Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

Abstract

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b5 (cyt b5) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b5 molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 ± 2 and 29 ± 4.5 s -1, respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P4502B4 catalyzes the oxidation of benzphetamine ≅ 100-fold (k = 0.15 ± 0.05 s-1) and cyclohexane ≅ 10-fold (k = 2.5 ± 0.35 s-1) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b5, and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b5 and CPR. Increasing the concentration of cyt b5 enhanced the amount of product formed by cyt b5 while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b5:cyt P450 molar ratios examined, cyt b5 inhibits the rate of NADPH consumption. Nevertheless, at low cyt b5:cyt P450 molar ratios ≤1:1, the rate of metabolism of cyclohexane and benzphetamine is enhanced, whereas at higher cyt b5:cyt P450 molar ratios, cyt b5 progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b5 to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b5 are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b5 and CPR compete for a binding site on cyt P450 2B4.