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Abstract

The S₂ state of the oxygen-evolving complex of photosystem II, which consists of a Mn₄O₅Ca cofactor, is EPR-active, typically displaying a multiline signal, which arises from a ground spin state of total spin S_T = 1/2. The precise appearance of the signal varies amongst different photosynthetic species, preparation and solvent conditions/compositions. Over the past five years, using the model species Thermosynechococcus elongatus, we have examined modifications that induce changes in the multiline signal, i.e. Ca²⁺/Sr²⁺-substitution and the binding of ammonia, to ascertain how structural perturbations of the cluster are reflected in its magnetic/electronic properties. This refined analysis, which now includes high-field (W-band) data, demonstrates that the electronic structure of the S₂ state is essentially invariant to these modifications. This assessment is based on spectroscopies that examine the metal centres themselves (EPR, ⁵⁵Mn-ENDOR) and their first coordination sphere ligands (¹⁴N/¹⁵N- and ¹⁷O-ESEEM, -HYSCORE and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry DFT now reproduce all EPR, ⁵⁵Mn and ¹⁴N experimental magnetic observables, with the inclusion of second coordination sphere ligands being crucial for accurately describing the interaction of NH₃ with the Mn tetramer. These results support a mechanism of multiline heterogeneity reported for species differences and the effect of methanol [Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 829], involving small changes in the magnetic connectivity of the solvent accessible outer Mn_A4 to the cuboidal unit Mn₃O₃Ca, resulting in predictable changes of the measured effective ⁵⁵Mn hyperfine tensors. Sr²⁺ and NH₃ replacement both affect the observed ¹⁷O-EDNMR signal envelope supporting the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site of substrate inclusion.