Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains

Graeme C.M. Smith, David G. Tew, C. Roland Wolf

    Research output: Contribution to journalArticlepeer-review

    140 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)8710-8714
    Number of pages5
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume91
    Issue number18
    DOIs
    Publication statusPublished - 30 Aug 1994

    Keywords

    • drug metabolism
    • electron transport
    • flavoproteins
    • nitric oxide synthase

    Fingerprint

    Dive into the research topics of 'Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains'. Together they form a unique fingerprint.

    Cite this