Murray James, Taylor Steven W, Zhang Bing, Ghosh Soumitra S, Capaldi Roderick A
Department of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
J Biol Chem. 2003 Sep 26;278(39):37223-30. doi: 10.1074/jbc.M305694200. Epub 2003 Jul 11.
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 Tyr122, while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr46, Tyr50, and Tyr51. 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.
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种，作为有害的副产物，这些副产物会破坏OXPHOS酶，随后增加自由基的产生。大脑中线粒体这种氧化损伤的积累被认为会导致神经元细胞死亡，从而导致神经退行性变。线粒体中主要的活性氮物种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线粒体膜反应，显著抑制复合物I、II和V的活性（50 - 80%），但对复合物IV的影响较小，对复合物III没有显著抑制作用。由于复合物I活性的抑制是帕金森病的一个已报道特征，我们对过氧亚硝酸盐诱导的来自富集复合物I制剂的蛋白质修饰进行了详细分析。免疫和质谱方法与二维聚丙烯酰胺凝胶电泳相结合，已用于表明导致3 - 硝基酪氨酸标记的过氧亚硝酸盐修饰主要与复合物I亚基、49 kDa亚基（NDUFS2）、TYKY（NDUFS8）、B17.2（17.2 kDa分化相关蛋白）、B15（NDUFB4）和B14（NDUFA6）相关。分别从MS/MS碎片图和提取离子色谱图中推断出这些亚基中最后三个以及两个共纯化蛋白F1F0 - ATP合酶的β和δ亚基的硝化位点和修饰产率估计值。亚基B15（NDUFB4）和B14（NDUFA6）的硝化程度最高。亚基B14中最具反应性的位点是Tyr122，而B15中最具反应性的区域包含3个紧密间隔的酪氨酸Tyr46、Tyr50和Tyr51。此外，在亚基B17.2中检测到色氨酸氧化位点，这增加了我们在复合物I亚基中检测到的翻译后修饰色氨酸的数量（泰勒，S.W.，法希，E.，默里，J.，卡帕尔迪，R.A.，和戈什，S.S.（2003年）《生物化学杂志》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。
