ATP synthase activity boosts membrane proton acceptance and lateral diffusion.

In most organisms, ATP synthesis is powered by the proton motive force (pmf) and catalyzed by ATP synthase. While the chemiosmotic theory originally proposed a "delocalized coupling" between proton pumps and consumers, growing evidence implicates the membrane in mediating localized proton transfer (PT). To directly track ultrafast PT at the membrane surface as a function of ATP synthase activity, we developed an in vitro system. We tethered a light-activated excited-state photoacid to the bilayer of unilamellar vesicles to confine PT to the membrane interface and co-reconstituted a thermophilic Bacillus TFOF1 ATP synthase. Using steady-state and time-resolved fluorescence spectroscopy, we quantified PT and lateral proton diffusion from the anchored photoacid under conditions with non-ATP- and ATP-producing enzymes. Our results show that the membrane accepts protons at its interface and that PT is enhanced only when ATP synthase is active. A comparison with soluble photoacid positioned near the membrane shows that protons consumed by ATP synthase do not equilibrate with the bulk aqueous phase. Instead, they are transferred directly along the two-dimensional membrane interface to the enzyme. This localized coupling can explain how ATP synthesis can proceed even when the apparent bulk pmf seems insufficient. Our results refine the proton translocation during ATP synthesis by revealing that the membrane itself is an active participant in PT, thereby strengthening the case for localized proton coupling in bioenergetics.