Biophysical and structural characterization of mitochondrial acyl-CoA dehydrogenases in the presence of polyoxometalate ruthenium compounds

Mitochondria are membrane-bound organelles integral to normal eukaryotic cell function, as they orchestrate cellular energy production, calcium homeostasis, apoptotic activation, and cell death. Mitochondrial defects occur in a wide variety of degenerative diseases, aging, and cancer. In particular, human inherited defects have been described for almost all enzymes and transporters involved in fatty acid oxidation (FAO), a process that requires the import of fatty acids (FA) into the mitochondria. All these defects have autosomal recessive inheritance and may be caused by mutations in a large set of genes. Among these genes is the one encoding the “moonlighting” protein homodimer ACAD9, which appears to play roles in both FAO and oxidative phosphorylation. Due to its multifunctional enzymatic properties, mutations in ACAD9 have atypical effects, making this enzyme an interesting subject of study. To date, no high-resolution structure of ACAD9 is available, largely due to its flexibility and relatively small size (167 kDa), which complicates structural studies, particularly through single-particle cryo-electron microscopy (cryo-EM). To enhance resolution, we employed a polyoxometalate ruthenium compound (Ru4POM) to reduce the protein’s flexibility and improve contrast in cryo-EM imaging. For this study, we used VLCAD, the homologous ancestor of ACAD9, as a control, performing parallel experiments on it. Given the direct relationship between structure and function in enzymes, we investigated the enzymatic activity of ACAD9 and VLCAD both with and without Ru4POM. The lack of a statistically significant difference in activity suggested that Ru4POM does not substantially alter the proteins’ structures. To assess the affinity of ACAD9 and VLCAD for Ru4POM, we conducted ITC experiments, which demonstrated the strong electrostatic nature of the interaction and revealed a different stoichiometry for the two proteins. Additionally, size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) was employed to confirm that the proteins used in the experiments were correctly folded. Cryo-EM data of the ACAD9-Ru4POM complex, previously collected by our research group, were subjected to a preliminary analysis, which led to an initial 3D map with an improved resolution relative to the deposited ACAD9 structures.