Structure/Function of Complex II Oxidoreductase

描述(申请人提供)：本研究项目描述了膜结合呼吸复合体II(琥珀酸：泛醌氧化还原酶/琥珀酸脱氢酶)及其细菌同系物的结构/功能。这项研究计划的长期目标是描述通过酶将电子从黄素转移到对苯二酚的机制。络合物II具有多个参与电子转移过程的氧化还原中心。它们包括一个共价结合的FAD辅因子、三个不同的铁-硫簇、一个膜结合的苯醌和一个b型血红素辅因子。除了血红素假体外，所有这些氧化还原中心都被认为是电子传递途径的重要组成部分。在线粒体中，复合体II是柠檬酸循环和膜结合电子传递链的重要组成部分。复合体II的特定突变导致疾病，包括肿瘤形成、神经退化、心脏功能障碍和过早衰老。这些突变中的大多数在结构上映射到复合体II的苯醌结合结构域。这些缺陷如何导致疾病的分子机制仍不清楚。本申请中描述的研究有三个总体目标。第一个是用来描述化合物II的苯二酚结合部位的基本成分，以及这个部位的结构如何影响催化活性。络合物II是这些研究的一个很好的模型，因为它能够与ubi-和napthoquone相互作用。因此，使用一系列定点突变和动力学分析、傅立叶变换红外光谱和脉冲EPR光谱以及X射线结晶学，将确定醌结合位点正常运行所需的组分。其次，利用脉冲辐解技术，结合其他方法，我们将测定野生型和突变型复合体II中特定氧化还原活性中心对之间的电子传递速率常数。假设电子传递动力学的改变有助于复合体II功能障碍，从而导致一系列导致疾病的代谢事件。因此，各种氧化还原中心，包括血红素在电子转移反应中的作用将被定义。最终目的是表征复合体II与其他蛋白质相互作用时的构象变化。最近的发现表明，富马酸氧化还原酶(QFR，富马酸还原酶)与细菌鞭毛开关复合体的一个成分FliG相互作用。这种相互作用对于控制鞭毛旋转和组装的方向很重要。这些研究将通过酶的诱变和动力学分析、QFR：FliG复合体的结构分析和定点自旋标记EPR光谱来完成。与公共健康相关：复合体II(琥珀酸：泛醌氧化还原酶)是参与线粒体新陈代谢的重要代谢成分。当复合体II功能不正常时，可能会导致神经退化、心脏病和肿瘤形成。本申请中描述的研究旨在了解Complex II突变(已知会导致疾病)如何影响Complex II的功能。这些研究还将使用线粒体对应的细菌模型描述Complex II如何与细胞中的其他蛋白质成分相互作用。这对于展示蛋白质复合体如何相互通信可能很重要。

DESCRIPTION (provided by applicant): This research program describes the structure/function of the membrane-bound respiratory Complex II (succinate:ubiquinone oxidoreductase/succinate dehydrogenase) and its bacterial homologues. The long-term objectives of this research program are to describe mechanisms of electron transfer through the enzyme to/from flavin to quinones. Complex II has a number of redox centers involved in the electron transfer process. These include a covalently-bound FAD cofactor, three distinct iron- sulfur clusters, a membrane-bound quinone, and a b heme cofactor. With the exception of the b heme prosthetic group all of these redox centers are known as essential components of the electron transfer pathway. In mitochondria Complex II is an essential component of both the citric acid cycle and the membrane-bound electron transport chain. Specific mutations in Complex II contribute to disease including, tumor formation, neurodegeneration, cardiac dysfunction, and premature aging. The majority of these mutations structurally map to the quinone-binding domain of Complex II. The molecular mechanisms of how these defects contribute to disease are still not understood. The studies described in this application have three general aims. The first is designed to describe the essential components of the quinone-binding site of Complex II and how the architecture of this site influences catalytic activity. Complex II is an excellent model for these studies since it has the ability to interact with both ubi- and napthoquinones. Thus, using a series of site-directed mutants and kinetic assays, Fourier transform infrared and pulsed EPR spectroscopy, and x-ray crystallography the necessary components for the proper functioning of the quinone-binding site will be defined. Second, by using the technique of pulse radiolysis, in conjunction with other methods, we will determine rate constants for electron transfer between specific pairs of redox-active centers in both wild-type and mutant forms of Complex II. It is hypothesized that altered kinetics of electron transfer contribute to Complex II dysfunction, which in turn leads to a cascade of metabolic events leading to disease. Thus, the role of the various redox centers, including the b heme in electron transfer reactions will be defined. The final aim is to characterize conformational changes of Complex II upon interaction with other proteins. Recent findings show that the Complex II homologue quinol:fumarate oxidoreductase (QFR, fumarate reductase) interacts with FliG, a component of the bacterial flagellar switch complex. This interaction is important for controlling the direction of flagellar rotation and assembly. These studies will be accomplished by mutagenesis and kinetic assays of the enzyme, structural analysi of the QFR:FliG complex, and site-directed spin labeling EPR spectroscopy. PUBLIC HEALTH RELEVANCE: Complex II (succinate:ubiquinone oxidoreductase) is an essential metabolic component involved in mitochondrial metabolism. When Complex II does not function properly, this can lead to neurodegeneration, heart disease, and tumor formation. The studies described in this application are designed to understand how Complex II mutations (known to cause disease) affect the function of Complex II. These studies will also describe how Complex II interacts with other protein components in the cell using bacterial models for their mitochondrial counterparts. This may be important to show how protein complexes communicate with one another.