Structure/Function of Complex II Oxidoreductase

描述（由申请人提供）：本研究项目描述膜结合呼吸复合物II（琥珀酸：泛醌氧化还原酶/琥珀酸脱氢酶）及其细菌同源物的结构/功能。本研究计划的长期目标是描述电子通过酶从黄素转移到醌的机制。配合物II有许多参与电子转移过程的氧化还原中心。这些包括一个共价结合的FAD辅助因子，三个不同的铁硫簇，一个膜结合的醌和一个b血红素辅助因子。除了b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，复合物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的特定突变可导致包括肿瘤形成、神经退行性变、心功能障碍和早衰在内的疾病。这些突变大部分在结构上映射到复合物II的醌结合结构域。这些缺陷如何导致疾病的分子机制尚不清楚。本应用程序中描述的研究有三个总体目标。第一篇文章旨在描述复合物II的醌结合位点的基本成分以及该位点的结构如何影响催化活性。配合物II是这些研究的一个很好的模型，因为它具有与乌比醌和萘醌相互作用的能力。因此，使用一系列位点导向突变体和动力学分析，傅里叶变换红外和脉冲EPR光谱，以及x射线晶体学，将定义醌结合位点正常运作的必要成分。其次，通过使用脉冲辐射分解技术，结合其他方法，我们将确定野生型和突变型络合物II中特定氧化还原活性中心对之间电子转移的速率常数。据推测，电子转移动力学的改变导致复合体II功能障碍，进而导致一系列代谢事件导致疾病。因此，各种氧化还原中心，包括b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。最近的研究表明，络合物II同源物富马酸喹啉氧化还原酶（QFR，富马酸还原酶）与细菌鞭毛开关络合物的组分FliG相互作用。这种相互作用对于控制鞭毛旋转和装配的方向是重要的。这些研究将通过酶的诱变和动力学分析、QFR:FliG复合物的结构分析和定点自旋标记EPR光谱来完成。公共卫生相关性：复合物II（琥珀酸：泛醌氧化还原酶）是参与线粒体代谢的重要代谢成分。当复合物II不能正常发挥作用时，就会导致神经退行性变、心脏病和肿瘤的形成。本申请中描述的研究旨在了解复合体II突变（已知会导致疾病）如何影响复合体II的功能。这些研究还将描述复合体II如何与细胞中其他蛋白质成分相互作用，使用细菌模型来模拟它们的线粒体对应物。这对于展示蛋白质复合物如何相互沟通可能很重要。

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.