Oxidation of water in photosynthetic organisms occurs in a chlorophyll containing enzyme, photosystem II. The catalytic manganese cluster of photosystem II cycles among five redox states called the S-n states. There are two forms of the S-2 state, which give rise to different EPR signals and which differ in magnetic coupling among the manganese atoms. We have used difference infrared spectroscopy to obtain more information about the environment of the manganese cluster in these two forms of the S-2 state. We present the 1600-1200 cm(-1) region of the difference spectrum associated with the generation of the multiline state and the g = 4.1 S-2 state. The difference spectrum associated with generation of the S-2 multiline state from the dark stable S-1 state shows broad spectral features in the 1500-1200 cm(-1) region at 1490 (negative), 1331 (negative), 1393 (positive), and 1267 (positive) cm(-1). Global N-15 labeling has little impact on intensities or frequencies in this region of the spectrum. These vibrational features are not observed in manganese-depleted, EDTA-treated photosystem II. Also, these features are not observed in oxygen-evolving photosystem II upon illumination at 80 K, a temperature where the manganese cluster is not oxidized. We conclude that these lines arise from amino acid residues that are close to or ligating to the cluster. From the frequencies and the lack of sensitivity to. N-15 labeling, we favor the assignment of these lines to the asymmetric and symmetric stretch of one or more glutamate and/or aspartate residue(s). The spectral breadth of the lines is consistent with either an inhomogeneous or a homogeneous broadening mechanism. When illumination conditions are used that generate the g = 4.1 S-2 EPR signal, these vibrational lines are not observed. These results are discussed in terms of current models for the catalytic site.