LAUSR

Significance As the terminal electron acceptor of our mitochondrial respiratory chains, complex IV drives and regulates oxidative phosphorylation, the process by which most of our ATP is produced. Complex IV forms supercomplexes (SCs) of different stoichiometries with other respiratory proteins, interacting via its subunits with tissue-specific or oxygen level-dependent expression isoforms, suggesting a link between SC assembly and metabolic/disease state. We investigated the effect of complex IV subunit isoform exchange in yeast using cryo-EM and biochemical assays and found no significant differences in overall SC formation, architecture, or catalytic activities. However, our structural work unexpectedly revealed the presence of a Hig1 protein which we propose is a stoichiometric subunit of complex IV, at least when within a SC with complex III. The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII–CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.