Energy Transduction in Cytochrome Oxidase

描述(申请人提供)：鉴于细胞色素c氧化酶对线粒体新陈代谢的决定性影响以及线粒体在控制细胞生命和死亡中的中心作用，更好地了解细胞色素c氧化酶的功能和调节的重要性已经变得越来越明显。在这笔资金的支持下，我们从线粒体模型系统球形红细菌的酶的一些新的高分辨率结构中衍生出了这个复杂的能量保存机制的新视角。这些揭示了与氧化还原状态改变相关的以前未观察到的构象变化，以及细菌和哺乳动物中保守的脂质和类固醇结合位点的存在。这一提议旨在通过进一步的结晶学努力以获得新的和更高分辨率的晶体形式，并通过研究脂类配体对酶的活性、稳定性和效率的影响，来确定新的结构发现的意义。其具体目标是：1)利用分子工程策略和机器人晶体筛选，产生更多的2和4亚单位红细菌氧化酶的晶体形式；2)创建、表征和结晶有助于捕获新的催化中间体或抑制灵活性的突变体，寻找新的构象状态并测试构象变化的重要性；3)筛选具有潜在生理意义或抑制或稳定作用的类固醇结合位点的替代配体。这些研究的一个主要工具将是结晶学，但我们全面分析氧化酶功能和光谱特征的能力，包括在线晶体光谱，将是解释结构发现的关键。预期的结果是对细胞色素氧化酶能量转换的分子机制有了新的理解，包括构象变化在门控和效率中的作用，以及脂类配体的调节作用。长期目标是通过结构/功能分析和发现作为生理效应器、结晶助剂、机械探针或可以调节氧化酶活性的药物的前体的化合物，更好地了解细胞色素氧化酶在包括癌症、肥胖、糖尿病和衰老在内的代谢性疾病状态中的作用。 与公共健康相关：细胞色素c氧化酶是正常生理功能中的关键角色，消耗我们呼吸的90%以上的氧气，并直接参与我们用来支持所有生命过程的大部分能量的产生；该研究计划的目标是更好地了解细胞色素氧化酶的功能和调节。鉴于细胞色素氧化酶对线粒体能量代谢的决定性影响以及线粒体在控制细胞生死中的中心作用，这一目标的重要性日益明显。长期目标是通过结构/功能分析和新化合物的发现，更好地了解细胞色素氧化酶在包括癌症、肥胖症、糖尿病和衰老在内的代谢性疾病状态中的作用，这些化合物是生理效应器、结晶助剂、机械探针或可以调节氧化酶活性和效率的药物的前体。

DESCRIPTION (provided by applicant): The importance of developing a better understanding of the function and regulation of cytochrome c oxidase has become increasingly apparent, given its decisive influence on mitochondrial metabolism and the central role of mitochondria in controlling cell life and death. Research supported by this grant has led us to a new perspective on this complex energy conserving machine, derived from a number of new high resolution structures of the enzyme from the mitochondrial model system, Rhodobacter sphaeroides. These reveal previously unobserved changes in conformation associated with altered redox state, and the presence of lipid and steroid binding sites conserved in bacteria and mammals. This proposal is aimed at determining the significance of the novel structural findings through further crystallographic efforts designed to obtain new and higher resolution crystal forms, and through studies of the effects of lipidic ligands on activity, stability and efficiency of oxidase. The Specific Aims are: 1) to generate additional crystal forms of two and four subunit Rhodobacter oxidase, using molecular engineering strategies and robotic crystal screening; 2) to create, characterize and crystallize mutants that facilitate the trapping of novel catalytic intermediates or that restrain flexibility, to look for new conformational states and test the importance of conformational change; 3) to screen for alternative ligands of a steroid binding site, with potential physiological significance, or inhibitory or stabilizing effects. A major tool in these studies will be crystallography, but our ability to comprehensively analyze oxidase function and spectral features, including on-line crystal spectra, will be crucial to interpreting the structural findings. The expected outcome is a new level of understanding of the molecular mechanism of energy conversion in cytochrome oxidase, including the role of conformational change in gating and efficiency, and the regulatory effects of lipidic ligands. The long term goal is to better understand the involvement of cytochrome oxidase in metabolic disease states including cancer, obesity, diabetes and aging, through structure/function analysis and the discovery of compounds that are physiological effectors, crystallization aids, mechanistic probes, or precursors to drugs that can modulate oxidase activity. PUBLIC HEALTH RELEVANCE: Cytochrome c oxidase is a critical player in normal physiological function, consuming more than 90% of the oxygen we breathe and being directly involved in the production of most of the energy we use to support all life processes; the goal of this research program is to develop a better understanding of cytochrome oxidase function and regulation. The importance of this objective has become increasingly apparent, given the decisive influence of cytochrome oxidase on mitochondrial energy metabolism and the central role of mitochondria in controlling cell life and death. The long term goal is to better understand the involvement of cytochrome oxidase in metabolic disease states including cancer, obesity, diabetes and aging, through structure/ function analysis and the discovery of new compounds that are physiological effectors, crystallization aids, mechanistic probes, or precursors to drugs that can modulate oxidase activity and efficiency.