HEK293T Coa6 knockout mitochondria

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in Coa6, leading to substitution of a conserved tryptophan to a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of Coa6 activity using gene-editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mtDNA encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in Coa6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore we find that Coa6 binds copper with a high affinity and also associates with mitochondrial copper chaperones, revealing that Coa6 is intricately involved in the copper-dependent biogenesis of COX2.