title: Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4.
abstract: We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s(-1). Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR-CYP3A4 complex can dissociate and reform between the first and second electron transfers.
authors.affiliation: Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, Henry Wellcome Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Yassar
authors.initials: Y
authors.lastname: Farooq
authors.forename: Gordon C K
authors.initials: GC
authors.lastname: Roberts
mesh_terms: D055162:Biocatalysis; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004579:Electron Transport; D006801:Humans; D006900:Hydroxylation; D006915:Hydroxytestosterones / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: C103238:1-palmitoyl-2-oleoyl-glycero-3-phosphatidic acid; D006915:Hydroxytestosterones; D008081:Liposomes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D013739:Testosterone; D009249:NADP; C025591:6 beta-hydroxytestosterone; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine