Chen Jingfeng, Chen Chwen-Lih, Alevriadou B Rita, Zweier Jay L, Chen Yeong-Renn
Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
Biochim Biophys Acta. 2011 May;1807(5):491-502. doi: 10.1016/j.bbabio.2011.03.001. Epub 2011 Mar 22.
Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(•-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(•)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(•-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (•)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.
线粒体衍生的氧自由基是细胞氧化损伤的重要介质。人们普遍推测，在某些病理条件下，过量的一氧化氮（NO）会增强线粒体产生的超氧阴离子（O₂•⁻）。在线粒体电子传递链中，琥珀酸 - 细胞色素c还原酶（SCR）催化电子从琥珀酸传递给细胞色素c。为深入了解NO过量产生如何介导SCR产生氧自由基的分子机制，我们使用分离的SCR、心肌成纤维细胞H9c2和内皮细胞，在体外和体内研究NO与SCR的相互作用。在NO供体（DEANO）存在的酶周转条件下，通过使用DEPMPO的电子顺磁共振（EPR）自旋捕获检测到SCR获得了产生羟基自由基的促氧化功能。在过氧化氢酶或铁螯合剂存在下，与DEPMPO/（•）OH加合物相关的EPR信号几乎完全消失，并且被超氧化物歧化酶（SOD）部分抑制，这表明涉及铁 - H₂O₂依赖性芬顿反应或O₂•⁻依赖性哈伯 - 韦斯机制。在77K下对SCR进行直接EPR测量表明形成了一种非血红素铁 - NO复合物，这意味着在黄素腺嘌呤二核苷酸（FAD）辅因子处电子向分子氧的泄漏增强，并且过量的NO使SCR易于产生（•）OH。在H9c2细胞中，DEANO释放的NO或细胞在暴露于缺氧/复氧后产生的NO刺激了SCR依赖性氧自由基的产生。在导致内皮细胞NO过量产生的剪切暴露下，在培养的血管内皮细胞中也检测到了SCR介导的氧自由基产生。
