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 ST = 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. Ca(2+)/Sr(2+)-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, (55)Mn-ENDOR)
and their first coordination sphere ligands ((14)N/(15)N- and (17)O-ESEEM, -HYSCORE
and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry
DFT now reproduce all EPR, (55)Mn and (14)N 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 MnA4 to the cuboidal unit Mn3O3Ca,
resulting in predictable changes of the measured effective (55)Mn hyperfine tensors.
Sr(2+) and NH3 replacement both affect the observed (17)O-EDNMR signal envelope supporting
the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site
of substrate inclusion.