Biochemical and computational approaches to dissect the effect of MT-CYB pathogenic mutations on respiratory chain activity and assembly

Complex III (ubiquinol:cytochrome c oxidoreductase) is a multisubunit membrane bound enzyme and in its native form is a symmetrical homodimer (CIII2). CIII2 is central for mitochondrial respiratory chain and is associated with different stoichiometry with Complex I and Complex IV to form supramolecular assemblies, called supercomplexes (SCs). Defects in CIII2 are rare and mostly associated with mutations in MT-CYB gene that encodes for one of the catalytic subunits, cytochrome b (cyt b). It has been suggested that pathogenic mutations in MT-CYB are mitigated when CIII2 is assembled in SCs [1]. Therefore, we applied biochemical approaches in human cellular models carrying pathogenic point mutations in cyt b to analyse the structural stability and enzymatic activity of CIII2. Our preliminary results showed that pathogenic mutation differently affected the kinetics of the assembly of CIII2 and its SCs after the treatment with a reversible mitochondrial translation inhibitor, suggesting a role of these mutations not only in CIII2 activity but also in its biogenesis. Interestingly, the rescue of the oxygen consumption profile was further delayed compared to the formation of enzymatic complexes. In addition, we applied the Protein Stability Prediction with a Gaussian Network Model (PSP-GNM) approach [2] to evaluate global changes in the unfolding Gibbs free energy change and study the effects of single amino acid mutations on cyt b stability on the available isolated and SC-bound CIII2 structures. Preliminary results indicate that some pathogenic mutations may affect the unfolding free energy of CIII2, stiffening the structure of the enzyme, in agreement with the reduction of CIII2 activity. This dual experimental and biocomputational approach may be very useful to dissect assembly processes and function of the respiratory chain to better understand the effect of these rare pathogenic mutations and to design new strategies for possible therapeutic options.