HEME A AND CYTOCHROME OXIDASE BIOSYNTHESIS

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Cytochrome oxidase promotes the transfer of electrons from reduced cytochrome c to molecular oxygen, the terminal step of the respiratory chains of mitochondria and aerobic microorganisms. In S. cerevisiae, this membrane complex is composed of 10 different subunit polypeptides of which three are encoded in mitochondrial DNA. The two genetically distinct groups of polypeptides are assembled into the holoenzyme by an elaborate process that requires the expression of some 40 nuclear genes. The products of two such genes, COX10 and COX11, have recently been implicated to function in the synthesis of heme A, a prosthetic group unique to cytochrome oxidase. One of the goals of this proposal is to characterize the roles of the COX10 and COX11 proteins in farnesylation of the 8-vinyl and oxidation of the 2-methyl substituents of the porphyrin ring and to screen for other heme A mutants. These strains will be used to used to identify the intermediates and enzymes of heme A biosynthesis, a pathway about which virtually nothing is known at present. A substantial number of non-maternally inherited human myopathies have been ascribed to decreased levels of cytochrome oxidase in muscle mitochondria. Despite extensive studies, none of these fatal diseases have been correlated with mutations in the structural or catalytic subunits of this respiratory complex. It is not unreasonable to think that in some cases the enzyme deficiency may stem from mutations affecting heme A biosynthesis. To assess this possibility, muscle biopsies from patients with cytochrome oxidase defects will be analyzed for the accumulation of heme A intermediates. This approach has been helpful in identifying a novel heme compound in a cox11 mutant of yeast. Secondly, the human cDNA homologs of COX10 and COX11 will be cloned in order to facilitate a molecular analysis of the comparable cDNAs from myopathic tissues. The cytochrome oxidase deficient phenotype of yeast strains assigned to approximated 11 complementation groups is elicited by mutations affecting late events in the enzyme assembly pathway. The third goal of this proposal is to complete the analysis of this set of genes and to clarify the functions supplied by the encoded proteins. These studies are anticipated to provide a blueprint of the genetic information and the molecular mechanisms governing assembly of the functional complex.