Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity.
NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors. Residues Tyr140 and Tyr178 are known to be involved in FMN binding. A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role. This role has been investigated by site-directed mutagenesis. Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50%. Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed. The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined. The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants. Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex. The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site. The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.
Paine
M J
MJ
ICRF Molecular Pharmacology Unit, Biomedical Research Centre, University of Dundee, Ninewells Hospital & Medical School, Dundee DD1 9SY, UK.
Ayivor
S
S
Munro
A
A
Tsan
P
P
Lian
L Y
LY
Roberts
G C
GC
Wolf
C R
CR
D019943:Amino Acid Substitution; D002384:Catalysis; D017124:Conserved Sequence; D003574:Cytochrome c Group / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*
D003574:Cytochrome c Group; D005292:Ferricyanides; C007931:hexacyanoferrate III; D010649:Phenylalanine; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase