Complexities of complex II: A versatile architecture for respiration

复合体 II 的复杂性：呼吸的多功能架构

• 批准号: 7767691
• 负责人: T M Iverson
• 金额: $ 26.29万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7767691
• 关键词: Active Sites; Acute; Adopted; Aerobic; Affect; Architecture; Aspartate; Bacteria; Binding; Binding Sites; Biochemical; Biological; Biological Models; Catalysis; Cell Respiration; Chemicals; Chemistry; Citric Acid Cycle; Complement; Complex; Computer Simulation; Computing Methodologies; Crystallization; DNA Sequence Rearrangement; Data; Disease; Electron Spin Resonance Spectroscopy; Electron Transport; Embryonic Lethal Mutation; Enzymes; Escherichia coli; Fumarates; Generations; Harvest; Homologous Gene; Human; Hydroquinones; Industrial fungicide; Location; Membrane; Mitochondria; Molecular Conformation; Motion; Mutation; Nerve Degeneration; Organism; Property; Proteins; Quinones; Reaction; Reactive Oxygen Species; Recycling; Research Personnel; Resolution; Respiration; Site; Spin Labels; Staging; Structure; Succinate dehydrogenase (ubiquinone); Succinates; System; Variant; Water; Work; Workplace; base; dicarboxylate; enzyme activity; improved; inhibitor/antagonist; insight; molecular recognition; mutant; oxidation; programs; quinol fumarate reductase; research study; respiratory enzyme; small molecule

DESCRIPTION (provided by applicant): Complex II is one of the four electron transfer enzymes that generate the transmembrane electrochemical gradient in mitochondrial aerobic respiration. Its genetic alteration or inhibition has a significant effect on survival of the organism. The complex II superfamily is perhaps the most versatile respiratory enzyme since it is involved in aerobic and anaerobic respiration and the Krebs cycle. The recycling of the complex II architecture for multiple purposes suggests that this fold is evolutionary ancient. Examination of this single enzyme can give insight into biological mechanisms of fumarate reduction, succinate oxidation, aspartate oxidation, quinol chemistry, electron transfer, and the formation of the transmembrane electrochemical gradient. In preliminary studies, the complex II homolog quinol:fumarate reductase has been overproduced, crystallized and the structure determined alone and in complex with inhibitors of the quinol-binding site. Further, the structures of several mutagenic variants have been determined, revealing unexpected conformational rearrangements that give insight into enzyme activity. These preliminary results allowed us to generate a hypothesis for the catalytic reaction mechanism and that includes accompanying conformational motion. This sets the stage for the Aims of this proposal: In Aim 1, we will describe the chemical details of catalysis at the dicarboxylate-binding site. To achieve this, we will co-crystallize the quinol:fumarate reductase (QFR) with multiple small molecules and structurally investigate mutants that alter the catalytic efficiency. In Aim 2, we will characterize the motions associated with catalysis. We have already classified enzyme motions into three distinct types - interdomain motions, motions of a catalytic loop, and global motions. We have begun structural characterization of a site-directed mutant that interrupts normal domain motion. Complemented by computational methods that evaluate the energies of each trapped state, we will further characterize the large, interdomain motion using a combination of site-directed spin labeling and electron paramagnetic resonance spectroscopy. In Aim 3, we will investigate the influence of environmental fungicides on the catalytic activity of complex II. Here, we will co-crystallize several quinol-binding inhibitors with both the wild type E. coli QFR and a variant of the E. coli QFR that has fungicide binding properties similar to the human enzyme. We will further work to express human complex II with a heterologous system for structural studies.

描述（由申请人提供）：复合物II是在线粒体有氧呼吸中产生跨膜电化学梯度的四种电子转移酶之一。它的遗传改变或抑制对生物体的存活具有显著影响。复合体II超家族可能是最通用的呼吸酶，因为它参与有氧和无氧呼吸以及克雷布斯循环。复合体II结构的再循环用于多种目的表明，这种褶皱是进化古老的。这种单一的酶的检查，可以给洞察反丁烯二酸盐还原，琥珀酸氧化，天冬氨酸氧化，醌醇化学，电子转移，和跨膜电化学梯度的形成的生物机制。在初步研究中，复杂的II同系物醌醇：延胡索酸还原酶已被过度生产，结晶和结构确定单独和复杂的抑制剂的喹啉结合位点。此外，已经确定了几种诱变变体的结构，揭示了意想不到的构象重排，从而深入了解酶的活性。这些初步结果使我们能够产生一个假设的催化反应机制，包括伴随的构象运动。这为该提案的目标奠定了基础：在目标1中，我们将描述在二羧酸结合位点催化的化学细节。为了实现这一目标，我们将使醌醇：富马酸还原酶（QFR）与多种小分子共结晶，并从结构上研究改变催化效率的突变体。在目标2中，我们将描述与催化相关的运动。我们已经将酶的运动分为三种不同的类型--域间运动、催化环运动和全局运动。我们已经开始了一个定点突变，中断正常域运动的结构特征。通过计算方法，评估每个被困状态的能量的补充，我们将进一步表征的大，域间运动使用的组合的定点自旋标记和电子顺磁共振光谱。在目标3中，我们将研究环境杀真菌剂对配合物II的催化活性的影响。在这里，我们将共结晶几个喹啉结合抑制剂与野生型E。coli QFR和E. coli QFR，其具有与人类酶类似的杀真菌剂结合特性。我们将进一步的工作，表达人类复合物II的异源系统的结构研究。