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J. Biol. Chem., Vol. 276, Issue 33, 30885-30891, August 17, 2001

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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^*

Hiroshi Yamazaki, Tsutomu Shimada^§, Martha V. Martin^¶, and F. Peter Guengerich^¶

From the  Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan, the ^§ Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan, and the ^¶ Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b₅ (b₅). Two major explanations are (i) direct electron transfer from b₅ and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b₅ or b₅ devoid of heme (apo-b₅), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b₅ but not by apo-b₅. Recently, a proposal has been made 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-b₅, enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6-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-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. 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, b₅ supported NADH-b₅ reductase/P450 3A4-dependent testosterone 6-hydroxylation, but apo-b₅ did not. Thus, apo-b₅ can stimulate P450 3A4 reactions as well as b₅ in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b₅ cannot be used to explain the catalytic stimulation.

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