NADPH-cytochrome P450 oxidoreductase - Structural basis for hydride and electron transfer

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from N ADPH, via two flavin cofactors, to various cytochrome P450s. The crystal st ructure of the rat reductase complexed with NADP(+) has revealed that nicot inamide access to FAD is blocked by an aromatic residue (Trp-677), which st acks against the reface of the isoalloxazine ring of the flavin. To investi gate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallo graphy. The first mutant, W677X, has the last two C-terminal residues, Trp- 677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, t he nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring w ith a tilt of similar to 30 degrees between the planes of the two rings. Th ese results, together with the S457A/C630A/D675N structure, allow us to pro pose a mechanism for hydride transfer regulated by changes in hydrogen bond ing and pi-pi interactions between the isoalloxazine ring and either the ni cotinamide ring or Trp-677 indole ring. Superimposition of the mutant and w ild-type structures shows significant mobility between the two flavin domai ns of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformatio nal changes occur during catalysis.