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 O₂-release and strongly perturb the water network surrounding the Mn₄Ca cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the H₂¹⁸O/H₂¹⁶O-exchange kinetics of the fast (W_f) and slow (W_s) exchanging substrate waters bound in the S₁, S₂ and S₃ states to the Mn₄Ca 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_s was increased in the S₁ and S₂ states, while both W_f and W_s exchange rates were decreased in the S₃ state. Additionally, we used EPR spectroscopy to characterize the Mn₄Ca cluster and its interaction with the redox active D1-Tyr161 (Y_Z). In the S₂ 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 S₁ state was not affected, while the split signal in the S₃ 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 Mn₄Ca cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.

在光合作用中，色素-蛋白质复合物光系统II（PSII）的析氧复合物（OEC）协调水的氧化。以前有报道称将V185N突变引入D1蛋白会极大地减慢O ₂释放并强烈干扰Mn ₄ Ca团簇周围的水网络。利用时间分辨膜入口质谱法，我们在这里测量了结合在S ₁，S ₂和S上的快速（W _f）和慢速（W _s）交换底物水的H ₂ ¹⁸ O / H ₂ ¹⁶ O交换动力学。S ₃表示Mn ₄Ca簇的PSII核心复合物分离自野生型和集藻的D1-V185N菌株。PCC 6803。我们发现，W _s的汇率在S ₁和S ₂状态下增加，而W _f和W _s汇率在S ₃状态下都下降。此外，我们使用EPR光谱来表征Mn ₄ Ca团簇及其与氧化还原活性D1-Tyr161（Y _Z）的相互作用。在S ₂状态下，我们观察到V185N-PSII中的多行信号大大减小，可以通过添加氨来恢复该信号。S中的分割信号D1-V185N突变体不影响₁状态，而S ₃状态的分裂信号不存在。这些发现通过N185残基通过氢键稳定了Mn ₄ Ca簇的Mn1位点处额外的水衍生配体的结合的提议而变得合理。讨论了底物水结合位点的含义。