al., 1 9 8 6 ; F U J I S A W A - S E H A R A et al., 1 9 8 7 ) . Tandem arrangement of the sequences was found to enhance remarkably the expression of the subordinate gene. F U J I S A W A - S E H A R A et al. ( 1 9 8 7 ) have found that the core sequence which is designated xenobiotic responsive element or X R E shows a significant homology with the sequence of glucocorticoid responsive elements (GRE) ( K A R I X et al., 1 9 8 4 ) of MMTV, human metallothionein and lysozvme genes as shown in Figure 9. Although X R E 1 and 2 have 1 and 2 base mismatches with the consensus sequence for the G R E (CNTGTT/CCT), respectively, the sequence similarity appears to extend to the flanking sequences on the both sides of the consensus. I t has been recently reported that a trans-acting factor for 3-MC (or TCDD) receptor which is believed to interact with X R E sequences has many physicochemical and biochemical properties in common with glucocorticoid receptor ( O K E Y et al., 1 9 8 0 ; W I L H E L M S S O N et al., 1 9 8 6 ; P O L A N D et al., 1 9 8 6 ; W H I T L O C K and G A L E A Z Z I , 1 9 8 4 ; C U T H I L L et al., 1 9 8 7 ) . Considered together, sequence homology between X R E s and G R E s leads us to a notion that the inducible system of X R E and 3-MC (or TCDD) receptor is evolutionarily related to that of G R E and glucocorticoid receptor. This notion will be finally tested by cloning the xenobiotic (3-MC or TCDD) receptor gene.

6.2.

Steroidogenic P - 4 5 0 c21 gene

A unique set of P-450s are localized mainly in adrenal cortex, testis and ovary. The activities of these P-450s are regulated by peptide hormones which are targeted to these organs, that is, ACTH for adrenal cortex, and F S H and L H for ovary and testis ( A D K S N T K and A T C H I S O N , 1986; W A T E R M A N et al., 1986). P-450 c21 is among them. Treatment of primary culture cells of adrenal cortex with ACTH causes approximately 15 fold increase in the synthesis of P-450 c21 within 36 hr ( F U N K E N S T E I N et al., 1983), together with enhanced synthesis of other P-450s including P-450 see (DuBois et al., 1981), P-450 11/3 ( K R A M E R et al., 1983), and P-450 17a ( M C C A R T H Y et al., 1983). The effect of ACTH is known to be mediated by cAMP ( W A T E R M A N et al., 1986). In vitro translation assay indicated that the enhanced rates of synthesis of these P-450s correlated with increases in the level of the corresponding mRNA ( F U N K E N S T E I N et al., 1983; D U B O I S et al., 1981; K R A M E R et al., 1983; M C C A R T H Y et al., 1983). These results are now being confirmed by RNA-DNA hybridization experiments using the cloned cDNAs. In order to investigate cw-acting DNA element responsive to ACTH treatment, P A R K E R et al. (1985) introduced cosmid DNAs bearing either the mouse P-450 c21A (active gene) or B (pseudogene) into Y 1 adrenocortical tumor cells, together with pSV2-neo to isolate transformant cells. Of 29 transfectants of P-450 c21A gene screened, 14 were shown to express P-450 c21 activity,

E v o l u t i o n , S t r u c t u r e , G e n e R e g u l a t i o n of P - 4 5 0

235

whereas no expression of the activity was observed with transfectants of P-450 c21B gene. Of the 14 positive clones, 8 were strongly responsive to ACTH for the expression of P-450 c21 activity with varying levels of the basal activity. RNA blot analysis and S I nuclease mapping clearly demonstrated that the elevated P-450 c21 activity by ACTH was mediated by the increase in the corresponding raRNA content. The external deletion of the 5' flanking region of the gene and subsequent transfection experiments revealed that an essential regulatory element for P-450 c21 expression is located between —230 and — 180 bp upstream of the transcription start site ( P A R K E R et al., 1986). The equivalent sequence of this element of the murine P-450 c21 gene was also found at the corresponding positions of —241 to —201 bp and —251 to —211 bp of the human ( H I G A S H I et al., 1986) and bovine ( C H U N G et al., 1986) genes, respectively, as shown in Figure 10. The striking conservation of the sequence in the region delineated by the deletion analysis of murine P-450 c21 gene provides further evidence for an important regulatory element in the region from —230 to —180 bp preceding the transcription start site. Mouse

P4 50C21

-210

AGGTCAGGGT CTTGCATCCC TTCCTTTCTT

Human

P4 5 0 c 2 1

-241

TGCATT

Bovine

P450c21

-251

T G

.

T T

. . . .

CTTGATGGAT G

,G T

G-

•

A

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The nature of this regulatory element has not yet been determined. I t is also not known how this element functions in the expression of the P-450 c21 gene or what trans-acting factor interacts with this element in the regulatory process. Recent studies have proposed that the consensus sequence, found in the promoter region of several genes that are regulated by cAMP, plays an important role in conferring cAMP response ( C O M B et al., 1986; S H O R T et al., 1986). This consensus does not appear to be included in the conserved sequence as described above. I t will be of interest to know whether the conserved sequence of the P-450 c21 gene plays a role in determining an adrenal-specific expression or has something to do with the ACTH-regulatory mechanism. Structural analysis and transfection experiments of the genes for other steroidogenic P-450s, such as P-450 see, P-450 11(3 and P-450 17c( in adrenal cortex will help us to elucidate the mechanism of the ACTH-specific and tissuespecific expression of the P-450s. Acknowledgment The authors would like to express their sincere thanks to Drs. T. IIZUKA, K. SOGAWA, A . F U J I S A W A - S E H A R A , T . IYANAGI, K . K A W A J I R I , a n d Y . TAGASHIRA f o r t h e i r

valu-

able advice and suggestions. The work performed in our laboratories was supported in

236

O . GOTOH; Y .

FUJII-KURIYAMA

part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of J a p a n , research grants f r o m the Ministry of Health a n d Welfare of J a p a n , and funds obtained under the Life Science Project from the I n s t i t u t e of Physical and Chemical Research, J a p a n .

7.

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Evolution, Structure, Gene Regulation of P-450

243

List of Authors A . I. ARCHAKOV

Department of Biochemistry Medico-Biological Faculty Ostrovitjanova la Moscow 117437 USSR

P . BATTIONI

Université René Descartes Laboratoire de Chimie et Biochimie 45 rue des Saints Pères F-75270 Paris Cedex 06 France

J . BLANCK

Central Institute of Molecular Biology Academy of Sciences of the GDR Robert-Rossle-StraBe 10 1115 Berlin DDR

Y . FITJII-KTTRIYAMA

Department of Biochemistry Tohoku University Sendai 980 Japan

O . GOTOH

Saitama Cancer Center Research Institute Ina-machi Saitama 862 Japan

F. P.

GUENGERICH

Vanderbilt University Department of Biochemistry School of Medicine Nashville Tennessee 37232 USA

244

J . - A . GTJSTAFSSON

Department of Medical Nutrition Karolinska Institute Huddinge University Hospital S-141 86 Huddinge Sweden

D . MANSUY

Université René Descartes Laboratoire de Chimie et Biochimie 45 rue des Saints Pères F-75270 Paris Cedex 06 France

E. T.

MOEGAN

Emory University School of Medicine Department of Pharmacology Atlanta Georgia 30822 USA

H.

REIN

Central Institute of Molecular Biology Academy of Sciences of the GDR Robert-Rossle-StraBe 10 1115 Berlin DDR

K.

RUCKPAUL

Central Institute of Molecular Biology Academy of Sciences of the GDR Robert-Rossle-StraBe 10 1115 Berlin DDR

A . A . ZHUKOV

Department of Biochemistry Medico-Biological Faculty Ostrovitjanova la Moscow 117437 USSR

Subject Index Acetaminophen 112 Acetanilide 108, 112, 114, 136 2-Acetylaminofluorene 112 Acetylene 132, 134 Active oxygen complexes of iron-porphyrins 71, 76, 77 Active oxygen complexes of Mn-porphyrins 78, 85, 87 Active site 22 — 24 A d a m a n t a n e 78, 88, 90, 92 Adrenal cytochrome P-450 116 Adrenocorticotropin 191 Alcohols 109, 111 Aldrin 121 Alkane hydroxylation — by alkylhydroperoxide 86 — by dioxygen 88, 90 — by hydrogen peroxide 87 — b y iodosylbenzene 78, 79 — by potassium hydrogen persulfate 86 — mechanism of 132 Alkene — epoxidation by alkylhydroperoxides 86 — epoxidation by amine-oxide 85 — epoxidation by dioxygen 89, 91 — epoxidation b y hydrogen peroxide 87 — epoxidation b y hypochlorite 84 — epoxidation b y iodosylbenzene 79 — oxidation into aldehydes 81, 92 Alkylhydroperoxides 72, 74, 86 N-Alkylporphyrin 80, 82 Allelic variants 179 Allelozymes 106 Allozymes 179 Allylic hydroxylation 82 Allylic rearrangement 82 Alternate p a t h w a y s 26 — 29, 47, 54, 55 Amine-oxide 85ff. Amino-terminal region 222 Amino acids functional significance 7,23,40 Amino acid replacement rate 212 2-Aminoanthracene 113 2-Aminofluorene 113 Aminopyrine 106, 134 Amplification of cytochrome P-450 genes 140 Androgen regulation 183 5»-Androstane-3a, 17^-diol-3,17-disulfate 108 Androstenedione 178 Antibodies, identification of cDNA clones 197

246

Aorta cytochrome P-450 115 Aromatase 116 Aromatic amine 108 Aromatic hydrocarbon 82 ff., 88, 103, 140 Aryl hydrocarbon 50 — 54 Ascorbate 89 Asymmetric epoxidation 95 Association, heterologous 32, 35, 36, 54, 55 Association, homologous 30 — 32, 35, 36, 39, 40, 42, 54, 55 Avermectin 132 Axial heme ligand 7, 8, 10 Azo dye reduction 109 Bacillus megaterium 42 Back donation 7, 8 Barbiturates 103 Basket-handless porphyrin 95 Benzanthracene 136 Benzene 111 Benzphetamine 106, 111, 115, 121 Benzo[a]pyrene 106, 111, 113, 114, 117, 120 Bladder cytochrome P-450 115 Borohydrides 88 Breast cytochrome P-450 116 Bufuralol 108, 119, 120, 135, 137 Calorimetric investigations 30 Carbon monoxide complex of cytochrome P-450 7, 31 Carbon tetrachloride 134, 109, 122 Carbonic anhydrase 191 Catalytic cycle, see reaction cycle Cell culture 142 Cell specificity 141 cDNA, see D N A Chemical mechanisms of cytochrome P-450 catalysis 132 ff. Chemical modification 23, 40 Chemical shift (substrate) 25 Chloramphenicol acetyltransferase activity 233 Chlorite 72 Cholesterol 18, 31, 44, 110, 112, 141 Cholestyramine 112 Chromium porphyrins 76 Chromosomal localization of P-450 genes 232 Circular dichroism 11, 18, 33, 34, 44 Clofibrate 105, 109

Cloning 187 Cof actors 141 Colon cytochrome P-450 118 Configuration entropy 15 Conformation 7, 11, 12, 16, 18, 2 2 - 2 6 , 3 2 - 3 4 , 37, 4 4 - 4 6 , 54, 55 Conformational flexibility 24 Coordination chemistry (cytochrome P-450) 69 Correlation — electron transfer/catalytic activity 34, 53, 54 — spin state/catalytic activity 43, 53, 54 — spin state/electron transfer 21, 22, 53 — spin state/redox potential 16, 18 — 20, 22, 5 3 - 5 5 Cortisol 121 Critical micellar concentration (CMC) 29, 33 Cross-hybridization 198, 228 Crossing over point 11 Cumylhydroperoxide 86 Cyclohexane 78, 86, 88 Cyclopropylamine 83 Cyclosporin 132 Cysteinate, role as ligand of cytochrome P-450 70, 72, 87, 93 Cytochrome b 5 -system — cytochrome b 5 6, 8, 31, 39, 43—47 in haloalkane reduction 168 in peroxidase reactions 166 in superoxide anion formation 158ff. in uncoupling 155, 163 — cytochrome b 5 reductase (NADH) 39, 44-46 — cytochrome P-450 system interactions 6, 39, 4 3 - 4 7 — effector function 46, 54 — immunochemical inhibiton 44 — organization 44 — random lateral distribution 44 — redox function 46, 54 — substrate specificity 44 — synergism 43 — 45, 54 Cytochrome c peroxidase compound I 71 Cytochrome P-450 CAM 153, 156, 159, 165 Cytochrome P-450 reductase — cluster association 30 — conformation 34 — hydrophilic domain 34, 41 — hydrophobic anchor 30, 34, 41 Dealkylation 83, 132 Debrisoquine 108, 119, 137, 143 Decaline 78

O-Demethylation 83 Derivative spectra (cytochrome P-450) 10, 33 Detergens 33, 35, 41, 37, 40 Detoxifying pathways 29 Deuterium retention 82 Dexamethason 107, 112 Dielectric constant (substrate) 25 Difference spectra — lipid/cytochrome P - 4 5 0 33 — reductase/cytochrome P - 4 5 0 39 — substrate 9 — 13, 15 Dihydropyridine 91, 121 Dimorphism 188 Dioxygen 88 ff. Dioxygenase activity of cytochrome P-450 167 l,l-Di(p-chlorophenyl)-2,2-dichloride ( D D E ) 106 Dissociation constant — cytochrome b 5 / c y t o c h r o m e P - 4 5 0 4 4 , 4 5 — lipid/cytochrome P - 4 5 0 34 — reductase/cytochrome P - 4 5 0 34, 38, 39, 42, 43 — substrate/cytochrome P - 4 5 0 9, 13 — 15, 25, 26, 34, 44, 46 DNA — cloning 197 — sequencing 187 Electrochemical reduction 91 Electron density (substrate) 25 Electronic structure (cytochrome P-450) 7 Electron microscopy 33 Electron transfer — cluster model 35, 36, 39, 40, 47, 54, 55 — cooperativity 36 — exchange complex 31, 32, 3 6 — 4 3 , 54 — first electron transfer 5, 21, 28, 34, 35 to 37, 42, 43, 45, 53, 226 — kinetic titration 38, 42 — Michaelis-Menten mechanism 37 — microsomal pathways 44, 46 — NADH supported 31, 4 3 — 4 5 , 54 — N A D P H supported 31, 34, 4 2 - 4 5 , 54 — partial reactions 34 — 36, 38, 39 — random distribution model 35, 36, 44 — redox state model 36 — second electron transfer 5, 28, 34, 35, 4 1 - 4 5 , 54 — sequential spin state model 20, 36, 37 — steady state approach 3 1 — 4 3 , 47 — stoichiometric profile 35, 37 — 39, 41 — transfer channel 221

247

Electrostatic interactions 39, 40, 45 E L I S A 185 Enantiomeric selectivity 135 Encainide O-demethylation 119 Enhancers 235 Enzyme-substrate complexes 9, 15, 23 Epoxidation 133 — rates 75, 85, 91, 92 E P R , ST-EPR-spectra 10, 11, 15, 24, 32 Erythromycin 121, 132 17^-Estradiol 106, 108, 111, 121, 141, 178, 183 Ethanol 105, 109, 111 7-Ethoxyresorufin 106, 135 l-[l-(4'-Ethyl)-phenoxyl]-3,7-dimethyl6,7-epoxy-trans-2-octane 110 Ethylmorphine 106 Evolution of cytochrome P-450 genes 215 E X A F S spectra 71 Expression of cytochrome P-450 genes 232 Extrahepatic cytochrome P-450 115 —118 Fatty acid hydroxylation 109, 117 Flavin mononucleotide 91 Fluorescence decay 31 Functional versatility 26, 29 Gene structure 228 Gene superfamily, family 184, 205 Genetic polymorphism 29, 48, 52, 53—55 Geranyl acetate, epoxidation 90 Glucaric acid 142 Glucocorticoid responsive element 235 Gonadal-hypothalomo-pituitary-liver-axis 188 Green pigments 80 Growth hormone 188 — human 189 — pretranslational 191 — regulation 180, 190 — secretion 188 Hammett constant 25 Hansch hydrophobicity constant 25 Heme binding sequences 123 Heme binding site 218 Heme insertion 141 Heme propionates 221 Hepatocytes 142 Heteroatom release 133 Heteroatom oxidation 132 Heterolytic cleavage 6, 7, 9 Hexadecane hydroxylation 117 Hexobarbital hydroxylation 120

248

High spin forms of cytochrome P-450 108, 112 Homolytic cleavage 6, 7, 9 Hormonal regulation 188ff. Horseradish peroxidase compound 1 7 1 H R 1 222 H R 2, see heme binding site Human liver cytochrome P-450 131 Hydrogen atom abstraction 132 Hydrogen peroxide 4, 9, 27 — 29, 47, 72, 74, 87, 155, 157, 163 Hydrogen persulfate 86 Hydropathy profile 217 Hydrophobic anchor 30, 40 Hydrophobic interactions (enzyme/substrate) 23, 24 Hydrophilic (domain) 30, 40 6/?-Hydroxycortisol 142 15/3-HydroxyIase 179, 184 — activity 177 — inhibition 186 — purification 179 16