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.

A novel heteroplasmic mitochondrial DNA (mtDNA) microdeletion affecting the cytochrome b gene (MT-CYB) was identified in an Italian female patient with a multisystem disease characterized by sensorineural deafness, cataracts, retinal pigmentary dystrophy, dysphagia, postural and gait instability, and myopathy with prominent exercise intolerance. The deletion is 18-base pair long and encompasses nucleotide positions 15,649-15,666, causing the loss of six amino acids (Ile-Leu-Ala-Met-Ile-Pro) in the protein, but leaving the remaining of the MT-CYB sequence in frame. The defective complex III function was cotransferred with mutant mtDNA in cybrids, thus unequivocally establishing its pathogenic role. Maternal relatives failed to show detectable levels of the deletion in blood and urinary epithelium, suggesting a de novo mutational event. This is the second report of an in-frame intragenic deletion in MT-CYB, which most likely occurred in early stages of embryonic development, associated with a severe multisystem disorder with prominent exercise intolerance.

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A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival.

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