Stimulation of cytochrome P450 reactions by apo-cytochrome b(5) - Evidenceagainst transfer of heme from cytochrome P450 3A4 to apo-cytochrome b5, orheme oxygenase

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimu lated by another microsomal protein, cytochrome b(5) (b(5)). Two major expl anations are (i) direct electron transfer from b(5) and (ii) a conformation al. 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 b5 but not by apo-b(5). Recently, a proposal has been mad e by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b5 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 6 beta -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 s imilar to5% of the heme can be transferred to apo-b(5), which cannot accoun t for the observed stimulation. The presence of the heme scavenger apomyogl obin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loo sely bound P450 3A4 heme was provided in experiments with apo-heme oxygenas e, in which only 3% of the P450 heme was converted to biliverdin. Finally, b(5) supported NADH-b(5) reductase/P450 3A4-dependent testosterone 6 beta - 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 tra nsfer from P450 3A4 to apo-b(5) cannot be used to explain the catalytic sti mulation.