XAS STUDIES OF HEME-COPPER ASSEMBLY PROTEINS

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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.