Oxidative damage to mitochondrial complex I due to peroxynitrite - Identification of reactive tyrosines by mass spectrometry
Oxidative damage to mitochondrial complex I due to peroxynitrite - Identification of reactive tyrosines by mass spectrometry
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DOI：
10.1074/jbc.m305694200
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发表时间：
2003-09-26
影响因子：
4.8
通讯作者：
Capaldi, RA
中科院分区：
文献类型：
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作者：
Murray, J;Taylor, SW;Capaldi, RA
越来越多的证据表明，氧化磷酸化（OXPHOS）在线粒体内产生活性氧和氮物质作为不需要的副产物，其可以损害OXPHOS酶，随后增强自由基的产生。这种对脑中线粒体的氧化损伤的积累被认为导致神经元细胞死亡，从而导致神经变性。线粒体中主要的活性氮物质是一氧化氮和过氧亚硝酸盐。在这里，我们表明，过氧亚硝酸盐与牛心脏线粒体膜反应，显着抑制复合物I，II和V（50 - 80%）的活动，但对复合物IV的影响较小，没有显着抑制复合物III。由于抑制复合物I的活性已被报道的帕金森氏病的功能，我们进行了详细的分析过氧亚硝酸盐诱导的修饰蛋白质从丰富的复合物I制剂。免疫学和质谱方法结合二维PAGE已被用于显示过氧亚硝酸盐修饰导致3-硝基酪氨酸签名主要与复合物I亚基，49-kDa亚基（NDUFS 2），TYKY（NDUFS 8），B17.2（17.2-kDa分化相关蛋白），B15（NDUFB 4）和B14（NDUFA 6）相关。硝化位点和估计的修改产量推导出MS/MS fragmentogram和提取的离子色谱图，分别为最后三个这些亚基以及两个共纯化蛋白质，β和D亚基的F1 F0-ATP合酶。亚基B15（NDUFB 4）和B14（NDUFA 6）的硝化程度最高。B14亚基中最具反应活性的位点是Tyr（122），而B15亚基中最具反应活性的区域包含3个紧密间隔的酪氨酸Tyr（46）、Tyr（50）和Tyr（51）。此外，在亚基B17.2中检测到色氨酸的氧化位点，增加了我们在复合物I亚基中检测到的后修饰的色氨酸的数量（Taylor，S. W.，Fahy，E.，默里，J.，卡帕尔迪河一、和Ghosh，S. S.（2003）J.Biol.Chem.278，19587 - 19590）。这些氧化和硝化位点可能是评估神经退行性疾病中氧化应激的有用生物标志物。
There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V ( 50 - 80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 ( 17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the beta and the d subunits of the F1F0-ATP synthase. Subunits B15 ( NDUFB4) and B14 ( NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr(122), while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr(46), Tyr(50), and Tyr(51). In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I subunits ( Taylor, S. W., Fahy, E., Murray, J., Capaldi, R. A., and Ghosh, S. S. (2003) J. Biol. Chem. 278, 19587 - 19590). These sites of oxidation and nitration may be useful biomarkers for assessing oxidative stress in neurodegenerative disorders.