Bakker P T, Albracht S P
Biochim Biophys Acta. 1986 Jul 23;850(3):413-22. doi: 10.1016/0005-2728(86)90109-x.
The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O2 of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6-6.5.
已运用冷冻淬灭技术研究了线粒体内膜亚基颗粒中由NADPH介导的NADH:Q氧化还原酶的还原作用。研究发现，在pH 6.5条件下，50%的铁硫簇2、3和4可在30毫秒内被NADPH还原。其余的簇，包括簇1，还原缓慢且不完全；由此得出结论，这些簇在NADPH氧化酶活性中不起作用。在厌氧条件下，pH 8时也得到了几乎相同的结果，这表明在这两个pH值下，NADPH与该酶的反应速率基本相同。在pH 8时，NADPH对一半的簇2的还原速率和程度不受氧气或鱼藤酮存在的影响。这意味着该酶的pH依赖性氧化是此pH下缺乏NADPH氧化酶活性的原因。提出了该酶的二聚体模型，其中一个原体含有化学计量的FMN和铁硫簇1 - 4，负责在pH 8时氧化NADH。这个原体不能与NADPH反应。另一个原体仅含有FMN和簇2、3和4，推测其催化NADPH的氧化。预计该原体被泛醌氧化强烈依赖于pH。这个原体也可能在pH 6 - 6.5时催化NADH的氧化。
