The purification and properties of the respiratory-chain reduced nicotinamide--adenine dinucleotide dehydrogenase of Torulopsis utilis.
1. An NADH-ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH-ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH-ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH-ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes.
D003580:Cytochromes / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D005292:Ferricyanides; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007097:Imines; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D009243:NAD; D009249:NADP; D010088:Oxidoreductases / Q000302:isolation & purification*; D010641:Phenotype; D011809:Quinones; D013053:Spectrophotometry; D013440:Sulfides / Q000032:analysis; D008063:Thioctic Acid; D014451:Ubiquinone; D014812:Vitamin K
D003580:Cytochromes; D005292:Ferricyanides; D007097:Imines; D011809:Quinones; D013440:Sulfides; D009243:NAD; D014812:Vitamin K; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D008063:Thioctic Acid; D005486:Flavin Mononucleotide; D007501:Iron; D010088:Oxidoreductases