Photoinhibition of photosystem II (PSII) activity and loss of the D1 reaction center protein were studied in PSII-enriched membrane fragments in which the water-splitting complex was inhibited by depletion of either calcium or chloride or by removing manganese. The Ca2+-depleted PSII was found to be the least susceptible to inhibition by light as reported previously (Krieger, A., and Rutherford, A. W. (1997) Biochim. Biophys. Acta 1319, 91-98). This different susceptibility to light was not reflected in the extent of D1 protein loss. In Mn-depleted PSII the loss of activity and the loss of the D1 protein were correlated, while in Cl-- and Ca2+-depleted PSII, there was very little loss of the D1 protein. The production of free radicals and singlet oxygen was measured by EPR spin-trapping techniques in the different samples. 1O2 and carbon-centered radicals could be detected after photoinhibition of active PSII, while hydroxyl radical formation dominated in all of the other samples. In addition, photoinhibition of PSII was investigated in which the functional Mn cluster was reconstituted (i. e., photoactivated). As expected this led to a protection against photoinhibition. When the photoactivation procedure was done in the absence of Ca2+ no activity was obtained although a nonfunctional Mn cluster was formed. Despite the lack of activity the binding of Mn partially protected against the loss of D1. These data demonstrate that, during photoinhibition, the extent of D1 loss is neither affected by the water-splitting activity of the sample nor correlated to the kinetics of PSII activity loss. D1 loss seems to be independent of the chemical nature of the reactive oxygen species formed during photoinhibition and seems to occur only in the absence of Mn. It is proposed that Mn binding protects against D1 loss by maintaining a protein structure which is not accessible to cleavage.