The interaction of cytochrome c and the heme domain of cytochrome P-450 BM-3 with the reductase domain of cytochrome P-450 BM-3

Abstract

Cytochrome P-450 BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-ocygenase that resembles the Class II P-450 systems of the eukaryotic endoplasmic reticulum. Its single polypeptide chain contains both a P-450 heme domain and an NADPH: P-450 reductase domain, each of which bears significant structural and functional homology with its microsomal coutnerparts. We report here that cytochrome c, which can accept NADPH-derived electrons from the reductase domain of P-450 BM-3, did not inhibit myristate hydroxylation catalyzed by P-450 BM-3 or by two reductase domain mutant enzymes (W574Y, W574F) which have diminished hydroxylase activity relative to wild-type enzyme but reatin cytochrome c reductase activity levels comparable to wild-type enzyme. Because reduced cytochrome c generated independently of the reductase domain of P-450 BM-3 did not support myristate hydroxylation, it seems likely that cytochrome c binds to a site on the reductase domain which does not overlap the site of the heme domain interaction. We also found that myristate did not inhibit 24 P-450 BM-3-mediated cytochrome c reduction. Since neither substrate inhibited the conversion of the other, we conclude that the rate-limiting steps for both myristate hydroxylation and cytochrome c reduction by P-450 BM-3 do not involve electron transfer through the reductase domain.