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 (S-0 to S-4) before water is oxidized and O-2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S-2 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 S-2(LS) and S-2(HS) is pH dependent, with a pK(a) approximate to 8.3 (n approximate to 4) for the native Mn4CaO5 and 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-S2LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S(3)Tyr(z) state, consistent with EPR data showing minor or no NH3-induced modification of S-3 and S-0. In both Ca-PSII and Sr-PSII, S-2(HS) was capable of advancing to S-3 at low temperature (198 K). This is an experimental demonstration that the S-2(LS) is formed first and advances to S-3 via the S-2(HS) state without detectable intermediates. We discuss the nature of the changes occurring in the S-2(LS) to S-2(HS) transition which allow the S-2(HS) to S-3 transition to occur below 200 K. This work also provides a protocol for generating S-3 in concentrated samples without the need for saturating flashes.