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） bc1膜蛋白复合物的催化作用。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。cyt bc1的主要生物学作用是在线粒体中进行能量转导。在一个被称为q循环的反应方案中，它将电子传递与质子在膜上的转导结合起来，从而帮助建立一个质子梯度或质子动力（pmf）来驱动ATP合成。也有争议的说法是，cyt bc1通过直接产生超氧化物（SO）和驱动过氧化氢、羟基自由基和过氧亚硝酸盐的产生，是衰老和许多疾病和病理生理过程的罪魁祸首。关于这些复合物如何起作用，人们提出了几个相互矛盾的模型，这些模型在电子转移、化学反应、中间体甚至活性酶底物复合物的结构等细节上存在差异。解决这些冲突对于理解正常功能和复杂的SO生产的基础是至关重要的。虽然线粒体的cyt bc1复合体和叶绿体相关的cyt b6f的许多晶体结构已经发表，但没有显示氧化活性位点的底物喹啉，留下了在该位点发生的特定反应的开放。在一定条件下，cyt bc1可以将流过它的电子的一半转移到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，这些目标将促进我们对以下方面的理解：a)半醌作为中间体在cyt bc1催化氧化喹啉中的作用；B) cyt bc1的整体反应机制；c) cyt bc1能否通过产生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.