DISSECTION OF NADPH-CYTOCHROME P450 OXIDOREDUCTASE INTO DISTINCT FUNCTIONAL DOMAINS

NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochrome P450 and catalyzes the one-electron reduction of many drug s and foreign compounds. This enzyme is a flavoprotein containing the cofactors FMN and FAD, which are essential for its function. We have e xpressed the putative FMN and FAD/NADPH binding domains of P450 reduct ase and show that these distinct peptides fold correctly to bind their respective cofactors. The FAD/NADPH domain catalyzed the one-electron reduction of a variety of substrates but did not efficiently reduce c ytochrome c or cytochrome P450 (as judged by the oxidation of the CYP1 A1 substrate 7-ethoxyresorufin). However, the domains could be combine d to provide a functional enzyme active in the reduction of cytochrome c and in transferring electrons to cytochrome P450. Both the reconsti tution of the domains and the direct binding of cytochrome c to the FM N domain were ionic-strength dependent. The PMN domain containing the hydrophobic membrane anchor sequence was a potent inhibitor of reconst ituted monooxygenase activity. These data strongly support the hypothe sis that FMN/FAD-containing proteins have evolved as a fusion of two a ncestral genes and provide fundamental insights into how this and stru cturally related proteins, such as nitric oxide synthase and sulfite r eductase, have evolved and function.