Reactive oxygen species production in cardiac mitochondria after complex I inhibition: Modulation by substrate-dependent regulation of the NADH/NAD(+) ratio.
Reactive oxygen species production in cardiac mitochondria after complex I inhibition: Modulation by substrate-dependent regulation of the NADH/NAD(+) ratio.
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DOI：
10.1016/j.freeradbiomed.2016.04.002
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发表时间：
2016-07
影响因子：
7.4
通讯作者：
Weiss JN
中科院分区：
文献类型：
--
作者：
Korge P;Calmettes G;Weiss JN
由分离的复合物I产生的活性氧（ROS）急剧依赖于NADH/NAD+比率。我们使用alamethicin-permeabilized线粒体研究基质NADH和ROS生产的底物依赖性时，复合物I抑制piericidin或鱼藤酮。当复合物I在苹果酸/谷氨酸存在下被抑制时，由于不受基质NAD（H）流出限制的NADH产生的快速增加，膜透化加速O2消耗和ROS产生。在抑制剂的存在下，苹果酸和谷氨酸都需要产生足够高的NADH/NAD+比率，以通过苹果酸脱氢酶（MDH）和天冬氨酸转氨酶（AST）的协调活性来支持ROS产生。与苹果酸和谷氨酸的存在下，ROS的生产率是密切相关的本地NADH的产生，而在没有底物，ROS的生产被加速增加[NADH]。单独使用苹果酸时，草酰乙酸积累限制了MDH的NADH生产，除非还添加谷氨酸以促进通过AST去除草酰乙酸。α-酮戊二酸（KG）以及AST抑制也逆转了NADH的产生并抑制了ROS的产生。如果苹果酸和谷氨酸之前，而不是之后提供piericidin或鱼藤酮，ROS的产生显着减少，由于CoA的时间依赖性流出。CoA消耗减少了α-酮戊二酸脱氢酶（KGDH）对KG的氧化，从而导致[KG]的增加抑制了AST对草酰乙酸的去除和MDH对NADH的生成。这些发现在完整线粒体中很大程度上是模糊的，这是由于强大的H2 O2清除和控制基质中底物浓度的能力有限。我们的结论是，在线粒体与抑制复合物I，苹果酸/谷氨酸刺激的活性氧生成强烈依赖于草酰乙酸的清除和KGDH的能力，以氧化由AST产生的KG。
Reactive oxygen species (ROS) production by isolated complex I is steeply dependent on the NADH/NAD+ ratio. We used alamethicin-permeabilized mitochondria to study the substrate-dependence of matrix NADH and ROS production when complex I is inhibited by piericidin or rotenone. When complex I was inhibited in the presence of malate/glutamate, membrane permeabilization accelerated O2 consumption and ROS production due to a rapid increase in NADH generation that was not limited by matrix NAD(H) efflux. In the presence of inhibitor, both malate and glutamate were required to generate a high enough NADH/NAD+ ratio to support ROS production through the coordinated activity of malate dehydrogenase (MDH) and aspartate aminotransferase (AST). With malate and glutamate present, the rate of ROS production was closely related to local NADH generation, whereas in the absence of substrates, ROS production was accelerated by increase in added [NADH]. With malate alone, oxaloacetate accumulation limited NADH production by MDH unless glutamate was also added to promote oxaloacetate removal via AST. α-ketoglutarate (KG) as well as AST inhibition also reversed NADH generation and inhibited ROS production. If malate and glutamate were provided before rather than after piericidin or rotenone, ROS generation was markedly reduced due to time-dependent efflux of CoA. CoA depletion decreased KG oxidation by α-ketoglutarate dehydrogenase (KGDH), such that the resulting increase in [KG] inhibited oxaloacetate removal by AST and NADH generation by MDH. These findings were largely obscured in intact mitochondria due to robust H2O2 scavenging and limited ability to control substrate concentrations in the matrix. We conclude that in mitochondria with inhibited complex I, malate/glutamate-stimulated ROS generation depends strongly on oxaloacetate removal and on the ability of KGDH to oxidize KG generated by AST.