The low spin - high spin equilibrium in the S₂-state of the water oxidizing enzyme

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S₂ using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S₂^LS and S₂^HS is pH dependent, with a pK_a ≈ 8.3 (n ≈ 4) for the native Mn₄CaO₅ and pK_a ≈ 7.5 (n ≈ 1) for Mn₄SrO₅. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pK_a upon the Ca/Sr exchange. EPR also showed that NH₃ addition reversed the effect of high pH, NH₃-S₂^LS being present at all pH values studied. Absorption spectroscopy indicates that NH₃ is no longer bound in the S₃Tyr_Z state, consistent with EPR data showing minor or no NH₃-induced modification of S₃ and S₀. In both Ca-PSII and Sr-PSII, S₂^HS was capable of advancing to S₃ at low temperature (198 K). This is an experimental demonstration that the S₂^LS is formed first and advances to S₃via the S₂^HS state without detectable intermediates. We discuss the nature of the changes occurring in the S₂^LS to S₂^HS transition which allow the S₂^HS to S₃ transition to occur below 200 K. This work also provides a protocol for generating S₃ in concentrated samples without the need for saturating flashes.