Infrared Determination of the Protonation State of a Key Histidine Residue in the Photosynthetic Water Oxidizing Center

Photosynthetic water oxidation is performed at the Mn₄CaO₅ cluster in photosystem II (PSII). The protonation structures of amino acid residues and water molecules around the Mn₄CaO₅ cluster are crucial in water oxidation reactions. In this study, we determined the protonation state of a key His residue in water oxidation, D1-H337, that is directly hydrogen-bonded with the oxygen atom of the Mn₄CaO₅ cluster, using polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Flash-induced polarized ATR-FTIR difference spectra upon the S₁ → S₂ transition of oriented PSII membranes showed broad negative and positive features at about 2600 and 2900 cm^-1, respectively, with large dichroic ratios, accompanied by several minor peaks attributable to the Fermi resonance of a His NH vibration. Quantum mechanics/molecular mechanics (QM/MM) calculations well reproduced the characteristics of these features as the N_τH stretching vibrations of D1-H337 in its protonated cation form. The spectral features were reversed in the S₃ → S₀ transition, indicating that this His remains protonated during the S-state cycle. The redox potential (E_m) of the Mn₄CaO₅ cluster in the S₁ → S₂ transition, which was estimated from the QM/MM calculations, was found to be comparable to that of water oxidation when D1-H337 is protonated cation. It was thus concluded that the positive charge on the protonated D1-H337 plays an important role in retaining a high E_m value of the Mn₄CaO₅ cluster throughout the reaction cycle to enable water oxidation.