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 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.
Smith
G C
GC
Imperial Cancer Research Fund, Molecular Pharmacology Unit, University of Dundee, United Kingdom.
Tew
D G
DG
Wolf
C R
CR
D003001:Cloning, Molecular; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D011994:Recombinant Proteins; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase