Spectroscopic Studies on Cytochrome P450 11-Beta-Hydroxylase and Model Compounds By Normand J. Cloutier Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in Biological Chemistry at the Massachusetts Institute of Technology February 1996 © 1996 Massachusetts Institute of Technology All rights reserved Signature of Author Department of Chemistry November 10, 1995 Certified by U',lliam H. Orme-Johnson Professor of Chemistry Thesis Supervisor Accepted by Dietmar Seyferth Committee on Graduate Students bAcs Tsi iChairman,J Departmental OF TECHNOLOGY MAR 0 4 1996 LIBRARIES This doctoral thesis has been examined by a Committee of the Department of Chemistry as follows: tZ CI ~C4 Professor John M. Essigmann Chairmnj Professor William H. Orme-Johnson Thesis ý_vlisor Professor Peter T. Lansbury Professor James R. Williamson Spectroscopic Studies on Cytochrome P450 11-Beta-Hydroxylase and Model Compounds By NORMAND J. CLOUTIER Submitted to the Department of Chemistry on November 10, 1995 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Chemistry ABSTRACT The partial synthesis of deuterated versions of corticosterone was performed. The C-3 and C-20 carbonyls of corticosterone were protected by forming ethylene glycol-derived ketals. This was followed by the protection of the C-21 hydroxy group with tert-butyldiphenyl silyl chloride, after which the C-11 beta-hydroxy group was oxidized to the ketone with a pyridine-chromic acid reagent. The C-21 silyl ether was then deprotected. Deuteriums were then incorporated, into the 11-keto steroid, at what are believed to be positions C-9 and C-12: (1) one compound with the C-9 and C-12 positions perdeuterated; (2) another with only one deuterium at the beta position of C-12; (3) and a third compound with deuteriums at C-9 and the alpha position of C-12. All non-deuterated intermediates have been characterized by NMR, IR, and MS. MS spectra of the deuterated steroids were consistent with the expected products. Purifications of adrenodoxin, adrenodoxin reductase, and cytochrome P450 110-hydroxylase (all from bovine adrenal glands) were performed. Preparation of pure 110-hydroxylase relied on the use of an adrenodoxin-Sepharose affinity matrix, resulting in a specific activity of 8.3 nmol of the P450 chromophore per mg of protein; this was free of contaminating cytochrome P450 cholesterol side chain cleavage enzyme. Each of these purified proteins gave single bands on silver- stained SDS PAGE gels. Three-pulse electron spin echo envelope modulation (ESEEM) studies on 14N and "1 N isotopically labeled bis (imidazole) and bis (ethyl thioglycolate) heme model compounds were performed. All measurements were conducted at g=2.25 (2900 Gauss, microwave frequency of 9.14 GHz) and a 250 nsec delay between the first and second pulses. A relatively unperturbed "5N-derived larmor frequency modulation at 1.27 MHz (from a sample containing s'N in both its porphyrin and imidazole ligands) was assigned to the axial imidazole. An Apple Macintosh computer program was written that converts 20 bit integer (Nicolet 1180E computer) ESEEM time-domain data to ASCII format. This program also allows the visualization of the data and manipulation of data points, on Apple Macintosh computers. Extended x-ray absorption fine structure (EXAFS) experiments (measured in fluorescence mode) were performed on bovine cytochrome P450 113-hydroxylase (with either 11-deoxycorticosterone or metyrapone, bound in the active site) and heme model compounds. Multiple data sets were acquired for each sample, using an energy-resolving 13-element Canberra x-ray fluorescence detector. The dead time for each detector element was determined, after which data from the multiple scans were added. Distinguishable backgrounds from the 13 different detector elements were found, dictating that future data analysis involve 13 separate background removals before all the data (for any one sample) can be accurately summed. Detector artifacts were found to be responsible for the low distance peaks found in previously reported EXAFS studies on the cytochrome P450 side chain cleavage enzyme. Thesis Supervisor: William H. Orme-Johnson Title: Professor of Chemistry DEDICATION This thesis is dedicated to those who suffer from steroid hormone disorders and for those who are working on helping the situation. ACKNOWLEDGMENTS I would like to thank my long-time friends who have stood by me through thick and thin. They are Fred Francis and Lorna Gotzmann. They approach life with the greatest excitement, wonder and determination. I am blessed by their friendship. And a big thank you to Marissa Asta, my best friend for this past year. My family has been a surprising source of strength for me as well. Although they are all very independent, I have never failed to feel their respect and compassion. I would especially like to thank my brother Marc who has helped me through many personal crises and whom I consider a model human being. I am also very grateful for my parents' support and for their constant faith in me. My lab mates have made my stay at MIT both more educational and colorful. David Wright earns a gold star in my book for his efforts at organizing us into a team. I am indebted to Jeremy Selengut for his help in getting me started in organic synthesis and for his helpful scientific criticisms; he is both a valued colleague and trusted friend. Patti Christie's friendship, straightforwardness and excellent experimental advice has also helped me 'stay on track'. My XAFS experiments in May of 1995 would not have been possible without the help of Rob Pollock. I am also indebted to the stewardship that I received from Emily Miao and Andrew Kolodziej. I would also like to thank Chun Zuo for taking the time to show me how to operate the Orme-Johnson electron spin echo machine. A great deal of the work described in this thesis was also made far easier with the enthusiastic and focused help of undergraduate students Kurt Snyder and Lisa VanDermark. I would like to thank all those from other institutes whom I have learned invaluable information from. They are Israel Hanukoglu from the Weizmann Institute and Richard Magliozzo, Jack Peisach, and Mark Chance from the Albert Einstein College of Medicine. My thanks also go the support staff of the MIT chemistry headquarters. In the final months of thesis preparation, they always treated me with the greatest amount of respect. Prof. Jamie Williamson has my vote for the next departmental chair because of his friendliness, accessibility, and for his efforts on my behalf. Finally, I would like to thank my thesis advisor, Prof. William (Bill) H. Orme- Johnson, for accepting me into his lab and encouraging my intellectual development. Bill often gave me the benefit of his experience, but always let met do things my own way. Table of Contents Abstract ------------------------------------------------- --------------- ------------------- 3 Dedication --------------------------------------------------------------------------------------- 5 Acknowledgments ---------------------------------------------------------------------------- 6 Table of Contents ------------------------------------------------------------ 7 CHAPTER 1: Placing Cytochrome P450 in Context ----------------------------------------- 10 1.1. P450 Chemical Mechanism ---------------------------------------- 13 1.2. Structural Studies On P450 Enzymes ----------------------------------- ------ 22 1.2.1. Crystallographic Studies --------------------------------------- 22 1.2.2. Non-Crystallographic Studies ----------------------------------- -------------- 24 1.3. Two Classes of Cytochromes P450 ------------------------------------------ 24 1.3.1. Class I Mammalian P450 Enzymes ----------------------------------- ------ 24 1.3.2. Class II Mammalian P450 Enzymes ---------------------------------------- 26 1.3.3. Similarities Between the Two Classes of Mammalian P450 Enzymes ---------- 27 1.4. Steroidogenesis -- ------------------------------ ------------------------------ 28 1.4.1. Description and Localization of Steroidogenic Cytochromes P450 --------------- 30 1.4.2. Physiological Regulation of Steroidogenesis ------------------------------------ 30 1.4.2.1. Hormonal Regulation --------------------------------------- 30 1.4.2.1.1. Adrenocorticotrophic Hormone (ACTH) ------------------------------------ 30 1.4.2.1.2. Angiotensin II ------------------------------------------------------------------ 32 1.4.2.2. Regulation by Post Translational Modification ---------------------------------- 34 1.4.2.2.1 Protein phosphorylation ---------------------------------------------------- 34 1.4.2.2.2. Protein Glycosylation -------- ------------------------ ----------------- 34 1.4.2.2.3. Limited Protein Hydrolysis ------------------------------------------------------- 35 1.4.2.3. Neuronal Regulation -------------------------------------------------------------------- 36 1.4.2.4. Redox Regulation-----------------------------------------------------------------------36 1.4.3. A Closer Look at P450scc ----------------------------------------- ------------------------ 38 1.4.3.1. P450e,, Catalyzed Reactions -------- -------------------------------------- 38 1.4.3.2. Importation Into the Mitochondrion and Proteolytic Processing -------------- 41 1.4.3.3. P450sce's Membrane Topology ----------------------------------- 42 1.4.3.3.1. P450sc 's Orientation in the Membrane ------------------------------------