The Roles of Cytochrome b₅ in Reconstituted Monooxygenase Systems Containing Various Forms of Hepatic Microsomal Cytochrome P-450

Abstract

The roles of cytochrome b₅ in NADPH-dependent monooxygenase reactions catalyzed by reconstituted systems containing four forms of hepatic microsomal cytochrome P-450, i.e. P-450₁, P-450₂, P-448₁, and P-448₂, were examined. Various substrates were metabolized actively in the absence of cytochrome b₅ by the system containing P-450₁, but the monooxygenase reactions were accompanied by oxidation of NADPH uncoupled to the product formation. When cytochrome b₅ was included in the system, the product formation increased to various extents, depending on the substrates used, while NADPH oxidation changed much less, resulting in an improvement of the coupling efficiency. The increase was large when a substrate metabolized at a low velocity was employed. Evidence is presented that the second of two electrons required for the monooxygenase reactions could be introduced into P-450₁ via cytochrome b₅. On the other hand, the rate of P-450₁ reduction was not affected by the addition of cytochrome b5 to the system and that of cytochrome b5 reduction by NADPH-cytochrome P-450 reductase was sufficient to support electron flow to cytochrome P-450 via cytochrome b₅ as the second electron. The stimulatory effect of cytochrome b₅ on the P-450₁-catalyzed monooxygenase reactions can be explained by assuming that the rate of the second electron supply to the oxygenated P-450₁-substrate complex from cytochrome b₅ is higher than that directly from NADPH-cytochrome P-450 reductase. An electron flow from NADH via cytochrome b₅ can be utilized as the second electron for the O-deethylase reaction of 7-ethoxycoumarin catalyzed by reconstituted systems containing P-450₂ and P-448₂ when both NADH-cytochrome b₅ reductase and cytochrome b₅ are included in the system, although the cytochrome has no stimulatory effect at all on the deethylase activity of these two cytochrome P-450's. It has been shown that the second electron for P-448₁ can also be supplied from NADH via cytochrome b₅ in a reconstituted acetanilide p-hydroxylase system containing P-448₁, cytochrome b₅, and the two reductases.