Effects of pH on the S(3) state of the oxygen evolving complex in photosystem II probed by EPR split signal induction

The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) 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 CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(•). Here we report on the pH dependence for the induction of S(2)'Y(Z)(•) between pH 4.0 and pH 8.7. At acidic pH the split S(3) 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 S(3) 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)(•).