Distinct Assembly Complexes Facilitate cbb₃-type Cytochrome Oxidase Maturation

Cytochrome oxidase (Cox) assembly is inherently challenging because subunit associations need to be tightly coordinated with cofactor insertions. In mitochondria, this process relies on large assembly machineries, whereas bacterial Cox assembly has been thought to involve mostly only brief interactions between assembly factors and subunits. However, here we identified three stable assembly complexes of 500, 390, and 150 kDa that are required for the maturation of cbb₃‑type Cox in the α‑proteobacterium R. capsulatus. These complexes mediate the stepwise insertion of heme and copper cofactors and are organized around the cupric reductase CcoG, which forms together with the ATPase CcoI and the chaperones SenC and CcoS a copper insertion module. Formation and stability of these complexes is determined by copper loading of CcoG and CcoI. Overall, our findings demonstrate that large multi-protein assembly machineries also exist in bacteria and are required for the maturation of cbb₃-Cox, the most ancient bacterial Cox.