Electronic structure of the oxygen evolving complex in photosystem II, as revealed by ⁵⁵Mn Davies ENDOR studies at 2.5 K

We report the first ⁵⁵Mn pulsed ENDOR studies on the S ₂ state multiline spin centre of the oxygen evolving complex (OEC) in Photosystem II (PS II), at temperatures below 4.2 K. These were performed on highly active samples of spinach PS II core complexes, developed previously in our laboratories for photosystem spectroscopic use, at temperatures down to 2.5 K. Under these conditions, relaxation effects which have previously hindered observation of most of the manganese ENDOR resonances from the OEC coupled Mn cluster are suppressed. ⁵⁵Mn ENDOR hyperfine couplings ranging from ∼50 to ∼680 MHz are now seen on the S₂ state multiline EPR signal. These, together with complementary high resolution X-band CW EPR measurements and detailed simulations, reveal that at least two and probably three Mn hyperfine couplings with large anisotropy are seen, indicating that three Mn^III ions are likely present in the functional S₂ state of the enzyme. This suggests a low oxidation state paradigm for the OEC (mean Mn oxidation level 3.0 in the S₁ state) and unexpected Mn exchange coupling in the S₂ state, with two Mn ions nearly magnetically silent. Our results rationalize a number of previous ligand ESEEM/ENDOR studies and labelled water exchange experiments on the S₂ state of the photosystem, in a common picture which is closely consistent with recent photo-assembly (Kolling et al., Biophys. J. 2012, 103, 313-322) and large scale computational studies on the OEC (Gatt et al., Angew. Chem., Int. Ed. 2012, 51, 12025-12028, Kurashige et al. Nat. Chem. 2013, 5, 660-666).