Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase

• Published in: The Journal of biological chemistry 1986-06, Vol.261 (17), p.7824-7830
• Main Authors: Kurzban, G P, Strobel, H W
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Enzymes and enzyme inhibitors; Flavin Mononucleotide - pharmacology; Flavin-Adenine Dinucleotide - pharmacology; Fundamental and applied biological sciences. Psychology; Kinetics; Male; Microsomes, Liver - enzymology; NADPH-Ferrihemoprotein Reductase - isolation & purification; NADPH-Ferrihemoprotein Reductase - metabolism; Oxidoreductases; Protein Binding; Rats; Rats, Inbred Strains
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)57476-2

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.