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. 1994 Aug 30;91(18):8710–8714. doi: 10.1073/pnas.91.18.8710

Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains.

G C Smith 1, D G Tew 1, C R Wolf 1
PMCID: PMC44676  PMID: 8078947

Abstract

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.

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