Structure/Function of Complex II Oxidoreductase

复合物 II 氧化还原酶的结构/功能

• 批准号: 7930990
• 负责人: Gary Cecchini
• 金额: $ 20.92万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7930990
• 关键词: Address; Affect; Amino Acids; Architecture; Bacterial Model; Behavior; Binding; Binding Sites; Biochemical; Bioenergetics; Biological; Biological Assay; Biological Models; Cardiac; Cell physiology; Cells; Citric Acid Cycle; Collaborations; Complex; Crystallography; DNA Sequence Rearrangement; Defect; Disease; Electron Spin Resonance Spectroscopy; Electron Transport; Enzymes; Escherichia coli; Event; Family; Flavins; Fourier Transform; Fumarates; Functional disorder; Goals; Heart Diseases; Heme; Homologous Gene; Human; Hydroquinones; Investigation; Iron; Kinetics; Laboratories; Lead; Link; Maps; Measures; Membrane; Membrane Structure and Function; Metabolic; Metabolism; Methods; Mitochondria; Modeling; Molecular; Motor; Multienzyme Complexes; Mutagenesis; Mutation; Nerve Degeneration; Oxidation-Reduction; Oxidoreductase; Paraganglioma; Pathway interactions; Pheochromocytoma; Physiologic pulse; Physiological; Premature aging syndrome; Process; Prosthesis; Proteins; Protons; Pulse Radiolysis; Quinones; Reaction; Research; Role; Rotation; Series; Side; Site; Site-Directed Mutagenesis; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spin Labels; Structure; Study models; Succinate Dehydrogenase; Succinate dehydrogenase (ubiquinone); Sulfur; Switching Complex; System; Techniques; Testing; Ubiquinone; Vertebral column; Vitamin K 2; Work; cofactor; design; dicarboxylate; genetic manipulation; insight; interest; mutant; programs; protein complex; protonation; public health relevance; respiratory complex II; tumor; tumorigenesis; water channel

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.

描述（由申请人提供）：本研究计划描述了膜结合呼吸复合物II（琥珀酸：泛醌氧化还原酶/琥珀酸脱氢酶）及其细菌同系物的结构/功能。这项研究计划的长期目标是描述电子转移的机制，通过酶/从黄素醌。络合物II具有许多参与电子转移过程的氧化还原中心。这些包括共价结合的FAD辅因子、三个不同的铁-硫簇、膜结合的醌和B血红素辅因子。除了B血红素辅基之外，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中的特定突变导致疾病，包括肿瘤形成、神经变性、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物II的醌结合结构域。这些缺陷如何导致疾病的分子机制仍然不清楚。本申请中描述的研究具有三个总体目标。第一个是设计来描述的醌结合位点的复合物II的基本组成部分，以及该网站的架构如何影响催化活性。复合物II是这些研究的一个很好的模型，因为它有能力与ubi-和napthoquinones相互作用。因此，使用一系列的定点突变体和动力学分析，傅立叶变换红外和脉冲EPR光谱，和X-射线晶体学的必要组成部分的醌结合位点的正常运作将被定义。其次，通过使用脉冲辐解技术，结合其他方法，我们将确定在野生型和突变形式的复合物II的氧化还原活性中心之间的电子转移速率常数。据推测，改变动力学的电子转移有助于复合物II功能障碍，这反过来又导致级联的代谢事件，导致疾病。因此，各种氧化还原中心的作用，包括电子转移反应中的B血红素将被定义。最终的目的是表征与其他蛋白质相互作用后的复合物II的构象变化。最近的研究结果表明，复合物II同系物醌醇：富马酸氧化还原酶（QFR，富马酸还原酶）与FliG，细菌鞭毛开关复合物的组分相互作用。这种相互作用对于控制鞭毛旋转和组装的方向是重要的。这些研究将通过酶的诱变和动力学测定、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组分。当复合物II不能正常工作时，这可能导致神经变性，心脏病和肿瘤形成。本申请中描述的研究旨在了解复合物II突变（已知引起疾病）如何影响复合物II的功能。这些研究还将描述复合物II如何与细胞中的其他蛋白质组分相互作用，使用线粒体对应物的细菌模型。这对于显示蛋白质复合物如何相互通信可能很重要。