ENERGY CONSERVATION AND TRANSFER IN MITOCHRONDRIA

线粒体中的能量守恒和转移

• 批准号: 3482912
• 负责人: YOUSSEF HATEFI
• 金额: $ 54.6万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-15 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3482912
• 关键词: NAD(H) phosphate; NAD(P)H dehydrogenase; adenosine triphosphate; adenosinetriphosphatase; antibody; bioenergetics; biological transport; chloroplasts; conformation; cow; cytochrome b; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; flavoproteins; hydrogen transporting ATP synthase; iron sulfur protein; laboratory rabbit; laboratory rat; membrane proteins; metalloproteins; mitochondrial membrane; oxidative phosphorylation; phosphotransferases; protonation; radiotracer; succinate dehydrogenase

This is an ongoing comprehensive program concerned with study of the structure, composition, function, regulation, and interaction of the enzyme complexes of the mitochondrial electron transport/oxidative phosphorylation system, which were previously isolated, purified and characterized by the applicant and coworkers. Those under study in recent years have been Complex I (NADH:ubiquinone oxidoreductase), Complex II (succinate:ubiquinone oxidoreductase), Complex III (ubiquinol:cytochrome c-oxidoreductase), and Complex V (ATP synthase). Studies planned for the next grant period include the following: Complex I: Structure of iron-sulfur flavoprotein (FP) and iron-sulfur protein (IP) fragments; study of electron transfer from FP to IP; isolation of iron-sulfur (FeS)-containing polypeptides of the hydrophobic protein fraction; delineation of the inhibition sites of dicyclohexylcarbodiimide and ethoxy-formic anhydride (EFA); attempt to differentiate the Em of FeS clusters N3 and N4; study possible effect of guanidine on site 1 coupling. Complex II: Separation of two subunits of cytochrome b560; study of role of each subunit (and of b560 heme) in electron transfer from succinate dehydrogenase (SDH) to ubiquinone (Q) and in reconstitution with Complex III; attempt at resolution of SDH into its two subunits without altering their FeS cluster structure for localization of clusters S1, S2 and S3. Complex III: Delineate inhibition sites of EFA, myxothiazol, stigmalellin, antimycin, etc.; test the individual steps of the Q-cycle hypothesis; study effect of various inhibitors on Complex III structure. Complex V: Isolate subunits 6 and 8 by electroelution; use antibodies to subunits 6 and 8 to study their possible roles in ATP synthesis and hydrolysis; check whether there is a polypeptide in mitochondria and Complex V that is structurally related to subunit-6; localize the F0 thiols whose modifications result in (a) uncoupling, and (b) inhibition of ATPase activity. Mechanism of Oxidative Phosphorylation: Continuation of studies on the mechanism of energy transfer in ATP synthase (we have evidence that, in the absence of proton translocation, modifications at specific F0 loci result in changes in the mode of ATP binding to F1); continuation of studies on the kinetic modalities of ATP synthesis and the role of electrochemical potential of Proton in interconversion of these kinetic modes; extension of these studies to E. coli membranes, chloroplasts and chromatophores.

这是一个持续进行的与学习有关的综合计划 的结构、组成、功能、调节和 线粒体酶复合物的相互作用 电子传递/氧化磷酸化系统， 申请人先前分离、纯化和表征 和同事。 近年来研究的对象是 复合物 I（NADH：泛醌氧化还原酶）、复合物 II （琥珀酸：泛醌氧化还原酶），复合物 III （泛醇：细胞色素 c-氧化还原酶）和复合物 V（ATP 合酶）。 计划下一个资助期的研究包括 如下：复合物 I：​​铁硫黄素蛋白 (FP) 的结构 和铁硫蛋白（IP）片段；电子转移研究 从FP到IP；含铁硫 (FeS) 的分离 疏水蛋白部分的多肽；的划定 二环己基碳二亚胺和乙氧基甲酸的抑制位点 酸酐（EFA）；尝试区分 FeS 团簇的 Em N3和N4；研究胍对位点 1 偶联的可能影响。 复合物 II：细胞色素 b560 两个亚基的分离；学习 每个亚基（和 b560 血红素）在电子转移中的作用 从琥珀酸脱氢酶 (SDH) 到泛醌 (Q) 以及 用复合物 III 重构；尝试将 SDH 解析为 它的两个亚基不改变它们的FeS簇结构 簇 S1、S2 和 S3 的定位。 情结 III：描绘 EFA、myxothiazol、stigmalellin、抗霉素等的抑制位点； 检验 Q 循环假设的各个步骤；研究效果 复合物 III 结构的各种抑制剂。 复合物 V：分离 通过电洗脱分离亚基 6 和 8；使用亚基 6 的抗体和 8 研究它们在ATP合成和水解中可能的作用； 检查线粒体中是否存在多肽 复合体 V 与 subunit-6 结构相关；本地化 F0 硫醇的修饰导致 (a) 解偶联，以及 (b) 抑制 ATP 酶活性。 氧化机制 磷酸化：继续研究其机制 ATP合酶中的能量转移（我们有证据表明，在 不存在质子易位、特定 F0 位点的修饰 导致 ATP 与 F1 结合的模式发生变化）；延续 ATP合成动力学模式及其作用的研究进展 质子在这些物质相互转化中的电化学势 动力学模式；将这些研究扩展到大肠杆菌膜， 叶绿体和色素细胞。