The microsomal and nuclear envelope flavoprotein NADPHcytochrome P450 oxidoreductase (P450R 1 ; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4; hereafter referred to as reductase), one of a family of FMN-and FAD-containing enzymes that includes nitric-oxide synthase, the sulfite reductase ␣-subunit, and P450BM-3 (for recent reviews, see Refs. 1 and 2), mediates the transfer of electrons from NADPH to cytochrome P450 and other microsomal proteins as well as to nonphysiological electron acceptors such as cytochrome c and ferricyanide (3). Electron transfer to electron acceptors such as cytochromes c or P450 proceeds from NADPH to FAD to FMN, while ferricyanide and 3-acetylpyridine adenine dinucleotide phosphate (AcPyrADP) accept electrons directly from FAD (4 -6). Sequence comparisons as well as a variety of biochemical studies (6 -11) have suggested the presence of independent FMN-and FAD-binding domains. The amino-terminal FMNbinding domain, with homology to the bacterial flavodoxins (7), is separated by an insertion sequence having no homology to any known protein from the FAD/NADPH binding domain, which is related to another class of flavoproteins, the transhydrogenases (1, 2, 12), and includes ferredoxin-NADP ϩ reductase (FNR), NADH-nitrate reductase, and NADH-cytochrome b 5 reductase as well as phthalate dioxygenase reductase (13). Both FNR and P450R abstract the pro-R (A-side) hydrogen of NADPH (12,14), with the conformation of the bound nicotinamide anti in P450R (14) but unknown for other members of this family. These sequence similarities have been confirmed by the recently described x-ray crystal structure of P450R, which also highlights the role of the insertion sequence in aligning the flavin isoalloxazine rings in a position for direct electron transfer (15). Finally, potential flavin-and NADPH-binding, as well as catalytic, residues identified by sequence comparisons of P450R with flavodoxin and FNR (2, 7) have been confirmed by biochemical studies, including site-directed mutagenesis and kinetic measurements (16 -18), as well as crystallographic studies (15).The crystal structures of FNR and P450R place three conserved residues, Ser 457 , Cys 630 , and Asp 675 , in close proximity to the isoalloxazine ring of FAD. Ser 457 is in a position to form a hydrogen bond with the oxidized or reduced flavin N-5 of P450R, while the homologous Ser 96 of FNR interacts with the reduced, but not the oxidized, flavin N-5, suggesting a role for these residues in hydride transfer and/or stabilization of the reduced flavin (12,15,19, 20