Analysis of Oligomerization Properties of Heme a Synthase Provides Insights into Its Function in Eukaryotes

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.

The Assembly Factor Pet117 Couples Heme a Synthase Activity to Cytochrome Oxidase Assembly

“…This heme a biosynthetic defect as well as the defects observed in CcO assembly and activity resulting from loss of Cox15 complex stability in the absence of Pet117 are consistent with our previous study of Cox15 that linked its multimerization to heme a biosynthesis and/or transfer of heme a to Cox1 during CcO assembly (8). The phenotype of pet117⌬ cells thus underscores the importance of Cox15 multimerization for Cox15 function.…”

“…3 indicating that Pet117 is a mitochondrial matrix protein loosely associated with the IM. Moreover, loss of this 20-amino acid-long region results in impaired Cox15 oligomerization and heme a synthase activity (8), which mimics the consequences of PET117 deletion. The Cox15-Pet117 interaction was undetectable in mitochondria from cells expressing the Cox15(L-20)-FLAG variant of heme a synthase lacking this unstructured linker region (Fig.…”

“…Because heme a synthase oligomers contain multiple copies of Cox15 (8), we tested whether loss of Pet117 disrupted Cox15-Cox15 homotypic interaction. To this end, mitochondria from cells expressing two functional, differentially tagged variants of heme a synthase used previously (8), Cox15-Myc and Cox15-FLAG, were used in co-immunoprecipitation (co-IP) experiments with anti-Myc-agarose beads (Fig.…”

“…In the yeast Saccharomyces cerevisiae, Cox10 and Cox15 are large (52 and 55 kDa, respectively) polytopic proteins with 7-9 predicted transmembrane segments (3). They form complexes of ϳ300 and ϳ250 kDa, respectively, which are probably homooligomeric and may also associate with additional proteins (8 -10), and the mammalian orthologs of these heme a biosynthetic enzymes appear to be organized in a similar fashion (8).…”

“…We recently determined that the matrix-exposed, unstructured linker region (of 20 amino acid residues) connecting the N-and C-terminal membrane-embedded halves of Cox15 is required for enzymatic oligomerization and activity and that activity is compromised and oligomerization is affected in the yeast analog of the R217W variant of Cox15 observed in patients with mitochondrial disease (8).…”