Mitochondrial disease can result from mutations in the enzymes responsible for biosynthesis of heme a and hemylation of respiratory complex IV of the electron transport chain, also known as cytochrome c oxidase (CcO). One of these enzymes, which is essential for assembly and function of CcO and thus function of the electron transport chain, is the heme a synthase, COX15. A previously unknown fatal missense mutation of COX15, c.232G>A (p.Gly78Arg), was recently described in a case report by Galvão de Oliveira et al. Here, we show that the p.Gly78Arg-mimicking substitution in the homologous Cox15 protein in Saccharomyces cerevisiae (G95R) causes Cox15 protein instability and recapitulates the CcO defect observed in the patient. We demonstrate that the CcO defect observed with this Cox15 variant stems from insufficient heme a synthesis, and consequently, inefficient CcO hemylation and decreased levels of CcO. Our results provide insights into the etiology of the disease caused by this variant, suggesting that Cox15 protein instability and consequent attenuation of heme a synthase function is the main molecular factor behind the resulting multisystemic mitochondrial disorder in humans.