Title: Unusual Cytochrome P450 Enzymes and Reactions
Abstract: Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. Details of the cytochrome P450 catalytic mechanism are described in the accompanying minireview by Green and co-workers (1Krest C.M. Onderko E.L. Yosca T.H. Calixto J.C. Karp R.F. Livada J. Rittle J. Green M.T. Reactive intermediates in cytochrome P450 catalysis.J. Biol. Chem. 2013; 288: 17074-17081Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Most of the P450 reactions can be rationalized in the context of the intermediate FeO3+, which corresponds to what was first described as “Compound I” in peroxidase chemistry (Fig. 1A, i). A variant mechanism has been proposed for some P450 reactions (Fig. 1A, ii), particularly those regarding aldehydes (4Ortiz de Montellano P.R. De Voss J.J. Substrate oxidation by cytochrome P450 enzymes.in: Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 183-245Crossref Scopus (211) Google Scholar). Another mode of P450 reactions is the rearrangement of some oxygenated substrates (e.g. prostaglandins) (Fig. 1A, iii), as exemplified by the P450s CYP5A1 and CYP8A1, the respective prostacyclin and thromboxane synthases (see below). Another mechanistic aspect is the distinction of reactions regarding low- and high-spin FeO3+, proposed by Shaik et al. (7Shaik S. Kumar D. de Visser S.P. Altun A. Thiel W. Theoretical perspective on the structure and mechanism of cytochrome P450 enzymes.Chem. Rev. 2005; 105: 2279-2328Crossref PubMed Scopus (1033) Google Scholar) to explain multiple reactions catalyzed by a P450. Although intriguing, this hypothesis has been examined only at the theoretical level. The experimental approaches described by Green and co-workers (1Krest C.M. Onderko E.L. Yosca T.H. Calixto J.C. Karp R.F. Livada J. Rittle J. Green M.T. Reactive intermediates in cytochrome P450 catalysis.J. Biol. Chem. 2013; 288: 17074-17081Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Rittle J. Green M.T. Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics.Science. 2010; 330: 933-937Crossref PubMed Scopus (1043) Google Scholar) can be applied to the questions posed by the spin hypothesis. Most of the unusual reactions of P450s can be understood in the context of rearrangements, either of the reaction products (due to instability) or within an enzymatic reaction intermediate. Examples of both will be considered. For a more extensive collection of unusual P450 reactions, see previous reviews (4Ortiz de Montellano P.R. De Voss J.J. Substrate oxidation by cytochrome P450 enzymes.in: Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 183-245Crossref Scopus (211) Google Scholar, 9Guengerich F.P. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.Chem. Res. Toxicol. 2001; 14: 611-650Crossref PubMed Scopus (1407) Google Scholar, 10Isin E.M. Guengerich F.P. Complex reactions catalyzed by cytochrome P450 enzymes.Biochim. Biophys. Acta. 2007; 1770: 314-329Crossref PubMed Scopus (368) Google Scholar11Guengerich F.P. Isin E.M. Unusual metabolic reactions and pathways.in: Prakash C. Gau L. Zhong D. Aizawa H. Lee P. Handbook of Metabolic Pathways of Xenobiotics. 1. John Wiley & Sons, Inc., New York2014Crossref Google Scholar). The literature of these unusual reaction products is dominated by contributions from the fields of drug metabolism and plant biochemistry (which are not unrelated, in that many drugs are plant secondary metabolites). Most drugs are complex molecules, and regulatory agencies require elucidation of chemical structures of metabolites prior to registration (12Humphreys W.G. Drug metabolism research as an integral part of the drug discovery process.in: Zhang D. Zhu M. Humphreys W.G. Drug Metabolism in Drug Design and Development. John Wiley & Sons, Inc., Hoboken, NJ2008: 239-260Google Scholar). Plants have large numbers of P450 genes (hundreds per species) and utilize these in the synthesis of complex secondary metabolites, e.g. alkaloids, modified terpenes, flavonoids, etc. Many examples are known of α-hydroxy heteroatom products that break down, e.g. carbinolamines, hemiacetals, and gem-halohydrins (Fig. 1B). However, some stable carbinolamines (5Kedderis G.L. Dwyer L.A. Rickert D.E. Hollenberg P.F. Source of the oxygen atom in the product of cytochrome P-450-catalyzed N-demethylation reactions.Mol. Pharmacol. 1983; 23: 758-760PubMed Google Scholar, 6Shea J.P. Valentine G.L. Nelson S.D. Source of oxygen in cytochrome P-450 catalyzed carbinolamine formation.Biochem. Biophys. Res. Commun. 1982; 109: 231-235Crossref PubMed Scopus (28) Google Scholar) and hemiaminals (13Ross D. Farmer P.B. Gescher A. Hickman J.A. Threadgill M.D. The metabolism of a stable N-hydroxymethyl derivative of a N-methylamide.Life Sci. 1983; 32: 597-604Crossref PubMed Scopus (5) Google Scholar) are known. In many cases, the initial products have not been observed directly, but indirect approaches have been used to demonstrate their existence, e.g. oxygenated halogen atoms (haloso compounds) (supplemental Fig. S1) (14He X. Cryle M.J. De Voss J.J. Ortiz de Montellano P.R. Calibration of the channel that determines the ω-hydroxylation regiospecificity of cytochrome P450 4A1. Catalytic oxidation of 12-halododecanoic acids.J. Biol. Chem. 2005; 280: 22697-22705Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Guengerich F.P. Oxidation of halogenated compounds by cytochrome P-450, peroxidases, and model metalloporphyrins.J. Biol. Chem. 1989; 264: 17098-17205Abstract Full Text PDF PubMed Google Scholar). Sometimes, alcohol products dehydrate to yield olefins. However, olefins can also be formed directly by an oxidative mechanism resembling those of recognized desaturases (9Guengerich F.P. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.Chem. Res. Toxicol. 2001; 14: 611-650Crossref PubMed Scopus (1407) Google Scholar, 16Rettie A.E. Rettenmeier A.W. Howald W.N. Baillie T.A. Cytochrome P-450 catalyzed formation of δ4-VPA, a toxic metabolite of valproic acid.Science. 1987; 235: 890-893Crossref PubMed Scopus (256) Google Scholar). Arene oxides (epoxides) rearrange to phenols (17Guroff G. Daly J.W. Jerina D.M. Renson J. Witkop B. Udenfriend S. Hydroxylation-induced migration: the NIH shift.Science. 1967; 157: 1524-1530Crossref PubMed Scopus (416) Google Scholar). In addition, vinyl compounds with good leaving groups can form epoxides that rearrange, e.g. to α-halocarbonyls (18Liebler D.C. Guengerich F.P. Olefin oxidation by cytochrome P-450: evidence for group migration in catalytic intermediates formed with vinylidene chloride and trans-1-phenyl-1-butene.Biochemistry. 1983; 22: 5482-5489Crossref PubMed Scopus (112) Google Scholar, 19Cai H. Guengerich F.P. Mechanism of aqueous decomposition of trichloroethylene oxide.J. Am. Chem. Soc. 1999; 121: 11656-11663Crossref Scopus (39) Google Scholar). In more complex (cellular) settings, leaving groups may be attached to the alcohols generated by P450s. Accordingly, the elimination or nucleophilic addition products may be recovered and identified. The latter type of reaction with a macromolecule (i.e. DNA and protein) is a major issue in chemical carcinogenesis and toxicity (20Rendic S. Guengerich F.P. Contributions of human enzymes in carcinogen metabolism.Chem. Res. Toxicol. 2012; 25: 1316-1383Crossref PubMed Scopus (214) Google Scholar). A dramatic example of rearrangement in a product involves the drug candidate BMS-690514 (supplemental Fig. S2). The reaction is rationalized by an electrophilic attack of a P450 (CYP3A4?) on a pyrrole ring to form a hydroxypyrrole, which rearranges to open the pyrrole ring and eventually involves reaction of a neighboring aniline ring to form a stable carbinolamine product (21Hong H. Caceres-Cortes J. Su H. Huang X. Roongta V. Bonacorsi Jr., S. Hong Y. Tian Y. Iyer R.A. Humphreys W.G. Christopher L.J. Mechanistic studies on a P450-mediated rearrangement of BMS-690514: conversion of a pyrrolotriazine to a hydroxypyridotriazine.Chem. Res. Toxicol. 2011; 24: 125-134Crossref PubMed Scopus (4) Google Scholar). In general, rearrangements of enzyme intermediates are more complex, more interesting, and often more difficult to rationalize. A brief discussion of kinetics and mechanism is in order before consideration of examples. Strained cycloalkyl entities have been utilized as “radical clocks” (22Austin R.N. Deng D. Jiang Y. Luddy K. van Beilen J.B. Ortiz de Montellano P.R. Groves J.T. The diagnostic substrate bicyclohexane reveals a radical mechanism for bacterial cytochrome P450 in whole cells.Angew. Chem. Int. Ed. Engl. 2006; 45: 8192-8194Crossref PubMed Scopus (30) Google Scholar, 23Auclair K. Hu Z. Little D.M. Ortiz de Montellano P.R. Groves J.T. Revisiting the mechanism of P450 enzymes with the radical clocks norcarane and spiro[2,5]octane.J. Am. Chem. Soc. 2002; 124: 6020-6027Crossref PubMed Scopus (140) Google Scholar). Abstraction of a hydrogen atom is an energetically unfavorable reaction, but, once formed, the “oxygen rebound” step (Fig. 1A, i) is very rapid, as recently demonstrated in direct experiments with P450 119A1 Compound I by Green and co-workers (1Krest C.M. Onderko E.L. Yosca T.H. Calixto J.C. Karp R.F. Livada J. Rittle J. Green M.T. Reactive intermediates in cytochrome P450 catalysis.J. Biol. Chem. 2013; 288: 17074-17081Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Rittle J. Green M.T. Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics.Science. 2010; 330: 933-937Crossref PubMed Scopus (1043) Google Scholar). The rates of rearrangement of some cycloalkyl radical systems are known (in solution) and can be used to estimate the rates of rearrangement and oxygen rebound (radical recombination, i.e. reaction of FeOH3+ + •C) in enzymes. Although this field has not been without controversy (24Toy P.H. Newcomb M. Hollenberg P.F. Hypersensitive mechanistic probe studies of cytochrome P450-catalyzed hydroxylation reactions. Implications for the cationic pathway.J. Am. Chem. Soc. 1998; 120: 7719-7729Crossref Scopus (102) Google Scholar), rates of recombination of ∼109 s−1 are considered to occur in several P450s (25Cooper H.L. Groves J.T. Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate.Arch. Biochem. Biophys. 2011; 507: 111-118Crossref PubMed Scopus (41) Google Scholar). Such studies have caveats regarding rates of rearrangement, in that atoms of these molecules are undoubtedly restrained in active sites through various bonding forces, and this can change the rates compared with those measured in simple chemical systems (26Frey P.A. Radicals in enzymatic reactions.Curr. Opin. Chem. Biol. 1997; 1: 347-356Crossref PubMed Scopus (37) Google Scholar). With this background, we can consider some of the rearrangements. Another aspect to consider is one already mentioned, that of desaturation resulting from the abstraction of a second hydrogen atom to achieve a net 2-electron loss. One recent example of an unusual rearrangement in a P450 complex involves the P450 enzyme CYP125 (from Mycobacterium tuberculosis and rhodococci) and the oxidation of cholesterol (Fig. 2). An unusual product is the formate ester (M2 in Fig. 2A). The reaction is shown as releasing formate in the proposed mechanism in Fig. 2A, presumably without extrusion into the medium (equilibration with any formate in the medium was not examined.) An alternative concerted Baeyer-Villiger reaction is shown in Fig. 2B. Many other P450 reactions are known in which rearrangements undoubtedly occur with a P450 enzyme-intermediate complex. Notable examples are the conversion of vinyl halides to α-haloacetaldehydes (18Liebler D.C. Guengerich F.P. Olefin oxidation by cytochrome P-450: evidence for group migration in catalytic intermediates formed with vinylidene chloride and trans-1-phenyl-1-butene.Biochemistry. 1983; 22: 5482-5489Crossref PubMed Scopus (112) Google Scholar, 28Miller R.E. Guengerich F.P. Oxidation of trichloroethylene by liver microsomal cytochrome P-450: evidence for chlorine migration in a transition state not involving trichloroethylene oxide.Biochemistry. 1982; 21: 1090-1097Crossref PubMed Scopus (207) Google Scholar). A recent similar example involves the “direct” conversion of 7,8-dehydrocholesterol to 7-ketocholesterol by the human P450 CYP7A1 (29Shinkyo R. Xu L. Tallman K.A. Cheng Q. Porter N.A. Guengerich F.P. Conversion of 7-dehydrocholesterol to 7-ketocholesterol is catalyzed by human cytochrome P450 7A1 and occurs by direct oxidation without an epoxide intermediate.J. Biol. Chem. 2011; 286: 33021-33028Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Strained cycloalkyl rings undergo rearrangements (2Macdonald T.L. Zirvi K. Burka L.T. Peyman P. Guengerich F.P. Mechanism of cytochrome P-450 inhibition by cyclopropylamines.J. Am. Chem. Soc. 1982; 104: 2050-2052Crossref Scopus (162) Google Scholar, 30Bondon A. Macdonald T.L. Harris T.M. Guengerich F.P. Oxidation of cycloalkylamines by cytochrome P-450. Mechanism-based inactivation, adduct formation, ring expansion, and nitrone formation.J. Biol. Chem. 1989; 264: 1988-1997Abstract Full Text PDF PubMed Google Scholar). Other examples in which cleavage of a substrate occurs in such a mechanism involve the ipso cleavage of bisphenol A (supplemental Fig. S3) (31Kolvenbach B. Schlaich N. Raoui Z. Prell J. Zühlke S. Schäfter A. Guengrich F.P. Corvini P.F.X. Degradation of bisphenol A: does ipso substitution apply to phenols containing a quartenary C-α structure in the para positions?.Appl. Environ. Microbiol. 2007; 73: 4476-4784Crossref Scopus (88) Google Scholar) and the conversion of nabumetone to 6-methoxy-2-naphthylacetic acid by the human P450 CYP1A2 (supplemental Fig. S4) (32Haddock R.E. Jeffery D.J. Lloyd J.A. Thawley A.R. Metabolism of nabumetone (BRL 14777) by various species including man.Xenobiotica. 1984; 14: 327-337Crossref PubMed Scopus (70) Google Scholar, 33Turpeinen M. Hofmann U. Klein K. Mürdter T. Schwab M. Zanger U.M. A predominate role of CYP1A2 for the metabolism of nabumetone to the active metabolite, 6-methoxy-2-naphthylacetic acid, in human liver microsomes.Drug Metab. Dispos. 2009; 37: 1017-1024Crossref PubMed Scopus (42) Google Scholar). P450-catalyzed C–C and C–O bond couplings are common in plant biosynthetic pathways (e.g. alkaloid biosynthesis) and in bacteria (e.g. antibiotic synthesis in Actinomycetes). These reactions are often critical in the biosynthesis of plant secondary metabolites. An interesting example from Taxol biosynthesis (plus a possible mechanism) is shown in Fig. 3A (34Rontein D. Onillon S. Herbette G. Lesot A. Werck-Reichhart D. Sallaud C. Tissier A. CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4(5),11(12)-diene into the cyclic ether 5(12)-oxa-3(11)-cyclotaxane.J. Biol. Chem. 2008; 283: 6067-6075Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Some other interesting examples include C–O bond coupling in the biosynthesis of grayanic acid (supplemental Fig. S5) (37Culberson C.F. Armaleo D. Induction of a complete secondary-product pathway in a cultured lichen fungus.Exp. Mycol. 1992; 16: 52-63Crossref Scopus (82) Google Scholar), coupling in the synthesis of isoquinoline alkaloids (supplemental Fig. S6) (38Ikezawa N. Iwasa K. Sato F. CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in Eschscholzia californica.Plant Cell Rep. 2009; 28: 123-133Crossref PubMed Scopus (78) Google Scholar), and oxidative coupling in early steps of morphine biosynthesis (supplemental Fig. S7) (39Ikezawa N. Iwasa K. Sato F. Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells.J. Biol. Chem. 2008; 283: 8810-8821Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 40Gesell A. Rolf M. Ziegler J. Díaz Chávez M.L. Huang F.C. Kutchan T.M. CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of morphine biosynthesis in opium poppy.J. Biol. Chem. 2009; 284: 24432-24442Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). These coupling reactions, generally believed to involve radical chemistry, are not restricted to plants and bacteria. Human P450s have been shown to also catalyze steps in morphine synthesis (supplemental Fig. S8) (41Grobe N. Zhang B. Fisinger U. Kutchan T.M. Zenk M.H. Guengerich F.P. Mammalian cytochrome P450 enzymes catalyze the phenol-coupling step in endogenous morphine biosynthesis.J. Biol. Chem. 2009; 284: 24425-24431Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Most of these reactions are internal couplings, in which rings are in juxtaposition to fuse if a radical pathway is initiated. The coupling of two flaviolin molecules together has been observed in a Streptomyces coelicolor CYP158A2 reaction, in which the two molecules are both bound in the active site (42Zhao B. Guengerich F.P. Bellamine A. Lamb D.C. Izumikawa M. Lei L. Podust L.M. Sundaramoorthy M. Kalaitzis J.A. Reddy L.M. Kelly S.L. Moore B.S. Stec D. Voehler M. Falck J.R. Shimada T. Waterman M.R. Binding of two flaviolin substrate molecules, oxidative coupling, and crystal structure of Streptomyces coelicolor A3(2) cytochrome P450 158A2.J. Biol. Chem. 2005; 280: 11599-11607Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). A dimer of capsaicin is formed during its oxidation by human liver microsomes (although which P450 is involved remains unknown) (43Reilly C.A. Henion F. Bugni T.S. Ethirajan M. Stockmann C. Pramanik K.C. Srivastava S.K. Yost G.S. Reactive intermediates produced from the metabolism of the vanilloid ring of capsaicinoids by P450 enzymes.Chem. Res. Toxicol. 2013; 26: 55-66Crossref PubMed Scopus (36) Google Scholar). The general reaction supports a view of an aromatic radical pathway but is rare among oxidations of unlinked substrates. Recently, the environmental contaminant BDE-47, a polybrominated bis-phenyl ether flame retardant, was reported to be converted to a (brominated) p-dioxin molecule by the introduction of a second ether oxygen (supplemental Fig. S9) (44Feo M.L. Gross M.S. McGarrigle B.P. Eljarrat E. Barceló D. Aga D.S. Olson J.R. Biotransformation of BDE-47 to potentially toxic metabolites is predominantly mediated by human CYP2B6.Environ. Health Perspect. 2013; 121: 440-446Crossref PubMed Google Scholar). A highly unusual reaction is involved in the synthesis of a phytotoxin, thaxtomin, in Streptomyces turgidiscabies (Fig. 3B) (35Barry S.M. Kers J.A. Johnson E.G. Song L. Aston P.R. Patel B. Krasnoff S.B. Crane B.R. Gibson D.M. Loria R. Challis G.L. Cytochrome P450-catalyzed l-tryptophan nitration in thaxtomin phytotoxin biosynthesis.Nat. Chem. Biol. 2012; 8: 814-816Crossref PubMed Scopus (155) Google Scholar). The FeO22+ form of the enzyme is proposed to react with nitric oxide to form an iron peroxynitrite-like species that can produce a nitrating species capable of nitrating tryptophan. This is not a physiological reaction but nevertheless demonstrates the versatility of P450s (this is not an oxidation per se but is included here in the context of mechanistic similarity). The ferrous form of a mutant of bacterial CYP102A1 (P450BM-3) reacted with ethyl diazoacetate to convert styrene to a cyclopropyl derivative (Fig. 3C) (36Coelho P.S. Brustad E.M. Kannan A. Arnold F.H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes.Science. 2013; 339: 307-310Crossref PubMed Scopus (585) Google Scholar). Multiple turnovers were observed, and the stereochemistry was distinct from that seen in reactions with only free heme. Although P450s are well known catalysts of alkane hydroxylations, the oxidation of methane presents a special challenge due to the strong C–H bond strength (104 kcal mol−1). Two groups have employed a strategy of “activating” CYP102A1 by using short perfluorinated fatty acids (with no C–H bonds available for oxidation) to “activate” the enzyme and then also adding short alkanes, which can be oxidized as substrates (45Zilly F.E. Acevedo J.P. Augustyniak W. Deege A. Häusig U.W. Reetz M.T. Tuning a P450 enzyme for methane oxidation.Angew. Chem. Int. Ed. Engl. 2011; 50: 2720-2724Crossref PubMed Scopus (120) Google Scholar, 46Kawakami N. Shoji O. Watanabe Y. Use of perfluorocarboxylic acids to trick cytochrome P450BM3 into initiating the hydroxylation of gaseous alkanes.Angew. Chem. Int. Ed. Engl. 2011; 50: 5315-5318Crossref PubMed Scopus (111) Google Scholar). The activation process includes both a spin-state change in the iron and a constriction in the size of the active site. Watanabe and co-workers (46Kawakami N. Shoji O. Watanabe Y. Use of perfluorocarboxylic acids to trick cytochrome P450BM3 into initiating the hydroxylation of gaseous alkanes.Angew. Chem. Int. Ed. Engl. 2011; 50: 5315-5318Crossref PubMed Scopus (111) Google Scholar) were not able to oxidize methane or ethane in their system, but Reetz and co-workers (45Zilly F.E. Acevedo J.P. Augustyniak W. Deege A. Häusig U.W. Reetz M.T. Tuning a P450 enzyme for methane oxidation.Angew. Chem. Int. Ed. Engl. 2011; 50: 2720-2724Crossref PubMed Scopus (120) Google Scholar) were able to achieve multiple turnovers of methane, an unusual feat. Although the vast majority of P450 reactions are oxidations, reductions are also known. Most are observed more readily under anaerobic or hypobaric conditions. For rather unclear reasons, the substrates for such reactions accept electrons from ferrous P450 at a rate that is competitive with the rates of binding and reduction of oxygen. These reactions can occur in cells with low oxygen tension, e.g. those most distant from capillary arteries in animals. The electrons must be transferred one at a time. The nature of the 1-electron reduced intermediates and, in some ways, the fate of an oxygen in a substrate remain unclear. One unusual case is the conversion of benzo[a]pyrene epoxides to the polycyclic hydrocarbon itself, i.e. benzo[a]pyrene (47Sugiura M. Yamazoe Y. Kamataki T. Kato R. Reduction of epoxy derivatives of benzo(a)pyrene by microsomal cytochrome P-450.Cancer Res. 1980; 40: 2910-2914PubMed Google Scholar). For reasons that are not clear, the only reactions of the human “orphan” P450 CYP2S1 that have been reproducibly observed to date are all reductions (48Nishida C.R. Lee M. Ortiz de Montellano P.R. Efficient hypoxic activation of the anticancer agent AQ4N by CYP2S1 and CYP2W1.Mol. Pharmacol. 2010; 78: 497-502Crossref PubMed Scopus (100) Google Scholar, 49Xiao Y. Shinkyo R. Guengerich F.P. Cytochrome P450 2S1 is reduced by NADPH-cytochrome P450 reductase.Drug Metab. Dispos. 2011; 39: 944-946Crossref PubMed Scopus (26) Google Scholar50Wang K. Guengerich F.P. Oxidation of fluorinated 2-aryl-benzothiazole antitumor molecules by human cytochromes P450 1A1 and 2W1. Deactivation by cytochrome P450 2S1.Chem. Res. Toxicol. 2012; 25: 1740-1751Crossref PubMed Scopus (64) Google Scholar). At least three non-redox P450 reactions have been reported, including a phospholipase D-type hydrolysis by several mammalian P450s (51Yun C.-H. Ahn T. Guengerich F.P. Yamazaki H. Shimada T. Phospholipase D activity of cytochromes P450 in human liver endoplasmic reticulum.Arch. Biochem. Biophys. 1999; 367: 81-88Crossref PubMed Scopus (28) Google Scholar), pyrophosphatase (hydrolytic) activity of S. coelicolor CYP170A1 (52Zhao B. Lei L. Vassylyev D.G. Lin X. Cane D.E. Kelly S.L. Yuan H. Lamb D.C. Waterman M.R. Crystal structure of albaflavenone monooxygenase containing a moonlighting terpene synthase active site.J. Biol. Chem. 2009; 284: 36711-36719Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and the rearrangement of a bicyclic pentaenone to an oxetane by CYP154A1 from the same bacterium (53Cheng Q. Lamb D.C. Kelly S.L. Lei L. Guengerich F.P. Cyclization of a cellular dipentaenone by Streptomyces coelicolor cytochrome P450 154A1 without oxidation-reduction.J. Am. Chem. Soc. 2010; 132: 15173-15175Crossref PubMed Scopus (30) Google Scholar). Those reactions are likely to involve elements of acid-base chemistry, but the details are unknown. In the case of CYP170A1, an alternative active site for the hydrolytic reaction has been identified (the P450 also catalyzes oxidative reactions). The “classical” eukaryotic P450 enzymes are membrane-associated P450s that interact either with the NADPH-dependent diflavin enzyme cytochrome P450 reductase (CPR 2The abbreviations used are: CPR, cytochrome P450 reductase; ADR, adrenodoxin reductase; ADx, adrenodoxin; FDx, ferredoxin; PG, prostaglandin; TXA2, thromboxane A2. ; for Class II microsomal P450s) or with the flavoprotein adrenodoxin reductase (ADR) and the iron-sulfur protein adrenodoxin (ADx; for Class I mitochondrial P450s) (Fig. 4). Typical prokaryotic Class I systems have similar flavoprotein reductase and ferredoxin (FDx) partners (60Munro A.W. Girvan H.M. McLean K.J. Variations on a (t)heme–novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily.Nat. Prod. Rep. 2007; 24: 585-609Crossref PubMed Scopus (225) Google Scholar). Interestingly, some of the mammalian microsomal P450s are also imported in the mitochondria and use ADx (an FDx) and its ADR (64Bajpai P. Sangar M.C. Singh S. Tang W. Bansal S. Chowdhury G. Cheng Q. Fang J.-K. Martin M.V. Guengerich F.P. Avadhani N.G. Metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by mitochondria-targeted cytochrome P450 2D6. Implications for Parkinson disease.J. Biol. Chem. 2013; 288: 4436-4451Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Recent years have seen the identification of several distinct types of P450s that do not fall into the general Class I/II types, many of which have been identified through characterization of P450 enzymes identified from genome sequencing projects. The discovery of a high-activity fatty acid hydroxylase in Bacillus megaterium led Narhi and Fulco (65Narhi L.O. Fulco A.J. Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium.J. Biol. Chem. 1987; 262: 6683-6690Abstract Full Text PDF PubMed Google Scholar) to identify the first P450 linked to its redox partner. P450BM-3 (CYP102A1) has a eukaryote-like CPR fused to the C terminus of the P450. Both domains lack the membrane anchor regions typical of their eukaryotic relatives, and P450BM-3 was the first example of a prokaryotic CPR (54Munro A.W. Leys D.G. McLean K.J. Marshall K.R. Ost T.W. Daff S. Miles C.S. Chapman S.K. Lysek D.A. Moser C.C. Page C.C. Dutton P.L. P450 BM3: the very model of a modern flavocytochrome.Trends Biochem. Sci. 2002; 27: 250-257Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The P450-CPR fusion arrangement of P450BM-3 facilitates rapid electron transfer from NADPH to the P450 heme, and the CPR portion (of P450BM-3) is also reduced by NADPH much faster than are eukaryotic CPRs (66Munro A.W. Lindsay J.G. Bacterial cytochromes P-450.Mol. Microbiol. 1996; 20: 1115-1125Crossref PubMed Scopus (136) Google Scholar), leading to P450BM-3 having the fastest substrate oxidation rate yet reported for a P450 enzyme, ∼250 s−1 (67Noble M.A. Miles C.S. Chapman S.K. Lysek D.A. MacKay A.C. Reid G.A. Hanzlik R.P. Munro A.W. Roles of key active-site residues in flavocytochrome P450 BM3.Biochem. J. 1999; 339: 371-379Crossref PubMed Scopus (244) Google Scholar). The catalytic proficiency of P450BM-3 has aroused great interest in its capacity for oxychemical synthesis, and notable successes in re-engineering P450BM-3 include transforming the enzyme from a fatty acid (ω-1, ω-2, and ω-3) hydroxylase into variants capable of oxidizing short chain alkanes and alkenes as well as steroids (68Glieder A. Farinas E.T. Arnold F.H. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase.Nat. Biotechnol. 2002; 20: 1135-1139Crossref PubMed Scopus (349) Google Scholar, 69Kille S. Zilly F.E. Acevedo J.P. Reetz M.T. Regio- and stereoselect