Beattie D S, Japa S, Howton M, Zhu Q S
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506.
Arch Biochem Biophys. 1992 Feb 1;292(2):499-505. doi: 10.1016/0003-9861(92)90022-o.
The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.
在具有良好呼吸控制率的完整酵母线粒体中，研究了烟酰胺腺嘌呤二核苷酸（NADH）和乙醇对杜醌（DQ）以及2,3 - 二甲氧基 - 5 - 甲基 - 6 - 癸基 - 1,4 - 苯醌（DB）的还原作用。在这些线粒体中，外源性NADH被定位在内膜外表面的NADH脱氢酶氧化，而线粒体基质中乙醇氧化产生的NADH则被定位在内膜内表面的NADH脱氢酶氧化。乙醇对DQ的还原作用被粘噻唑抑制了86%；然而，NADH对DQ的还原作用仅被粘噻唑抑制了18%，这表明内部（而非外部）NADH：泛醌氧化还原酶与泛醇：细胞色素c氧化还原酶（细胞色素bc1复合物）之间的蛋白质 - 蛋白质相互作用参与了NADH对DQ的还原过程。还在缺乏细胞色素b、铁硫蛋白和泛醌的酵母突变体中研究了NADH和乙醇对DQ和DB的还原作用。外源性NADH对这两种醌类似物的还原作用降低到了野生型线粒体中观察到水平的10%至20%；然而，突变体中乙醇对它们的还原速率等于或高于野生型线粒体中观察到的速率。此外，缺乏细胞色素b和铁硫蛋白的线粒体中DQ的还原对粘噻唑敏感，这表明这两种蛋白质都不是粘噻唑的必需结合位点。这三个突变体的线粒体还含有大量对抗霉素和粘噻唑不敏感的NADH：细胞色素c还原酶活性，但未检测到琥珀酸：细胞色素c还原酶活性。这些结果表明，缺乏功能性细胞色素bc1复合物的突变体已经适应了氧化NADH。
