Roles of cytochrome b(5) in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes

NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b(5) (b(5)), NADH-b(5) reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b(5) reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b(5) from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-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 b(5). In the presence of testosterone and MgCl2, P450 3A4 was reduced by b(5) and NADH-b(5) reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b(5) immunoglobulin G (IgG) (purified using rabbit b(5) affinity chromatography) inhibited testosterone 6 beta-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. MgCl2 stimulated rates of reduction of b(5) by NADPH-P450 reductase, but not by NADH-b(5) reductase, in reconstituted systems. These results suggest that b(5) is 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 b(5) is supported in this study. (C) 1996 Academic Press, Inc.