Binding of Acetate in the S₂ State of the Oxygen-Evolving Complex in Photosystem II

Photosynthetic water oxidation is catalyzed by the Mn₄CaO₅ oxygen-evolving complex (OEC) of photosystem II (PSII), where hydrogen-bonding and ion-binding networks regulate proton transfer, substrate-water delivery, and S-state advancement. Acetate binding inhibits oxygen evolution, competes with chloride, and stabilizes the S=5/2 spin isomer of the S₂ state, but its donor-side binding site remains unresolved. Here, we combine EPR spectroscopy, pH-dependent oxygen-evolution measurements, mutagenesis, and QM/MM calculations to support a donor-side acetate-binding model and determine how acetate perturbs the OEC environment. Acetate increases the ratio of the g=4.1 to g=2 S₂-state EPR signals in spinach PSII membranes and cyanobacterial PSII core complexes, with stronger stabilization persisting to higher pH in spinach PSII. The D1-N87A Synechocystis PSII variant exhibits spinach-like acetate sensitivity and pH-dependent oxygen-evolution behavior, with an effective acidic pKa of approximately 5.3, versus 4.2 for wild-type cyanobacterial PSII, implicating long-range perturbations of the narrow-channel hydrogen-bonding network. QM/MM calculations support acetate binding near the D1-D61/W1 region, where the acetate-bound S=5/2 isomer is only 1.0 kcal mol^-1 higher in free energy than the S=1/2 isomer, consistent with the observed spin-isomer equilibrium shift. These results reveal how acetate perturbs proton-transfer and chloride-binding processes in PSII.