Heme A and cytochrome oxidase biosynthesis

DESCRIPTION (provided by applicant): Yeast cytochrome c oxidase (COX), the terminal complex of the mitochondrial respiratory chain, is composed of 12 different polypeptides. The three subunits constituting the catalytic core of the complex are encoded by mitochondrial genes. The remaining 8 subunits are products of the nuclear genome. Biogenesis of this important enzymes is governed by more than three dozen genes that intercede at all stages of the assembly pathway. The functions of these genes have been and continue to be the focus of this project. During the past grant period we have 1) completed studies on Scolp and Cox15p that function in maturation of the CuA center in subunit 2 and biosynthesis of heme A, respectively, 2) identified and studied the functions of 4 new COX-specific genes, 3) shown that cytochrome c is required in a structural capacity for COX assembly, 4) identified a ternary complex of Cox14p, Mss51p, and Coxlp involved in regulation of Coxlp translation, 5) collaborated with Dr. Pierre Rustin on studies of patients with COX dysfunctions. In the present application we propose to continue investigations of COX deficient mutants to gain a better understanding of the process by which COX is assembled. These studies will aim to clarify how Shylp functions in the regulatory circuit that coordinates the synthesis of subunit 1 to its utilization for COX assembly. Another aim is to explore the roles of the mitochondrial intermembrane metallo-proteins Cox23p, Cox19p, and Pet191 p in maturation of the copper centers of COX. These studies will examine copper transfer from these intermembrane proteins to Cox17p and whether any of them are present in a common complexe(s)? Studies on Cox15p, the heme O hydroxylase of mitochondria, were hampered by problems encountered in our attempts to purify the protein. The most serious of these difficulties have been resolved and we expect to obtain a purified Cox15p suitable for the in vitro enzymatic studies that had been planned earlier. Concurrently, we will extend our functional analyses to new and partially characterized COX-specific genes by combined biochemical and genetic approaches used in the past. Finally, we will continue to collaborate with the Rustin lab on studies of human disorders stemming from defective COX. These studies will strive to use an alternative oxidase to rescue COX mutants and to improve on current methods for assigning human mutations to known COX genes.