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

Haoming Zhang,Lucy Waskell,Sang-Choul Im,Djemel Hamdane

doi:10.1074/jbc.m709094200

Abstract

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 b(5):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.

Highlights

Microsomal cytochromes3 P450, functioning as monooxygenases, catalyze the oxidative biotransformation of numerous pharmaceuticals, carcinogens, pro-carcinogens, and endogenous compounds like fatty acids and steroids
The results presented in this work demonstrate the following: 1) that Mn-cyt b5 does not stimulate the activity of cyt P450 2B4 under steady-state conditions, and 2) that Mn-cyt b5 inhibits the reduction of ferric cyt P450 but not cyt c by cytochrome P450 reductase (CPR), confirming that, under steady-state conditions, cyt b5 stimulates activity by enhancing the rate of catalysis by cyt P450 2B4 and that cyt b5 inhibits activity by binding to cyt P450 2B4 and preventing CPR from binding and reducing it
We have demonstrated that cyt b5 and Mn-cyt b5 substantially hinder the reduction of ferric cyt P450 2B4 by CPR, a critical step in the oxidative transformation of substrates

Summary

Introduction

Microsomal cytochromes (cyt) P450, functioning as monooxygenases, catalyze the oxidative biotransformation of numerous pharmaceuticals, carcinogens, pro-carcinogens, and endogenous compounds like fatty acids and steroids. It was possible to demonstrate under single turnover conditions that catalysis by cyt P450 2B4 occurred faster in the presence of cyt b5 than with CPR and that at high concentrations cyt b5 appeared to displace CPR from cyt P450 2B4 These observations suggested an explanation for the results under steady-state conditions where cyt b5 stimulated product formation at low concentrations but inhibited activity at higher concentrations. Inhibition of product formation and NADPH consumption at high levels of cyt b5 was attributed to the ability of cyt b5 to bind to the proximal surface of cyt P450 2B4 and prevent CPR from binding and reducing ferric cyt P450 2B4. Mn-cyt b5 should significantly decrease the reduction of ferric cyt P450 2B4 by CPR, but should not decrease the ability of CPR to reduce its redox partner cyt c

Methods

Results

Conclusion