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A Professional and Personal Odyssey

2009; Elsevier BV; Volume: 284; Issue: 30 Linguagem: Inglês

10.1074/jbc.x109.007518

1083-351X

Bettie Sue Siler Masters,

Health Sciences Research and Education

As I began to write this article reflecting on my professional career in biomedical research, many memories of people, activities, situations, and experiences were swirling in my head. Because of the extraordinary support that I have received from my family, they are never far from my thoughts. I believe that sharing a few of these memories will be helpful for those who find themselves in similar situations or periods of professional development. This is not meant to be a comprehensive review of the fields to which I have had the privilege of contributing but a personal stroll down a path in which I have found extraordinary pleasure and from which I have derived a sense of accomplishment. I was born just prior to World War II into a middle class family in the historic town of Lexington, VA. My father was a radio announcer, a nightclub singer (he had a beautiful baritone voice), and the chosen master of ceremonies for most of the local shows in several cities in Southwest Virginia. Because my mother was a full-time parent, he had to hold several jobs to pay the bills. Because he had small children and was working in a vital industry (public information), he was not drafted during the war. Neither he nor my mother was college-educated, although both were well read and surprisingly cosmopolitan to have been raised in southwestern Virginia. Because I was his firstborn and my sister came along 2 years later, all of his dreams were wrapped up in what we would accomplish in our lifetimes. One of his oft-repeated admonitions was, “I don't care what you become as an adult, as long as you do your job well.” However, it never escaped my attention that he wanted us to become professionals. Hence, the fact that we lived next door to a physician, his wife, and two boys had the inevitable effect that we observed that their somewhat higher grade of lifestyle (they owned their home, but we rented our two-bedroom apartment) was achievable. Both of my parents wanted desperately for their daughters to obtain college educations and they both insisted that we could “be anything we wanted to be.” Unfortunately, I was not born with the artistic talent with which my sister and my brother (who was to arrive 22 years later during my first year in graduate school) were endowed, so my talents had to be sought elsewhere. In those days, we were known as “tomboys,” and there was little hope for our interests to be more feminine, despite my mother's attempts, because our only playmates were boys on either side of us. We loved to climb trees, especially the cherry tree between our houses, and to play football and baseball and, best of all, cowboys and Indians. When my parents were vehemently against my getting a football for Christmas, I told my grandmother, and she made sure it was among her gifts for me, much to my mother's dismay. I loved to collect things, and I particularly loved outdoor activities. By the time I reached high school age, I had read Sir Arthur Conan Doyle's “The Adventures of Sherlock Holmes”; I admired Holmes' and Dr. Watson's analytical skills as sleuths, and perhaps this was an early indication that I would like to solve mysteries of another type. As I matured and entered high school, the only one in Radford, VA, where my father took the position of Program Director for a new radio station, my interests in sports remained, but my teachers perceived other talents. While taking ballet and tap lessons, when I could work them in around varsity basketball practice, I was able to maintain the highest scholastic average in the school and graduated as valedictorian of my class. In my sophomore year, my chemistry teacher discovered my interest in science and encouraged it. He was a very quiet, “Mr. Peepers”-type man, but he had a twinkle in his eye, and he knew how to draw out his students if they were at all sensitive to his guidance and teaching. In my junior year in high school, I entered the Westinghouse Science Talent Search and placed such that I was given a scholarship to the college of my choice. At that time, I had decided, for some reason unbeknownst to me now, that I wanted to attend William and Mary College in Williamsburg, the oldest land grant college in the United States. Whereas I was readily accepted there, the Dean refused to honor a science scholarship for a woman, so my hopes of attending were dashed. Instead, Roanoke College, a small liberal arts college located in Salem, VA, honored the scholarship and enabled me to complete a double major in biology and chemistry. As fate would have it, the Chairman of the Classics and Fine Arts Department, Dr. Miles S. Masters, took notice, and when his youngest son, 1st Lt. Robert Masters, arrived home for a leave from active duty as a Marine jet pilot, we met: no matchmaking there! It did not take long for me to realize that “this was the guy,” and when he was stationed for active duty in Japan for a year, the letters flowed back and forth while I completed my freshman year and began my sophomore year. My years at Roanoke College seemed to speed by, with labs scheduled 4 days per week leaving very little leisure time, except for playing varsity basketball for a couple of years, participating in biology and chemistry club activities, and editing the school yearbook with a college friend. I graduated as salutatorian of my college class. The pursuit of a medical degree had been foremost in my mind since I became a teenager, but the economics of my family situation held no hope of this coming to fruition. The next step in my life became very clear when my comparative anatomy professor urged me to consider a career in biomedical research with a major in biochemistry. This was great advice, except that I had no idea what biochemistry was because Roanoke College did not have a biochemistry course in its curriculum. Not being afraid of challenges, I applied with the help and encouragement of my professors and a competitive score on the Graduate Record Examination Aptitude Test to the institutions in the United States best known for their biochemistry programs. At that time, the University of Wisconsin, The Johns Hopkins University, Ohio State University, and Duke University were among the forerunners, and I was accepted into all of the programs with some support from three of them. I entered the Duke University Biochemistry Graduate Program during Dr. Philip Handler's chairmanship and enjoyed the outstanding teaching of a number of notables, among them, Dr. Handler, Dr. Irwin Fridovich, Dr. Salih Wakil, Dr. Eugene Davidson, and Dr. Henry Kamin. These men (there were no women on the faculty at that time) challenged all of the students as they redesigned the graduate curriculum with our class as guinea pigs. To qualify for Ph.D. candidacy, we had to pass a series of preliminary examinations based on different areas of biochemistry, known only to those who generated the examinations. The students found this guessing game somewhat frustrating, but it did force us to read everything that was coming out in the biochemical literature, as well as to review our notes. I then interviewed with those faculty members whom I believed I could work well with and chose Dr. Henry Kamin as my mentor. This was probably one of the defining decisions of my life insofar as my career was concerned. Dr. Kamin was not only a great teacher and an innovative thinker, but he was truly a Renaissance man who was a lover of history, music, and art; a gourmet cook; and a golf enthusiast. He was known to play golf in faraway places such as Ireland and Australia, but his widow, Dottie, says never very well. He was a master of the English language, and his ability to dictate a manuscript over a Dictaphone directly from my data notebooks, with me at his side discussing the experiments, was unbelievable. The most incredible thing was that these manuscripts were accepted for publication, without further revision, by the Journal of Biological Chemistry on several occasions. His wife, Dottie, a graduate of the University of North Carolina School of Nursing, had become a research associate in pharmacology by this time but remained the “nurse” for numerous faculty members and their families, including me. She was present in the delivery room when our first daughter, Diane, was born in Duke Hospital, trying to keep me calm and cool during the non-air-conditioned July heat. When I arrived at Duke, Charles Williams had just completed his Ph.D. training under Dr. Kamin and had taken a postdoctoral fellowship position with Prof. Vincent Massey at the University of Sheffield in England. Dr. Williams' Ph.D. dissertation addressed the cellular localization of TPNH (NADPH)-cytochrome c reductase in the microsomal fraction of pig liver, and Williams and Kamin (1Williams Jr., C.H. Kamin H. Microsomal triphosphopyridine nucleotide cytochrome c reductase of liver.J. Biol. Chem. 1962; 237: 587-595Abstract Full Text PDF PubMed Google Scholar) and Phillips and Langdon (2Phillips A.H. Langdon R.G. Hepatic triphosphopyridine nucleotide- cytochrome c reductase: isolation, characterization, and kinetic studies.J. Biol. Chem. 1962; 237: 2652-2660Abstract Full Text PDF PubMed Google Scholar) published the localization of this activity simultaneously in the Journal of Biological Chemistry. In 1950, Horecker (3Horecker B.L. Triphosphopyridine nucleotide-cytochrome c reductase in liver.J. Biol. Chem. 1950; 183: 593-605Abstract Full Text PDF Google Scholar) had first identified this NADPH-cytochrome c reductase activity in whole liver acetone powder but could not determine its cellular localization from his studies. In 1955 and 1957, respectively, La Du et al. (4La Du B.N. Gaudette L. Trousof N. Brodie B.B. Enzymatic dealkylation of aminopyrine (pyramidon) and other alkylamines.J. Biol. Chem. 1955; 214: 741-745Abstract Full Text PDF PubMed Google Scholar) and Gillette et al. (5Gillette J.R. Brodie B.B. La Du B.N. The oxidation of drugs by liver microsomes: on the role of TPNH and oxygen.J. Pharmacol. Exp. Ther. 1957; 119: 532-540PubMed Google Scholar) reported that the addition of cytochrome c inhibited the TPNH (NADPH)-mediated oxidative dealkylation of monomethyl-4-aminoantipyrine and other alkylamines catalyzed by liver microsomal fractions and that this process required oxygen. In the latter article, Gillette et al. (5Gillette J.R. Brodie B.B. La Du B.N. The oxidation of drugs by liver microsomes: on the role of TPNH and oxygen.J. Pharmacol. Exp. Ther. 1957; 119: 532-540PubMed Google Scholar) were the first to report the generation of hydrogen peroxide in the absence of substrate in these microsomal fractions, an observation that was to have significance much later in the interpretation of data from studies of microsomal oxidases and oxygenases. The discovery of oxygenases, the enzymes that catalyze the incorporation of the atom(s) of molecular oxygen into organic molecules, was made by Hayaishi et al. (6Hayaishi O. Katagiri M. Rothberg S. Mechanism of the pyrocatechase reaction.J. Am. Chem. Soc. 1955; 77: 5450-5451Crossref Scopus (227) Google Scholar) with pyrocatechase from a pseudomonad and by Mason et al. (7Mason H.S. Fowlks W.L. Peterson E. Oxygen transfer and electron transport by the phenolase complex.J. Am. Chem. Soc. 1955; 77: 2914-2915Crossref Scopus (168) Google Scholar) with a phenolase complex from mushrooms in 1955, but the connection had not yet been made to the NADPH-requiring system in liver microsomes. Following the reports by Klingenberg (8Klingenberg M. Pigments of rat liver microsomes.Arch. Biochem. Biophys. 1958; 74: 379-386Google Scholar) and Garfinkel (9Garfinkel D. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions.Arch. Biochem. Biophys. 1958; 77: 493-509Crossref PubMed Scopus (276) Google Scholar) in 1958 of a reduced carbon monoxide-binding pigment in mammalian liver microsomes with an absorbance at 450 nm, Omura and Sato (10Omura T. Sato R. A new cytochrome in liver microsomes.J. Biol. Chem. 1962; 237: PC1375-PC1376Abstract Full Text PDF Google Scholar) published the purification and characterization of a heme-binding protein from rabbit liver microsomes that they determined to be a b-type cytochrome. Although the absorbance maximum of the reduced CO difference spectrum of this purified hemeprotein was at 420 nm, future studies were to show that this was the CO-binding pigment observed by Klingenberg and Garfinkel with an altered absorbance maximum. Thus, the groundwork was laid for a connection to be made between the existence of this hemeprotein and the fixation of O2. This connection was made by Estabrook, Cooper, and Rosenthal (11Estabrook R.W. Cooper D.Y. Rosenthal O. The light-reversible carbon monoxide inhibition of the steroid C-21 hydroxylation system of the adrenal cortex.Biochem. Z. 1963; 338: 741-755PubMed Google Scholar) and Cooper et al. (12Cooper D.Y. Levin S. Narasimhulu S. Rosenthal O. Estabrook R.W. Photochemical action spectrum of the terminal oxidase of mixed function oxidase systems.Science. 1965; 147: 400-402Crossref PubMed Scopus (297) Google Scholar) using the photochemical action spectrum methodology of Warburg. By reversing the CO inhibition of the hydroxylation of 17α-hydroxyprogesterone by adrenal microsomes and the oxygenation of codeine, acetanilide, and testosterone by liver microsomes by light with maximal absorption at 450 nm, they proved that this hemeprotein was the terminal oxidase in these reactions. Following this period, between 1960 and 1965, I was working on the mechanism of the protease-solubilized microsomal NADPH-specific (NADPH, nicotinamide-adenine dinucleotide phosphate, was called triphosphopyridine nucleotide then) flavoprotein (missing its N-terminal membrane anchor) that reduced cytochrome c and several artificial electron acceptors but whose role in cytochrome P450-mediated reactions remained unproven (13Masters B.S. Kamin H. Gibson Q.H. Williams Jr., C.H. Studies on the mechanism of microsomal triphosphopyridine nucleotide-cytochrome c reductase.J. Biol. Chem. 1965; 240: 921-931Abstract Full Text PDF PubMed Google Scholar, 14Masters B.S. Bilimoria M.H. Kamin H. Gibson Q.H. The mechanism of 1- and 2-electron transfers catalyzed by reduced triphosphopyridine nucleotide-cytochrome c reductase.J. Biol. Chem. 1965; 240: 4081-4088Abstract Full Text PDF PubMed Google Scholar). During this time, I was challenged by Dr. Kamin to purify sufficient quantities (∼100 mg from porcine liver, not an easy task) of the reductase to take to Sheffield, England, where I would be able to work with Drs. Quentin Gibson and Vincent Massey, gurus in stopped-flow and static titration spectrophotometry of heme- and flavin-containing proteins, to characterize it kinetically and spectrally. The gauntlet was dropped, and I worked night and day for 6 months to meet this challenge. In the spring of 1962, I left for the University of Sheffield, where Charles Williams was a postdoctoral fellow with Dr. Massey, to perform a raft of experiments that would become the backbone of my dissertation and the subject of two papers in the Journal of Biological Chemistry (Fig. 1). As a graduate student and then a postdoctoral fellow, supported by the American Cancer Society and later by the American Heart Association, I was able to show that the enzyme contained 2 mol of flavin/mol of protein and that its catalytic mechanism required one of those flavins to be fully reduced; the 1-electron-reduced semiquinone form of the enzyme was inactive toward any of the electron acceptors tried (13Masters B.S. Kamin H. Gibson Q.H. Williams Jr., C.H. Studies on the mechanism of microsomal triphosphopyridine nucleotide-cytochrome c reductase.J. Biol. Chem. 1965; 240: 921-931Abstract Full Text PDF PubMed Google Scholar, 14Masters B.S. Bilimoria M.H. Kamin H. Gibson Q.H. The mechanism of 1- and 2-electron transfers catalyzed by reduced triphosphopyridine nucleotide-cytochrome c reductase.J. Biol. Chem. 1965; 240: 4081-4088Abstract Full Text PDF PubMed Google Scholar). I had also contributed to the Williams and Kamin publication (1Williams Jr., C.H. Kamin H. Microsomal triphosphopyridine nucleotide cytochrome c reductase of liver.J. Biol. Chem. 1962; 237: 587-595Abstract Full Text PDF PubMed Google Scholar) with observations that NADPH reduced cytochrome b5 in microsomal preparations but the protease-solubilized, purified enzyme did not. This suggested that the preparation was defective or that a factor was missing. It was shown later that the full-length detergent-solubilized enzyme reduced cytochrome b5. Because my predecessor, Charles Williams, had shown that FAD was a component of his preparations, we were remiss in not re-examining my preparations of reductase, which eliminated the acid precipitation and high ionic strength ammonium sulfate fractionation steps (13Masters B.S. Kamin H. Gibson Q.H. Williams Jr., C.H. Studies on the mechanism of microsomal triphosphopyridine nucleotide-cytochrome c reductase.J. Biol. Chem. 1965; 240: 921-931Abstract Full Text PDF PubMed Google Scholar) that tend to release noncovalently bound flavins from their apoproteins. Somewhat later, in 1973, Iyanagi and Mason (15Iyanagi T. Mason H.S. Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase.Biochemistry. 1973; 12: 2297-2308Crossref PubMed Scopus (280) Google Scholar) showed definitively that NADPH-cytochrome c reductase, in both the proteolytically solubilized form and the full-length, detergent-solubilized form, contained 1 mole each of FAD and FMN. In 1974, Iyanagi et al. (16Iyanagi T. Makino N. Mason H.S. Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b5 reductases.Biochemistry. 1974; 13: 1701-1710Crossref PubMed Scopus (158) Google Scholar) determined from the redox potentials that one of these flavins acted as the entrance flavin and that the other was involved in the exit of electrons, although they did not identify which flavin had the higher potential. Vermilion and Coon (17Vermilion J.L. Coon M.J. Identification of the high and low potential flavins of liver microsomal NADPH-cytochrome P-450 reductase.J. Biol. Chem. 1978; 253: 8812-8819Abstract Full Text PDF PubMed Google Scholar) showed that FAD accepted electrons from NADPH and then shuttled the electrons one-by-one into the FMN of the reductase, from which the electrons exited to various electron acceptors. Bob Masters and I were married after our first year in graduate school, and after he earned his MBA at the University of North Carolina, he joined the young company Scientific Products of American Hospital Supply Corp. After a very productive graduate training period, lasting 3 years and 9 months, I decided to remain with Dr. Kamin for additional postdoctoral training and to expand on the studies that I had begun as a student. Our first daughter, Diane, was born barely a month after I was awarded my Ph.D. degree at Duke; my husband and I moved into our first home in Durham that same summer, and a new phase of our lives was to begin. My American Cancer Society postdoctoral fellowship was to begin immediately, so there was little time to enjoy those first months with our baby daughter. During this period, our second daughter, Deborah, was born, and our days were filled with our family and career commitments as I began an advanced research fellowship with the American Heart Association. My husband was traveling most of the week, and so we engaged full-time help with our toddlers. These were very busy but extremely happy times as we made time for trips to Virginia to visit family and enjoy tent camping by lakes, in the mountains, and at the North Carolina seashore. By the time this training period was to end and my husband, Bob, was ready to move ahead in his company, I had successfully obtained an American Heart Association established investigatorship and a grant-in-aid. Let the games begin. It was now time for me to prove to myself and others that I could become an independent scientist and contribute to my research area in a meaningful way. When my husband and I were looking at positions that would be mutually compatible, the choices boiled down to Atlanta or Dallas. During a phone call with Dr. Ronald W. Estabrook, who was to become the new Chairman of Biochemistry at The University of Texas Southwestern Medical School in Dallas, Dr. Kamin mentioned that I had a choice between the two cities. A job offer was made on the spot. Dr. Estabrook had heard me present at a Federation meeting in Atlantic City, and we had met later in Philadelphia at the Johnson Foundation, where I was performing some stopped-flow kinetics studies with Dr. Quentin Gibson, the co-inventor of this technology. My good fortune was to receive the blessing of Dr. Kamin, as I flexed my muscles and tried my wings, and to be given the reductase “problem” to pursue as an independent investigator. Armed with American Heart Association support for salary and a research grant, I arrived in Dallas in the summer of 1968, among a group of four new recruits, to join Dr. Estabrook's department. I was extremely proud of my first laboratory, all 400 square feet of it, and turned a few heads when I decided to paint the walls above the tile wainscoting a peachy pink. These years were to be highly motivating and productive, extremely busy with a young family, and very exciting as the young Southwestern Medical School gained national prominence due to its visionary leadership, beginning with Drs. Charles C. Sprague and Donald M. Seldin in the late 1960s and their wise decision to build up the basic sciences there. Evidence accumulated from several laboratories in the late 1960s and early 1970s that the microsomal flavoprotein that I had been studying was the physiological electron donor to cytochrome P450 in liver microsomes (18Kuriyama Y. Omura T. Siekevitz P. Palade G.E. Effects of phenobarbital on the synthesis and degradation of the protein components of rat liver microsomal membranes.J. Biol. Chem. 1969; 244: 2017-2026Abstract Full Text PDF PubMed Google Scholar, 19Omura T. Gillette J.R. Conney A.H. Cosmides G.J. Estabrook R.W. Fouts J.R. Mannering G.J. Microsomes, Drug Oxidations, and Chemical Carcinogenesis. Academic Press, New York1969: 160-161Google Scholar, 20Wada F. Shibata H. Goto M. Sakamoto Y. Participation of the microsomal electron transport system involving cytochrome P-450 in ω-oxidation of fatty acids.Biochim. Biophys. Acta. 1968; 162: 518-524Crossref PubMed Scopus (63) Google Scholar, 21Masters B.S. Baron J. Taylor W.E. Isaacson E.L. LoSpalluto J. Immunochemical studies on electron transport chains involving cytochrome P-450. I. Effects of antibodies to pig liver microsomal reduced triphosphopyridine nucleotide-cytochrome c reductase and the non-heme iron protein from bovine adrenocortical mitochondria.J. Biol. Chem. 1971; 246: 4143-4150Abstract Full Text PDF PubMed Google Scholar). Utilizing antibodies prepared against the proteolytically solubilized NADPH-cytochrome c reductase, these laboratories demonstrated that inhibition of NADPH-dependent, cytochrome P450-mediated oxygenation of drugs and steroids was obtained. Having in hand the purified preparations I had brought from Duke, I was able to prepare antibodies and to begin our studies on the microsomal metabolism of drugs and steroids immediately (21Masters B.S. Baron J. Taylor W.E. Isaacson E.L. LoSpalluto J. Immunochemical studies on electron transport chains involving cytochrome P-450. I. Effects of antibodies to pig liver microsomal reduced triphosphopyridine nucleotide-cytochrome c reductase and the non-heme iron protein from bovine adrenocortical mitochondria.J. Biol. Chem. 1971; 246: 4143-4150Abstract Full Text PDF PubMed Google Scholar). Because steroid metabolism was catalyzed by adrenal cortical tissues, we performed immunochemical titration studies on microsomes and mitochondria from the adrenal cortex as well. We decided to prepare antibodies to adrenodoxin, a mitochondrial iron-sulfur protein electron carrier, to compare the effects in the two organs and subcellular fractions. These data showed unequivocally that concomitant inhibition of TPNH (NADPH)-cytochrome c reductase and ethylmorphine demethylation in pig liver microsomes (Fig. 2) was obtained upon titration with anti-reductase γ-globulin. As a bonus to these studies, which confirmed that NADPH-cytochrome c reductase was indeed NADPH-cytochrome P450 oxidoreductase (CYPOR), we were able to show that adrenal microsomes contained a similar activity. Antibodies to the reductase inhibited both liver and adrenal microsomal cytochrome c reductase activity but had no effect on NADPH-mediated cytochrome c or P450 reduction by adrenal mitochondria. In addition, because adrenal cortical mitochondria had been shown to catalyze steroid hydroxylations, we determined that the source of electrons was not NADPH-cytochrome P450 oxidoreductase but was dependent, instead, upon the NADPH-mediated pathway in mitochondria involving adrenodoxin. Antibodies to adrenodoxin inhibited only mitochondrial cytochrome P450 reduction. These results demonstrated that the microsomal and mitochondrial P450-mediated pathways involved different electron transport enzymes. These studies supported the conclusions of Lu, Junk, and Coon (22Lu A.Y. Junk K.W. Coon M.J. Resolution of the cytochrome P-450-containing omega-hydroxylation system of liver microsomes into three components.J. Biol. Chem. 1969; 244: 3714-3721Abstract Full Text PDF PubMed Google Scholar), who had reconstituted the cytochrome P450-mediated hydroxylation of lauric acid with purified CYPOR, cytochrome P450, and a lipid fraction from rabbit liver microsomes. This was the first successful reconstitution of any cytochrome P450-mediated oxygenation system, and although the substrate was not a drug or a steroid, their beautiful data strongly supported the role of NADPH-cytochrome P450 reductase. In their second publication, however, Lu et al. (23Lu A.Y. Strobel H.W. Coon M.J. Hydroxylation of benzphetamine and other drugs by a solubilized form of cytochrome P-450 from liver microsomes: lipid requirement for drug demethylation.Biochem. Biophys. Res. Commun. 1969; 36: 545-551Crossref PubMed Scopus (109) Google Scholar) showed that benzphetamine, aminopyrine, ethylmorphine, and hexobarbital were all metabolized by their reconstitution system. Laurate and benzphetamine were found to be mutually inhibitory, as would be expected if a common “methyl hydroxylase” was involved. The second publication identified the necessary third fraction as a heat-stable, non-protein component that was soluble in organic solvents and behaved like a lipid, which would be expected for optimal activity of membrane-bound components. It is interesting that Lu et al. (23Lu A.Y. Strobel H.W. Coon M.J. Hydroxylation of benzphetamine and other drugs by a solubilized form of cytochrome P-450 from liver microsomes: lipid requirement for drug demethylation.Biochem. Biophys. Res. Commun. 1969; 36: 545-551Crossref PubMed Scopus (109) Google Scholar) made the statement that “Although the existence of multiple distinct forms would account for the broad specificity (of the cytochrome P450-mediated reactions), no more than one form of P450 has been identified with certainty by spectral methods.” Whereas this statement was certainly true at that time, to those of us who have labored in this area of research for many years, this has become the ultimate understatement in that thousands of cytochrome P450 genes have now been identified throughout phylogeny, and no fewer than 57 P450 genes exist throughout the human body. Another microsomal activity that attracted much interest during this time was the enzyme system that degraded heme to bilirubin. It was shown by Rudi Schmid's group (24Tenhunen R. Marver H.S. Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc. Natl. Acad. Sci. U.S.A. 1968; 61: 748-755Crossref PubMed Scopus (1511) Google Scholar, 25Tenhunen R. Marver H.S. Schmid R. The enzymatic catabolism of hemoglobin: stimulation of microsomal heme oxygenase by hemin.J. Lab. Clin. Med. 1970; 75: 410-421PubMed Google Scholar) that the system required NADPH and molecular O2, and because of the function of cytochrome P450 in microsomal oxygenation reactions, the prevailing thought was that cytochrome P450 could serve as the terminal oxidase in the formation of bilirubin. In fact, this group published an article in 1972 that seemed to provide evidence via a photochemical action spectrum that heme oxygenation was catalyzed by cytochrome P450 (26Tenhunen R. Marver H. Pimstone N.R. Trager W.F. Cooper D.Y. Schmid R. Enzymatic degradation of heme: oxygenative cleavage requiring cytochrome P-450.Biochemistry. 1972; 11: 1716-1720Crossref PubMed Scopus (85) Google Scholar). In 1972, my laboratory collaborated with Schacter and Marver (27Schacter B.A. Nelson E.B. Marver H.S. Masters B.S. Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system.J. Biol. Chem. 1972; 247: 3601-3607Abstract Full Text PDF PubMed Google Scholar) and showed that CYPOR was required as a source of electrons for heme degradation catalyzed by rat and pig liver and spleen microsomes, which gave credence to the idea that cytochrome P450 could serve as a heme oxygenase. This was, however, not the case, and the actual isolation and purification by Maines et al. (28Maines M.D. Ibrahim N.G. Kappas A. Solubilization and partial purification of heme oxygenase from rat liver.J. Biol. Chem. 1977; 252: 5900-5903Abstract Full Text PDF PubMed Google Scholar) of an enzyme that performed this function put this hypothesis to permanent rest. So, an additional physi