The low spin - high spin equilibrium in the S 2 -state of the water oxidizing enzyme.

In Photosystem II (PSII), the Mn4 CaO5 -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 pKa  ≈ 8.3 (n ≈ 4) for the native Mn4 CaO5 and pKa  ≈ 7.5 (n ≈ 1) for Mn4 SrO5 . 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 -S2 LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3 TyrZ 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 S3 via 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.