Proton Transfer Dynamics in Heme-Copper Oxidases

血红素铜氧化酶中的质子转移动力学

• 批准号: 6893238
• 负责人: RICHARD BRIAN DYER
• 金额: $ 12.54万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6893238
• 关键词: Raman spectrometry; amine oxidase (copper); bioenergetics; chemical transfer reaction; cytochrome oxidase; electron transport; enzyme activity; enzyme structure; heme; hydrogen ions; infrared spectrometry; interferometry; intermolecular interaction; ion transport; lasers; membrane channels; oxidation reduction reaction; protein structure function; site directed mutagenesis; ultraviolet spectrometry

The goal of this subproject, entitled Proton Transfer Dynamics in Heme-Copper Oxidases, is to understand the fundamental mechanisms of energy transduction in the heme-copper oxidases, with a particular emphasis on the role of the protein dynamics in effecting redox driven proton pumping. The heme-copper oxidases are a class of molecular machines responsible for energy transduction in aerobic respiration by the conversion of redox energy into a transmembrane proton gradient. Recent developments provide the opportunity for the full elucidation of the molecular mechanism of redox-linked proton pumping by the heme-copper oxidases. The determination of high-resolution structures for two closely related enzymes in this family provides an essential starting point for interpreting functional studies. These structures can guide the development of testable hypotheses regarding the mechanism(s) of coupling of redox energy to proton translocation. However, the structures by themselves cannot resolve this issue, for they provide only a static picture and thus no information on the intermediate states in the redox cycle and inherently lack the resolution required to determine changes in protonation states. Another key element is the availability of specific mutations in bacterial heme-copper oxidases. These mutations allow for direct evaluation of the role of particular residues in proton pumping. A final component that contributes to the current opportunity is provided by the substantial recent technical advances that have been made in the field of time-resolved vibrational spectroscopy. These advances, which were in significant part pioneered in our labs, now provide the capability of observing protonation state changes of single amino acid residues, or redox state changes in specific structures and coupled protein conformational changes, in an enzyme of the size of cytochrome c oxidase, and following the dynamics of these changes over many decades of time. Isotope edited FTIR difference techniques coupled with site directed mutagenesis provide definitive assignment of the vibrational observables to specific, functionally related structures. Vibrational spectroscopy can thus provide the detailed structural and dynamic information about redox and protonation state changes necessary to define the molecular mechanisms of redox coupled proton transport. We propose to apply equilibrium and time-resolved vibrational techniques to elucidate the structures and dynamics of the proton conduction pathways and to elucidate the mechanism of coupling the redox reactions to proton translocation. Specifically, we plan to (1) elucidate the nature of the proton conduction pathways in CcO and (2) determine the role of protein dynamics in coupling electron and proton transfer reactions in CcO, using vibrational spectroscopy.

这个子项目的目标，题为血红素铜氧化酶的质子转移动力学，是 理解血红素-铜氧化酶中能量转导的基本机制，特别强调蛋白质动力学在影响氧化还原驱动质子泵中的作用。血红素铜氧化酶是一类负责有氧呼吸中能量转换的分子机器，通过将氧化还原能量转化为跨膜质子梯度。最近的发展提供了机会，充分阐明的氧化还原连接的质子泵的血红素铜氧化酶的分子机制。在这个家庭中的两个密切相关的酶的高分辨率结构的测定提供了一个重要的起点，解释功能的研究。这些结构可以指导关于氧化还原能量与质子移位的耦合机制的可检验假设的发展。然而，结构本身不能解决这个问题，因为它们仅提供静态图像，因此没有关于氧化还原循环中的中间状态的信息，并且固有地缺乏确定质子化状态变化所需的分辨率。另一个关键因素是细菌血红素铜氧化酶中特定突变的可用性。这些突变允许直接评估特定残基在质子泵中的作用。最后一个促成目前机会的因素是，最近在时间分辨领域取得的重大技术进展， 振动光谱学这些进展，这是在我们的实验室开创了显着的一部分，现在提供的能力，观察质子化状态变化的单个氨基酸残基，或氧化还原状态的变化，在特定的结构和耦合蛋白质构象的变化，在酶的细胞色素c氧化酶的大小，并遵循这些变化的动力学在几十年的时间。同位素编辑的FTIR差异技术加上定点诱变提供明确的分配的振动观测到特定的，功能相关的结构。因此，振动光谱学可以提供关于氧化还原和质子化状态变化的详细结构和动态信息，这些信息是定义分子结构所必需的。 氧化还原耦合质子传输的分子机制。我们建议应用平衡和时间分辨振动技术来阐明质子传导途径的结构和动力学，并阐明耦合的氧化还原反应的质子易位的机制。具体来说，我们计划（1）阐明质子传导途径的性质在CcO和（2）确定蛋白质动力学的作用，在耦合电子和质子转移反应在CcO，使用振动光谱。