Pulse EPR Spectroscopic Characterization of the S3 State of the Oxygen-Evolving Complex of Photosystem II Isolated from Synechocystis

The S3 state is the last semi-stable state in the water splitting reaction that is catalyzed by the Mn4O5Ca 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 S3 state. These signals are ascribed to an S3 form that contains a five-coordinate MnIV center bridged to a cuboidal MnIV 3O4Ca unit. High-resolution X-ray free-electron laser studies of the S3 state have observed the OEC with all-octahedrally coordinated MnIV in what is described as an open cuboid-like cluster. No five-coordinate MnIV centers have been reported in these S3 state structures. Here, we report high-field/frequency (130 GHz) pulse EPR of the S3 state in Synechocystis sp. PCC 6803 PSII as isolated in the presence of glycerol. The S3 state of PSII from Synechocystis exhibits multiple broadened forms (≈69% of the total signal) similar to those seen in the chemically modified S3 centers from T. elongatus. Field-dependent ELDOR-detected nuclear magnetic resonance resolves two classes of 55Mn 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-MnIV 4 open cubane structure of the S3 state and suggest that the broadened S3 signals arise from a perturbation of Mn4A and/or Mn3B, possibly induced by the presence of glycerol in the as-isolated Synechocystis PSII.