Spectroscopic evidence for Ca²⁺ involvement in the assembly of the Mn₄Ca cluster in the photosynthetic water-oxidizing complex

Biogenesis and repair of the inorganic core (Mn₄CaO _xCl_y), 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 (g_eff), ⁵⁵Mn hyperfine (A_Z) 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 g_eff) and A_Z (ΔA_Z = 22%), attributable to a protein conformational change. Binding of Ca²⁺ to its effector site eliminates this pH dependence and locks both g_eff and A_Z 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.