Roles of cytochrome b5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes

Arch Biochem Biophys. 1996 Jan 15;325(2):174-82. doi: 10.1006/abbi.1996.0022.


NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 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 b5 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 b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 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 b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 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 b5 is supported in this study.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / metabolism*
  • Cytochromes b5 / metabolism*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Magnesium Chloride / pharmacology
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / metabolism*
  • Mixed Function Oxygenases / metabolism*
  • NAD / metabolism
  • NADP / metabolism
  • Nifedipine / metabolism*
  • Oxidation-Reduction
  • Rabbits
  • Recombinant Proteins / metabolism
  • Testosterone / metabolism*


  • Recombinant Proteins
  • Magnesium Chloride
  • NAD
  • Testosterone
  • NADP
  • Cytochromes b5
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • Nifedipine