The S₀ state of the water oxidizing complex in photosystem II: pH dependence of the EPR split signal induction and mechanistic implications

Water oxidation in photosystem II is catalyzed by the CaMn₄ cluster. The electrons extracted from the CaMn₄ cluster are transferred to P₆₈₀⁺ via the redox-active tyrosine residue D1-Tyr161 (Y_Z). The oxidation of Y_Z is coupled to a deprotonation creating the neutral radical Y_Z^•. Light-induced oxidation of Y_Z is possible down to extreme temperatures. This can be observed as 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 PSII. Here we have used the split S₀ EPR signal to study the mechanism of Y_Z oxidation at 5 K in the S₀ state. The state of the hydrogen bond between Y_Z and its proposed hydrogen bond partner D1-His190 is investigated by varying the pH. The split S₀ EPR signal was induced by illumination at 5 K between pH 3.9 and pH 9.0. Maximum signal intensity was observed between pH 6 and pH 7. On both the acidic and alkaline sides the signal intensity decreased with the apparent pK_as (pK_app) ∼4.8 and ∼7.9, respectively. The illumination protocol used to induce the split S₀ EPR signal also induces a mixed radical signal in the g ∼ 2 region. One part of this signal decays with similar kinetics as the split S₀ EPR signal (∼ 3 min, at 5 K) and is easily distinguished froma stable radical originating from Car/Chl. We suggest that this fast-decaying radical originates from Y_Z^•. The pH dependence of the light-induced fast-decaying radical was measured in the same pH range as for the split S₀ EPR signal. The pK_app for the light-induced fast-decaying radical was identical at acidic pH (∼4.8). At alkaline pH the behavior was more complex. Between pH 6.6 and pH 7.7 the signal decreased with pK_app ∼7.2. However, above pH 7.7 the induction of the radical species was pH independent. We compare our results with the pH dependence of the split S₁ EPR signal induced at 5 K and the S ₀ → S₁ and S₁ → S₂ transitions at room temperature. The result allows mechanistic conclusions concerning differences between the hydrogen bond pattern around Y_Z in the S₀ and S₁ states.