Pulse EPR Spectroscopic Characterization of the S 3 State of the Oxygen-Evolving Complex of Photosystem II Isolated from Synechocystis .

The S ₃ state is the last semi-stable state in the water splitting reaction that is catalyzed by the Mn ₄ O ₅ Ca cluster that makes up the oxygen-evolving complex (OEC) of photosystem II (PSII). Recent high-field/frequency (95 GHz) electron paramagnetic resonance (EPR) studies of PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus have found broadened signals induced by chemical modification of the S ₃ state. These signals are ascribed to an S ₃ form that contains a five-coordinate Mn ^IV center bridged to a cuboidal Mn ^IV ₃ O ₄ Ca unit. High-resolution X-ray free-electron laser studies of the S ₃ state have observed the OEC with all-octahedrally coordinated Mn ^IV in what is described as an open cuboid-like cluster. No five-coordinate Mn ^IV centers have been reported in these S ₃ state structures. Here, we report high-field/frequency (130 GHz) pulse EPR of the S ₃ state in Synechocystis sp. PCC 6803 PSII as isolated in the presence of glycerol. The S ₃ state of PSII from Synechocystis exhibits multiple broadened forms (≈69% of the total signal) similar to those seen in the chemically modified S ₃ centers from T. elongatus . Field-dependent ELDOR-detected nuclear magnetic resonance resolves two classes of ⁵⁵ Mn nuclear spin transitions: one class with small hyperfine couplings (| A| ≈ 1-7 MHz) and another with larger hyperfine couplings (| A| ≈ 100 MHz). These results are consistent with an all-Mn ^IV ₄ open cubane structure of the S ₃ state and suggest that the broadened S ₃ signals arise from a perturbation of Mn4A and/or Mn3B, possibly induced by the presence of glycerol in the as-isolated Synechocystis PSII.