X-ray Spectroscopic Studies on Heme-Copper Oxidases

说明(申请人提供)：血红素-铜氧化物酶是哺乳动物和多种真核和原核生物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，最终驱动三磷酸腺苷的内源合成，在细胞生物能量学和人类健康中发挥着关键作用。随着人们对氧化还原中心的结构和催化机理有了更好的了解，导致关键氧化还原中心的组装和金属化的途径将成为下一个 边疆。这项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中心的组装和金属化的蛋白质上。我们的实验计划分为 4个方面。(I)我们将表征参与铜转移到CUA中心的氧化和还原形式的Sco蛋白家族的铜中心。(Ii)我们将利用硒蛋氨酸取代来探索Sco与其伴侣蛋白(含CUA的CCO亚基II)之间金属转移的机制，以提供位置特异性的金属转移反应的XAS探针。(Iii)我们将通过体内铜(I)和铜(II)定点突变的检测来探讨枯草杆菌Sco同源物的功能。(Iv)我们将应用表达蛋白连接作为一种工具，在Sco、CUA和COX 11的活性中心掺入硒半胱氨酸，作为额外的XAS光谱功能探针。这些研究将提供参与细胞色素氧化酶组装的蛋白质的急需的生物物理数据，并将补充将CCO组装缺陷与呼吸系统疾病联系起来的广泛的遗传学证据。

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.