Nitric oxide-induced persistent inhibition and nitrosylation of active site cysteine residues of mitochondrial cytochrome-c oxidase in lung endothelial cells.
Nitric oxide-induced persistent inhibition and nitrosylation of active site cysteine residues of mitochondrial cytochrome-c oxidase in lung endothelial cells.
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一氧化氮诱导肺内皮细胞线粒体细胞色素 C 氧化酶活性位点半胱氨酸残基的持续抑制和亚硝基化。
DOI：
10.1152/ajpcell.00325.2004
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Patel,JawaharlalM
中科院分区：
文献类型：
--
作者：
Zhang,Jianliang;Jin,Bilian;Li,Liuzhe;Block,EdwardR;Patel,JawaharlalM
细胞色素氧化酶是线粒体电子传递链的末端酶，炎症、污染的空气和烟草烟雾产生的过量一氧化氮（NO）对细胞色素氧化酶的持续抑制有助于增强氧化剂的产生和肺细胞的程序性死亡或凋亡。我们试图确定肺动脉内皮细胞（PAEC）长期暴露于病理生理浓度的NO是否会通过氧化还原修饰复合体IV的关键半胱氨酸残基（位于假定的NO敏感基序中）导致复合体IV的持续抑制。猪PAEC长期暴露于1 mM 2,2 ' -（羟基亚硝基肼）-双乙胺（NOC-18；缓释NO供体，相当于1 - 5 μM NO）中，导致复合物IV的逐渐持续抑制，同时线粒体GSH和GSSG的比例降低。PAEC线粒体中硫氧还蛋白的过度表达减弱了no诱导的复合物IV活性的丧失，提示复合物IV活性的氧化还原调节。复合体IV亚基的序列分析揭示了一个新的假定的no敏感基序在亚基II （S2）。在猪复合体IV S2中，只有两个半胱氨酸残基位于假定的基序中。免疫沉淀、Western blot分析和“生物素开关”实验表明，PAEC暴露于1mm NOC-18后，复合物IV S2的s -亚硝基化增加了200%。对复合体IV S2的这两种半胱氨酸进行定点诱变，减弱了no，增加了复合体IV S2的亚硝基化。这些结果首次证明NO亚基化复合体IV的活性位点半胱氨酸，这与复合体IV的持续抑制有关。通过NO敏感半胱氨酸残基的亚基化来抑制复合体IV可能是NO复合物IV信号传导中肺内皮细胞NO毒性的一个新的上游事件。
Persistent inhibition of cytochrome-coxidase, a terminal enzyme of the mitochondrial electron transport chain, by excessive nitric oxide (NO) derived from inflammation, polluted air, and tobacco smoke contributes to enhanced oxidant production and programmed cell death or apoptosis of lung cells. We sought to determine whether the long-term exposure of pulmonary artery endothelial cells (PAEC) to pathophysiological concentrations of NO causes persistent inhibition of complex IV through redox modification of its key cysteine residues located in a putative NO-sensitive motif. Prolonged exposure of porcine PAEC to 1 mM 2,2′-(hydroxynitrosohydrazino)-bis-ethanamine (NOC-18; slow-releasing NO donor, equivalent to 1–5 μM NO) resulted in a gradual, persistent inhibition of complex IV concomitant with a reduction in ratios of mitochondrial GSH and GSSG. Overexpression of thioredoxin in mitochondria of PAEC attenuated NO-induced loss of complex IV activities, suggesting redox regulation of complex IV activity. Sequence analysis of complex IV subunits revealed a novel putative NO-sensitive motif in subunit II (S2). There are only two cysteine residues in porcine complex IV S2, located in the putative motif. Immunoprecipitation and Western blot analysis and “biotin switch” assay demonstrated that exposure of PAEC to 1 mM NOC-18 increasedS-nitrosylation of complex IV S2 by 200%. Site-directed mutagenesis of these two cysteines of complex IV S2 attenuated NO-increased nitrosylation of complex IV S2. These results demonstrate for the first time that NO nitrosylates active site cysteines of complex IV, which is associated with persistent inhibition of complex IV. NO inhibition of complex IV via nitrosylation of NO-sensitive cysteine residues can be a novel upstream event in NO-complex IV signaling for NO toxicity in lung endothelial cells.