X-ray Spectroscopic Studies on Heme-Copper Oxidases

描述：(申请人提供)血红素-铜氧化酶的核心作用 在生物能源生产方面已经使它们成为 全球各地的许多实验室。因为晶体结构现在已经 定义了氧化还原中心的配位和质子的同一性 对于抽水渠道，人们的注意力集中在确定机械细节上。 要解开这些机制，需要应用专门的 光谱和生物物理技术。我们的贡献是 应用X射线吸收光谱(XAS)唯一确定 几种不同衍生物中铜中心的配位作用 氧化酶家族的成员。缺乏来自幼崽的光或磁信号 使XAS特别适用于测试建议的休眠结构和 双核中心的中间态。在此续期申请中，我们 将扩大我们的研究范围，以包括氧化酶家族的新成员和 催化中间体。首先，我们将调查幼崽的休息状态 枯草芽孢杆菌、环状酸杆菌、 Thioalcalommicrobium Aerophilum和Thermus CuA缺失突变体 嗜热菌。减少的表格将被仔细检查，以寻找证据 功能重要的组氨酸解离和氯离子连接 观察b(03)酶。接下来我们将确定KEY的结构 反应途径中的中间体，再次寻找证据 幼崽的组氨酸解离被认为是质子的重要元素 抽水。这些数据将得到无机物和工程学研究的支持 细胞色素氧化酶的蛋白质模型。最后，我们将开始探索 细胞色素氧化酶与新近发现的相互作用机制 金属配位伴侣，被认为是正常成熟所必需的 膜上完整的氧化物酶。

DESCRIPTION: (provided by applicant) The central role of heme-copper oxidases in biological energy production has made them the targets of intense study in many laboratories across the globe. Since the crystal structures have now defined the coordination of the redox centers and the identity of proton pumping channels, attention has focused on determining mechanistic details. Unraveling these mechanisms has required application of specialized spectroscopic and biophysical techniques. Our contribution has been the application of X-ray absorption spectroscopy (XAS) to uniquely define the coordination of the copper centers in a number of derivatives of different members of the oxidase family. The lack of optical or magnetic signals from CuB makes XAS particularly relevant to testing proposed structures for resting and intermediate states of the binuclear center. In this renewal application, we will extend our studies to include novel members of the oxidase family and catalytic intermediates. First, we will investigate resting states of the CuB centers of quinol oxidases from Bacillus subtilis, Acidianus ambivalens, Thioalcalomicrobium aerophilum, and a CuA-deleted mutant of Thermus thermophilus. The reduced forms will be examined carefully for evidence of functionally important histidine dissociation and chloride ligation as recently observed for the b(03) enzyme. We will next determine the structures of key intermediates in the reaction pathway, again searching for evidence of histidine dissociation from CuB proposed to be an important element of proton pumping. These data will be backed up by studies on inorganic and engineered protein models for cytochrome oxidase. Finally, we will begin to explore the mechanism of interaction of cytochrome oxidase with recently discovered metallochaperones, which are believed to be essential for proper maturation of the intact oxidase in the membrane.