Effects of pH on the S₃ state of the oxygen evolving complex in photosystem II probed by EPR split signal induction

The electrons extracted from the CaMn₄ cluster during water oxidation in photosystem II are transferred to P₆₈₀⁺ via the redox-active tyrosine D1-Tyr161 (Y_Z). Upon Y_Z oxidation a proton moves in a hydrogen bond toward D1-His190 (His_Z). The deprotonation and reprotonation mechanism of Y_Z-OH/Y _Z-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (∼5 K) Y_Z can be oxidized to its neutral radical, Y_Z^•. This can be followed by the induction of a split EPR signal from Y_Z ^• in a magnetic interaction with the CaMn₄ cluster, offering a way to probe for Y_Z oxidation in active photosystem II. In the S₃ state, light in the near-infrared region induces the split S₃ EPR signal, S₂?Y_Z^•. Here we report on the pH dependence for the induction of S₂?Y _Z^• between pH 4.0 and pH 8.7. At acidic pH the split S₃ EPR signal decreases with the apparent pK_a (pK _app) ∼ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y_Z-His_Z motif. At alkaline pH, the split S₃ EPR signal decreases with the pK _app ∼ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK_app ∼ 8.3) promoted by a change in the redox potential of Y_Z. Our results allow dissection of the proton-coupled electron transfer reactions in the S ₃ state and provide further evidence that the radical involved in the split EPR signals is indeed Y_Z^•.