Water Bridges Conduct Sequential Proton Transfer in Photosynthetic Oxygen Evolution

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 Å 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 S₂ to S₃ step of this cycle. Here, we study the effect of acetate on a previous step of the cycle, the S₁ to S₂ 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 S₁ to the S₂ state. Isotope-editing of acetate reveals direct contributions to the S₂-minus-S₁ infrared spectrum consistent with protonation of bound acetate in PSII. In the acetate-derived S₂-minus-S₁ 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.