External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide.
Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I. To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria. The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A. Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate. Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64%. Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin. Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production. Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed. These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S. cerevisiae mitochondria.
Fang
Jing
J
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, WV 26506-9142, USA.
Beattie
Diana S
DS
D000935:Antifungal Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D014450:Electron Transport Complex III / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008689:Methacrylates; D008928:Mitochondria / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D012402:Rotenone / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure; D019802:Succinic Acid / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology
D000935:Antifungal Agents; D008293:Malates; D008689:Methacrylates; D011090:Polyenes; D013844:Thiazoles; D012402:Rotenone; D009243:NAD; D013481:Superoxides; D000968:Antimycin A; C030517:myxothiazol; C030298:malic acid; C041573:stigmatellin; D019802:Succinic Acid; D006861:Hydrogen Peroxide; D009245:NADH Dehydrogenase; D014450:Electron Transport Complex III