Respiratory chain complexes can super-assemble into quaternary structures called supercomplexes that optimize cellular metabolism. The interaction between complexes III (CIII) and IV (CIV) is modulated by supercomplex assembly factor 1 (SCAF1, also known as COX7A2L). The discovery of SCAF1 represented strong genetic evidence that supercomplexes exist in vivo. SCAF1 is present as a long isoform (113 amino acids) or a short isoform (111 amino acids) in different mouse strains. Only the long isoform can induce the super-assembly of CIII and CIV, but it is not clear whether SCAF1 is required for the formation of the respirasome (a supercomplex of CI, CIII2 and CIV). Here we show, by combining deep proteomics and immunodetection analysis, that SCAF1 is always required for the interaction between CIII and CIV and that the respirasome is absent from most tissues of animals containing the short isoform of SCAF1, with the exception of heart and skeletal muscle. We used directed mutagenesis to characterize SCAF1 regions that interact with CIII and CIV and discovered that this interaction requires the correct orientation of a histidine residue at position 73 that is altered in the short isoform of SCAF1, explaining its inability to interact with CIV. Furthermore, we find that the CIV subunit COX7A2 is replaced by SCAF1 in supercomplexes containing CIII and CIV and by COX7A1 in CIV dimers, and that dimers seem to be more stable when they include COX6A2 rather than the COX6A1 isoform.