In photosystem II, oxygen evolution occurs by the accumulation of photo-induced oxidizing equivalents at the oxygen-evolving complex (OEC). The sequentially oxidized states are called the S(0)-S(4) states, and the dark stable state is S(1). Hydrogen bonds to water form a network around the OEC; this network is predicted to involve multiple peptide carbonyl groups. In this work, we tested the idea that a network of hydrogen bonded water molecules plays a catalytic role in water oxidation. As probes, we used OEC peptide carbonyl frequencies, the substrate-based inhibitor, ammonia, and the sugar, trehalose. Reaction-induced FT-IR spectroscopy was used to describe the protein dynamics associated with the S(1) to S(2) transition. A shift in an amide CO vibrational frequency (1664 (S(1)) to 1653 (S(2)) cm(-1)) was observed, consistent with an increase in hydrogen bond strength when the OEC is oxidized. Treatment with ammonia/ammonium altered these CO vibrational frequencies. The ammonia-induced spectral changes are attributed to alterations in hydrogen bonding, when ammonia/ammonium is incorporated into the OEC hydrogen bond network. The ammonia-induced changes in CO frequency were reversed or blocked when trehalose was substituted for sucrose. This trehalose effect is attributed to a displacement of ammonia molecules from the hydrogen bond network. These results imply that ammonia, and by extension water, participate in a catalytically essential hydrogen bond network, which involves OEC peptide CO groups. Comparison to the ammonia transporter, AmtB, reveals structural similarities with the bound water network in the OEC.