Hoefnagel M H, Millar A H, Wiskich J T, Day D A
Botany Department, University of Adelaide, Australia.
Arch Biochem Biophys. 1995 Apr 20;318(2):394-400. doi: 10.1006/abbi.1995.1245.
The partitioning of electrons between the alternative oxidase and the cytochrome pathway of soybean mitochondria has been reassessed in the presence of the alternative oxidase activator pyruvate. In the presence of pyruvate and with succinate as substrate, the alternative oxidase became active at a much lower level of ubiquinone reduction than in the absence of pyruvate. Under state 4 (no ADP present) conditions, activation of the alternative oxidase with pyruvate resulted in an oxidation of b cytochromes, demonstrating switching of electrons away from the cytochrome chain. In the presence of ferricyanide and the cytochrome oxidase inhibitor KCN, cytochrome chain activity could be followed spectrophotometrically and that of the alternative pathway with an oxygen electrode. Under these conditions, the addition of pyruvate diverted electron flow from the cytochrome chain to the alternative pathway; subsequent inhibition of the alternative oxidase increased electron flow via the cytochrome chain. This indicates that electrons can be switched from one pathway to the other when the cytochrome chain is not saturated and this was confirmed by n-propylgallate titrations (p plots) of mitochondria oxidizing succinate. Decreases in ADP/O ratios and phosphorylation rate upon addition of pyruvate indicated that the alternative pathway could also contribute to respiration under state 3 conditions. The results indicate that when the alternative oxidase is activated by pyruvate, it can compete for electrons with the cytochrome chain and does not act as an overflow pathway. The significance of these observations for in vivo respiration is discussed.
在交替氧化酶激活剂丙酮酸存在的情况下，对大豆线粒体中电子在交替氧化酶和细胞色素途径之间的分配进行了重新评估。在丙酮酸存在且以琥珀酸为底物的情况下，交替氧化酶在泛醌还原水平比无丙酮酸时低得多的情况下就变得活跃。在状态4（无ADP存在）条件下，用丙酮酸激活交替氧化酶导致b细胞色素氧化，表明电子从细胞色素链转移。在铁氰化物和细胞色素氧化酶抑制剂KCN存在的情况下，可通过分光光度法追踪细胞色素链活性，并用氧电极追踪交替途径的活性。在这些条件下，添加丙酮酸会使电子流从细胞色素链转向交替途径；随后抑制交替氧化酶会增加通过细胞色素链的电子流。这表明当细胞色素链未饱和时，电子可以从一条途径切换到另一条途径，这通过对氧化琥珀酸的线粒体进行正丙基没食子酸滴定（p图）得到了证实。添加丙酮酸后ADP/O比值和磷酸化速率降低，表明交替途径在状态3条件下也可能对呼吸作用有贡献。结果表明，当交替氧化酶被丙酮酸激活时，它可以与细胞色素链竞争电子，而不是作为一条溢流途径。讨论了这些观察结果对体内呼吸作用的意义。
