Mechanisms of Respiration

描述（由申请人提供）：细胞色素c氧化酶是为细胞提供能量的主要酶之一。它是通过偶联分子氧还原质子泵和最终氧化磷酸化。嗜热栖热菌的细胞色素ba 3是一种同源物，含有一个天然突变库，这些突变也赋予蛋白质新的化学行为。这些可用于识别能量转换机制的一般特征。在过去的资助期间，我们已经开发了表达系统的栖热菌细胞色素c552，ba 3的天然底物，并为ba 3本身。我们也有这些分子的内部X射线结构。这些工具使我们能够开始研究酶的机制。我们的实验方法包括研究Fea 3-CuB位点的详细化学，探索酶的机制，探索靠近CuB的非金属配体氨基酸的作用，并探索远离酶的氧化还原中心的氨基酸残基在质子转移中的作用。具体目的有四个：[1]确定Fea 3-O-CuB桥的化学性质。[2]为了确定细胞色素ba 3减少双氧水的机制，包括有关电子转移事件的详细信息，Fea 3-氧中间体的化学性质，其出现和消失的特征时间，以及在此过程中质子交换的相应动力学和化学计量。[3]我们将确定新的酪氨酸（I-Y237），是共价连接到一个组氨酸（I-H233）配体的作用。[4]我们将使用定点诱变来评估细胞色素ba 3的结构所建议的三种新的质子转移途径中的每一种的作用。在整个拟议的工作中，我们使用了各种各样的生物化学，光谱和X射线结构工作，对这种重要的酶进行了积极的，多管齐下的研究。这项工作对人类健康的意义在于提供有关生物系统能量守恒的基本信息。

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.