Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I)
Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I)
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DOI：
10.1074/jbc.m406576200
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发表时间：
2004-09-17
影响因子：
4.8
通讯作者：
Brand, MD
中科院分区：
文献类型：
--
作者：
Lambert, AJ;Brand, MD
线粒体复合物 I 中电子传输的途径和超氧化物产生的位点或机制均尚未确定。我们检查了大鼠骨骼肌线粒体在各种条件下的超氧化物生成率（以过氧化氢生成量衡量）。即使当复合物 I 在复合物 III 抑制剂豆豆菌素存在下被丙酮酸加苹果酸完全还原时，在 NADH 连接的正向电子传输过程中复合物 I 产生的超氧化物的速率还不到琥珀酸连接的反向电子传输过程中的 10%。这种不对称性不能用质子动势或其分量的差异来解释。然而，当在 ATP 存在下添加复合物 I 醌结合位点的抑制剂以产生 pH 梯度时，通过正向电子传输产生超氧化物的速率与相同 pH 梯度下反向电子传输所见的速率一样快。这些观察结果表明醌结合位点抑制剂可以使复合物 I 采用反向电子传输过程中发生的高度产生自由基的状态。尽管在每种情况下完全抑制NADH：泛醌氧化还原酶活性，但不同类别的醌结合位点抑制剂（鱼藤酮、杀粉虫素和高浓度的粘噻唑）在正向电子传输过程中产生不同的超氧化物产生速率（粘噻唑的速率是鱼藤酮的两倍），这表明复合物I快速产生超氧化物的位点位于泛半醌结合位点区域，而不是在上游。黄素或低电位 FeS 中心。
Neither the route of electron transport nor the sites or mechanism of superoxide production in mitochondrial complex I has been established. We examined the rates of superoxide generation (measured as hydrogen peroxide production) by rat skeletal muscle mitochondria under a variety of conditions. The rate of superoxide production by complex I during NADH-linked forward electron transport was less than 10% of that during succinate-linked reverse electron transport even when complex I was fully reduced by pyruvate plus malate in the presence of the complex III inhibitor, stigmatellin. This asymmetry was not explained by differences in protonmotive force or its components. However, when inhibitors of the quinone-binding site of complex I were added in the presence of ATP to generate a pH gradient, there was a rapid rate of superoxide production by forward electron transport that was as great as the rate seen with reverse electron transport at the same pH gradient. These observations suggest that quinone-binding site inhibitors can make complex I adopt the highly radical-producing state that occurs during reverse electron transport. Despite complete inhibition of NADH: ubiquinone oxidoreductase activity in each case, different classes of quinone-binding site inhibitor (rotenone, piericidin, and high concentrations of myxothiazol) gave different rates of superoxide production during forward electron transport (the rate with myxothiazol was twice that with rotenone) suggesting that the site of rapid superoxide generation by complex I is in the region of the ubisemiquinone-binding sites and not upstream at the flavin or low potential FeS centers.