Mechanisms of Energy Transduction in Heme-Copper Oxidases

描述(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转移链中的末端酶，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O2的四电子还原为H2O，维持氧化磷酸化的电子流；(Ii)通过将氧还原化学与质子转移相耦合，为ATP的产生创造质子梯度。虽然氧还原化学相对较好，但氧化还原连接的氧还原反应的能量被利用来进行质子转移的机制还没有解决。它仍然是生物能量学中尚未解决的主要问题之一。这种知识差距在一定程度上是因为难以在酶的巨大蛋白质基质中检测质子。我们的假设是，血红素外围基团的振动模式可以作为质子在酶中占据和移动的报告。基于这一假设，在我们的初步数据的支持下，我们将发展一种新的方法，氢/氚交换共振拉曼光谱，并用于研究CcO中质子转移的关键驱动因素。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转移的理解。为了实现这一目标，提出了三个具体的目标：(I)定义对溶剂H/D交换敏感的外围血红素基团的共振拉曼标记；(Ii)确定溶剂H/D敏感的共振拉曼模式是如何被氧化还原过程调制的；以及(Iii)确定参与氧化学与质子转移耦合的关键残基的作用。为了实现这些目标，这项新技术将与快速动力学技术和诱变方法相结合，以研究一种哺乳动物酶，以及其具有不同类型的血红素的细菌类似物。实验结果将得到计算模型的补充，以在分子水平上加深我们对CCOO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提供新的线索。从这种多方面的方法获得的信息是其他技术无法获得的，将为针对CCOO相关疾病的治疗方案的合理设计提供基础。 与公共健康相关：拟议的研究路线将提供氧还原化学和细胞色素c氧化酶质子转移之间耦合的机制细节，细胞色素c氧化酶是生理学中最重要的酶之一。这与美国国立卫生研究院的使命有关，即发展基础知识，最终将有助于减轻人类疾病的负担。

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.