; THE PEROXIDASE PROPERfiES OF CYTOCHROME I> 450 CENTRE FOR NEWFOUNDLAND STUDIES TOTAL OF 10 PAGES ONLY MAY BE XEROXED • (Without Author's Permission) F.U ~ G. HRYCA Y C . I FEB 251974 THE PEROXIDASE PROPERTIES OF CYTOCHROME P-450 A thesis submitted to the Faculty of Graduate Studies of Memorial University of Newfoundland in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry by © Eugene G. Hrycay May, 1973 (i) ACKNOWLEDGEMENTS The major portion of this work was carried out in the Department of Biochemistry, Memorial University of Newfoundland. The author expresses gratitude to Dr. Peter J. O'Brien for his overall supervision of the work. Thanks are also due to Dr. Johan E. van Lier, Biochemistry Laboratories, Centre Hospitalier Universitaire, Sherbrooke, Quebec, for supplying the steroid hydroperoxides and for use of his facilities in carrying out part of this work. The author would like to express his gratitude to Professor George S. Boyd, Department of Biochemistry, Edinburgh University, Scotland, for his many stimulating discussions, for use of his research facilities, and for providing moral support in times of stress. Finally, thanks go out to Ms. Barbara Hennessey and Mrs. Kay Cherian for assistance in certain enzyme preparations and to Mrs. Helen Kennedy for typing the manuscript. (ii) ABSTRACT The decomposition of steroid and other organic hydroperoxides by microsomes from rat liver and bovine adrenal cortex has been examined using TMPD as an electron donor. A comparison of the hydroperoxide specificity of microsomal peroxidase revealed the 17a-hydroperoxide derivatives of progesterone and pregnenolone to be very effective sub- strates. Treatment of rats with phenobarbital or 3-methylcholanthrene enhanced the specific activity of microsomal peroxidase and the cytochrome P-450 content to a similar extent. Microsomal "P-450 par- ticles" devoid of cytochrome b showed the same peroxidase activity -5 per mole of P-450 as did original microsomes. Further evidence for P-450 being a microsomal peroxidase was the inhibition of peroxidase activity by type I, type II, and modified type II compounds and by reagents converting P-450 to P~420. The rate of NADPH or NADH oxidation by microsomal fractions was markedly enhanced by the hydroperoxides. Evidence for the involve- ment of NADPH-cytochrome ~ reductase in NADPH-peroxidase activity indluded a similar K for NADPH; inhibition by £-mercuribenzoate m with partial protection by NADPH; inh~&ition by NADP+; and inactivation by antiserum to the flavoenzyme. Evidence of a role for NADH-cytochrome b reductase in NADH-peroxidase activity included a similar Km for NADH; 5 inhibition by £-mercuribenzoate with partial protection by NADH; and inactivation by antibody to the flavoprotein. Evidence for the involvement of P-450 in the NAD(P)H-peroxidase (iii) reaction included inhibition by type I, type II, and modified type II compounds; inhibition by reagents converting P-450 to P-420; and marked stimulation by in vivo phenobarbital treatment. The NADPH- and NADH­ reduced forms of P-450 were oxidized very rapidly by cumene hydroperoxide under a CO atmosphere. Incubation of the hydroperoxides with microsomal fractions gave rise to the corresponding hydroxy derivatives as the major products. Mechanisms for the P-450-dependent reduction of hydroperoxides are proposed and the role of hydroperoxides as possible intermediates in biological hydroxylation reactions discussed. (iv) ABBREVIATIONS NADPH - reduced nicotinamide adenine dinucleotide phosphate NADP+ - nicotinamide adenine dinucleotide phosphate NADH reduced nicotinamide adenine dinucleotide NAD+ nicotinamide a4enine dinucleotide GSH reduced glutathione TMPD N,N,N' ,N'-tetramethyl-£-phenylenediamine gc gas chromatography ms - mass spectrometry tlc thin layer chromatography EDTA - ethylenediaminetetraacetate P-450 cytochrome P-450 (v) Systematic nomenclature for compounds given trivial names in the text include: pregn-4-ene-3,20-dione progesterone 3S-hydroxypregn-5-en-20-one pregnenolone 17a,21-dihydroxypregn-4-ene-3,20-dione 17a,21-dihydroxyprogesterone 17a-hydroxypregn-4-ene-3,20-dione 17a-hydroxyprogesterone 3S,l7a-dihydroxypregn-5-en-20-one 17a-hydroxypregnenolone androst-4-ene-3,17-dione androstenedione 3S-hydroxyandrost-5-en-17-one dehydroepiandrosterone 3S-hydroxy-5a-pregnan-20-one allopregnanolone 17S-hydroxyandrost-4-en-3-one --- testosterone cholest-5-en-3S-ol cholesterol 4,4,14a-trimethyl-5a-cholesta-8,24-dien-3S-ol lanosterol 2-phenyl-2-hydroperoxypropane cumene hydroperoxide £-ethoxyacetanilide --- phenacetin o-biphenylenemethane fluorene 1,2,3,4-tetrahydronaphthalene tetralin 1,2,3,4-tetrahydro-2-naphthol tetralol (vi) TABLE OF CONTENTS INTRODUCTION - I. Mixed-function oxidase reactions in liver microsomes 1 II. Mixed-function oxidase reactions in non-hepatic tissues. 3 III. Cytochrome P-450 and its role in hydroxylation reactions • . 4 CHAPTER I: CYTOCHROME P-450 AS A MICROSOMAL PEROXIDASE IN STEROID HYDROPEROXIDE DECOMPOSITION Materials and Methods 19 The TMPD-peroxidase assay 22 Kinetic properties of microsomal peroxidase 22 Hydroperoxide specificity of microsomal peroxidase 25 Tissue distribution of microsomal peroxidase activity 29 Inhibition of microsomal peroxidase activity by various reagents . 29 Inhibition of peroxidase activity by reagents that convert cytochrome P-450 to cytochrome P-420 • . • . 32 Inhibition of peroxidase activity by type I and type II compounds 32 Inhibition of peroxidase activity by cyanide . 35 Spectral changes during hydroperoxide interaction with microsomal fractions . • . • • . • . 38 Comparison of adrenocortical microsomal peroxidase activity with the rate of progesterone 17a-hydroperoxide reduction by microsomes . • • . • • • . • . 40 Effect of phenobarbital or 3-methylcholanthrene pre­ treatment of rats on microsomal peroxidase activity 41 Effectiveness of purified cytochrome P-450 and various heme catalysts for peroxidase activity . 43 Destruction of cytochrome P-450 by hydroperoxides. 45 (vii) CHAPTER II: PRODUCTS OF DECOMPOSITION OF PREGNENE 17a-HYDRO­ PEROXIDES AND CUMENE HYDROPEROXIDE BY MICROSOMAL FRACTIONS Materials and Methods 49 Conversion products of progesterone 17a-hydroperoxide • 54 Conversion metabolites of pregnenolone 17a-hydroperoxide. 59 Products of decomposition of cumene hydroperoxide . • • • • 60 CHAPTER III: THE MICROSOMAL NADPH-PEROXIDASE ELECTRON TRANSPORT SYSTEM Materials and Methods . • • 64 The NADPH-peroxidase assay . 68 Hydroperoxide specificity of microsomal NADPH-peroxidase. 68 Tissue distribution of microsomal NADPH-peroxidase activity . • . • • • . • • • . 71 Efficiency of NADPH oxidation by hydroperoxides . 71 Inhibition of NADPH-peroxidase activity by various re- agents 74 Evidence for the involvement of NADPH-cytochrome ~ reductase in NADPH-peroxidase activity . • . • . 74 Inhibition of NADPH-peroxidase activity by modifiers of cytochrome P-450 • . • • . • . • • • • 81 Inhibition of NADPH-peroxidase activity by cyanide 84 Inhibition of microsomal N-demethylase and NADPH­ peroxidase activity by steroids • . • • • . 84 Cytochrome b and microsomal NADPH-peroxidase activity 86 5 Synergistic effect of NADH on the NADPH-peroxidase reaction rate . • . • . • • . • • . 86 Inhibition of NADPH-peroxidase activity by trypsin 88 (viii) Reconstitution of the microsomal NADPH-peroxidase activity ... 91 Stimulation of NADPH-peroxidase activity by phenobarbital treatment 95 Oxidation of NADPH-reduced cytochrome P-450 by hydroper- oxides . 95 CHAPTER IV: THE MICROSOMAL NADH-PEROXIDASE ELECTRON TRANSPORT SYSTEM Materials and Methods 100 The NADH-peroxidase assay 103 Hydroperoxide specificity of microsomal NADH-peroxidase. 103 Tissue distribution of microsomal NADH-peroxidase activity . 106 Efficiency of NADH oxidation by hydroperoxides . 106 Effect of pH and ionic strength on NADH-peroxidase activity . • . 109 Inhibition of NADH-peroxidase activity by various reagents . 109 Evidence for the involvement of NADH-cytochrome ~ reductase in NADH-peroxidase activity . • . 109 Inhibition of NADH-peroxidase activity by modifiers of cytochrome P-450 • . • . • . 113 Inhibition of NADH-peroxidase activity by cyanide 116 Stimulation of NADH-peroxidase activity by phenobarbital treatment 119 Cytochrome b and microsomal NADH-peroxidase activity 119 5 NADPH-cytochrome c reductase and NADH-peroxidase activity . • . • . .••..•. 121 Effect of trypsin on microsomal NADH-peroxidase activity .....•.............. 121 (ix) Reconstitution of the microsomal NADH-peroxidase activity . 122 Oxidation of NADH-reduced cytochrome P-450 by hydroperoxides . • . • . 127 DISCUSSION 130 REFERENCES 152 PUBLICATIONS ARISING FROM THIS WORK . 159 (x) LIST OF TABLES Number Name 1 Some reactions catalyzed by cytochrome P-450- dependent enzyme systems . • . 5 2 Hydroperoxide specificity of microsomal peroxidase . 28 3 Tissue distribution of microsomal peroxidase activity. 30 4 Effect of various modifiers on hepatic microsomal peroxidase activity . 31 5 Inhibition of hepatic microsomal peroxidase activity by reagents that convert cytochrome P-450 to cytochrome P-420 • . • . • . 33 6 Inhibition of hepatic microsomal peroxidase activity by type I and type II ligands . • . 34 7 Inhibition of adrenocortical microsomal peroxidase activity by type I and type II ligands • • 36 8 Effect of phenobarbital or 3-methylcholanthrene pretreatment of rats on microsomal peroxidase activity 42 9 Comparative efficiencies of heme catalysts for peroxidase activity . •