Arrested Substrate Binding Resolves Catalytic Intermediates in Higher-Plant Water Oxidation

Among the intermediate catalytic steps of the water-oxidizing Mn₄ CaO₅ cluster of photosystem II (PSII), the final metastable S₃ state is critically important because it binds one substrate and precedes O₂ evolution. Herein, we combine X- and Q-band EPR experiments on native and methanol-treated PSII of Spinacia oleracea and show that methanol-treated PSII preparations of the S₃ state correspond to a previously uncharacterized high-spin (S=6) species. This is confirmed as a major component also in intact photosynthetic membranes, coexisting with the previously known intermediate-spin conformation (S=3). The high-spin intermediate is assigned to a water-unbound form, with a Mn^IV₃ subunit interacting ferromagnetically via anisotropic exchange with a coordinatively unsaturated Mn^IV ion. These results resolve and define the structural heterogeneity of the S₃ state, providing constraints on the S₃ to S₄ transition, on substrate identity and delivery pathways, and on the mechanism of O-O bond formation.