Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae , CIV is found solely in a SC with cytochrome bc 1 (complex III, CIII). Here, we present the cryo-EM structure of S. cerevisiae CIV in a III 2 IV 2 SC at 3.3 Å resolution. While overall similarity to mammalian homologues is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A – involved in allosteric inhibition by ATP – may arise from its association with CIII. The CIII–CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.

SummaryThe spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.