Photosystem Ii oxidizes water to form molecular oxygen; this process occurs at a multinuclear manganese cluster. Treatment with reductants, such as hydroxylamine, leads to release of manganese and loss of oxygen evolution. The manganese cluster can be reassembled through a process called photoactivation. The first step of photoactivation consists of binding of Mn2+, which is then photooxidized to form Mn3+. We have used difference Fourier transform infrared spectroscopy to study the oxidation of a partially assembled manganese cluster; this partially assembled cluster may represent an intermediate in photoassembly and contains, on average, one manganese per reaction center, Difference infrared spectra, associated with the photooxidation of this partially assembled cluster at 200 K, are similar to difference infrared spectra associated with the formation of the S-2 multiline state. In particular, spectral features in the 1500-1200 cm(-1) region, previously assigned to the asymmetric and symmetric stretching vibrations of aspartate and/or glutamate residues, are observed in both cases and are essentially identical, These data indicate that the first, high-affinity binding site for manganese in photosystem ll has the same coordination as the manganese atom, which is oxidized upon formation of the S-2 multiline state.