Metal Ion Probes Membrane Protein Structure and Function

描述（由申请人提供）：本提案的总体目标是了解细胞色素（cyt）bc 1膜蛋白复合物中的催化作用。这是我们更大目标的一部分，即了解这些膜蛋白在人类，病原体和病理过程中的多种生物学作用。细胞色素bc 1的主要生物学作用是在细胞内进行能量传递。它将电子传递耦合到质子跨膜转导的反应方案中称为Q循环，从而帮助建立质子梯度或质子动力（pmf）来驱动ATP合成。也有争议的说法，细胞色素bc 1是一个罪魁祸首，在老化和一些疾病和病理生理过程中，通过直接产生超氧化物（SO）和驱动生产过氧化物，羟基自由基和过氧亚硝酸盐。关于这些复合物如何发挥作用，已经提出了几个相互矛盾的模型，在电子转移、化学反应、中间体甚至活性酶底物复合物的结构的细节上都有不同。这些冲突的解决是至关重要的理解的基础上的正常功能和SO生产的复杂。虽然已经发表了许多线粒体的cyt bc 1复合物和叶绿体中相关的cyt b6 f的晶体结构，但没有一个显示氧化活性位点中的底物醌醇，这使得在该位点发生的特异性反应保持开放状态。在某些条件下，cyt bc 1可以将流经它的电子转移到SO中。SO的产生机制与其正常的生理反应密切相关。该提案提出了一系列具体目标，这些目标将共同促进我们对以下问题的理解：a）半醌作为cyt bc 1催化氧化醌醇的中间体的作用; B）cyt bc 1的总体反应机制;以及c）cyt bc 1是否可以通过产生SO在人类疾病中发挥作用。

DESCRIPTION (provided by applicant): The overall aim of this proposal is to understand catalysis in the cytochrome (cyt) bc1 membrane protein complexes. This is a part of our larger goal of understanding the multiple biological roles of these membrane proteins in humans, pathogens and pathological processes. The main biological role of cyt bc1 is in energy transduction in the mitochondrion. It couples electron transport to the transduction of protons across a membrane in a reaction scheme known as the Q-cycle, thereby helping establish a proton gradient or proton motive force (pmf) to drive ATP synthesis. There are also controversial claims that cyt bc1 is a culprit in aging and a number of diseases and pathophysiological processes by directly generating superoxide (SO) and driving production of peroxide, hydroxyl radicals and peroxynitrite. Several conflicting models of how these complexes function have been proposed, differing in the details of the electron transfers, chemical reactions, intermediates, and even the structure of the active enzyme substrate complex. Resolution of these conflicts is crucial for understanding the basis of normal function and SO production by the complex. Although a number of crystal structures of the cyt bc1 complex of mitochondria and the related cyt b6f from chloroplasts have been published, none show the substrate quinol in the oxidative active site, leaving open the specific reactions that occur at this site. Under certain conditions, cyt bc1 can divert up to half the electrons that flow through it into SO. The mechanism of SO production is very closely related to its normal physiological reactions. This proposal puts forth a set of Specific Aims that, taken together, will advance our understanding of: a) the role of semiquinone as an intermediate in the catalytic oxidation of quinol by cyt bc1; b) the overall reaction mechanism in cyt bc1; and c) whether cyt bc1 can play a role in human diseases through production of SO.