Energy Transduction in Cytochrome Oxidase

描述（由申请人提供）：鉴于细胞色素c氧化酶对线粒体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核心作用，更好地了解细胞色素c氧化酶的功能和调控的重要性已经变得越来越明显。这项拨款支持的研究使我们对这种复杂的节能机器有了新的看法，这种机器源于线粒体模型系统球形红杆菌中酶的许多新的高分辨率结构。这些揭示了以前未观察到的与氧化还原状态改变相关的构象变化，以及在细菌和哺乳动物中保守的脂质和类固醇结合位点的存在。该建议旨在通过进一步的晶体学努力来确定新结构发现的意义，旨在获得新的和更高分辨率的晶体形式，并通过研究脂质配体对氧化酶的活性，稳定性和效率的影响。具体目标是：1)利用分子工程策略和机器人晶体筛选，生成2亚基和4亚基红杆菌氧化酶的额外晶体形式；2)创建、表征和结晶有利于捕获新型催化中间体或抑制柔性化的突变体，寻找新的构象状态并测试构象变化的重要性；3)筛选类固醇结合位点的替代配体，具有潜在的生理意义，或抑制或稳定作用。这些研究的主要工具将是晶体学，但我们综合分析氧化酶功能和光谱特征的能力，包括在线晶体光谱，将对解释结构发现至关重要。预期的结果是对细胞色素氧化酶能量转化的分子机制有了新的认识，包括构象变化在门控和效率中的作用，以及脂质配体的调节作用。长期目标是通过结构/功能分析和发现生理效应物、结晶辅助物、机制探针或调节氧化酶活性的药物前体化合物，更好地了解细胞色素氧化酶在代谢疾病状态（包括癌症、肥胖、糖尿病和衰老）中的作用。

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.