Properties of the stable aerobic and anaerobic half-reduced states of NADPH-cytochrome c reductase.
The microsomal flavoprotein, NADPH-cytochrome c reductase, has been reexamined to determine: (1) the nature of the flavine bound to the enzyme and (2) the oxidation-reduction state of the "half-reduced" form of the flavoprotein. Iyanagi and Mason (Iyanagi, T., and Mason, H.S. (1973), Biochemistry 12, 2297) have recently proposed that NADPH-cytochrome c reductase contains both FAD and FMN as prosthetic groups in lieu of FAD as the sole constituent, as suggested by all previous studies of this enzyme. The data presented herein, utilizing the recently published fluorometric procedure of Faeder and Siegel (Faeder, E. J., and Siegle, L. M. (1973), Anal. Biochem. 53, 332) for the determination of FAD and FMN in mixtures, confirm the conclusions of Iyanagi and Mason for both rat and pig liver reductase preparations. Data for other flavoproteins are also presented. Iyanagi and Mason have also concluded that the air-stable "semiquinone" is a form of NADPH-cytochrome c reductase reduced by one electron per two falvines (F-FH). The present studies, however, do not agree with this conclusion, but instead support our previous results which indicate that both the aerobic and anaerobic half-reduced states of this flavoprotein exist in the two-electron reduced form (FH-FH). Removal of NADP+ does not affect the spectrum of the air-stable half-reduced form of the flavoprotein, nor does it affect the back titration of this intermediate by potassium ferricyanide. The possible implications of these observations on the catalytic cycle of the flavines of NADPH-cytochrome c reductase are discussed.
D000818:Animals; D002606:Charcoal / Q000494:pharmacology; D003579:Cytochrome Reductases / Q000378:metabolism*; D005292:Ferricyanides / Q000494:pharmacology; D005486:Flavin Mononucleotide; D005182:Flavin-Adenine Dinucleotide; D008099:Liver / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D009249:NADP; D010100:Oxygen; D011487:Protein Conformation; D051381:Rats; D013552:Swine
D005292:Ferricyanides; D005182:Flavin-Adenine Dinucleotide; D002606:Charcoal; D009249:NADP; D005486:Flavin Mononucleotide; D003579:Cytochrome Reductases; D010100:Oxygen