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J. Biol. Chem., Vol. 276, Issue 31, 29163-29170, August 3, 2001

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NADPH-Cytochrome P450 Oxidoreductase
STRUCTURAL BASIS FOR HYDRIDE AND ELECTRON TRANSFER^*

Paul A. Hubbard, Anna L. Shen^§, Rosemary Paschke, Charles B. Kasper^§, and Jung-Ja P. Kim^¶

From the  Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the ^§ McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP⁺ has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. 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, the nicotinamide moiety of NADP⁺ lies against the FAD isoalloxazine ring with a tilt of ~30° between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and - interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.

The atomic coordinates and the structure factors (code 1JA0, 1J9Z, and 1JA1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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