Heme a synthase: Mutagenesis, complex formation and protein-protein interactions

Heme a is a cofactor that is found only in the terminal oxidases of the respiratory chain in mitochondria and some bacteria. Heme a is absolutely required for cytochrome c oxidase (CcO) stability and function in eukaryotes. Despite considerable progress toward elucidating the details of heme a biosynthesis and CcO assembly, there is still much remaining to be resolved. The reaction mechanism of heme a synthase (HAS), the maturation of the heme a centers in C cO, and protein-protein interactions involving HAS were investigated, and the findings are compiled in this dissertation. The HAS genes from Bacillus subtilis (ctaA) and Saccharomyces cerevisiae (COX15) were expressed in E. coli and S. cerevisiae (yeast), respectively. Mutant derivatives of the invariant amino acids in the active site of recombinant CtaA and Cox15p were generated, and heme a production by these mutants was analyzed via high performance liquid chromatography. Affinity chromatography was utilized for the purification of recombinant Cox15p. Blue native polyacrylamide gel electrophoresis was utilized to investigate chromosomally tagged Cox15p, Mss51p, Coa1p, and shy1p complexes in yeast. Protein-protein interactions involving Cox15p were assessed via affinity chromatography and immunoprecipitation studies. Mutagenesis studies showed that the invariant amino acids in the N-terminal half of HAS are more important for activity than the invariant amino acids in the C-terminal half. For the first time, Cox15p was purified and shown to be in complexes ranging in size from 250-600 kDa. Formation of the Cox15p complexes may represent an early step in C cO assembly. CcO assembly starts with the synthesis of subunit I from CcO, followed by formation of Mss51p complexes in which the heme a and a3 centers in subunit I remain vacant. The activation of Cox15p and synthesis of heme a could elicit a signaling event that begins the progression of subunit I maturation from the Mss51p complexes to the downstream Coa1p and Shy1p complexes. Hemes a and a3 insertions into subunit I likely occur simultaneously in Shy1p-related complexes. Cox15p could also have a secondary function involving formation of a supercomplex in yeast as components of complexes V, III and IV were identified in the Cox15p complexes.