Mechanisms of Respiration

说明(申请人提供)：细胞色素c氧化酶是为细胞提供能量的主要酶之一。它通过将氧气还原与质子泵和最终氧化磷酸化相结合来实现这一点。嗜热菌的细胞色素Ba3是一种含有天然突变的同系物，这些突变也赋予了蛋白质新的化学行为。这些可以用来识别能量转导机制的一般特征。在过去的资助期间，我们已经开发了Ba3的天然底物Thermus细胞色素C552和Ba3本身的表达系统。我们也有这两种分子的内部X射线结构。这些工具使我们能够开始研究这种酶的机制。我们的实验方法包括研究Fea3-CUB位点的详细化学结构，探索酶的机制，探索接近CUB的非金属配体氨基酸的作用，以及探索远离酶氧化还原中心的氨基酸残基在质子转移中的作用。有四个具体的目标：[1]确定FeA3-O-Cub桥的化学性质。[2]确定细胞色素Ba3将氧气还原为水的机理，包括关于电子转移事件的详细信息，FeA3-氧中间体的化学性质，它们出现和消失的特征时间，以及在这一过程中质子交换的相应动力学和化学计量。[3]我们将确定与组氨酸(I-H233)配体共价连接的新型酪氨酸(I-Y237)在CUB中的作用。[4]我们将使用定点突变来评估细胞色素Ba3结构提示的三个新的质子转移途径中的每一个的作用。在整个拟议的工作中，我们使用了广泛的生化、光谱和X射线结构工作来对这种重要的酶进行积极的、多管齐下的研究。这项工作对人类健康的意义在于提供关于生物系统节约能源的基本信息。

DESCRIPTION (provided by applicant): Cytochrome c oxidase is one of the principal enzymes that provides energy for the cell. It does so by coupling dioxygen reduction to proton pumping and ultimately oxidative phophorylation. Cytochrome ba3 from Thermus thermophilus is a homolog that contains a storehouse of natural mutations that also endow the protein with novel chemical behaviors. These can be used to identify general features of energy transduction mechanisms. During past funding periods, we have developed expression systems for Thermus cytochrome c552, the natural substrate of ba3, and for ba3 itself. We also have in-house X-ray structures of both these molecules. These tools have permitted us to begin studies of the enzyme's mechanism. Our experimental approach involves study of the detailed chemistry of the Fea3-CuB site, exploration of the enzyme's mechanism, probing the roles of non-metal liganding amino acids close to CuB, and exploring the roles of amino acid residues, distant from the enzyme's redox centers, in proton transfer. There are four Specific Aims: [1] To determine the chemical nature of the Fea3-O-CuB bridge. [2] To determine the mechanism whereby cytochrome ba3 reduces dioxygen to water, including detailed information about electron transfer events, the chemical nature of Fea3-oxygen intermediates, their characteristic times of appearance and disappearance, and the corresponding dynamics and stoichiometry of proton exchanges during this process. [3] We will determine the role of the novel tyrosine (I-Y237) that is covalently linked to a histidine (I-H233) ligand to CuB. [4] We will use site-directed mutagenesis to assess the role of each of three novel proton transfer pathways suggested by the structure of cytochrome ba3. Throughout the proposed work, we use a wide variety of biochemical, spectral, and X-ray structural work to mount an aggressive, multi-pronged study of this important enzyme. The significance of this work to human health lies in providing fundamental information about the conservation of energy by biological systems.