Mechanisms of Energy Transduction in Heme-Copper Oxidases

描述（由申请人提供）：细胞色素c氧化酶（Cytochrome c Oxidase, CcO）是真核生物和原核生物电子传递链的末端酶，负责生物圈90%以上的氧气利用。该酶具有双重作用：(i)通过催化氧还原成水的四电子还原来维持氧化磷酸化的电子流；（ii）通过耦合氧还原化学和质子易位来产生ATP的质子梯度。尽管对氧还原化学的理解相对较好，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不清楚。它仍然是生物能量学中未解决的主要问题之一。这种知识差距部分是由于难以在酶的巨大蛋白质基质中检测质子。我们假设血红素外周基团的振动模式可以作为酶中质子占用和运动的报告者。基于这一假设，基于我们的初步数据，我们将开发一种新的方法——氢/氘交换共振拉曼光谱，并将其用于研究CcO中质子移位的关键驱动因素。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被氧化还原过程调制；（iii）确定耦合氧化学与质子易位的关键残基的作用。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，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，从而在分子水平上进一步了解氧化酶超家族的质子泵送机制，并为氧化酶超家族的结构和功能的进化保护提供新的思路。从这种多方面的方法中获得的信息是其他技术无法获得的，将为合理设计针对CcO相关疾病的治疗方法提供基础。

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. PUBLIC HEALTH RELEVANCE: The proposed line of research will provide the mechanistic details underlying the coupling between the oxygen reduction chemistry and proton translocation in cytochrome c oxidase, one of the most important enzymes in physiology. It is relevant to the part of the NIH's mission that pertains to developing fundamental knowledge that will ultimately help reduce the burden of human disease.