ROLE OF ACIDIC RESIDUES IN THE INTERACTION OF NADPH-CYTOCHROME-P450 OXIDOREDUCTASE WITH CYTOCHROME-P450 AND CYTOCHROME-C

Site-directed mutagenesis of the acidic clusters (207)Asp-Asp-Asp(209) and (213)Glu-Glu-Asp(215) of NADPH cytochrome P450 oxidoreductase dem onstrates that both cytochrome c and cytochrome P450 interact with thi s region; however, the sites and mechanisms of interaction of the two substrates are clearly distinct, Substitutions in the first acidic clu ster did not affect cytochrome c or ferricyanide reductase activity, b ut substitution of as paragine for aspartate at position 208 reduced c yto chrome P450-dependent benzphetamine N-demethylase activity by 63% with no effect on K-m(P450) or K-m(NADPH). Substitutions in the second acidic cluster affected cytochrome c reduction but not benzphetamine N-demethylase or ferricyanide reductase activity, The E214Q enzyme exh ibited a 59% reduction in cytochrome c reductase activity and a 47% re duction in KC,yt c under standard conditions (x0.27 M potassium phosph ate, pH 7.7), as well as a decreased K-m(Cytc) at every ionic strength and a shift of the salt dependence of cytochrome c reductase activity toward lower ionic strengths, The E214Q substitution did not affect c ytochrome c reductase activity under standard conditions, but shifted the salt dependence of cytochrome c reductase activity toward higher i onic strengths, Measurements of the effect of ionic strength on steady state kinetic properties indicated that increasing ionic strength des tabilized and reductase-cytochrome c(3+) ground state and reductase-cy tochrome c transition state complexes for the wild type, E213Q, and E2 14Q enzymes, suggesting the presence of electrostatic interactions inv olving Glu(213) and G1u(214) as well as additional residues outside th is region, The ionic strength dependence of k(cat)/K-m(Cytc) for the w ild-type and E214Q enzymes is consistent with the presence of charge-p airing interactions in the transition state and removal of a weak ioni c interaction in the reductase-cytochrome c transition-state complex b y the E214Q substitution, The ionic strength dependence of the E213Q e nzyme, however, is not consistent with a simple electrostatic model, E ffects of ionic strength on kinetic properties of E213Q suggest that s ubstitution of glutamine stabilizes the reductase cytochrome c(3+) gro und-state complex, leading to a net increase in activation energy and decrease in k(cat). Glu(213) is also involved in a repulsive interacti on with cytochrome c(3)+. Cytochrome c(2+) K-i for the wild-type enzym e was 82.4 mu M at 118 mM ionic strength and 10.8 mu M at 749 mM ionic strength; similar values were observed for the E214Q enzyme, Cytochro me c Ki for the E213Q enzyme was 17.6 mu M at 118 mM and 15.7 mu M at 749 mM ionic strength, consistent with removal of an electrostatic rep ulsion between the reductase and cytochrome c(2+).