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

Abstract

Among the intermediate catalytic steps of the water‐oxidizing Mn4CaO5 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 MnIV 3 subunit interacting ferromagnetically via anisotropic exchange with a coordinatively unsaturated MnIV 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.