Photosystem II oxygen-evolving complex photoassembly displays an inverse H/D solvent isotope effect under...

Photosystem II (PSII) performs the solar-driven oxidation of water used to fuel oxygenic photosynthesis. The active site of water oxidation is the oxygen-evolving complex (OEC), a Mn₄CaO₅ cluster. PSII requires degradation of key subunits and reassembly of the OEC as frequently as every 20 to 40 min. The metals for the OEC are assembled within the PSII protein environment via a series of binding events and photochemically induced oxidation events, but the full mechanism is unknown. A role of proton release in this mechanism is suggested here by the observation that the yield of in vitro OEC photoassembly is higher in deuterated water, D₂O, compared with H₂O when chloride is limiting. In kinetic studies, OEC photoassembly shows a significant lag phase in H₂O at limiting chloride concentrations with an apparent H/D solvent isotope effect of 0.14 ± 0.05. The growth phase of OEC photoassembly shows an H/D solvent isotope effect of 1.5 ± 0.2. We analyzed the protonation states of the OEC protein environment using classical Multiconformer Continuum Electrostatics. Combining experiments and simulations leads to a model in which protons are lost from amino acid that will serve as OEC ligands as metals are bound. Chloride and D₂O increase the proton affinities of key amino acid residues. These residues tune the binding affinity of Mn^2+/3+ and facilitate the deprotonation of water to form a proposed μ-hydroxo bridged Mn²⁺Mn³⁺ intermediate.