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; 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）：该研究计划描述了膜结合的呼吸复合物II的结构/功能（琥珀酸酯：泛素酮氧化还原酶/琥珀酸酯脱氢酶）及其细菌同源物。该研究计划的长期目标是描述通过从黄素到奎因酮的酶转移电子传递的机制。复合物II在电子传输过程中涉及许多氧化还原中心。其中包括一个共价结合的时尚辅助因子，三个不同的铁硫簇，一个结合膜的醌和B血红素辅因子。除B血红素假体组外，所有这些氧化还原中心都称为电子转移途径的必需组成部分。在线粒体中，II是柠檬酸周期和膜结合的电子传输链的重要组成部分。复合物II的特异性突变有助于疾病，包括肿瘤形成，神经退行性，心脏功能障碍和过早衰老。这些突变的大多数在结构上映射到复合物II的喹酮结合结构域。这些缺陷如何促进疾病的分子机制仍不清楚。本应用程序中描述的研究具有三个一般目标。第一个旨在描述复合物II的喹酮结合位点的基本组成部分，以及该站点的结构如何影响催化活性。复合物II是这些研究的绝佳模型，因为它具有与Ubi-和Napthoquinones相互作用的能力。因此，使用一系列位置定向的突变体和动力学测定，傅立叶变换红外和脉冲的EPR光谱和X射线晶体学将定义用于正确功能的必要组件。其次，通过使用脉冲辐射分解技术，与其他方法结合使用，我们将确定野生型和突变体形式的复合物II的特定氧化还原活性中心之间电子传递的速率常数。假设电子转移的动力学改变会导致复杂的II功能障碍，这又导致一系列代谢事件导致疾病。因此，将定义各种氧化还原中心（包括B血红素）在电子转移反应中的作用。最终目的是表征与其他蛋白质相互作用后复合物II的构象变化。最近的发现表明，复合物II同源物喹酚：富马酸氧化还原酶（QFR，富马酸盐还原酶）与Flig相互作用，Flig是细菌鞭毛开关复合物的成分。这种相互作用对于控制鞭毛旋转和组装方向很重要。这些研究将通过诱变和动力学测定酶，QFR：Flig复合物的结构分析以及位置定向的自旋标记EPR光谱法来完成。公共卫生相关性：复合物II（琥珀酸酯：泛酮氧化还原酶）是与线粒体代谢有关的必不可少的代谢成分。当复合物II无法正常运行时，这可能导致神经变性，心脏病和肿瘤形成。本应用中描述的研究旨在了解复杂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使用线粒体对应物的细菌模型如何与细胞中其他蛋白质成分相互作用。这可能表明蛋白质复合物如何相互通信可能很重要。