Stimulation of Cytochrome P450 Reactions by Apo-cytochromeb 5

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 eitherb 5 or b 5 devoid of heme (apo-b 5), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated byb 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 6β-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 ∼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 5reductase/P450 3A4-dependent testosterone 6β-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.