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Cytochrome b5 Increases the Rate of Product Formation by Cytochrome P450 2B4 and Competes with Cytochrome P450 Reductase for a Binding Site on Cytochrome P450 2B4

2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês

10.1074/jbc.m703845200

1083-351X

Haoming Zhang, Sang‐Choul Im, Lucy Waskell,

Cancer Treatment and Pharmacology

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b5 (cyt b5) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b5 molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 ± 2 and 29 ± 4.5 s–1, respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine ≅100-fold (k = 0.15 ± 0.05 s–1) and cyclohexane ≅10-fold (k = 2.5 ± 0.35 s–1) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b5, and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b5 and CPR. Increasing the concentration of cyt b5 enhanced the amount of product formed by cyt b5 while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b5:cyt P450 molar ratios examined, cyt b5 inhibits the rate of NADPH consumption. Nevertheless, at low cyt b5:cyt P450 molar ratios ≤1:1, the rate of metabolism of cyclohexane and benzphetamine is enhanced, whereas at higher cyt b5:cyt P450 molar ratios, cyt b5 progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b5 to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b5 are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b5 and CPR compete for a binding site on cyt P450 2B4. The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b5 (cyt b5) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b5 molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 ± 2 and 29 ± 4.5 s–1, respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine ≅100-fold (k = 0.15 ± 0.05 s–1) and cyclohexane ≅10-fold (k = 2.5 ± 0.35 s–1) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b5, and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b5 and CPR. Increasing the concentration of cyt b5 enhanced the amount of product formed by cyt b5 while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b5:cyt P450 molar ratios examined, cyt b5 inhibits the rate of NADPH consumption. Nevertheless, at low cyt b5:cyt P450 molar ratios ≤1:1, the rate of metabolism of cyclohexane and benzphetamine is enhanced, whereas at higher cyt b5:cyt P450 molar ratios, cyt b5 progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b5 to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b5 are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b5 and CPR compete for a binding site on cyt P450 2B4. The cytochromes (cyt) 2The abbreviations used are: cyt, cytochrome; CPR, NADPH-cytochrome P450 reductase; DLPC, dilauroylphosphatidylcholine; GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry. 2The abbreviations used are: cyt, cytochrome; CPR, NADPH-cytochrome P450 reductase; DLPC, dilauroylphosphatidylcholine; GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry. P450 are a superfamily of heme-containing enzymes that catalyze the biotransformation of a large number of endogenous and xenobiotic compounds by utilizing reducing equivalents from NADPH. The mammalian hepatic microsomal cytochromes P450 receive electrons from their redox partner, NADPH-cyt P450 reductase (CPR). To be able to catalyze the oxidation of substrates, cyt P450 requires two electrons and two protons. Its catalytic cycle is complex with multiple steps (1Denisov I.G. Makris T.M. Sligar S.G. Schlichting I. Chem. Rev. 2005; 105: 2253-2277Crossref PubMed Scopus (1551) Google Scholar). The first step is the binding of substrate, which may increase the redox potential of the heme. CPR, not cyt b5, then delivers the first electron to reduce the ferric heme to the ferrous state. The ferrous iron binds oxygen to form the oxyferrous complex (Fe3+OO–), whose potential is high enough to accept a second electron from either CPR or cyt b5 to yield the peroxo intermediate (Fe3+OO2–). The peroxo intermediate is rapidly protonated (first proton) to form the hydroperoxo intermediate (Fe3+OOH–). A second proton is delivered to the distal oxygen, resulting in cleavage of the oxygen molecule to form water and the oxyferryl intermediate [FeIV = O], generally considered to be the active hydroxylating species. An atom of oxygen is inserted into the substrate, and product release follows.It has been recognized for 3 decades that under steady-state conditions, cyt b5 alters the rate of catalysis by selected cytochromes P450 (for recent reviews see Refs. 2Porter T.D. J. Biochem. Mol. Toxicol. 2002; 16: 311-316Crossref PubMed Scopus (150) Google Scholar, 3Schenkman J.B. Jansson I. Pharmacol. Ther. 2003; 97: 139-152Crossref PubMed Scopus (373) Google Scholar, 4Zhang H. Myshkin E. Waskell L. Biochem. Biophys. Res. Commun. 2005; 338: 499-506Crossref PubMed Scopus (57) Google Scholar, 5Bosterling B. Trudell J.R. Trevor A.J. Bendix M. J. Biol. Chem. 1982; 257: 4375-4380Abstract Full Text PDF PubMed Google Scholar). Cyt b5 has been reported to affect the catalytic activity of more than 20 cyt P450 isoforms, including the majority of the human drug-metabolizing cyt P450 isoforms like cyt P450 3A4, 2B6, 2C9, 2C19, and 2E1 (6Mokashi V. Li L. Porter T.D. Arch. Biochem. Biophys. 2003; 412: 147-152Crossref PubMed Scopus (16) Google Scholar, 7Yamaori S. Yamazaki H. Suzuki A. Yamada A. Tani H. Kamidate T. Fujita K. Kamataki T. Biochem. Pharmacol. 2003; 66: 2333-2340Crossref PubMed Scopus (53) Google Scholar, 8Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (129) Google Scholar, 9Yamazaki H. Nakamura M. Komatsu T. Ohyama K. Hatanaka N. Asahi S. Shimada N. Guengerich F.P. Shimada T. Nakajima M. Yokoi T. Protein Expression Purif. 2002; 24: 329-337Crossref PubMed Scopus (208) Google Scholar). Cyt b5 may enhance, inhibit, or not alter catalysis by microsomal cyt P450 depending on the specific cyt P450 isoform, the substrate, and the experimental conditions (10Gruenke L.D. Konopka K. Cadieu M. Waskell L. J. Biol. Chem. 1995; 270: 24707-24718Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 11Morgan E.T. Coon M.J. Drug Metab. Dispos. 1984; 12: 358-364PubMed Google Scholar). It has been suggested that cyt b5 modulates cyt P450 catalysis by donating the second electron to cyt P450 and/or by acting as an allosteric modifier of the oxygenase (3Schenkman J.B. Jansson I. Pharmacol. Ther. 2003; 97: 139-152Crossref PubMed Scopus (373) Google Scholar). In the case of cyt P450 2B4, it has been shown that the electron-donating properties of cyt b5 are required for its stimulatory activity (10Gruenke L.D. Konopka K. Cadieu M. Waskell L. J. Biol. Chem. 1995; 270: 24707-24718Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 11Morgan E.T. Coon M.J. Drug Metab. Dispos. 1984; 12: 358-364PubMed Google Scholar, 12Canova-Davis E. Waskell L. J. Biol. Chem. 1984; 259: 2541-2546Abstract Full Text PDF PubMed Google Scholar). Its inhibitory properties, which are also manifest by manganese-protoporphyrin IX cyt b5, are likely related to its ability to displace CPR from its binding site on cyt P450 2B4 (11Morgan E.T. Coon M.J. Drug Metab. Dispos. 1984; 12: 358-364PubMed Google Scholar, 12Canova-Davis E. Waskell L. J. Biol. Chem. 1984; 259: 2541-2546Abstract Full Text PDF PubMed Google Scholar, 13Bridges A. Gruenke L. Chang Y.T. Vakser I.A. Loew G. Waskell L. J. Biol. Chem. 1998; 273: 17036-17049Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Recent experiments performed under single turnover conditions suggest that both the electron-donating ability and effector function of cyt b5 may be operating to enhance catalysis by cyt P450 2B4 (14Zhang H. Gruenke L. Arscott D. Harris D.L. Glavanovich M. Johnson R. Waskell L. Biochemistry. 2003; 42: 11594-11603Crossref PubMed Scopus (43) Google Scholar).It has also been reported that apo-cyt b5 devoid of heme can stimulate catalysis (9Yamazaki H. Nakamura M. Komatsu T. Ohyama K. Hatanaka N. Asahi S. Shimada N. Guengerich F.P. Shimada T. Nakajima M. Yokoi T. Protein Expression Purif. 2002; 24: 329-337Crossref PubMed Scopus (208) Google Scholar, 15Yamazaki H. Johnson W.W. Ueng Y.F. Shimada T. Guengerich F.P. J. Biol. Chem. 1996; 271: 27438-27444Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 16Loughran P.A. Roman L.J. Aitken A.E. Miller R.T. Masters B.S. Biochemistry. 2000; 39: 15110-15120Crossref PubMed Scopus (21) Google Scholar, 17Loughran P.A. Roman L.J. Miller R.T. Masters B.S. Arch. Biochem. Biophys. 2001; 385: 311-321Crossref PubMed Scopus (51) Google Scholar, 18Auchus R.J. Lee T.C. Miller W.L. J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). However, some investigators have disputed that apo-cyt b5 can stimulate cyt P450 catalysis (19Guryev O.L. Gilep A.A. Usanov S.A. Estabrook R.W. Biochemistry. 2001; 40: 5018-5031Crossref PubMed Scopus (80) Google Scholar). These conflicting reports indicate that the mechanism by which cyt b5 affects the catalysis of cyt P450 remains poorly understood. Hildebrandt and Estabrook (20Hildebrandt A. Estabrook R.W. Arch. Biochem. Biophys. 1971; 143: 66-79Crossref PubMed Scopus (449) Google Scholar) proposed that cyt b5 functions by transferring one of the two reducing equivalents to cyt P450 based on the observation in microsomes that cyt b5 partially reoxidized upon the addition of NADH to a microsomal drug-metabolizing reaction under steady-state conditions. This proposal is supported by in vitro experiments demonstrating that ferrous cyt b5 is an efficient electron donor to oxyferrous cyt P450 (Fe3+OO–) (21Bonfils C. Balny C. Maurel P. J. Biol. Chem. 1981; 256: 9457-9465Abstract Full Text PDF PubMed Google Scholar, 22Pompon D. Coon M.J. J. Biol. Chem. 1984; 259: 15377-15385Abstract Full Text PDF PubMed Google Scholar). Furthermore, stimulation of benzphetamine and methoxyflurane metabolism by cyt P450 2B4 is only observed in the presence of holo-cyt b5 but not in the presence of apo-cyt b5 or cyt b5 containing manganese protoporphyrin IX (11Morgan E.T. Coon M.J. Drug Metab. Dispos. 1984; 12: 358-364PubMed Google Scholar, 12Canova-Davis E. Waskell L. J. Biol. Chem. 1984; 259: 2541-2546Abstract Full Text PDF PubMed Google Scholar). Chlorzoxazone metabolism by cyt P450 2E1 is also stimulated by cyt b5, but not by apo-cyt b5, in a purified reconstituted system as well as in membranes in which cyt P450 2E1 and CPR have been coexpressed (8Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (129) Google Scholar, 9Yamazaki H. Nakamura M. Komatsu T. Ohyama K. Hatanaka N. Asahi S. Shimada N. Guengerich F.P. Shimada T. Nakajima M. Yokoi T. Protein Expression Purif. 2002; 24: 329-337Crossref PubMed Scopus (208) Google Scholar). These results are supportive of an electron transfer role for cyt b5 in cyt P450 catalysis. Stimulation of cyt P450 activity was originally attributed to more rapid electron transfer to oxyferrous cyt P450 by cyt b5 than CPR. However, data from our laboratory have recently demonstrated that cyt b5 and CPR reduce cyt P450 at a similar rate but that catalysis nevertheless occurs more slowly in the presence of reductase (14Zhang H. Gruenke L. Arscott D. Harris D.L. Glavanovich M. Johnson R. Waskell L. Biochemistry. 2003; 42: 11594-11603Crossref PubMed Scopus (43) Google Scholar). Presumably, oxyferrous cyt P450 exists in different conformations in the presence of cyt b5 and CPR.Attempts to characterize the interaction of cyt P450 at the individual steps in its reaction cycle with its amphipathic redox partners have been extremely challenging because membrane-binding proteins form heterogeneous aggregates between and among themselves in aqueous solution. It is also likely that cyt P450 changes conformation during its reaction cycle and that its various conformations may react differently with cyt b5 and CPR (23French J.S. Guengerich F.P. Coon M.J. J. Biol. Chem. 1980; 255: 4112-4119Abstract Full Text PDF PubMed Google Scholar). Cyt P450 2B4 forms a 1:1 complex with CPR and with cyt b5 in reconstituted systems. The binding of the redox partner is enhanced in the presence of substrates, and substrates enhance the affinity for the redox partner (21Bonfils C. Balny C. Maurel P. J. Biol. Chem. 1981; 256: 9457-9465Abstract Full Text PDF PubMed Google Scholar, 23French J.S. Guengerich F.P. Coon M.J. J. Biol. Chem. 1980; 255: 4112-4119Abstract Full Text PDF PubMed Google Scholar, 24Miwa G.T. West S.B. Huang M.T. Lu A.Y. J. Biol. Chem. 1979; 254: 5695-5700Abstract Full Text PDF PubMed Google Scholar, 25Tamburini P.P. White R.E. Schenkman J.B. J. Biol. Chem. 1985; 260: 4007-4015Abstract Full Text PDF PubMed Google Scholar). A site-directed mutagenesis study of the interactions of cyt P450 2B4 with CPR and cyt b5 has identified residues, primarily in the C-helix on the proximal side of cyt P450 2B4, that participate in binding both CPR and cyt b5 (13Bridges A. Gruenke L. Chang Y.T. Vakser I.A. Loew G. Waskell L. J. Biol. Chem. 1998; 273: 17036-17049Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). These data demonstrate that CPR and cyt b5 have nonidentical but nevertheless overlapping binding sites on the proximal surface of cyt P450 2B4 and predict that cyt b5 and CPR will compete for this binding site on cyt P450. Model complexes of cyt P450 2B4 with cyt b5 and CPR also support the hypothesis of an overlapping binding site (4Zhang H. Myshkin E. Waskell L. Biochem. Biophys. Res. Commun. 2005; 338: 499-506Crossref PubMed Scopus (57) Google Scholar). On the contrary, Schenkman and co-workers (26Tamburini P.P. Schenkman J.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 11-15Crossref PubMed Scopus (53) Google Scholar) have proposed a ternary complex of cyt P450, cyt b5, and CPR based on the finding that a carbodiimide cross-linked cyt b5-cyt P450 2B4 complex was able to accept electrons from CPR for the metabolism of para-nitroanisole. The experiment with the cross-linked complex led to the conclusion that cyt b5 and CPR had distinct binding sites on cyt P450 2B4 and that it was possible for cyt P450, cyt b5, and CPR to form an active ternary complex. The experiments described in this work address this issue using a recently developed procedure that directly measures product formation from a single catalytic cycle of cyt P450 2B4. They also enable us to compare the rate of product formation with the rate of reduction of cyt P450 2B4 determined spectrophotometrically (14Zhang H. Gruenke L. Arscott D. Harris D.L. Glavanovich M. Johnson R. Waskell L. Biochemistry. 2003; 42: 11594-11603Crossref PubMed Scopus (43) Google Scholar).In view of the complexity of the multistep reaction cycle of cyt P450, the fact that the first electron to the ferric cyt P450 can only be efficiently delivered by CPR, and the heterogeneously aggregated state of the three membrane proteins in the reconstituted system, we elected to bypass the early steps in the catalytic cycle and to examine product formation by oxyferrous cyt P450 2B4 under single turnover conditions. The ferric cyt P450 2B4 was stoichiometrically reduced by dithionite, eliminating the requirement for CPR for the delivery of the first electron. Catalysis was initiated by mixing the reduced cyt P450 complexes with an oxygen-containing solution. By directly measuring the rate at which product formation occurred during a single catalytic cycle, it was possible to determine whether cyt b5 or CPR was responsible for catalysis. These experimental conditions circumvent many of the problems associated with investigating enzymatic activity under steady-state conditions and enabled us to show that increasing the concentration of cyt b5 leads to more catalysis by cyt b5 at the expense of catalysis by CPR. The data were interpreted to indicate that cyt b5 and CPR are competing for a mutually exclusive binding site on cyt P450 2B4.EXPERIMENTAL PROCEDURESChemicals—All chemicals used are of ACS grade unless otherwise specified. Cyclohexane, cyclohexanol-d12, NADPH, benzphetamine, and sodium dithionite were purchased from Sigma. Dilauroylphosphatidyl-choline (DLPC) was purchased from Doosan Serdary Research Laboratory (Toronto, Canada).Protein Expression and Purification—The membrane-bound forms of cyt P450 2B4, rabbit cyt b5, and rat CPR were expressed and purified from E. coli as described previously (14Zhang H. Gruenke L. Arscott D. Harris D.L. Glavanovich M. Johnson R. Waskell L. Biochemistry. 2003; 42: 11594-11603Crossref PubMed Scopus (43) Google Scholar). The concentration of cyt P450 was determined using an extinction coefficient of Δϵ450–490 nm of 91 mm–1 cm–1 for the cyt P450-CO complex, as described by Omura and Sato (27Omura T. Sato R. J. Biol. Chem. 1964; 239: 2379-2385Abstract Full Text PDF PubMed Google Scholar). The concentration of CPR was determined using an extinction coefficient of 21 mm–1 cm–1 at 456 nm for oxidized enzyme (28Vermilion J.L. Coon M.J. J. Biol. Chem. 1978; 253: 2694-2704Abstract Full Text PDF PubMed Google Scholar). The concentration of cyt b5 was determined using an extinction coefficient of 185 mm–1 cm–1 for the absorbance change at 426 minus 409 nm between ferrous and ferric cyt b5, respectively (29Estabrook R.W. Werringloer J. Methods Enzymol. 1978; 52: 212-220Crossref PubMed Scopus (302) Google Scholar).Determination of the Rate of Product Formation under Single Turnover Conditions in a Rapid Chemical Quench Apparatus—Measurements of the kinetics of product formation were performed on a QFM-400 chemical quench flow apparatus (Molecular Kinetics, Inc., Indianapolis, IN). The QFM-400 apparatus was housed in an anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, MI), where the oxygen level was maintained at ∼5 ppm. The temperature of the reaction chamber of the QFM-400 apparatus was maintained with a circulating water bath.The complexes of cyt P450 with its redox partners were preformed by incubating cyt P450 with either CPR or cyt b5 or both overnight at ∼4 °C in an anaerobic chamber in Buffer A (0.1 m potassium phosphate, pH 7.4, 15% glycerol). The overnight incubation prevents precipitation and ensures the sample is anaerobic. DLPC was included in the protein mixture at a molar ratio of DLPC:cyt P450 of 60:1. Methyl viologen was present at a concentration of 0.1 μm. The substrate of cyt P450, either cyclohexane or benzphetamine, was added to a final concentration of 1 mm. Cyclohexane was added from a stock of methanolic solution, and the methanol concentration in the reaction mixture was ≤0.25% (v/v). The cyt P450-cyt b5 complex was reduced stoichiometrically in a tonometer under anaerobic conditions, using a standardized solution of dithionite. The reduction was monitored by following absorbance changes at 422 and 438 nm for cyt b5 and cyt P450, respectively. Likewise, the cyt P450-CPR complex was reduced with dithionite, and the reduction was monitored at 585 and 385 nm for CPR and cyt P450, respectively. The cyt P450-cyt b5 complex was reduced to a ferrous state (cyt P4502+ and cyt b 2+5), whereas the cyt P450-CPR complex was reduced by 3-electron equivalents (ferrous cyt P450 and 2e-reduced CPR). When all three proteins were present, the preformed complexes were reduced with 4-electron equivalents to ferrous cyt P450, ferrous cyt b5, and 2e-reduced CPR.The reduced protein complex was loaded into Syringe A of the chemical quench flow apparatus inside the anaerobic chamber. With cyclohexane as the substrate, the protein concentration in Syringe A was 10–15 μm for the cyt P450-cyt b5 complex and 15 μm for the cyt P450-CPR complex. The concentration of the cyt P450-cyt b5 and cyt P450-CPR complex was 30 μm when benzphetamine was used as substrate. Syringe B contained air-saturated Buffer A and 1 mm substrate. The reaction was initiated by mixing the contents of Syringe A (containing the reduced proteins) with an equal volume from Syringe B (containing substrate and air-saturated Buffer A ([O2] =∼250 μm). At designated times the reaction was rapidly quenched with an equal volume of 1 m NaOH. The reaction temperature was 30 °C with cyclohexane and 15 °C with benzphetamine. The last sample was collected ∼10 s after mixing in the presence of cyclohexane and at ∼60 s in the presence of benzphetamine. The sample at time = 0 was prepared by first mixing the reduced protein complex with 1 m NaOH to inactivate the enzymes and then adding air-saturated buffer. Typically 15–30 samples were collected during the time course of a reaction. The amount of cyclohexanol in each reaction mixture was quantified by gas chromatography-mass spectrometry (GC-MS), whereas the amount of norbenzphetamine was quantified by liquid chromatography-mass spectrometry (LC-MS). The kinetics of product formation were determined from a plot of the amount of product formed versus time.Quantification of Cyclohexanol with Gas Chromatography-Mass Spectrometry (GC-MS)—The amount of cyclohexanol produced by cyt P450 was quantified with a GC-MS method developed in this laboratory with a detection limit of 6 pmol at a signal-to-noise ratio of ≥3. At higher concentrations of cyclohexanol, the error was ±10–15%. Immediately after the reaction was quenched, it was spiked with 267 pmol of the internal standard, cyclohexanol-d12. The samples containing cyclohexanol and perdeuterated cyclohexanol were then extracted twice with 3 ml of dichloromethane. The extracts were concentrated with a stream of nitrogen to ∼1–5 μl and redissolved in 50 μl of methanol. Standard solutions containing known amounts of both the internal standard and cyclohexanol (6–500 pmol) were prepared and processed under the same conditions as the quenched unknown samples each time an experiment was performed. The only difference was that the proteins in the samples used for the standard curve had not been reduced by dithionite.Quantitative analysis was performed on an HP6890/MSD5893 GC-MS instrument (Agilent Technologies). A 2-μl aliquot of the methanolic solution containing cyclohexanol and cyclohexanol-d12 was injected at 250 °C in the pulse-splitless mode. Cyclohexanol and its perdeuterated analogue were chromatographed on a capillary DB-210 column (0.20 mm × 20 m × 0.18 μm; J & W Scientific) using helium as carrier gas. A deactivated fused silica guard column (0.2 mm × 10 m; Agilent Technologies) was placed in front of the capillary DB-210 column. Following injection of the samples, the oven temperature was maintained at 50 °C for 3 min and then increased to 100 °C at a rate of 10 °C/min. Both cyclohexanol and cyclohexanol-d12 eluted at ∼7.4 min. The mass spectrometer detector was operated in the selective ion monitoring mode with electronic ionization at 70 eV. In the selective ion monitoring mode, the intensities of only four ions were recorded, i.e. m/z 57 and 82 from fragmentation of cyclohexanol and m/z 61 and 92 from fragmentation of cyclohexanol-d12. Product was quantified by generating a standard curve with 6–500 pmol of cyclohexanol, using cyclohexanol-d12 as the internal standard. It gave rise to a straight line with R2 ≥ 0.98 in the range of calibration. The amount of cyclohexanol in the unknown samples was calculated by using the ratio of the area under the curve from the chromatogram of the selected ions at m/z 82 and m/z 92 based on the calibration curve performed on the same day.Quantification of Norbenzphetamine with LC-MS/MS—The amount of norbenzphetamine produced under single turnover conditions was quantified with an LC-MS/MS method developed in this laboratory. After the reaction was quenched with NaOH, 0.3 nmol of the internal standard, norbenzphetamine-d5, was spiked into each sample. Norbenzphetamine and norbenzphetamine-d5 were extracted twice with 3 ml of methyl-tert-butyl ether. The extracts were dried with a stream of nitrogen gas and redissolved in 0.4 ml of a solution of 50% acetonitrile, 50% H2O for LC-MS/MS analyses. An aliquot of 10 μl of the solution was injected into the LC-MS for quantification. Standard solutions containing a known amount of norbenzphetamine (0.02–5 nmol) and 0.3 nmol of norbenzphetamine-d5 were prepared under the same conditions as the quenched samples except that the protein complex was not reduced.LC-MS/MS analysis was performed on a Thermo Finnigan TSQ Quantum mass spectrometer (Thermo Scientific, MA). Norbenzphetamine was separated from benzphetamine on a Zorbax SB-C18 column (2.1 × 150 mm, 3.5 μm; Agilent Technologies). The column temperature was maintained at 35 °C. The mobile phase consisted of 100% acetonitrile (A) and 20 mm ammonium formate (B). The column was equilibrated with 50% A, 50% B for 6 min at a flow rate of 0.4 ml/min. Norbenzphetamine and norbenzphetamine-d5 were eluted with a linear gradient from 50% A, 50% B to 90% A, 10% B in 5 min, and the mobile phase was held at 90% A, 10% B for another 2 min. Norbenzphetamine and norbenzphetamine-d5 eluted at ∼3.2 min, whereas benzphetamine eluted at ∼4.3 min. It is of note that commercially available benzphetamine is contaminated with ≅0.01% norbenzphetamine that gives a background signal during LC-MS/MS analysis of the samples from the single turnover experiments. This background norbenzphetamine was quantified in the time = 0 sample and subtracted from the experimental samples. With a typical norbenzphetamine background ≅10 pmol of norbenzphetamine can be detected with a signal:noise ratio of 3.The mass spectrometer was operated in the selected reaction monitoring mode. Norbenzphetamine and norbenzphetamine-d5 were ionized by electrospray ionization at ∼3500 V. Protonated parental ions ([M + H]+) of norbenzphetamine (m/z 226.1) and norbenzphetamine-d5 (m/z 231.1) were admitted into the first quadrupole and fragmented by collision with nitrogen gas in the second quadrupole. The fragment ions of m/z 91.1 from parental ion of m/z 226.1 and m/z 96.1 from parental ion of m/z 231.1 were detected in the third quadrupole, and the integrated area of the two fragment ions, m/z 91.1 versus m/z 96.1, were used for quantification.Determination of the Rate of Product Formation under Pre-steady-state Conditions in a Rapid Chemical Quench Apparatus—The kinetics of product formation were measured under pre-steady-state conditions using the chemical quench flow apparatus to determine whether product release was the rate-limiting step in catalysis. The experiments were conducted at 30 °C in a manner identical to that described for the single turnover conditions except that Syringe A contained the preformed (at 4 °C overnight) oxidized complexes in aerobic Buffer A at a concentration of 10 μm for cyt P450 and CPR and 30 μm for cyt b5 when it was present. Syringe B contained 1 mm cyclohexane and 0.3 mm NADPH in aerobic Buffer A. Cyclohexanol was analyzed using the GC-MS assay as described above.Measurement of NADPH Consumption and Substrate Metabolism under Steady-state Conditions—The rate of NADPH consumption and cyclohexane metabolism by cyt P450 2B4 under steady-state conditions was measured at 30 °C in a spectrophotometric quartz cuvette. Cyt P450 (0.3 nmol), CPR (0.3 nmol), varying amounts of cyt b5 (0–1.5 nmol), and DLPC (68 nmol) were added in the given order to an Eppendorf tube. The mixture was incubated at room temperature for 1 h. Potassium phosphate buffer, pH 7.4, was added to a final concentration of 50 mm in a volume of 1 ml. Cyclohexane was added as a methanolic solution to a final concentration of 1 mm cyclohexane and ≤0.25% (v/v) methanol. The reaction mixture was incubated in the cuvette for 5 min at 30 °C to achieve thermal equilibrium before NADPH was added to a final concentration of 0.3 mm to initiate the reaction. Consumption of NADPH was continually monitored at 340 nm for 5 min, at which time the reaction was terminated by mixing 0.2 ml of the reaction mixture with an equal volume of 1 m NaOH. The rate of NADPH consumption was calculated using an extinction coefficient of 6.2 mm–1 cm–1 at 340 nm. The amount of cyclohexanol was quantified with the GC-MS assay method as described previously. Cyclohexanol was extracted once with 5 ml of dichloromethane