Roles of Cytochrome b5in the Oxidation of Testosterone and Nifedipine by Recombinant Cytochrome P450 3A4 and by Human Liver Microsomes

NADH-dependent testosterone 6β-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5(b5), NADH–b5reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH–b5reductase with NADPH–P450 reductase produced an eightfold increase in testosterone 6β-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6β-hydroxylation activities in the presence of NADPH–P450 reductase but resulted in complete loss of the activities in the absence of NADPH–P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH–P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5and NADH–b5reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH–P450 reductase. Anti-human b5immunoglobulin G (IgG) (purified using rabbit b5affinity chromatography) inhibited testosterone 6β-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH–P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2stimulated rates of reduction of b5by NADPH–P450 reductase, but not by NADH–b5reductase, in reconstituted systems. These results suggest that b5is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH–P450 reductase are accelerated by complexation with substrates and b5is supported in this study.