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This work elucidates in molecular detail the role of the MCIA complex in CI assembly and its functional role in mitochondrial bioenergetics. By using a combination of biochemical and biophysical approaches, including SAXS at beamline BM29, it was discovered that ECSIT is the bridging component in the MCIA complex. The modular nature of ECSIT, which is organised into N- and C-terminal binding domains, enables recruitment of its MCIA partners NDUFAF1 and ACAD9 using independent interaction interfaces. Furthermore, the three-dimensional architecture of an MCIA subcomplex comprising the ECSIT C-terminal region bound to ACAD9 was determined by single-particle

Fig. 31: The MCIA subcomplex molecular architecture. Left: Cryo-EM 2D class averages of the complex reveal a compact core with diffuse densities indicated with arrowheads. Right: Top and side views of the 15 Å resolution cryo-EM envelope of the complex at two thresholds. The extra densities protruding out of the central ACAD9 dimer core (each monomer in a different colour) correspond to segments of ECSIT (arrowheads).

Fig. 32: Proposed model of how deflavination of ACAD9 by ECSIT permits the coordinated regulation of mitochondrial energy metabolism pathways.

cryo-EM at beamline CM01. The cryo-EM map revealed that the subcomplex possesses a defined core formed by an ACAD9 dimer with extra densities that protrude out from the ACAD9, corresponding to ordered segments of ECSIT (Figure 31). The fact that only a small proportion of the bound ECSIT is visible in the cryo-EM map is compatible with SAXS data, indicating that the subcomplex is dynamic. Strikingly, interaction with ECSIT induces ACAD9 to eject its FAD cofactor from its catalytic site. Deflavination of ACAD9 implies the shutdown of its enzymatic activity and hence, its redirection towards the action on the assembly of CI (Figure 32).