Influence of Ionic Strength on the P450 Monooxygenase Reaction and Role of Cytochrome b 5 in the Process

Abstract

A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the monooxygenase reaction with benzphetamine as substrate. Cytochrome b 5 also can increase the monooxygenase coupling efficiency. At low ionic strength cytochrome b 5 did not much influence the reduction of P450, but the rate constant of the cytochrome b 5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b 5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b 5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b 5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b 5 on the cytochrome P450-mediated monooxygenation reaction is due to an increase in the efficiency of the electron transfer reaction: With cytochrome b 5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b 5 complex in a single interaction, obviating the need for a second interaction with the reductase.