We investigated the biochemical phenotype of the
mtDNA T8993G point mutation in the
ATPase 6 gene, associated with
neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of
succinate-sustained
ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the
succinate-sustained
ATP synthesis rate of the particles was found, but both the
ATP hydrolysis rate and
ATP-driven
proton translocation (when the
protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the
ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze
proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human
F(1)F(0)-ATPase that causes a severe impairment of
ATP synthesis. This is possibly due to a defect in either the vectorial
proton transport from the cytosol to the mitochondrial matrix or the coupling of
proton flow through F(0) to
ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired
ATP synthesis. On the other hand,
ATP hydrolysis that involves
proton flow from the matrix to the cytosol is essentially unaffected.