ABSTRACT The classical view of oxidative phosphorylation is that a proton motive force PMF generated by the respiratory chain complexes fuels ATP synthesis. Under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected the two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions in detail. pH profiles of mitochondrial sub-compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH-sensor directly at F 1 and F O of ATP synthase, complex IV and in the matrix. Our results clearly show that ATP synthase activity is substantially controlling the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. GRAPHICAL ABSTRACT HIGHLIGHTS The ΔpH along and across the inner mitochondrial membrane is not homogeneous The proton motive force at cristae tips is controlled by F 1 F O ATP synthase Under OXPHOS conditions, the pH difference between F O and F 1 of active ATP synthase is almost negligible (1.2 proton vs. 1 proton equivalent) IF1 is required to prevent the onset of ATP hydrolysis under OXPHOS conditions

Mitochondrial F1FO ATP synthase determines the local proton motive force in cristae tips