The cytochrome b p.278Y>C mutation causative of a multisystem disorder enhances superoxide production and alters supramolecular interactions of respiratory chain complexes.

Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.

Fine-tuning of the respiratory complexes stability and supercomplexes assembly in cells defective of complex III.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.