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 S2LS and S2HS is pH dependent, with a pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5 and pKa ≈ 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 pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of high pH, NH3-S2LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3TyrZ state, consistent with EPR data showing minor or no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2HS was capable of advancing to S3 at low temperature (198 K). This is an experimental demonstration that the S2LS is formed first and advances to S3via the S2HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2LS to S2HS transition which allow the S2HS 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.
Keywords: DFT; EPR; Mn(4)CaO(5) cluster; Oxygen evolution; Photosystem II; Spin state.
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