Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

The active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)CaO(5) cluster and Tyr(Z), a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7Å away from the Mn(4)CaO(5) cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I(-) for Cl(-) in the cyanobacterium Thermosynechococcus elongatus with either Ca(2+) or Sr(2+) biosynthetically assembled into the Mn(4) cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV-visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl(-)/I(-) exchange considerably slowed down the two S(3)Tyr(Z)(•)→(S(3)Tyr(Z)(•))'→S(0) reactions in which the fast phase, S(3)Tyr(Z)(•)→(S(3)Tyr(Z)(•))', reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on Tyr(Z)(•) and the slow phase corresponds to the S(0) and O(2) formations and to a second proton release. The t(1/2) for S(0) formation increased from 1.1 ms in Ca/Cl-PSII to ≈6 ms in Ca/I-PSII and from 4.8 ms in Sr/Cl-PSII to ≈45 ms in Sr/I-PSII. In all cases the Tyr(Z)(•) reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca(2+) binding site and the Cl(-) binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl(-) and Ca(2+) binding sites by which proton release may be channelled.