Biogenesis and repair of the inorganic core (Mn4CaO xCly), in the water-oxidizing complex of photosystem II (WOC-PSII), occurs through the light-induced (re)assembly of its free elementary ions and the apo-WOC-PSII protein, a reaction known as photoactivation. Herein, we use electron paramagnetic resonance (EPR) spectroscopy to characterize changes in the ligand coordination environment of the first photoactivation intermediate, the photo-oxidized Mn3+ bound to apo-WOC-PSII. On the basis of the observed changes in electron Zeeman (geff), 55Mn hyperfine (AZ) interaction, and the EPR transition probabilities, the photogenerated Mn3+ is shown to exist in two pH-dependent forms, differing in terms of strength and symmetry of their ligand fields. The transition from an EPR-invisible low-pH form to an EPR-active high-pH form occurs by deprotonation of an ionizable ligand bound to Mn 3+, implicated to be a water molecule: [Mn3+(OH 2)] ↔ [Mn3+(OH-)]. In the absence of Ca2+, the EPR-active Mn3+ exhibits a strong pH dependence (pH ∼6.5-9) of its ligand-field symmetry (rhombicity Δδ = 10%, derived from geff) and AZ (ΔAZ = 22%), attributable to a protein conformational change. Binding of Ca2+ to its effector site eliminates this pH dependence and locks both geff and AZ at values observed in the absence of Ca2+ at alkaline pH. Thus, Ca2+ directly controls the coordination environment and binds close to the high-affinity Mn3+, probably sharing a bridging ligand. This Ca2+ effect and the pH-induced changes are consistent with the ionization of the bridging water molecule, predicting that [Mn3+-(μ-O-2)-Ca2+] or [Mn3+-(μ-OH-2)2-Ca2+] is the first light intermediate in the presence of Ca2+. The formation of this intermediate templates the apo-WOC-PSII for the subsequent rapid cooperative binding and photo-oxidation of three additional Mn2+ ions, forming the active water oxidase.
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