Role of Cytochrome b5 in Hydroxylation by a Reconstituted Cytochrome P-450-containing System

Abstract

1. The role of cytochrome b5 in the oxidative metabolism of benzphetamine and 3,4-benzpyrene in the presence of NADPH was studied with the reconstituted hydroxylation system isolated from rat liver microsomes whose components were cytochrome P-450 or P-448, NADPH-cytochrome c reductase, and lipid. In the absence of cytochrome b5, the turnover number for benzphetamine and 3,4-benzpyrene (nanomoles per min per nmole of P-450 or P-448) in the reconstituted system was at least as good as that in liver microsomes, indicating that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation system. 2. The addition of cytochrome b5 at a ratio of cytochrome P-450 to b5 normally found in microsomes inhibited NADPH-dependent benzphetamine N-demethylation by the three components (cytochrome P-450, NADPH-cytochrome c reductase, and lipid). This inhibition was shown to be due to competition between cytochrome b5 and cytochrome P-450 for NADPH-cytochrome c reductase. The addition of NADH and cytochrome b5 reductase to the reconstituted system reversed the inhibition of NADPH-dependent benzphetamine N-demethylation by cytochrome b5. Thus, the NADH synergistic effect observed in liver microsomes with benzphetamine as the substrate and NADPH as electron donor could be due to NADH and cytochrome b5 reductase preventing the inhibition by cytochrome b5. 3. The addition of cytochrome b5 at a ratio of cytochrome P-448 to b5 normally found in microsomes did not inhibit the NADPH-dependent hydroxylation of 3,4-benzpyrene by the three components (cytochrome P-448, NADPH-cytochrome c reductase, and lipid). In the presence of NADH, NADPH, cytochrome b5, cytochrome b5 reductase, and the three components, the rate of 3,4-benzpyrene hydroxylation was significantly increased. This increased rate was due to a NADH-dependent hydroxylation pathway via cytochrome b5 and cytochrome b5 reductase. 4. It is concluded that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation systems. However, this cytochrome plays an important role in regulating NADPH-dependent hydroxylation of these substrates and is also an essential component of the NADH-dependent hydroxylation pathway.