Proton transfer using water bridges has been observed in bulk water, acid-base reactions, and several proton-translocating biological systems. In the photosynthetic water-oxidizing enzyme, photosystem II (PSII), protons from substrate water are transferred 35 angstrom from the Mn4CaO5 catalytic site to the chloroplast lumen. This process leads to acidification of the lumen and ATP synthesis. Water oxidation occurs in a flash-induced, five-step S-n state cycle; acetate is a chloride-dependent inhibitor of the S-2 to S-3 step of this cycle. Here, we study the effect of acetate on a previous step of the cycle, the S-1 to S-2 transition, using reaction-induced infrared spectroscopy. PSII was isolated from spinach, and experiments were conducted at pH 7.5, using 532 nm laser flashes to advance the cycle from the dark-adapted state S-1 to the S-2 state. Isotope-editing of acetate reveals direct contributions to the S-2 minus-S-1 infrared spectrum consistent with protonation of bound acetate in PSII. In the acetate-derived S-2-minus-S-1 PSII spectra, an accompanying decrease in the intensity of a 2830 cm(-1) band is observed when compared to the chloride control. The 2830 cm(-1) band has been assigned previously to a stretching vibration of an internal, hydrated hydronium ion, W-n(+). Density functional studies of a catalytic site model predict the spontaneous transfer of a proton from this internal hydronium ion to acetate, when acetate is substituted at a chloride-binding site. Taken together, the results show that the mechanism of PSII proton transfer at pH 7.5 involves proton hopping through an internal, water-containing network.