Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I
Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I
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DOI：
10.1021/bi7009822
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发表时间：
2007-09-25
期刊：
影响因子：
2.9
通讯作者：
Andrei, D. Vinoaradov
中科院分区：
文献类型：
--
作者：
Grivermikova, Vera G.;Kotlyar, Alexander B.;Andrei, D. Vinoaradov
最近发现了一种非常有效且特异性的线粒体 NADH 抑制剂：泛醌氧化还原酶（复合物 1），它是 NADH (NADH-OH) 的衍生物（Kotlyar, A. B.、Karliner, J. S. 和 Cecchini, G. (2005) FEBS Lett. 579, 4861-4866）。在这里，我们对紧密耦合的亚软骨颗粒中 NADH-OH 和其他核苷酸与氧化和还原复合物 I 的相互作用进行了定量分析。琥珀酸存在时，NADH-OH 结合率及其对复合物 I 的亲和力都会大大降低。鱼藤酮、抗霉素 A 和解偶联剂可完全逆转琥珀酸盐的作用。 ADP-核糖（NADH 氧化的竞争性抑制剂）的相对亲和力也受到酶还原的显着影响（氧化酶和琥珀酸还原酶的 K-D 分别为 30 和 500 μM）。 NADH-OH 的结合被证明可以消除复合物 I 产生的琥珀酸支持的超氧化物。逐渐抑制鱼藤酮敏感的未偶联 NADH 氧化酶和 NADH-OH 的反向电子转移活性产生相同的最终滴定点（类似于 0.1 nmol/mg 蛋白质）。 NADH氧化酶的滴定呈直线，而逆反应的滴定呈凸曲线。简要讨论了解释正向和反向反应的不同滴定模式的可能模型。
A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex 1), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (K-D of 30 and 500 mu M for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (similar to 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.