Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications.
Diphenyliodonium has been shown to be an irreversible, time-dependent inhibitor of NADPH cytochrome P450 oxidoreductase (EC 1.6.2.4) with the Ki for diphenyliodonium chloride being 2.8 mM. Kinetic studies have indicated that diphenyliodonium interacts with the reduced enzyme and NADPH is essential for inactivation to take place. Cytochrome c acts as a competitive substrate. The use of radiolabeled diphenyliodonium has enabled two sites of covalent modification to be identified. Isolation of radiolabeled cofactor followed by mass spectrometry has shown that a phenyl group is added to FMN while the FMN is effectively trapped in the reduced state. Trypsin digestion of S-carboxymethylated P450 reductase after inhibition with radiolabeled inhibitor shows covalent modification of the protein. Purification of a single radiolabelled peptide followed by automated Edman degradation has enabled identification of the second site of covalent attachment as Trp 419.
SmithKline Beecham Research, Welwyn, Hertfordshire, U.K.
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D000818:Animals; D001667:Binding, Competitive; D001713:Biphenyl Compounds / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000378:metabolism / Q000494:pharmacology; D004790:Enzyme Induction; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008433:Mathematics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D009861:Onium Compounds / Q000494:pharmacology*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D016339:Spectrometry, Mass, Fast Atom Bombardment
D001713:Biphenyl Compounds; D003574:Cytochrome c Group; D009861:Onium Compounds; C031291:diphenyliodonium; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital