Difference infrared spectroscopy is used to study the manganese-containing catalytic site of photosystem II. Vibrational spectroscopy can test the hypothesis that there are protein conformational differences between the two EPR detectable forms of the S-2 state, which are known as the g = 4.1 and the multiline state. A light-minus-dark difference spectrum is constructed at 200, 130, and 80 K. These illumination temperatures generate the S-2 multiline state, the S-2 g = 4.1 state, and a chlorophyll cation radical, respectively. Our data show that a unique protein conformation is associated with the formation of the g = 4.1 S-2 state. Also, difference infrared spectroscopy demonstrates that formation of the S-2 multiline state perturbs the vibrational spectrum of one or more carboxylic acid residues that may be in the vicinity of the manganese cluster. Tile perturbation is probably due to a:change in hydrogen bonding or effective dielectric constant upon formation of the S-2 state. Further, this carboxylate residue is conserved in plant and in cyanobacterial photosystem II.