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: 7576701
• 负责人: T M Iverson
• 金额: $ 26.56万
• 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/7576701
• 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。其基因改变或抑制对生物体的生存具有显着影响。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。复杂的 II 结构的回收用于多种目的表明这种折叠是进化古老的。对这种单一酶的检查可以深入了解富马酸还原、琥珀酸氧化、天冬氨酸氧化、对苯二酚化学、电子转移和跨膜电化学梯度形成的生物学机制。在初步研究中，复合物 II 同系物对苯二酚：富马酸还原酶已过量产生、结晶，并单独确定其结构，并与对苯二酚结合位点的抑制剂复合。此外，几种诱变变体的结构已经确定，揭示了意想不到的构象重排，从而深入了解酶活性。这些初步结果使我们能够对催化反应机制产生假设，其中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，我们将描述二羧酸盐结合位点催化的化学细节。为了实现这一目标，我们将把对苯二酚：富马酸还原酶（QFR）与多个小分子共结晶，并在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。我们已经开始对中断正常结构域运动的定点突变体进行结构表征。辅以评估每个俘获态能量的计算方法，我们将结合定点自旋标记和电子顺磁共振波谱进一步表征大的域间运动。在目标3中，我们将研究环境杀菌剂对配合物II催化活性的影响。在这里，我们将与野生型大肠杆菌 QFR 和具有与人类酶相似的杀菌剂结合特性的大肠杆菌 QFR 变体共结晶几种对苯二酚结合抑制剂。我们将进一步致力于用异源系统表达人类复合物II以进行结构研究。