Structure and assembly of the mammalian mitochondrial supercomplex CIII₂CIV

The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes¹, whose precise role is debated. Supercomplexes CIII₂CIV_1-2 (refs. ^2,3), CICIII₂ (ref. ⁴) and CICIII₂CIV (respirasome)^5-10 exist in mammals, but in contrast to CICIII₂ and the respirasome, to date the only known eukaryotic structures of CIII₂CIV_1-2 come from Saccharomyces cerevisiae^11,12 and plants¹³, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII₂CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII₂CIV from the CIII₂ precursor to the final CIII₂CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. ¹⁴) deep into CIII₂, while its C terminus is integrated into CIV. Our structures (which include CICIII₂ and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII₂CIV and has no role in the assembly of the respirasome. We show that CIII₂ is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII₂, explaining the presence of one copy of CIV in CIII₂CIV in mammals. Finally, we show that CIII₂ and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII₂CIV in fine tuning the efficiency of electron transfer in the electron transport chain.