Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains

Journal article

NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochrome P450 and catalyzes the one-electron reduction of many drugs and foreign compounds. This enzyme is a flavoprotein containing the cofactors FMN and FAD, which are essential for its function. We have expressed the putative FMN and FAD/NADPH binding domains of P450 reductase 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 cytochrome c or cytochrome P450 (as judged by the oxidation of the CYP1A1 substrate 7-ethoxyresorufin). However, the domains could be combined to provide a functional enzyme active in the reduction of cytochrome c and in transferring electrons to cytochrome P450. Both the reconstitution of the domains and the direct binding of cytochrome c to the FMN domain were ionic-strength dependent. The FMN domain containing the hydrophobic membrane anchor sequence was a potent inhibitor of reconstituted monooxygenase activity. These data strongly support the hypothesis that FMN/FAD-containing proteins have evolved as a fusion of two ancestral genes and provide fundamental insights into how this and structurally related proteins, such as nitric oxide synthase and sulfite reductase, have evolved and function.

Authors: Smith, GC; Tew, DG; Wolf, CR

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: National Academy of Sciences
• Journal: Proceedings of the National Academy of Sciences
• Volume: 91
• Issue: 18
• Pages: 8710-8714
• Publication date: 1994-08-30
• DOI: 10.1073/pnas.91.18.8710
• EISSN: 1091-6490
• ISSN: 0027-8424
• Language: English
• Keywords: Cytochrome P450; Cytochrome; Oxidoreductase; Biochemistry; Enzyme; Chemistry; Reductase; Cofactor; Cytochrome P450 reductase; Flavoprotein; Biology; Monooxygenase; Cytochrome c; Flavin group; Coenzyme Q – cytochrome c reductase