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

Among the intermediate catalytic steps of the water‐oxidizing Mn(4)CaO(5) cluster of photosystem II (PSII), the final metastable S(3) state is critically important because it binds one substrate and precedes O(2) 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 S(3) 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(IV) (3) subunit interacting ferromagnetically via anisotropic exchange with a coordinatively unsaturated Mn(IV) ion. These results resolve and define the structural heterogeneity of the S(3) state, providing constraints on the S(3) to S(4) transition, on substrate identity and delivery pathways, and on the mechanism of O−O bond formation.