Photosystem II (PSII) is the only enzyme in nature that can catalyze the challenging catalytic photooxidation of H(2)O into four protons, four electrons, and O(2). Slowing down turnover of the O(2)-evolving complex (OEC) is a plausible approach to gain mechanistic information on the reaction. However, modulating the kinetics of the reaction without perturbing the active site is a challenge. In this study, it is shown that the steady-state activity of cyanobacterial PSII is inhibited by small zwitterions, such as glycine betaine and β-alanine. We show that the binding of zwitterions is nondenaturing, is highly reversible, and results in the decrease of the rate of catalytic turnover by ∼50% in the presence of excess zwitterion. Control measurements of photoinduced electron transfer in O(2)-inactive PSII show that the inhibition by zwitterions is the result of a specific decrease in the rate of catalytic turnover of the OEC. Recovery of activity upon addition of an exogenous proton carrier (HCO(3)(-)) provides evidence that proton-transfer pathways, thought to be essential for the relay of protons from the OEC to the lumen, are affected. Interestingly, no inhibition is observed for spinach PSII, suggesting that zwitterions act specifically by binding to the extrinsic proteins on the lumenal side of PSII, which differ significantly between plants and cyanobacteria, to slow proton transfer on the electron donor side of PSII.