The photosynthetic protein, Photosystem II (PSII) found in both plants and cyanobacteria is the center for the intricate processes of water oxidation and oxygen evolution. At the oxygen evolving complex (OEC) is a four manganese and calcium (Mn4Ca) cluster and surrounding carboxylate and histidine residues, which help control the active site environment and oxidizing water molecules through a series of electron transfer steps. The OEC accrues oxidizing equivalents in several steps during the catalytic cycle called storage states, or S-states, resulting in protons, electrons, and dioxygen, which is then released and carried out from the active site through a channel within the protein. Identifying the residues involved in this procedure as well as those which stabilize the catalytic cycle through a series of hydrogen bonds is important in understanding the mechanism as a whole. Spectroscopic methods of analysis such as Fourier transform infrared (FTIR) difference spectroscopy can illustrate the dynamic nature of bond creation/destruction that occurs in structural rearrangement. FTIR spectroscopy analysis reproducibly shows many vibrational modes for each S-state throughout the cycle, indicating an arranged routine in water oxidation, are best viewed in the midfrequency region for amide I/II bending, symmetric/asymmetric carboxylate stretching, and carbonyl of carboxylic acid stretching. Changes in vibrational modes, shown as positive or negative peaks, indicate residue participation either at the active site or possibly a water or proton channel.Data have been collected for two ligands to the Mn4Ca cluster, CP43-Glu354 and D1-Glu333 characterizing the former as a ligand which does not change its coordination mode during the S1 to S2 transition but does show evidence that ligation takes place at Mn3 and Mn4 ions. The latter plays an integral role in structure reinforcement as well as dynamic interactions, though the specific activity is not yet clear. Two different channels, an oxygen channel (large) and water or proton egress channel (broad) were observed. Of the large, CP43-Glu354 and D1-Glu329 were included. Of the broad, D1-Glu65, D2-Glu312, D1-Glu333, D2-Glu323, and D2-Lys317 were observed. For the large channel, CP43-Glu354 gave evidence of controlling a hydrogen-bonding network but not directly engaging unlike the D1-Glu329 which does. For the broad, only D1-Glu65 and D2-Glu312 showed clear signs of participating in the network. D1-Glu333 and D2-Lys317 showed evidence of important interaction in structure stability, a likely situation considering proximity to a chloride ion. This study shows the complex and sensitive nature for activity throughout PSII and not just in the OEC during the catalytic cycle
