Mechanisms of Energy Transduction in Heme-Copper Oxidases

血红素铜氧化酶的能量转换机制

• 批准号: 8729494
• 负责人: DENIS L. ROUSSEAU
• 金额: $ 26.55万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8729494
• 关键词: Automobile Driving; Bioenergetics; Catalytic Domain; Cattle; Chemistry; Complement; Computer Simulation; Consensus; Copper; Coupled; Coupling; Data; Deuterium; Disease; Electron Transport; Electrons; Elements; Enzymes; Eukaryota; Foundations; Generations; Goals; Heme; Heme Group; Human; Hydrogen; Iron; Kinetics; Knowledge; Light; Link; Membrane; Methodology; Methods; Mission; Mitochondria; Molecular; Molecular Conformation; Monitor; Movement; Mutagenesis; Mutate; Oxidases; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen; Pathway interactions; Peripheral; Physiology; Process; Production; Prokaryotic Cells; Proteins; Proton Pump; Proton-Motive Force; Protons; Pump; Raman Spectrum Analysis; Reaction; Reporter; Research; Role; Side; Signal Transduction; Site; Solvents; Structure; Techniques; Water; analog; base; copper oxidase; cytochrome c; cytochrome c oxidase; design; heme a; heme a3; human disease; improved; mutant; new technology; prevent; therapeutic target

DESCRIPTION (provided by applicant): Cytochrome c Oxidase (CcO), the terminal enzyme in the electron transfer chain of eukaryotes and prokaryotes, is responsible for over 90% of the oxygen utilization in the biosphere. The enzyme serves a dual role of (i) maintaining electron flow for oxidative phosphorylation, by catalyzing the four- electron reduction of O2 to H2O and (ii) creating a proton gradient for ATP production, by coupling the oxygen reduction chemistry to proton translocation. Although the oxygen reduction chemistry is relatively well understood, the mechanism by which the energy of the redox-linked oxygen reduction reaction is harnessed for proton translocation is unresolved. It remains as one of the major unsolved issues in bioenergetics. This knowledge gap is in part a result of the difficulty in detecting protons in the vast protein matrix of the enzyme. It is our hypothesis that the vibrational modes of the heme peripheral groups can serve as reporters of proton occupancy and movement in the enzyme. Based on this hypothesis, as supported by our preliminary data, a new methodology, hydrogen/deuterium exchange resonance Raman spectroscopy, will be developed and used to investigate the critical driving elements for proton translocation in CcO. The objective of this project is to improve our understanding of how the electron transfer and oxygen reduction chemistry regulates proton translocation. To achieve this objective three Specific Aims are proposed: (i) Define the resonance Raman markers of the peripheral heme groups that are sensitive to solvent H/D exchange; (ii) Determine how the solvent H/D sensitive resonance Raman modes are modulated by the redox processes; and (iii) Identify the roles of critical residues involved in coupling oxygen chemistry to proton translocation. To accomplish these Aims, the new technology will be combined with fast kinetic techniques and mutagenesis methods to investigate a mammalian enzyme, as well as its bacterial analogs with differing heme types. The experimental results will be complemented by computational modeling to advance our understanding of the proton pumping mechanism in CcO at the molecular level, as well as to shed new light on the evolutionary conservation of the structure and function of the oxidase superfamily of enzymes. The information derived from this multifaceted approach, which is unattainable by other techniques, will provide a foundation for the rational design of therapeutics targeting CcO related diseases.

描述（由申请人提供）：细胞色素c氧化酶（CcO）是真核生物和原核生物电子传递链中的末端酶，负责生物圈中90%以上的氧气利用。该酶具有双重作用：(i) 通过催化 O2 到 H2O 的四电子还原来维持氧化磷酸化的电子流，以及 (ii) 通过将氧还原化学与质子易位耦合来为 ATP 产生创建质子梯度。尽管氧还原化学相对较好地理解，但利用氧化还原连接的氧还原反应的能量进行质子易位的机制尚未解决。它仍然是生物能学中未解决的主要问题之一。这种知识差距的部分原因是难以检测酶的巨大蛋白质基质中的质子。我们的假设是，血红素外周基团的振动模式可以充当酶中质子占据和运动的报告者。基于这一假设，并得到我们初步数据的支持，我们将开发一种新的方法，即氢/氘交换共振拉曼光谱，并用于研究 CcO 中质子易位的关键驱动元件。该项目的目的是提高我们对电子转移和氧还原化学如何调节质子易位的理解。为了实现这一目标，提出了三个具体目标： (i) 定义对溶剂 H/D 交换敏感的外周血红素基团的共振拉曼标记； (ii) 确定溶剂 H/D 敏感共振拉曼模式如何通过氧化还原过程进行调制； (iii) 确定参与氧化学与质子易位耦合的关键残基的作用。为了实现这些目标，新技术将与快速动力学技术和诱变方法相结合，以研究哺乳动物酶及其具有不同血红素类型的细菌类似物。实验结果将通过计算模型得到补充，以促进我们在分子水平上对 CcO 中质子泵送机制的理解，并为酶氧化酶超家族的结构和功能的进化保守性提供新的线索。从这种多方面方法获得的信息是其他技术无法获得的，将为合理设计针对 CcO 相关疾病的疗法提供基础。