JBC INTERFERin siRNA transfection reagent

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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.

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 Dagger , William W. Johnson , Yune-Fang Ueng , Tsutomu Shimada Dagger and F. Peter Guengerich

From the Dagger  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 6beta 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 6beta 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 min-1) to support testosterone 6beta hydroxylation (~14 min-1) unless b5 or apo-b5 was present, when rates of ~700 min-1 were measured. The oxidation-reduction potential (Em,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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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.