*6.1. The CI+CIII2+CIV SC or Respirasome*

The structures of the CI+CIII2+CIV SC isolated from different mammalian mitochondria have been determined by single-particle electron cryo-EM at resolutions ranging from ~30 to ~4 Å [67–70] or by electron cryotomography (cryo-ET) at ~30 Å resolution [71,72].

In the structure of respirasome, CIII<sup>2</sup> borders the concave arc of CI membrane arm, and CIV is located near CIII<sup>2</sup> at the distal end of the CI membrane arm, with cardiolipin molecules filling the gaps between the individual complexes [67,71]. In the respirasome, two distinct arrangements have been identified, a major "tight" and a minor "loose" form, which mainly diverge for the position of CIV. As illustrated in the cartoon of Figure 2, the most extensive and stable interactions take place between CI and CIII2.

**Figure 2.** Proposed interactions between the respiratory complexes of the respirasome (according to [69]). Nomenclature of human subunits is indicated. The number of tight contacts between CI and CIII is greater than those between CI–CIV and CIII–CIV.

Two are the major interaction points: in the inner membrane between CI subunit NDUFA11 and the CIII subunits UQCRB, UQCRQ and UQCRH, and at the matrix between the CI subunits NDUFB4 and NDUFB9 and CIII subunits UQCRC1 and UQCRFS1 [69]. Another important interaction occurs between CI subunit NDUFB7 and subunit UQCRH on CIII2. Of note is that both subunits contain disulphide bonds, suggesting that redox regulation might modulate the interactions between the respiratory complexes [69].

Few interactions occur between CI and CIV, the most important linking the CI ND5 subunit to COX7C at the interface between matrix and inner membrane, the other between NDUFB3 subunit and COX8B. The contacts between CIII and CIV mainly involve the CIV COX7A subunit with the UQCRC1 and UQCR11 subunits and COX5B subunits with the UQCRC1 subunit [69].

Interestingly, high-molecular-weight bands above the CI+CIII2+CIV SC were previously described by BN-PAGE analysis [63]. More recently, mass spectrometry analysis has suggested that the main components of these bands are subunits of CI, CIII and CIV, and EM analysis detected a minor population of particles with circular arrangements. This led to proposing a higher oligomeric state named megacomplex CI2+CIII2+CIV2. This assembled structure is shaped by a central CIII<sup>2</sup> surrounded by two copies each of CI and CIV. This arrangement may be an oligomerization form of respiratory complexes operating under the most efficient emergency conditions, because both monomers of the CIII dimer could receive CoQH<sup>2</sup> from each CI and pass reduced cytochrome *c* to one adjacent CIV. Further analysis by cryo-EM allowed to better define the architecture of the megacomplex [73], although these results were intensely debated, mainly due to limitations of cryo-ET technology in the reconstruction of supramolecular assemblies.
