X-ray Spectroscopic Studies on Heme-Copper Oxidases

描述：（由申请人提供）血红素铜氧化酶的核心作用 在生物能源生产中的作用使它们成为了 遍布地球仪的许多实验室。由于晶体结构现在 确定了氧化还原中心的配位和质子的身份 泵送通道，注意力集中在确定机械细节。 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 X射线吸收光谱（XAS）的应用，以独特地定义 铜中心在许多不同的衍生物中的配位 氧化酶家族的成员。缺乏来自CuB的光或磁信号 使XAS特别适用于测试建议的休息结构， 双核中心的中间态。在此更新申请中，我们 将扩大我们的研究，包括氧化酶家族的新成员， 催化中间体首先，我们将研究Cub的静息状态， 来自枯草芽孢杆菌，Acidianus ambivalens， 嗜气硫碱微菌和栖热菌的一个CuA缺失突变体 嗜热菌将仔细检查简化形式，以获得以下证据： 功能上重要的组氨酸解离和氯连接， 观察到B（03）酶。我们接下来将确定关键的结构 反应途径中的中间体，再次寻找 组氨酸与CuB解离被认为是质子的重要元素 抽气这些数据将得到无机和工程研究的支持。 细胞色素氧化酶的蛋白质模型。最后，我们将开始探索 细胞色素氧化酶与最近发现的 金属伴侣，这被认为是必要的适当成熟的 完整的氧化酶。

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.