In photosystem II (PSII), photosynthetic water oxidation occurs at the O2-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S0–S4) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S2 to S1 transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7–2.7× slower rate of charge recombination with QA− relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an Ea of 5.87 ± 1.15 kcal mol−1 for decay back to the S1 state, compared to 0.80 ± 0.13 kcal mol−1 for the wild-type S2 state. In addition, we find that ammonia does not affect the S2-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S2 state of D1-S169A PSII. The altered S2 state of D1-S169A PSII provides insight into the S2➔S3 state transition.