Abstract

The role of cytochrome b₅ (b₅) in the α-naphthoflavone (α-NF)-mediated inhibition of H₂O₂-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although α-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H₂O₂ system. Analysis of the effect of various constituents of a standard reconstituted system on H₂O₂-supported activity showed that b₅ alone resulted in a 2.5-fold increase in the k_cat value and reversed the inhibitory effect of α-NF. In addition, titration with b₅ suggested that only 65% of the CYP3A4 participated in the interaction with b₅, consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b₅ on the kinetics of H₂O₂-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b₅, 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b₅ decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b₅ action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b₅. Ser-119 is in the B′-C loop, a predicted b₅-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b₅ with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool.