XAS STUDIES OF HEME-COPPER ASSEMBLY PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 血红素铜氧化酶是哺乳动物和多种真核生物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关键作用。随着对结构和催化机制的更好理解，导致关键氧化还原中心的组装和代谢的途径正在成为下一个前沿。该提案的目的将集中在参与CuA中心的组装和代谢的蛋白质上。我们的实验计划分为两个方面。(i)我们将探讨B的Sco同源物的功能。枯草杆菌通过XAS结构的研究，再加上在体内功能测定的天然蛋白质和Cu（I）和Cu（II）-位点特异性突变;（ii）我们将探讨机制的金属转移之间的Sco和它的伙伴蛋白质，含铜的CCO亚基II，使用硒代甲硫氨酸取代，以提供一个位点特异性XAS探针的金属转移反应。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。

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.