The roles of cytochrome b5 in reconstituted monooxygenase systems containing various forms of hepatic microsomal cytochrome P-450.

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-450(1), P-450(2), P-448(1), and P-448(2), were examined. Various substrates were metabolized actively in the absence of cytochrome b5 by the system containing P-450(1), 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-450(1) via cytochrome b5. On the other hand, the rate of P-450(1) 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-450(1)-catalyzed monooxygenase reactions can be explained by assuming that the rate of the second electron supply to the oxygenated P-450(1)-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-450(2) and P-448(2) 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-448(1) can also be supplied from NADH via cytochrome b5 in a reconstituted acetanilide p-hydroxylase system containing P-448(1), cytochrome b5, and the two reductases.