Defects in ATP synthase functioning due to the substitutions in its two mitochondrially encoded subunitsaand8lead to untreatable mitochondrial diseases. Defining the character of variants in genes encoding these subunits is challenging due to their low frequency, heteroplasmy of mitochondrial DNA in patients’ cells and polymorphisms of mitochondrial genome. We successfully used yeastS. cerevisiaeas a model to study the effects of variants inMT-ATP6gene and our research led to understand how eight amino acid residues substitutions impact the proton translocation through the channel formed by subunitaandc-ring of ATP synthase at the molecular level. Here we applied this approach to study the effects of the m.8403T>C variant inMT-ATP8gene. The biochemical data from yeast mitochondria indicate that equivalent mutation is not detrimental for the yeast enzyme functioning. The structural analysis of substitutions in subunit8introduced by m.8403T>C and five other variants inMT-ATP8provides indications about the role of subunit8in the membrane domain of ATP synthase and potential structural consequences of substitutions in this subunit.