Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4
2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês
10.1074/jbc.m709094200
ISSN1083-351X
AutoresHaoming Zhang, Djemel Hamdane, Sang‐Choul Im, Lucy Waskell,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoExperiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766–29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b5:cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis. Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766–29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b5:cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis. Microsomal cytochromes (cyt) 3The abbreviations used are:cytcytochrome P450cyt b5cytochrome b5CPRNADPH-cytochrome P450 reductaseDLPCdilauroylphosphatidylcholineMn-cyt b5manganese protoporphyrin IX cytochrome b5NHEnormal hydrogen electrodeeqequivalent. P450, functioning as monooxygenases, catalyze the oxidative biotransformation of numerous pharmaceuticals, carcinogens, pro-carcinogens, and endogenous compounds like fatty acids and steroids. Cyts P450 require two electrons and two protons to carry out catalysis that leads to insertion of a single oxygen atom into the substrate. In the mammalian microsomal cyt P450 system, the two electrons are delivered to cyt P450 by NADPH-dependent cytochrome P450 reductase (CPR). Like cyt P450, CPR is membrane-bound and located in the membrane of the endoplasmic reticulum. CPR contains two flavin molecules, FMN and FAD. The diflavin moiety of CPR is essential for sequential electron transfer to cyt P450 as it permits CPR to accept two electrons from NADPH and transfer one electron at a time to cyt P450. The first electron from CPR reduces ferric cyt P450 to ferrous cyt P450, which rapidly binds oxygen to form oxyferrous cyt P450. The second electron is then delivered to oxyferrous cyt P450. This is followed by protonation of the reduced oxyferrous intermediate leading to heterolytic cleavage of the oxygen bond to form water and an oxyferryl intermediate, the putative, active, oxidizing species of cyt P450. An oxygen atom is inserted into the substrate, and the more hydrophilic product dissociates from the enzyme. Readers are referred to a recent review for further details about the cyt P450 reaction cycle (1Denisov I.G. Makris T.M. Sligar S.G. Schlichting I. Chem. Rev. 2005; 105: 2253-2277Crossref PubMed Scopus (1606) Google Scholar). cytochrome P450 cytochrome b5 NADPH-cytochrome P450 reductase dilauroylphosphatidylcholine manganese protoporphyrin IX cytochrome b5 normal hydrogen electrode equivalent. An alternative electron donor to cyt P450 is cyt b5, another microsomal hemoprotein also located in the endoplasmic reticulum membrane. Because of its relatively high mid-point redox potential (+25 mV versus NHE), cyt b5 can deliver only the second electron to oxyferrous cyt P450 but not the first electron to ferric cyt P450. It has been recognized for 3 decades that cyt b5 may either increase, decrease, or not alter the activity of selected cyts P450 (2Schenkman J.B. Jansson I. Pharmacol. Ther. 2003; 97: 139-152Crossref PubMed Scopus (383) Google Scholar, 3Zhang H. Myshkin E. Waskell L. Biochem. Biophys. Res. Commun. 2005; 338: 499-506Crossref PubMed Scopus (57) 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 (1Denisov I.G. Makris T.M. Sligar S.G. Schlichting I. Chem. Rev. 2005; 105: 2253-2277Crossref PubMed Scopus (1606) Google Scholar, 4Mokashi V. Li L. Porter T.D. Arch. Biochem. Biophys. 2003; 412: 147-152Crossref PubMed Scopus (16) Google Scholar, 5Yamaori S. Yamazaki H. Suzuki A. Yamada A. Tani H. Kamidate T. Fujita K. Kamataki T. Biochem. Pharmacol. 2003; 66: 2333-2340Crossref PubMed Scopus (54) Google Scholar, 6Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (130) Google Scholar, 7Yamazaki 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 (216) Google Scholar, 8Locuson C.W. Wienkers L.C. Jones J.P. Tracy T.S. Drug Metab. Dispos. 2007; 35: 1174-1181Crossref PubMed Scopus (47) Google Scholar). The effect of cyt b5 has also been shown to depend on the cyt P450 isozyme and substrate (2Schenkman J.B. Jansson I. Pharmacol. Ther. 2003; 97: 139-152Crossref PubMed Scopus (383) Google Scholar, 9Canova-Davis E. Waskell L. J. Biol. Chem. 1984; 259: 2541-2546Abstract Full Text PDF PubMed Google Scholar). In the case of cyt P450 2B4 and 2E1, the electron donating properties of cyt b5 are required for its stimulatory activity (6Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (130) Google Scholar, 7Yamazaki 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 (216) Google Scholar, 9Canova-Davis E. Waskell L. J. Biol. Chem. 1984; 259: 2541-2546Abstract Full Text PDF PubMed Google Scholar, 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), although some studies suggest that apo-cyt b5 can stimulate the activity of cyt P450 3A4 via an allosteric effect (12Yamazaki 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 (192) Google Scholar). At present, the ability of apo-cyt b5 to stimulate cyt P450 3A4 is controversial (13Guryev O.L. Gilep A.A. Usanov S.A. Estabrook R.W. Biochemistry. 2001; 40: 5018-5031Crossref PubMed Scopus (81) Google Scholar). Experiments performed in the reconstituted system with purified proteins have demonstrated that ferrous cyt b5 can rapidly reduce oxyferrous cyt P450 2B4 (14Bonfils C. Balny C. Maurel P. J. Biol. Chem. 1981; 256: 9457-9465Abstract Full Text PDF PubMed Google Scholar, 15Pompon D. Coon M.J. J. Biol. Chem. 1984; 259: 15377-15385Abstract Full Text PDF PubMed Google Scholar). It is known that cyt P450 2B4 forms a 1:1 complex with CPR and with cyt b5 in a purified, reconstituted system (16Tamburini P.P. White R.E. Schenkman J.B. J. Biol. Chem. 1985; 260: 4007-4015Abstract Full Text PDF PubMed Google Scholar, 17French J.S. Guengerich F.P. Coon M.J. J. Biol. Chem. 1980; 255: 4112-4119Abstract 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 (18Bridges 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 (260) 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 the basis of a cross-linking study with a carbodiimide, Schenkman and co-workers (19Tamburini P.P. Schenkman J.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 11-15Crossref PubMed Scopus (53) Google Scholar) have proposed a two-site model, hypothesizing that cyt b5 and CPR bind at two distinct, functional sites on cyt P450 and form a ternary complex. Recently we investigated the interaction of cyt P450 2B4 with CPR and cyt b5 by examining product formation under both single turnover and steady-state conditions in an effort to understand the complex effects of cyt b5 on cyt P450 2B4 catalysis (20Zhang H. Im S.-C. Waskell L. J. Biol. Chem. 2007; 282: 29766-29776Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It was possible to demonstrate under single turnover conditions that catalysis by cyt P450 2B4 occurred faster in the presence of cyt b5 than with CPR and that at high concentrations cyt b5 appeared to displace CPR from cyt P450 2B4. These observations suggested an explanation for the results under steady-state conditions where cyt b5 stimulated product formation at low concentrations but inhibited activity at higher concentrations. The stimulatory activity at low cyt b5 concentrations was attributed to the ability of cyt b5 to mediate a more rapid formation of product, thereby decreasing side product (superoxide and hydrogen peroxide) formation. This meant that the efficiency of catalysis increased, i.e. more NADPH was used to form product rather than side products. Inhibition of product formation and NADPH consumption at high levels of cyt b5 was attributed to the ability of cyt b5 to bind to the proximal surface of cyt P450 2B4 and prevent CPR from binding and reducing ferric cyt P450 2B4. Site-directed mutagenesis data demonstrating that cyt b5 and CPR shared an overlapping binding site on cyt P450 2B4 supported the hypothesis that cyt b5 and CPR compete for a binding site on cyt P450 2B4 (18Bridges 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 (260) Google Scholar). Nevertheless, because CPR is also known to reduce cyt b5, it could be argued that cyt b5 inhibited cyt P450 activity by binding directly to CPR and preventing CPR from reducing cyt P450 2B4. If cyt b5 stimulates activity by enhancing the rate of catalysis compared with CPR and inhibits cyt P450 activity by binding directly to cyt P450 but not CPR as hypothesized, then Mn-cyt b5, which does not undergo oxidation or reduction under our experimental conditions, should only inhibit, not stimulate, activity in the purified, reconstituted system. Furthermore, Mn-cyt b5 should significantly decrease the reduction of ferric cyt P450 2B4 by CPR, but should not decrease the ability of CPR to reduce its redox partner cyt c. The results presented in this work demonstrate the following: 1) that Mn-cyt b5 does not stimulate the activity of cyt P450 2B4 under steady-state conditions, and 2) that Mn-cyt b5 inhibits the reduction of ferric cyt P450 but not cyt c by CPR, confirming that, under steady-state conditions, cyt b5 stimulates activity by enhancing the rate of catalysis by cyt P450 2B4 and that cyt b5 inhibits activity by binding to cyt P450 2B4 and preventing CPR from binding and reducing it. Chemicals—All chemicals were of the highest purity available unless otherwise specified. NADPH, benzphetamine, sodium dithionite, and horse heart cytochrome c were purchased from Sigma. Dilauroylphosphatidylcholine (DLPC) was purchased from Doosan Serdary Research Laboratory (Toronto, Canada). Carbon monoxide gas (purity > 99.5%) was purchased from Matheson Tri-Gas (Parsippany, NJ). Mn(III) protoporphyrin IX chloride was purchased from Frontier Scientific Inc. (Logan, UT). Re-distilled glycerol was purchased from Roche Diagnostics. Protein Expression and Purification—Cyt P450 2B4, cyt b5, and CPR were expressed and purified from Escherichia coli as described previously (20Zhang H. Im S.-C. Waskell L. J. Biol. Chem. 2007; 282: 29766-29776Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The concentration of cyt P450 was determined using an extinction coefficient of Δϵ450–490 nm of 91 mm cm–1 as described by Omura and Sato (21Omura 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 cm–1 at 456 nm for the oxidized enzyme (22Vermilion 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 between ferrous and ferric cyt b5 (23Estabrook R.W. Werringloer J. Methods Enzymol. 1978; 52: 212-220Crossref PubMed Scopus (303) Google Scholar). Kinetics of the Reduction of Ferric Cyt P450 2B4 by CPR in the Presence of Various Concentrations of Holo-cyt b5—The rate of electron transfer from CPR to ferric cyt P450 2B4, or the first electron transfer, was measured to probe the interaction between cyt P450 2B4, CPR, and cyt b5. The kinetics were determined with stopped-flow spectrophotometry by monitoring the absorbance increase at 450 nm as a result of formation of the ferrous cyt P450-CO adduct following reduction of ferric cyt P450 by CPR. The stopped-flow experiments were performed with a Hi-Tech SF61DX2 stopped-flow spectrophotometer (Hi-Tech, Wiltshire, UK) housed in an anaerobic chamber (Belle Technology, Dorset, UK) as reported previously (24Oprian D.D. Coon M.J. J. Biol. Chem. 1982; 257: 8935-8944Abstract Full Text PDF PubMed Google Scholar). The temperature of the stopped-flow spectrophotometer reaction chamber and observation cell was maintained at 30 °C using a circulating water bath. Cyt P450 2B4, CPR, and cyt b5 (when present) were pre-mixed by incubating cyt P450 (3 μm), CPR (3 μm), and various concentrations of cyt b5 (0–15 μm) in 0.1 m potassium phosphate, pH 7.4, buffer containing 15% glycerol, 0.18 mm DLPC, and 1 mm benzphetamine at 4 °C overnight. The anaerobic protein mixture was rapidly mixed with 0.1 m potassium phosphate buffer, pH 7.4, containing 15% glycerol, 1 mm benzphetamine, and 3 μm NADPH. Both solutions had been saturated with CO by blowing CO gas over the sample solutions. The absorbance change at 450 nm was recorded as a function of time. Reduction of Ferric Cyt b5 by Ferrous Cyt P450-CO—The kinetics of the reduction of ferric cyt b5 by ferrous cyt P450-CO were determined with a stopped-flow spectrophotometer, basically as described for reduction of ferric cyt P450 2B4 by CPR. The experiment was performed under anaerobic conditions to avoid possible side reactions involving oxygen. Cyt P450 2B4 was reduced with a stoichiometric amount of dithionite in a tonometer. The ferrous cyt P450 solution was saturated with CO gas to form the cyt P450-CO adduct. Cyt P450-CO and ferric cyt b5 were loaded into separate syringes in the stopped-flow instrument. The transient spectra were recorded with a photodiode array detector following rapid mixing of ferric cyt b5 with cyt P450-CO. The absorbance was also measured in the single wavelength mode at 450 nm. The final concentration of cyt b5 and cyt P450 after mixing was 5 μm in 0.1 m potassium phosphate buffer, pH 7.4, 15% glycerol. Preparation of Full-length and Soluble Mn-Cyt b5—The full-length Mn-cyt b5 was prepared by reconstituting full-length apo-cyt b5 with Mn(III) protoporphyrin IX as described by Morgan and Coon (11Morgan E.T. Coon M.J. Drug Metab. Dispos. 1984; 12: 358-364PubMed Google Scholar). The heme of cyt b5 was removed from holo-cyt b5 by acidifying the cyt b5-containing solution to pH 2.0, and the dissociated heme was extracted from the aqueous solution with 2-butanone. Apo-cyt b5 was then reconstituted with Mn(III) protoporphyrin IX at molar ratio of 2:1 to apo-cyt b5. Mn-cyt b5 was recovered from a Sephadex G-25 size-exclusion column where free Mn(III) protoporphyrin was bound. Soluble Mn-cyt b5 was prepared by reconstitution of soluble bovine apocytochrome b5 with Mn(III) protoporphyrin IX as described previously (25Gruenke L.D. Sun J. Loehr T.M. Waskell L. Biochemistry. 1997; 36: 7114-7125Crossref PubMed Scopus (22) Google Scholar). The concentration of Mn-cyt b5 was determined using an extinction coefficient of 57 mm–1cm–1 at 469 nm (25Gruenke L.D. Sun J. Loehr T.M. Waskell L. Biochemistry. 1997; 36: 7114-7125Crossref PubMed Scopus (22) Google Scholar). Kinetics of Reduction of Ferric Cyt P450 2B4 by CPR in the Presence of Full-length Mn-Cyt b5—The kinetics of reduction of ferric cyt P450 by CPR in the presence of Mn-cyt b5 was measured in the same way as in the presence of holo-cyt b5 as described above. Because it is redox-silent under our experimental conditions, Mn-cyt b5 does not participate in the electron transfer processes and does not undergo spectral changes under the experimental conditions. It is therefore possible to deconvolute the end point spectra recorded in the stopped-flow spectrophotometer as only 1 m eq of NADPH was used in the reaction. Deconvolution of the spectra by linear regression gives the concentration of each species at the end of the reaction. The concentration of each species at the end of the reaction was obtained by iterative regression of the observed spectrum as a linear combination of the standard spectrum of each species. When all three proteins were present, theoretically there are six possible species at the end of the reaction, including cyt P450-CO, ferric cyt P450 in the presence of 1 mm benzphetamine, oxidized Mn-cyt b5, reduced Mn-cyt b5, 1-electron-reduced CPR, and 2-electron-reduced CPR. The observed spectrum, Aobs, is expressed as in Equation 1, Aobs=∑i=1i=6∈i×Ci×I(Eq. 1) where ϵi and Ci represent the extinction coefficient and concentration of each of the six species, and l is the 1-cm path length. Linear regression was performed with SigmaPlot software (Systat Inc., San Jose, CA). Because of the low extinction coefficient of the 1- and 2-electron-reduced forms of CPR, it was not possible to obtain a reliable estimate of the amount present. Kinetics of Reduction of Cyt c by CPR in the Presence of Full-length Mn-Cyt b5—The effect of Mn-cyt b5 on reduction of cyt c by CPR was studied to examine whether Mn-cyt b5 forms a tight complex with CPR, capable of diminishing the ability of CPR to transfer electrons to cyt P450. A solution containing 8 μm of CPR and full-length Mn-cyt b5 (4, 8, 16, 24, and 40 μm) was preincubated at 4 °C overnight at the specified concentration in a glove box in 0.1 m potassium phosphate buffer, pH 7.4, that contained 15% glycerol and a 60-fold molar excess of DLPC with respect to CPR. The concentration of CPR in syringe 1 of the stopped-flow spectrophotometer was 8 μm before mixing, whereas the concentration of Mn-cyt b5 varied from 0 to 40 μm. The pre-mixed CPR and Mn-cyt b5 were rapidly mixed with an equal volume of the 0.1 m potassium phosphate buffer, pH 7.4, 15% glycerol, that contained cyt c (8 μm) and NADPH (8 μm). The kinetics of cyt c reduction were monitored at 550 nm, using an Δϵ of 21.1 mm–1cm–1 at 550 nm between ferric and ferrous cyt c (26Van G.B. Slater E.C. Biochim. Biophys. Acta. 1962; 58: 593-595Crossref PubMed Scopus (557) Google Scholar). Measurement of the Activity of Cyt P450 2B4 Under Steady-state Conditions in the Presence of Full-length Mn-Cyt b5—The rates of NADPH consumption and benzphetamine metabolism in the presence of Mn-cyt b5 were determined under steady-state conditions in the purified reconstituted cyt P450 2B4 system at 30 °C as described earlier (20Zhang H. Im S.-C. Waskell L. J. Biol. Chem. 2007; 282: 29766-29776Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The reaction was initiated by adding excess NADPH to a final concentration of 0.3 mm and terminated after 5 min by adding 70% trifluoroacetic acid to a final concentration of 5%. The amount of formaldehyde produced from metabolism of benzphetamine was analyzed with Nash's reagent as described (27Nash T. Biochem. J. 1953; 55: 416-421Crossref PubMed Scopus (4127) Google Scholar). NADPH consumption was determined by measuring the decrease in absorbance at 340 nm using an extinction coefficient of 6.2 mm–1cm–1. Data Analysis—Apparent rate constants and amplitudes for the rate of electron transfer from CPR to ferric cyt P450 and cyt c were obtained by fitting the absorbance changes at the selected wavelength as a single or double exponential function using SigmaPlot software (Systat Inc.). Electron Transfer from CPR to Ferric Cyt P450 in the Presence of Holo-cyt b5—In an attempt to test our hypothesis that cyt b5 inhibited the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to and reducing cyt P450 2B4, the rate of electron transfer from CPR to ferric cyt P450 was measured in the presence of cyt b5. The reduction of ferric cyt P450 2B4 was measured in the presence of carbon monoxide and 1 m eq of NADPH in the stopped-flow spectrophotometer. Reduction of CPR by NADPH (k ≅ 28 s–1) and the binding of CO to ferrous cyt P450 (k ≅ 100 s–1) occur significantly faster than reduction of cyt P450 2B4. Therefore, the rate of electron transfer to cyt P450 2B4 can be determined by observing the absorbance changes at 450 nm after mixing the pre-formed cyt P450-CPR complex with NADPH in the presence of CO (28Gray R.D. J. Biol. Chem. 1978; 253: 4364-4369Abstract Full Text PDF PubMed Google Scholar, 29Vatsis K.P. Oprian D.D. Coon M.J. Acta Biol. Med. Ger. 1979; 38: 459-473PubMed Google Scholar). Fig. 1A shows the absorbance changes at 450 nm during the course of the first electron transfer to cyt P450 in the presence of various concentrations of holo-cyt b5. Reduction of ferric cyt b5, which also occurs under these conditions, was monitored at 422 nm as shown in Fig. 1B. As expected, ferric cyt P450 was reduced by CPR biphasically in the absence of cyt b5. The biphasic rate constants are 4.1 and 0.51 s–1, and the amplitude of the fast phase, k1, is 81% (see Table 1). This result is similar to that reported by other investigators (29Vatsis K.P. Oprian D.D. Coon M.J. Acta Biol. Med. Ger. 1979; 38: 459-473PubMed Google Scholar, 30Backes W.L. Reker-Backes C.E. J. Biol. Chem. 1988; 263: 247-253Abstract Full Text PDF PubMed Google Scholar). In the presence of holo-cyt b5, the rate of reduction of ferric cyt P450 2B4 decreases as observed for cyt P450 1A2 and 2E1 (31Guengerich F.P. Johnson W.W. Biochemistry. 1997; 36: 14741-14750Crossref PubMed Scopus (155) Google Scholar). A 5-fold molar excess of cyt b5 completely eliminated formation of the cyt P450-CO complex as evidenced by the absence of an absorbance increase at 450 nm. In fact, a small decrease in absorbance was observed at this concentration, which reflects partial reduction of the excess cyt b5 (32Spatz L. Strittmatter P. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1042-1046Crossref PubMed Scopus (484) Google Scholar). With higher concentrations of cyt b5, the absorbance at 422 nm increased rapidly. The absorbance change at 422 nm in the presence of cyt b5 is a net result of reduction of cyt b5 and formation of cyt P450-CO. The former process results in an absorbance increase at 422 nm (Δϵ = 110 mm–1cm–1), whereas the latter results in an absorbance decrease (Δϵ =–20 mm–1cm–1) as shown by the absorbance decrease in the absence of cyt b5 (Fig. 1B, solid line). The increase in absorbance at 422 nm demonstrates that cyt b5 is reduced. In the presence of a 5-fold excess of cyt b5, cyt b5 is reduced at an apparent rate constant of 1.2 s–1.TABLE 1The rate constants and amplitudes observed during the electron transfer process from CPR to ferric cyt P450 in the presence of varying amounts of the membrane form cyt b5 at 30 °CConcentrationλobsPhase IPhase IIP450CPRCyt b5k1A1k2A2μmμmμm%%1.51.504504.1 ± 0.3281 ± 70.51 ± 0.05719 ± 3.51.51.50.754504.0 ± 0.5260 ± 50.73 ± 0.09140 ± 54220.39 ± 0.04100 ± 111.51.51.54501.7 ± 0.1349 ± 60.54 ± 0.06551 ± 84220.60 ± 0.04100 ± 121.51.53.04500.38 ± 0.05100 ± 84220.69 ± 0.09100 ± 131.51.54.54500.25 ± 0.042100 ± 114220.87 ± 0.056100 ± 141.51.57.54504221.2 ± 0.23100 ± 12 Open table in a new tab The decrease in the rate of reduction of ferric cyt P450 in the presence of cyt b5 is the net result of the following processes: 1) cyt b5 binding to the proximal surface of cyt P450 2B4, which inhibits the binding of CPR; 2) oxidation of cyt P450-CO by ferric cyt b5; and 3) reduction of cyt b5 by CPR. The three simultaneous reactions occurring under the experimental conditions are illustrated in Scheme 1. It is well documented that CPR reduces cyt b5 (33Guengerich F.P. Arch. Biochem. Biophys. 2005; 440: 204-211Crossref PubMed Scopus (68) Google Scholar, 34Wu F.F. Vergeres G. Waskell L. Arch. Biochem. Biophys. 1994; 308: 380-386Crossref PubMed Scopus (12) Google Scholar). Peterson et al. (35Peterson J.A. White R.E. Yasukochi Y. Coomes M.L. O'Keeffe D.H. Ebel R.E. Masters B.S. Ballou D.P. Coon M.J. J. Biol. Chem. 1977; 252: 4431-4434Abstract Full Text PDF PubMed Google Scholar) have reported that cyt b5 is capable of oxidizing ferrous carbon monoxy cyt P450, but the rate was not determined. A rate of 45 s–1 was calculated for oxidation of the CO adduct of cyt P450 1A2 by ferric cyt b5 based on kinetic simulation (31Guengerich F.P. Johnson W.W. Biochemistry. 1997; 36: 14741-14750Crossref PubMed Scopus (155) Google Scholar). To better understand the relative importance of the different electron transfer processes, we have directly measured the rate of reduction of ferric cyt b5 by cyt P450 2B4-CO. Reduction of Ferric Cyt b5 by Ferrous Cyt P450-CO—Experiments were performed anaerobically in the stopped-flow spectrophotometer to directly measure the rate of reduction of ferric cyt b5 by ferrous cyt P450-CO by mixing equimolar amounts of cyt P450-CO with ferric cyt b5. The results are presented in Fig. 2. As shown, the absorbance at 450 nm bleaches whereas the absorbance at 422 and 555 nm increases over time (Fig. 2A). The bleaching at 450 nm indicates that CO dissociates from cyt P450, presumably because of oxidation of ferrous cyt P450 to ferric cyt P450, whereas the increase in intensity at 422 and 555 nm is indicative of reduction of cyt b5. The absorbance change at 450 nm is fit to give biphasic rate constants of 0.6 and 0.1 s–1 (fast phase, 45%). Fitting the kinetic trace at 422 nm gave rate constants of 0.8 and 0.2 s–1 (fast phase, 50%). The simultaneous change in absorbance at 450 and 422 nm indicates that electron transfer from ferrous cyt P450-CO to ferric cyt b5 occurs without an observable intermediate. Approximately 40% of the cyt P450-CO is oxidized under these conditions. The rate of electron transfer from ferrous cyt P450 2B4 to ferric cyt b5 in the absence of CO was reported to be ∼2.7 and 0.44 s–1 at 5 °C (15Pompon D. Coon M.J. J. Biol. Chem. 1984; 259: 15377-15385Abstract Full Text PDF PubMed Google Scholar). The slower oxidation of cyt P450-CO by ferric cyt b5 may result from the increased redox potential of cyt P450-CO or the slow dissociation of CO from cyt P450-CO and subsequent reduction of cyt b5 by ferrous cyt P450. It has been estimated that th
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