ISCHEMIC/REPERFUSION DAMAGE TO THE RIESKE IRON PROTEIN

缺血和再灌注期间的心肌损伤增加， 老年人的心两个串联的缺陷是目前在复杂的III的时间 衰老心脏再灌注损伤：Rieske铁硫 蛋白质（ISP）和细胞色素c结合位点的衰老相关缺陷。 我们的初步证据已经确定了ISP作为一个突出的网站， 与线粒体功能缺陷相关的损伤 缺血开发了一种分层方法来识别化学品 再灌注期间ISP遭受的氧化损伤的性质， 可能是化学性质的反应物种负责的 蛋白质修饰，以及观察到的修饰的相关性 再灌注引起的心肌损伤。四个具体目标是 描述了实现这些目标。一是发展和 展示了生物化学和质谱方法， 氧化损伤结构鉴定第二个具体目标 研究了氧化损伤的光谱， ISP的可溶性催化域，称为铁硫 片段，然后在综合体内的ISP的集成水平上 三.第三个具体目标是研究这些氧化剂如何改变功能 当ISP在完整的线粒体内发挥作用时，这一具体目标 允许表征来自潜在的 脂质产生的生理相关氧化剂和活性物质 过氧化作用氧化修饰的模式将在以下情况下进行检查 电子流会随着表征良好的抑制剂而变化 电子传输的。第四个具体目标是确定 在缺血-再灌注期间产生的氧化修饰。使用 /180/2将允许检测到的氧化修饰 在再灌注后分离的ISP中， 再灌注损伤鉴定蛋白的比较 根据前三个目标积累的数据进行修改， 识别再灌注产生的可能反应性物质，以及 提示和线粒体一个线粒体位点用于其产生， 蛋白质损伤机制这些信息将用于第四次 旨在设计和评估基于机制的治疗干预措施， 改善衰老心脏缺血和再灌注期间的损伤。

Myocardial damage during ischemia and reperfusion is increased in the elderly heart. Two tandem defects are present in complex III at the time of reperfusion in the aging heart: damage to the Rieske iron-sulfur protein (ISP) and a aging-related defect in the cytochrome c binding site. Our preliminary evidence has identified the ISP as a prominent site of damage associated with mitochondrial functional deficits following ischemia. A hierarchical approach is developed to identify the chemical nature of oxidative damage suffered by the ISP during reperfusion, the probably chemical nature of the reactive species responsible for the protein modification, and the correlation of the observed modification with reperfusion induced myocardial injury. Four specific aims are described to accomplish these goals. The first is to develop and demonstrate biochemical and mass spectrometric methods for efficient structural identification of oxidative-damage. The second specific aim investigates the spectrum of oxidative damage that can be inflicted on the soluble catalytic domain of the ISP, referred to as the iron-sulfur fragment, and then at the level of integration of the ISP within complex III. The third specific aim examines how these oxidants alter the function of the ISP when it functions within intact mitochondria. This specific aim permits the characterization of the anticipated products from potentially physiologically relevant oxidants and reactive species generated by lipid peroxidation. The pattern of oxidative modifications will be examined when the electron flow is varied in response to well characterized inhibitors of electron transport. The fourth specific aim is to determine the oxidative modifications arising during ischemia-reperfusion. The use of /180/2 during reperfusion will permit the oxidative modifications detected in the ISP isolated following reperfusion to be unequivocally correlated with the reperfusion injury. Comparison of the identified protein modifications with the data accumulated from the first 3 aims will identify the probably reactive species generated by reperfusion, and suggest and mitochondrial a mitochondrial site for its generation and mechanism of protein damage. This information will be used in the fourth aim to design and evaluate mechanistic-based therapeutic interventions to ameliorate damage during ischemia and reperfusion in the aging heart.