Cryo-EM reveals the central steps of mitochondrial complex III assembly and the cooperative assembly of supercomplex CIII2CIV

Mitochondrial complex III is the central component of the respiratory chain and is conserved across eukaryotes. Complex III is an obligate dimer which assembles through a stepwise mechanism, involving 20 subunits and other assembly factors. Defects in the assembly of complex III are associated with metabolic diseases. The assembly mechanism of complex III has long been investigated using biochemical approaches, which suggested a process where folded subunits are added sequentially and in parallel for the two protomers after dimerization. Our structural investigation of CIII2 assembly challenges these assumptions: we observe using cryo-EM that incorporation and folding of its subunits can be uncoupled (as we show for cytochrome c1) and that after dimerization the assembly of the complex does not proceed in parallel for the two protomers (as we show for the folding of the intermembrane space domain). Our structures also reveal the mechanism of formation of supercomplex CIII2CIV for non-vertebrates, intertwined with the assembly of CIII2. This work reshapes our knowledge of complex III assembly and proposes a generalizable model for the maturation of the complex, alongside a model for supercomplex formation that supports the cooperative assembly model. Furthermore, as the observed assembly steps cannot be predicted by AlphaFold, our work also showcases the central role of cryo-EM in the study of assembly mechanism of protein complexes.