Assembly of nuclear DNA‐encoded subunits into mitochondrial complex IV, and their preferential integration into supercomplex forms in patient mitochondria

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Abstract
Complex IV is the terminal enzyme of the mitochondrial respiratory chain. In humans, biogenesis of complex IV involves the coordinated assembly of 13 subunits encoded by both mitochondrial and nuclear genomes. The early stages of complex IV assembly involving mitochondrial DNA‐encoded subunits CO1 and CO2 have been well studied. However, the latter stages, during which many of the nuclear DNA‐encoded subunits are incorporated, are less well understood. Using in vitro import and assembly assays, we found that subunits Cox6a, Cox6b and Cox7a assembled into pre‐existing complex IV, while Cox4‐1 and Cox6c subunits assembled into subcomplexes that may represent rate‐limiting intermediates. We also found that Cox6a and Cox7a are incorporated into a novel intermediate complex of approximately 250 kDa, and that transition of subunits from this complex to the mature holoenzyme had stalled in the mitochondria of patients with isolated complex IV deficiency. A number of complex IV subunits were also found to integrate into supercomplexes containing combinations of complex I, dimeric complex III and complex IV. Subunit assembly into these supercomplexes was also observed in mitochondria of patients in whom monomeric complex IV was selectively reduced. We conclude that newly imported nuclear DNA‐encoded subunits can integrate into the complex IV holoenzyme and supercomplex forms by associating with pre‐existing subunits and intermediate assembly complexes.