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膜蛋白复合体中的催化作用。这是我们更大目标的一部分，目的是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。它将电子传输耦合到质子在膜上的传递，这一反应机制被称为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。也有争议的说法是，Cyt Bc1是衰老和许多疾病的罪魁祸首，通过直接产生超氧化物(SO)并推动过氧化氢、羟基自由基和过氧亚硝酸根的产生。关于这些复合体如何发挥作用，人们提出了几种相互矛盾的模型，在电子转移、化学反应、中间体甚至活性酶底物复合体的结构等细节上存在差异。这些冲突的解决对于理解正常功能的基础以及由此产生的复合体是至关重要的。尽管线粒体的细胞色素bc1复合体和相关的叶绿体细胞色素b6f的一些晶体结构已经发表，但没有一种结构显示氧化活性部位的底物喹酚，留下了在这个部位发生的特定反应的可能性。在某些条件下，Cyt Bc1可以将流经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。这一建议提出了一系列具体的目标，这些目标综合起来将有助于我们理解：a)半喹酮作为细胞色素Bc1催化氧化对苯二酚的中间体的作用；b)细胞色素bc1的整体反应机制；以及c)细胞色素bc1是否通过产生SO而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.