X-ray Spectroscopic Studies on Heme-Copper Oxidases

描述（由申请人提供）：血红素-铜氧化酶是哺乳动物和多种真核生物和原核生物的电子传递链的末端氧化酶。它们通过将氧还原的氧化还原能转化为质子动力，最终用于驱动ATP的内源性合成，在细胞生物能量学和人类健康中发挥着关键作用。随着对结构和催化机理的深入了解，导致关键氧化还原中心组装和代谢的途径正在成为下一个研究热点。 边疆该建议旨在将我们对血红素铜氧化酶的研究扩展到这一领域，并将重点放在参与CuA和CuB中心组装和代谢的蛋白质上。我们的实验项目分为 4个领域。(i)我们将表征铜中心的氧化和还原形式的家庭参与铜转移到CuA中心的Sco蛋白。(ii)我们将探测Sco和它的伙伴蛋白，含铜CCO亚基II，使用硒代甲硫氨酸取代提供一个位点特异性的XAS探针的金属转移反应性之间的金属转移的机制。(iii)我们将探讨B的Sco同源物的功能。通过Cu（I）和Cu（II）位点特异性突变的体内测定，在枯草芽孢杆菌中表达。(iv)我们将应用表达的蛋白质连接作为工具，用于在Sco、CuA和考克斯11的活性中心掺入硒代半胱氨酸，作为功能的附加XAS光谱探针。这些研究将提供急需的生物物理数据的蛋白质参与细胞色素氧化酶的组装，并将补充广泛的遗传证据连接缺陷CCO大会与呼吸系统疾病。

DESCRIPTION (provided by applicant): Hemme-copper oxidases are the terminal oxidases of the electron transport chains of mammals and a wide variety of eukaryotic and prokaryotic organisms. They play a pivotal role in cellular bioenergetics and human health by converting the redox energy of oxygen reduction into a protonmotive force, which is ultimately used to drive the endogonic synthesis of ATP. As the structure and catalytic mechanism have become better understood, the pathways leading to the assembly and metallation of the critical redox centers are emerging as the next frontier. This proposal aims to extend our investigations on heme-copper oxidases into this area and will focus on the proteins involved in the assembly and metallation of the CuA and CuB centers. Our experimental program is divided into 4 areas. (i) We will characterize the copper centers of the oxidized and reduced forms of the family of Sco proteins involved in copper transfer to the CuA center. (ii) We will probe the mechanism of metal transfer between Sco and its partner protein, the CuA-containing CCO subunit II, using selenomethionine substitution to provide a site-specific XAS probe of metal transfer reactivity. (iii) We will probe the function of the Sco homologue of B. subtilis via in vivo assay of Cu(I) and Cu(II) site-specific mutations. (iv) We will apply expressed protein ligation as a tool for the incorporation of selenocysteine at the active centers of Sco, CuA and Cox 11 as an additional XAS spectroscopic probe of function. These studies will provide much-needed biophysical data on the proteins involved in cytochrome oxidase assembly, and will complement the extensive genetic evidence linking defects in CCO assembly with respiratory disease.