Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer.
Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer.
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DOI：
10.1074/jbc.m110.186841
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发表时间：
2011-05-20
期刊：
影响因子：
--
通讯作者：
Hirst J
中科院分区：
文献类型：
--
作者：
Pryde KR;Hirst J
NADH：泛醌氧化还原酶（复合物I）是线粒体中活性氧的主要来源，也是细胞氧化应激的贡献者。在分离的复合物I中，还原的黄素已知与分子氧反应，主要形成超氧化物，但使用完整线粒体的研究认为，超氧化物可能来自半醌物质，该物质也响应质子动力（Δp）。在这里，我们使用牛心submitochondrial颗粒表明，一个单一的机制描述了超氧化物的生产复杂的I在所有条件下（在两个NADH氧化和反向电子转移）。NADH诱导的超氧阴离子产生可被复合物I黄素位点抑制剂抑制，但不被泛醌还原抑制剂抑制，且与Δp无关。在琥珀酸氧化和ATP水解产生的Δp的驱动下，通过亚线粒体颗粒中复合物I的反向电子转移（RET）减少了黄素，导致NAD+和O2还原。RET诱导的超氧化物生成被黄素位点和泛醌还原抑制剂抑制。NADH诱导的超氧化物生成的电位依赖性（由NAD+电位设定）与RET诱导的超氧化物生成的电位依赖性（由琥珀酸电位和Δp设定）相匹配，并且它们都与黄素的电位依赖性相匹配。因此，根据相同的分子机制，NADH和RET诱导的超氧化物均由黄素产生。统一的机制描述了活性氧物质的生产复杂的I响应细胞条件的变化。它建立了一个路线，以了解酶和其病理作用之间的因果关系，并制定合理的策略来解决这些问题。
NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a contributor to cellular oxidative stress. In isolated complex I the reduced flavin is known to react with molecular oxygen to form predominantly superoxide, but studies using intact mitochondria contend that superoxide may result from a semiquinone species that responds to the proton-motive force (Δp) also. Here, we use bovine heart submitochondrial particles to show that a single mechanism describes superoxide production by complex I under all conditions (during both NADH oxidation and reverse electron transfer). NADH-induced superoxide production is inhibited by complex I flavin-site inhibitors but not by inhibitors of ubiquinone reduction, and it is independent of Δp. Reverse electron transfer (RET) through complex I in submitochondrial particles, driven by succinate oxidation and the Δp created by ATP hydrolysis, reduces the flavin, leading to NAD+ and O2 reduction. RET-induced superoxide production is inhibited by both flavin-site and ubiquinone-reduction inhibitors. The potential dependence of NADH-induced superoxide production (set by the NAD+ potential) matches that of RET-induced superoxide production (set by the succinate potential and Δp), and they both match the potential dependence of the flavin. Therefore, both NADH- and RET-induced superoxide are produced by the flavin, according to the same molecular mechanism. The unified mechanism describes how reactive oxygen species production by complex I responds to changes in cellular conditions. It establishes a route to understanding causative connections between the enzyme and its pathological effects and to developing rational strategies for addressing them.