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. Here we combine X- and Q-band EPR experiments on native and methanol-treated PSII of Spinacia oleracea (spinach) 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.