XAS STUDIES OF HEME-COPPER ASSEMBLY PROTEINS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。血红素-铜氧化酶是哺乳动物和多种真核及原核生物电子传递链的末端氧化酶。它们在细胞生物能量学和人类健康中发挥着关键作用，将氧还原的氧化还原能转化为质子动力，最终用于驱动三磷酸腺苷的内源合成。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组装和金属化的途径正在成为下一个前沿。这项提案旨在将重点放在参与CUA中心组装和金属化的蛋白质上。我们的实验计划分为两个区域。(I)我们将通过XAS结构研究结合天然蛋白的体内功能分析和铜(I)和铜(II)位点特异性突变来探讨枯草杆菌Sco同源物的功能；(Ii)我们将利用硒蛋氨酸取代来探索Sco与其含有CUA的CCO亚基II之间金属转移的机制，以提供金属转移活性的位点特异性XAS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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heme-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 will focus on the proteins involved in the assembly and metallation of the CuA center. Our experimental program is divided into two areas. (i) We will probe the function of the Sco homologue of B. subtilis via XAS structural studies coupled to in vivo functional assays of native proteins and Cu(I) and Cu(II)-site specific mutations; (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. 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.