title: Stimulation of cytochrome P450 reactions by apo-cytochrome b5: evidence against transfer of heme from cytochrome P450 3A4 to apo-cytochrome b5 or heme oxygenase.
abstract: Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b(5) (b(5)). Two major explanations are (i) direct electron transfer from b(5) and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b(5) or b(5) devoid of heme (apo-b(5)), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b(5) but not by apo-b(5). Recently, a proposal has been made by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b(5) can be explained only by transfer of heme from P450 preparations to apo-b(5), enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6beta-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only approximately 5% of the heme can be transferred to apo-b(5), which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally, b(5) supported NADH-b(5) reductase/P450 3A4-dependent testosterone 6beta-hydroxylation, but apo-b(5) did not. Thus, apo-b(5) can stimulate P450 3A4 reactions as well as b(5) in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b(5) cannot be used to explain the catalytic stimulation.
authors: Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan.
authors: H
authors: H
authors: Yamazaki
authors: T
authors: T
authors: Shimada
authors: M V
authors: MV
authors: Martin
authors: F P
authors: FP
authors: Guengerich
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D002384:Catalysis; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D045303:Cytochromes b; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009211:Myoglobin / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D001059:Apoproteins; D003573:Cytochrome b Group; D009211:Myoglobin; C010857:apomyoglobin; D013739:Testosterone; D006418:Heme; D045303:Cytochromes b; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D006419:Heme Oxygenase (Decyclizing); D009251:NADPH-Ferrihemoprotein Reductase