To probe the involvement of amino acid residues of the D2 protein in the water-splitting process in photosystem II, site-directed mutagenesis was applied to identify D2 residues that might contribute to binding the Mn cluster involved in oxygen evolution. Mutation of Glu-69 to Gln or Val in D2 of the cyanobacterium Synechocystis sp. PCC 6803 was found to lead to a loss of photoautotrophic growth. However, in cells of the Gln mutant (E69Q) a significant Hill reaction rate could be observed upon the start of illumination, but the oxygen evolution rate declined with a half-time of approximately 1 min. Addition of 1 mM Mn2+ stabilized oxygen evolution in E69Q thylakoids. Other divalent cations were ineffective in specific stabilization. When the water-splitting system was bypassed, the rate of electron transport remained stable during illumination, indicating that the inactivation of oxygen evolution is localized in the water-splitting complex. We interpret these observations to indicate that Glu-69 is a Mn ligand and that the loss of oxygen evolution in the E69Q mutant upon turnover of PS II is initiated by changes in the Mn cluster, possibly leading to Mn release from the water-splitting complex. The addition of exogenous Mn to E69Q thylakoids may help to keep the Mn cluster active for a longer time, perhaps by providing Mn to rebind in the cluster after release of one Mn and before the Mn cluster had disintegrated.(ABSTRACT TRUNCATED AT 250 WORDS)