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Enzyme Ingenuity in Biological Oxidations: a Trail Leading to Cytochrome P450

2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês

10.1074/jbc.r200015200

1083-351X

Minor J. Coon,

Drug Transport and Resistance Mechanisms

Those unfamiliar with basic research in biochemistry and related fields may assume that important discoveries are the result of brilliant ideas that are single mindedly pursued until, many years later, the answer is obtained, perhaps along with important biomedical applications. The progress of science is almost always more haphazard, as ambitious young scientists are influenced by their teachers, by the cooperative or competitive work of others, the availability of new techniques, and chance findings that may lead to different goals. Sixty years ago as an undergraduate at the University of Colorado, I took my first biochemistry course in the Chemistry Department taught by Professor Reuben Gustavson and had the good fortune to be invited by him to join his small research group studying steroid hormones. I had a tremendous amount to learn but was fascinated from then on with research and the possibility of making new discoveries. In these reflections the influence of my mentors/teachers, whom I much admired for their personal qualities and achievements, is acknowledged. Although my research over the years has taken many unexpected turns, a common thread has been an interest in biological oxidations, particularly those not readily explainable according to the predictions of organic chemistry. This curiosity has led to fundamental studies on the properties and mechanism of action of cytochrome P450, now often described as the most versatile biological catalyst known. Although this was not my original goal, the mammalian isoforms of this enzyme have turned out to be of biomedical importance because of their central involvement in the metabolism of steroids, drugs, and chemical carcinogens. William Cumming Rose was a dedicated and inspiring teacher and an outstanding pioneer in biochemistry and nutritional science who spent most of his career at the University of Illinois (1Carter H.E. Coon M.J. William Cumming Rose, a biographical memoir..Biogr. Mem. Natl. Acad. Sci. 1995; 68: 3-21Google Scholar). Young Will attended schools in small communities in North Carolina and South Carolina until the age of 14, when the inadequacy of the education caused his father to remove him from school and tutor him at home. He had been introduced to Latin, Greek, and Hebrew and was well prepared by the time he entered college. Will wished to attend a large University, but his father thought his son at age 16 was too young and convinced him to attend Davidson College in North Carolina, a school for which he developed a lifelong affection. While in graduate school at Yale University, Rose decided on the branch of chemistry he would pursue, which was biochemistry, under the guidance of Lafayette Mendel in the Sheffield Scientific School. In 1911, upon completion of his Ph.D. thesis, Rose left Yale for an instructorship in physiological chemistry at the University of Pennsylvania, followed by advanced study with Franz Knoop at the University of Freiburg and then a faculty position at the University of Texas in Galveston before he became professor and head of the Division of Biochemistry in the Chemistry Department at the University of Illinois. This provided a permanent and very supportive home for his scientific career for the next 35 years. In research Rose displayed a gift for meticulous experimentation and for thoroughness and clarity in his publications. As a teacher he imbued students who attended his carefully prepared lectures with enthusiasm for biochemistry. The subject came alive with his engrossing stories about the early history of the field and the personalities involved. No mention of his remarkable ability as a teacher would be complete without reference to the seminars and lectures at which he imparted scientific knowledge and also entertained his audience as an incomparable raconteur. His research interests included the intermediary metabolism of amino acids, creatine, uric acid, and related compounds, and he was renowned for the discovery, isolation, and identification of a new amino acid as α-amino-β-hydroxy-n-butyric acid, which he named threonine (2McCoy R.H. Meyer C.E. Rose W.C. Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid..J. Biol. Chem. 1935; 112: 283-302Abstract Full Text PDF Google Scholar). This was the culmination of experiments in which rats failed to grow on diets containing the 19 previously known amino acids. Thus, painstaking efforts over many years led to the missing growth factor found in proteins and in hydrolysate fractions therefrom. When I arrived in Urbana to undertake graduate study in 1943, the identity of the 10 amino acids essential for growth in rats and the 8 essential for the maintenance of nitrogen equilibrium in the human (that is, male graduate students) was already known (3Rose W.C. Haines W.J. Johnson J.E. The role of the amino acids in human nutrition..J. Biol. Chem. 1942; 146: 683-684Abstract Full Text PDF Google Scholar, 4Rose W.C. The role of the amino acids in human nutrition..Proc. Am. Philos. Soc. 1947; 91: 112-116PubMed Google Scholar). It fell my lot to isolate, purify, and analyze amino acids and then feed them to fellow students enlisted as human guinea pigs in experiments involving daily nitrogen balance determinations. The diets consisted of the mixture of amino acids under study, the known vitamins, cornstarch, corn oil, sucrose, butter fat, inorganic salts, and Celluflour (a product providing roughage but no nutritive value, nitrogen, or flavor). The only taste thrill in this otherwise bland fare was a large brown “candy” containing a bitter liver extract as a possible source of unknown vitamins flavored with peppermint oil and sweetened with sugar. In those days the recruits were grateful for the free rations, the dollar a day they were paid, and the prospect of seeing their initials in print in Rose's widely read publications. The resulting papers established the quantitative requirements for the essential amino acids, the availability of some of the D-isomers orN-acetyl derivatives, and the role of cysteine and tyrosine in sparing methionine and phenylalanine, respectively. The morale of the subjects was maintained over many weeks by the prospect of collecting data for doctoral theses, the obvious importance of our findings for human welfare, and the infectious enthusiasm of Dr. Rose. An added benefit in my case was that, while consuming these daily rations, I had ample time to think about experiments on the metabolism of the essential amino acids I might pursue later in my career, as described below. Rose's students were somewhat in awe of the professor, perhaps wondering whether they could meet his exacting standards or hope to emulate the seeming ease with which he succeeded in all of his professional endeavors. They learned in time that behind his somewhat reserved and formal manner was a genuine warmth and an understanding that young scientists develop their full potential only by profiting from their mistakes. His research achievements earned him wide recognition and many honors. On the occasion of his 90th birthday his former students, colleagues, and friends assembled in Urbana to join him in the celebration. He was much surprised when presented with a handsome bronze plaque announcing the establishment of the William C. Rose Award and Lectureship. As indicated in Fig.1, the plaque to be given to all awardees shows his likeness and a sketch of the Noyes Laboratory with the structures of the essential amino acids and the stereochemistry and crystal structure of threonine, with a quotation and chart from his classical 1935 paper published in the Journal of Biological Chemistry (2McCoy R.H. Meyer C.E. Rose W.C. Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid..J. Biol. Chem. 1935; 112: 283-302Abstract Full Text PDF Google Scholar). This award, now administered by the American Society for Biochemistry and Molecular Biology, has been given annually, and the lectures are presented at the Society's national meetings. Until his death at age 98, Will Rose took a keen interest in those selected for the award named for him. He and his wife Zula exerted a wonderfully positive influence on all who knew them and took a personal interest in the 90 graduate students who studied under him, of whom 56 received the Ph.D. degree. In later years he often commented on his happy family life until his wife's death in 1965, his exciting professional life, and the thrill of watching his students grow into professional stature. In the first paper of this series on Reflections, Arthur Kornberg (5Kornberg A. Remembering our teachers..J. Biol. Chem. 2001; 276: 3-11Abstract Full Text Full Text PDF PubMed Google Scholar) has written perceptively of his deep admiration for Severo Ochoa, whom he knew particularly well from his stay in that laboratory in 1946 and their close friendship until Severo's death in 1993. Accordingly, I will comment only briefly on my exposure to that exciting New York University laboratory during the year 1952. After a few years as a junior faculty member at the University of Pennsylvania I had not yet earned an official sabbatical but came to realize that a knowledge of enzymology was crucial to further progress in my studies on amino acid metabolism. My colleague Jack Buchanan at the University of Pennsylvania advised me that the Ochoa laboratory was possibly the world's finest in enzymology at that time, and I acted on that sound advice and was most fortunate to have an acceptance. My research there involved studying the details of acetoacetate synthesis and breakdown and led to the purification and characterization of coenzyme A transferase, now called acetoacetyl-succinic thiophorase, from heart muscle (6Stern J.R. Coon M.J. del Campillo A. Schneider M.C. Enzymes of fatty acid metabolism. IV. Preparation and properties of coenzyme A transferase..J. Biol. Chem. 1956; 221: 15-31Abstract Full Text PDF PubMed Google Scholar). The Ochoa laboratory was crowded and still in the Pharmacology Department in an old building on First Avenue, with limited equipment, including the single Beckman DU spectrophotometer mentioned by Kornberg. Nevertheless it was an exciting place to pursue research, with Severo's ever optimistic support, intense lunchtime and afternoon discussions that included such luminaries as Otto Loewi, Ephraim Racker, and occasionally Sarah Ratner, and a legion of visiting postdoctoral fellows, present and former students, and sabbatical guests from every corner of the world. Combined with an ethic of unremitting experimental work, the environment was ideal for a visitor to master enzymology as an essential tool in understanding carbohydrate and lipid metabolism. Unlike other scientific departments I had been exposed to at American universities, Ochoa's department was more in the European or Japanese tradition of a group revolving around “the professor.” Ochoa (shown in Fig. 2) was ambitious and inspiring, exceptionally well informed, and completely dedicated to science. In describing his career in Europe and the United States (8Ochoa S. The pursuit of a hobby..Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Scopus (20) Google Scholar), he stated that biochemistry had been his “only and real hobby,” but he greatly appreciated art and music and fully enjoyed their availability in New York City. In the laboratory he talked only of science, but under more relaxed circumstances his very broad cultural interests came to the fore. Because of my increasing interest in mechanistic aspects of enzyme action, I subsequently took advantage of a sabbatical leave to improve my knowledge of organic chemistry and spent 1961–1962 with Professor Vladimir Prelog, Director of the Organic Chemistry Laboratory of the Eidgenössische Technische Hochschule (Swiss Federal Institute of Technology) in Zürich. Widely known for his studies on natural products and his outstanding contributions to stereochemistry (9Arigoni D. Dunitz J.D. Eschenmoser A. Vladimir Prelog, 23 July 1906–7 January 1998..Biogr. Mem. Fellows R. Soc. 2000; 46: 445-464Google Scholar), Prelog had developed an interest in enzyme stereoselectivity, and I began working on oxidoreductases inCurvularia falcata. The goal was to establish the absolute stereochemical course of hydride transfer to carbonyl groups of substrates such as decalin-1-one, decalin-2-one, and decalin-1,4-diones. Of interest, all the stereogenic carbon atoms formed by microbial reduction possess the sameS-configuration, independent of the configuration of the other stereogenic centers in the molecule or whether the hydroxyls are in the axial or the equatorial positions. Two Curvulariaenzymes, one believed to favor transfer of hydrogen into the axial position and another to favor the equatorial position, proved to be very difficult to purify. We found, however, an enzyme with oxidoreductase activity toward alicyclic ketones in pig liver that could be purified and was shown to be the 3-oxoacyl-acyl carrier protein reductase component of a fatty acid synthetase (10Dutler H. Coon M.J. Kull A. Vogel H. Waldvogel G. Prelog V. Fatty acid synthetase from pig liver. 1. Isolation of the enzyme complex and characterization of the component with oxidoreductase activity for alicyclic ketones..Eur. J. Biochem. 1971; 22: 203-212Crossref PubMed Scopus (41) Google Scholar). Vlado Prelog (see Fig. 3) was a frequent visitor to the United States and was elected as a Foreign Associate of the National Academy of Sciences. He said that he preferred the academic system in which scientific departments had a number of independent full professors. After he succeeded the famous Leopold Ruzicka in 1957 at the Eidgenössische Technische Hochschule, he established a “collegiate leadership” in which all appointed professors participated, surely an unusual arrangement at that time in Continental Europe. This gave him more time for research, for which he received innumerable honors, culminating in the 1975 Nobel Prize in Chemistry, which he shared with John Cornforth. After his mandatory retirement the following year, Prelog was required to have the title of postdoctoral student (Fachhörer) to continue his work, thus eventually leading to his autobiography entitled “My 137 Semesters of Chemistry Studies” (11Prelog V. My 132 semesters of chemistry studies.in: Seeman J.I. Autobiographies of Eminent Chemists. American Chemical Society, Washington, D. C.1991Google Scholar). In addition to his legendary pleasure in scientific study, he was widely known for his charming and witty personality. I can hardly recall a meeting with him, even a research conference, where he didn't regale us with his never ending supply of anecdotes about almost every famous chemist or biochemist (including those from the past), jokes, and humorous comments about the shortcomings of totalitarian political regimes, which he deplored. In the fall of 1947 I had joined the faculty of the Department of Physiological Chemistry at the University of Pennsylvania, where I undertook studies on amino acid metabolism. The department was one of the first in this country to work with radioactive carbon-14 as a tracer in intermediary metabolism, and several of the senior faculty were widely known for their studies on this subject: Jack Buchanan on purine biosynthesis, D. Wright Wilson on pyrimidine biosynthesis, and Samuel Gurin on fatty acid oxidation. So little was known about amino acid metabolism in general at that time that it was difficult to make a specific choice, but I was intrigued by the branched chain compounds leucine, isoleucine, and valine. The main reason was that their metabolic fates might throw some light on the origin of the branched carbon structures of numerous biologically occurring compounds, including steroids, vitamins A, E, and K, and a variety of products in plants, thought to be derived from five-carbon units according to the biogenetic isoprene rule (12Ruzicka L. The isoprene rule and the biogenesis of terpenic compounds..Experientia. 1953; 9: 357-396Crossref PubMed Scopus (406) Google Scholar). Another reason to study leucine in particular was its known ketogenic property (acetoacetate production) in animals, because the intermediate thought to be formed by deamination and oxidative decarboxylation, isovaleric acid, was obviously blocked in the β-position from the entry of a carbonyl group and, therefore, could not undergo the classical β-oxidation that occurs with straight chain fatty acids. I undertook the chemical synthesis of the substrates labeled with radioactivity in specific positions, no easy task because14C-labeled barium carbonate was the only commercially available starting material, incubated the purified compounds with liver slices, and analyzed the acetoacetate formed. To my surprise, the isopropyl group of leucine (and isovaleric acid) provided the terminal three carbons of this product, but the carboxyl carbon was unaccounted for. Radioactive CO2 was then employed in other experiments and found to provide the missing carbon atom. These results (13Coon M.J. Gurin S. Studies on the conversion of radioactive leucine to acetoacetate..J. Biol. Chem. 1949; 180: 1159-1167Abstract Full Text PDF PubMed Google Scholar, 14Coon M.J. The metabolic fate of the isopropyl group of leucine..J. Biol. Chem. 1950; 187: 71-82Abstract Full Text PDF PubMed Google Scholar) and subsequent experiments with heart extracts (15Bachhawat B.K. Robinson W.G. Coon M.J. Carbon dioxide fixation in heart extracts by β-hydroxyisovaleryl coenzyme A..J. Am. Chem. Soc. 1954; 76: 3098Crossref Scopus (0) Google Scholar) thus led to the discovery of a new ATP-dependent carbon dioxide fixation in mammalian metabolism. The scheme in Fig. 4 shows our knowledge of leucine metabolism as we became aware of the role of coenzyme A and identified the involvement of the β-hydroxy-β-methylglutaryl-CoA cleavage enzyme in generating acetoacetate (16Bachhawat B.K. Robinson W.G. Coon M.J. The enzymatic cleavage of β-hydroxy-β-methylglutaryl coenzyme A to acetoacetate and acetyl coenzyme A..J. Biol. Chem. 1955; 216: 727-736Abstract Full Text PDF PubMed Google Scholar). We had originally thought from experiments with crude enzyme preparations that the substrate in the carboxylation reaction was β-hydroxyisovaleryl-CoA, but it was later correctly identified as the unsaturated compound β-methylcrotonyl-CoA (17Hilz H. Knappe J. Ringelmann E. Lynen F. Methylglutaconase, a new hydratase involved in the metabolism of branched chain carboxylic acids..Biochem. Z. 1958; 329: 476-489PubMed Google Scholar, 18del Campillo-Campbell A. Dekker E.E. Coon M.J. Carboxylation of β-methylcrotonyl coenzyme A by a purified enzyme from chicken liver..Biochim. Biophys. Acta. 1959; 31: 290-292Crossref PubMed Scopus (11) Google Scholar). In summary, we found that nature had solved the problem of a difficult oxidative reaction by introduction of an energy-dependent carboxylation as a crucial step. Of additional interest, our results showed how leucine metabolism is integrated into the main pathways of lipid metabolism and steroid biosynthesis. As is well known, the details of cholesterol biosynthesis were elegantly elucidated by Konrad Bloch and Feodor Lynen. After moving to a faculty position at the University of Michigan and then returning from sabbatical leave in the Prelog laboratory some years later, I decided to work on the oxidation of hydrocarbons, which (because of their poor chemical reactivity) might be an even greater challenge than leucine for enzymatic degradation. James Baptist, a postdoctoral associate from Illinois, agreed to undertake this problem and set about isolating a suitable bacterium from soil samples by an enrichment culture technique with hexane as the carbon source (19Baptist J.N. Gholson R.K. Coon M.J. Hydrocarbon oxidation by a bacterial enzyme system. I. Products of octane oxidation..Biochim. Biophys. Acta. 1963; 69: 40-47Crossref PubMed Scopus (95) Google Scholar). The organism eventually obtained, a strain ofPseudomonas oleovorans that was dubbed the “gasoline bug” by our colleagues, grew well on several straight chain alkanes (or on leucine) but not on cyclohexane or methylbutane. Cell-free extracts were obtained that required the addition of NADH for the aerobic conversion of radioactive octane to octanol (20Gholson R.K. Baptist J.N. Coon M.J. Hydrocarbon oxidation by a bacterial enzyme system. II. Cofactor requirements for octanol formation from octane..Biochemistry. 1963; 2: 1155-1159Crossref PubMed Scopus (34) Google Scholar). Thus, it was evident that alkane oxidation at a terminal methyl group involved oxygenation as the initial step rather than an ATP-dependent carboxylation reaction, as in leucine metabolism. We subsequently found that, when presented with fatty acids as substrates, the bacterial system preferred to attack the terminal methyl carbon atom to give the ω-hydroxy acids (21Kusunose M. Kusunose E. Coon M.J. Enzymatic ω-oxidation of fatty acids. II. Substrate specificity and other properties of the enzyme system..J. Biol. Chem. 1964; 239: 2135-2139Abstract Full Text PDF PubMed Google Scholar) rather than to utilize a more chemically feasible α- or β-oxidation pathway. More will be said about ω-oxidation below in connection with related mammalian enzyme systems. By preferential extraction of the bacterial cells and column chromatography, three enzyme components were separated and found to be required for the conversion of octane to octanol or of laurate to ω-hydroxylaurate in the reconstituted enzyme system (22Peterson J.A. Basu D. Coon M.J. Enzymatic ω-oxidation. I. Electron carriers in fatty acid and hydrocarbon hydroxylation..J. Biol. Chem. 1966; 241: 5162-5163Abstract Full Text PDF PubMed Google Scholar) (Fig.5). These were purified to homogeneity and characterized as follows: a red, nonheme iron protein containing two iron atoms but no labile sulfide and identified spectrally as rubredoxin (23Peterson J.A. Coon M.J. Enzymatic ω-oxidation. III. Purification and properties of rubredoxin, a component of the ω-hydroxylation system of Pseudomonas oleovorans..J. Biol. Chem. 1968; 243: 329-334Abstract Full Text PDF PubMed Google Scholar), previously found only in anaerobes; a flavoprotein containing one molecule of FAD and found to be the NADH-rubredoxin reductase (24Ueda T. Coon M.J. Enzymatic ω-oxidation. VII. Reduced diphosphopyridine nucleotide-rubredoxin reductase: properties and function as an electron carrier in ω-hydroxylation..J. Biol. Chem. 1972; 247: 5010-5016Abstract Full Text PDF PubMed Google Scholar); and the ω-hydroxylase, which was relatively insoluble in that it formed aggregates of very high molecular weight, had an indistinct spectrum, and lost activity upon dialysis that was restored by the addition of ferrous ions (25McKenna E.J. Coon M.J. Enzymatic ω-oxidation. IV. Purification and properties of the ω-hydroxylase of Pseudomonas oleovorans..J. Biol. Chem. 1970; 245: 3882-3889Abstract Full Text PDF PubMed Google Scholar, 26Ruettinger R.T. Olson S.T. Boyer R.F. Coon M.J. Identification of the ω-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein requiring phospholipid for catalytic activity..Biochem. Biophys. Res. Commun. 1974; 57: 1011-1017Crossref PubMed Scopus (43) Google Scholar). The instability and other properties of the hydroxylase made it a difficult candidate for detailed mechanistic studies, but this enzyme system in P. oleovorans has continued to be investigated by others. Verkade et al. (27Verkade P.E. Elzas M. van der Lee J. de Wolff H.H. Verkade-Sandbergen A. van der Sande D. Studies on the metabolism of fats..Proc. K. Ned. Akad. Wet. 1932; 35: 251-266Google Scholar) in the Netherlands discovered ω-oxidation when they fed fatty acids of intermediate chain length (or their esters or glycerides) to dogs and human subjects and isolated the resulting urinary dicarboxylic acids, and Carter (28Carter H.E. Lewis H.B. The oxidation of branched-chain fatty acids. In Biological Symposia. 5. Jacques Cattell Press, Lancaster, PA1941: 47-63Google Scholar) and Bergström et al. (29Bergström S. Borgström B. Tryding N. Westöö G. Intestinal absorption and metabolism of 2:2-dimethylstearic acid in the rat..Biochem. J. 1954; 58: 604-608Crossref PubMed Scopus (34) Google Scholar) later reported that some α- and β-substituted fatty acids undergo a similar attack in animals. We undertook a study to determine the enzymatic mechanism of this intriguing oxidative process in the late 1950s, but the instability and insolubility of the liver microsomal enzyme system prevented further progress, and we turned our attention to the more tractable bacterial system, as described above. Then, almost 10 years later, Anthony Lu joined our research group as a postdoctoral associate after completion of his graduate studies at the University of North Carolina. He impressed me as a highly talented and enthusiastic young scientist who would welcome a challenging problem, and I suggested that we again attempt to characterize the fatty acid ω-hydroxylating enzyme system of liver microsomes, making use of what we had learned about the bacterial system. The rest of this story is now well known. The hepatic system was resistant to the isolation and purification methods employed with the pseudomonad, but fortunately, we were not discouraged by the lack of knowledge at that time about membrane-bound enzymes in general and microsomal enzymes in particular. Thanks to Anthony's painstaking efforts for more than 2 years, the hydroxylating system eventually yielded to solubilization with various detergents in the presence of agents to protect against enzyme denaturation by the detergents. Column chromatography of the resulting preparations (again with detergents and protective agents) yielded a red fraction containing cytochrome P450 (identified by the spectral change upon addition of carbon monoxide to the reduced protein), a yellow fraction containing the flavoprotein NADPH-cytochrome P450 reductase (assayed by reduction of cytochrome c as an artificial electron acceptor), and a colorless, heat-stable fraction (30Lu A.Y.H. Coon M.J. Role of hemoprotein P-450 in fatty acid ω-hydroxylation in a soluble enzyme system from liver microsomes..J. Biol. Chem. 1968; 243: 1331-1332Abstract Full Text PDF PubMed Google Scholar, 31Lu A.Y.H. Junk K.W. Coon M.J. Resolution of the cytochrome P-450-containing ω-hydroxylation system of liver microsomes into three components..J. Biol. Chem. 1969; 244: 3714-3721Abstract Full Text PDF PubMed Google Scholar). The last of these was shown by Henry Strobel, another postdoctoral associate from North Carolina, to contain phospholipids, of which phosphatidylcholine was especially active (32Strobel H.W. Lu A.Y.H. Heidema J. Coon M.J. Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation..J. Biol. Chem. 1970; 245: 4851-4854Abstract Full Text PDF PubMed Google Scholar). When mixed and incubated together under precise conditions, the three components yielded a reconstituted enzyme system that converted lauric acid to ω-hydroxylauric acid in the presence of NADPH and oxygen, as shown in Fig. 6 (31Lu A.Y.H. Junk K.W. Coon M.J. Resolution of the cytochrome P-450-containing ω-hydroxylation system of liver microsomes into three components..J. Biol. Chem. 1969; 244: 3714-3721Abstract Full Text PDF PubMed Google Scholar). In the progress of science we all build on previous findings, and we had the benefit of knowing that microsomes contain a carbon monoxide-binding pigment of unknown function (33Ryan K.J. Engel L.L. Hydroxylation of steroids at carbon 21..J. Biol. Chem. 1957; 225: 103-114Abstract Full Text PDF PubMed Google Scholar, 34Klingenberg M. Pigments of rat liver microsomes..Arch. Biochem. Biophys. 1958; 75: 376-386Crossref PubMed Scopus (456) Google Scholar, 35Garfinkel D. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions..Arch. Biochem. Biophys. 1958; 77: 493-509Crossref PubMed Scopus (281) Google Scholar), which was identified as a hemeprotein and designated “P-450” by Omura and Sato (36Omura T. Sato R. A new cytochrome in liver microsomes..J. Biol. Chem. 1962; 237: 1375-1376Abstract Full Text PDF PubMed Google Scholar). Furthermore, the groundbreaking work of Omura, Sato, Cooper, Rosenthal, and Estabrook (37Omura T. Sato R. Cooper D.Y. Rosenthal O. Estabrook R.W. Function of cytochrome P-450 of microsomes..Fed. Proc. 1965; 24: 1181-1189PubMed Google Scholar) had shown by photochemical action spectroscopy that this pigment in hepatic microsomes is responsible for the hydroxylation of several steroids and drugs. Thus, we had in our hands the solubilized hemeprotein P450 from rabbit liver microsomes capable of oxidizing not only fatty acids at the terminal position but a huge variety of other substrates of much greater biochemical and pharmacological interest. The same methods led to the successful solubilization and resolution of the P450-containing enzyme system of human liver microsomes (38Kaschnitz R.M. Coon M.J. Drug and fatty acid hydroxylation by solubilized human liver microsomal cytochrome P-450-phospholipid requirement..Biochem. Pharmacol. 1975; 24: 295-297Crossref PubMed Scopus (22) Google Scholar). In addition, a visitor from France, Jean-Michel Lebeault, brought a strain of Candida tropicalis to my laboratory, and we found that, when grown on the long chain hydrocarbon tetradecane, it produced cytochrome P450 as the lauric acid ω-oxygenating catalyst that could be solubilized and reconstituted into a functional enzyme system (39Lebeault J.M. Lode E.T. Coon M.J. Fatty acid a