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.
Highlights
Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b5 (b5)
The following year, in the course of doing what were intended to be controls for other experiments, we found that apo-b5 was as effective as b5 in stimulating P450 3A4-catalyzed testosterone 6-hydroxylation and nifedipine oxidation [22]
Stimulation of P450 3A4 Catalytic Activity by Apo-b5—Since our first report of the stimulation of P450 3A4 activities by b5 and apo-b5 [22], the preparation of recombinant P450 3A4 has been changed in this laboratory
Summary
Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b5 (b5). Spectral analysis of mixtures indicated that only ϳ5% of the heme can be transferred to apo-b5, 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.
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