Cryogenic Trapping and Isotope Editing Identify a Protonated Water Cluster as an Intermediate in the Photosynthetic Oxygen-Evolving Reaction

Internal water is known to play a catalytic role in several enzymes. In photosystem II (PSII), water is the substrate. To oxidize water, the PSII Mn₄CaO₅ cluster or oxygen evolving center (OEC) cycles through five oxidation states, termed S_<i>n</i> states. As reaction products, molecular oxygen is released, and protons are transferred through a ∼25 Å hydrogen-bonded network from the OEC to the thylakoid lumen. Previously, it was reported that a broad infrared band at 2880 cm^–1 is produced during the S₁-to-S₂ transition and accompanies flash-induced, S state cycling at pH 7.5. Here, we report that when the S₂ state is trapped by continuous illumination under cryogenic conditions (190 K), an analogous 2740/2900 cm^–1 band is observed. The frequency depended on the sodium chloride concentration. This band is unambiguously assigned to a normal mode of water by D₂¹⁶O and H₂¹⁸O solvent exchange. Its large, apparent H₂¹⁸O isotope shift, ammonia sensitivity, frequency, and intensity support assignment to a stretching vibration of a hydronium cation, H₃O⁺, in a small, protonated internal water cluster, <i>n</i>H₂O­(H₃O⁺). Water OH stretching bands, which may be derived from the hydration shell of the hydronium ion, are also identified. Using the 2740 cm^–1 infrared marker, the results of calcium depletion and strontium reconstitution on the protonated water cluster are found to be pH dependent. This change is attributed to protonation of an amino acid side chain and a possible change in <i>n</i>H₂O­(H₃O)⁺ localization in the hydrogen-bonding network. These results are consistent with an internal water cluster functioning as a proton acceptor and an intermediate during the S₁-to-S₂ transition. Our experiments demonstrate the utility of this infrared signal as a novel functional probe in PSII