The D1-V185N mutation alters substrate water exchange by stabilizing alternative structures of the MnCa-cluster in photosystem II.

In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O-release and strongly perturb the water network surrounding the MnCa cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the HO/HO-exchange kinetics of the fast (W) and slow (W) exchanging substrate waters bound in the S, S and S states to the MnCa cluster of PSII core complexes isolated from wild type and D1-V185N strains of Synechocystis sp. PCC 6803. We found that the rate of exchange for W was increased in the S and S states, while both W and W exchange rates were decreased in the S state. Additionally, we used EPR spectroscopy to characterize the MnCa cluster and its interaction with the redox active D1-Tyr161 (Y). In the S state, we observed a greatly diminished multiline signal in the V185N-PSII that could be recovered by addition of ammonia. The split signal in the S state was not affected, while the split signal in the S state was absent in the D1-V185N mutant. These findings are rationalized by the proposal that the N185 residue stabilizes the binding of an additional water-derived ligand at the Mn1 site of the MnCa cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.