The interaction of cytochrome c and the heme domain of cytochrome P-450BM-3 with the reductase domain of cytochrome P-450BM-3.
Cytochrome P-450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase 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 counterparts. 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-450BM-3 or by two reductase domain mutant enzymes (W574Y, W574F) which have diminished hydroxylase activity relative to wild-type enzyme but retain cytochrome c reductase activity levels comparable to wild-type enzyme. Because reduced cytochrome c generated independently of the reductase domain of P-450BM-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 P-450BM-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-450BM-3 do not involve electron transfer through the reductase domain.
Klein
M L
ML
Department of Biological Chemistry, UCLA School of Medicine.
Fulco
A J
AJ
D001410:Bacillus megaterium / Q000201:enzymology*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D006418:Heme / Q000737:chemistry*; D006900:Hydroxylation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction
D003574:Cytochrome c Group; D009227:Myristic Acids; D019814:Myristic Acid; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase