Substrate water exchange in the S2 state of photosystem II is dependent on the conformation of the Mn4Ca cluster

Abstract

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn₄Ca cluster. During catalysis, the OEC cycles through five redox states, S₀ to S₄. In the S₂ state, the Mn₄Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H₂¹⁸O/H₂¹⁶O exchange between bulk water and the two substrate waters bound at the Mn₄Ca cluster in the S^LS₂, S^HS₂, and the S₃ states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10 times faster in the S^HS₂ than in the S^LS₂ state, and that the S^LS₂ → S^HS₂ conversion has at physiological temperature an activation barrier of 17 ± 1 kcal mol⁻¹. Of the presently suggested S^HS₂ models, our findings are best in agreement with a water exchange pathway involving a S^HS₂ state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S₃ state is governed by a different equilibrium than in S₂, and that the exchange of the fast substrate water in the S₂ state is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining possibilities for O–O bond formation in PSII are discussed.