Abstract
Heme a is an essential cofactor for function of cytochrome c oxidase in the mitochondrial electron transport chain. Several evolutionarily conserved enzymes have been implicated in the biosynthesis of heme a, including the heme a synthase Cox15. However, the structure of Cox15 is unknown, its enzymatic mechanism and the role of active site residues remain debated, and recent discoveries suggest additional chaperone-like roles for this enzyme. Here, we investigated Cox15 in the model eukaryote Saccharomyces cerevisiae via several approaches to examine its oligomeric states and determine the effects of active site and human pathogenic mutations. Our results indicate that Cox15 exhibits homotypic interactions, forming highly stable complexes dependent upon hydrophobic interactions. This multimerization is evolutionarily conserved and independent of heme levels and heme a synthase catalytic activity. Four conserved histidine residues are demonstrated to be critical for eukaryotic heme a synthase activity and cannot be substituted with other heme-ligating amino acids. The 20-residue linker region connecting the two conserved domains of Cox15 is also important; removal of this linker impairs both Cox15 multimerization and enzymatic activity. Mutations of COX15 causing single amino acid conversions associated with fatal infantile hypertrophic cardiomyopathy and the neurological disorder Leigh syndrome result in impaired stability (S344P) or catalytic function (R217W), and the latter mutation affects oligomeric properties of the enzyme. Structural modeling of Cox15 suggests these two mutations affect protein folding and heme binding, respectively. We conclude that Cox15 multimerization is important for heme a biosynthesis and/or transfer to maturing cytochrome c oxidase.