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各种真核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量转化为质子动力，最终用于驱动ATP的内源性合成，在细胞生物能量学和人体健康中发挥关键作用。随着结构和催化机制的深入研究，关键氧化还原中心的组装和金属化途径将成为下一个研究方向

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.