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Volume 271, Number 44, Issue of November 1, 1996 pp. 27438-27444
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Lack of Electron Transfer from Cytochrome b₅ in Stimulation of Catalytic Activities of Cytochrome P450 3A4
CHARACTERIZATION OF A RECONSTITUTED CYTOCHROME P450 3A4/NADPH-CYTOCHROME P450 REDUCTASE SYSTEM AND STUDIES WITH APO-CYTOCHROME b₅

(Received for publication, June 27, 1996)

Hiroshi Yamazaki , William W. Johnson ^¶ , Yune-Fang Ueng , Tsutomu Shimada and F. Peter Guengerich

From the  Osaka Prefectural Institute of Public Health, Osaka 537, Japan and the ^¶ Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b₅ (b₅) for optimal rates. The stimulatory effect of b₅ on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b₅ to the ferrous P450-O₂-substrate complex. We found that apo-b₅, devoid of heme, could substitute for b₅ in stimulating two prototypic activities, testosterone 6 hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b₅ could not substitute for b₅ in a testosterone 6 hydroxylation system composed of NADH-b₅ reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min¹) to support testosterone 6 hydroxylation (~14 min¹) unless b₅ or apo-b₅ was present, when rates of ~700 min¹ were measured. The oxidation-reduction potential (E_m,7) of the ferric/ferrous couple of P450 3A4 was unchanged (~310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b₅. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b₅; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b₅ can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.

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