Mechanisms of Energy Transduction in Heme-Copper Oxidases

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至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偶联来产生ATP产生的质子梯度。虽然氧还原化学是相对较好的理解，机制，其中的氧化还原连接的氧还原反应的能量是利用质子易位是未解决的。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大量蛋白质基质中检测质子。这是我们的假设，血红素周边基团的振动模式可以作为记者的质子占有率和运动的酶。基于这一假设，我们的初步数据的支持下，一种新的方法，氢/氘交换共振拉曼光谱，将被开发和用于调查的关键驱动元素的质子易位在CcO。这个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，提出了三个具体的目标：（i）定义的共振拉曼标记的周边血红素基团是敏感的溶剂H/D交换;（ii）确定如何溶剂H/D敏感的共振拉曼模式调制的氧化还原过程;和（iii）确定参与耦合氧化学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，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建模来补充，以促进我们在分子水平上对CcO中质子泵机制的理解，并为氧化酶超家族的结构和功能的进化保守性提供新的见解。从这种多方面的方法，这是无法达到的其他技术，将提供一个基础，合理设计的治疗靶向CcO相关疾病。 公共卫生相关性：拟议的研究线将提供的机制细节之间的耦合的氧还原化学和质子易位的细胞色素c氧化酶，在生理学中最重要的酶之一。它与NIH的使命的一部分有关，即开发最终有助于减轻人类疾病负担的基础知识。

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.