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J. Biol. Chem., Vol. 283, Issue 9, 5217-5225, February 29, 2008

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Cytochrome b₅ Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4^*

From the Department of Anesthesiology, University of Michigan Medical School and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105

Experiments demonstrating that cytochrome (cyt) b₅ inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b₅ was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766–29776). In this work experiments were undertaken with manganese-containing cyt b₅ (Mn-cyt b₅) to test this hypothesis. Because Mn-cyt b₅ does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b₅ in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b₅. At a Mn-cyt b₅:cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b₅ had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b₅ was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b₅, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b₅ only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b₅ competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b₅ inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.

Received for publication, November 6, 2007 , and in revised form, December 14, 2007.

^* This work was supported by National Institutes of Health Grant GM35533 and a Veterans Affairs merit review grant (to L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1, Table 1, and acknowledgments.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Anesthesiology, University of Michigan, Research Service, 2215 Fuller Rd., Ann Arbor, MI 48105. Tel.: 734-845-5858; Fax: 734-845-3096; E-mail: waskell{at}umich.edu.

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