The small membrane protein CcoS is involved in cofactor insertion into the cbb₃-type cytochrome c oxidase

Respiratory complexes, such as cytochrome oxidases, are cofactor-containing multi-subunit protein complexes that are critically important for energy metabolism in all domains of life. Their intricate assembly strictly depends on accessory proteins, which coordinate subunit associations and cofactor deliveries. The small membrane protein CcoS was previously identified as an essential assembly factor to produce an active cbb₃-type cytochrome oxidase (cbb₃-Cox) in Rhodobacter capsulatus, but its function remained unknown. Here we show that the ΔccoS strain assembles a heme b deficient cbb₃-Cox, in which the CcoN-CcoO subunit association is impaired. Chemical crosslinking demonstrates that CcoS interacts with the CcoN and CcoP subunits of cbb₃-Cox, and that it stabilizes the interaction of the Cu-chaperone SenC with cbb₃-Cox. CcoS lacks heme- or Cu-binding motifs, and we did not find evidence for direct heme or Cu binding; rather our data indicate that CcoS, together with SenC, coordinates heme and Cu insertion into cbb₃-Cox.