Intermediate Formation via Proton Release during the Photoassembly of the Water-Oxidizing Mn₄CaO₅ Cluster in Photosystem II

The early stages of the photoassembly of the water-oxidizing Mn₄CaO₅ cluster in spinach photosystem II (PSII) were monitored using rapid-scan time-resolved Fourier transform infrared (FTIR) spectroscopy. Carboxylate stretching and the amide I bands, which appeared upon the flash-induced oxidation of a Mn²⁺ ion, changed their features during the subsequent dark rearrangement process, indicating the relocation of the Mn³⁺ ion concomitant with protein conformational changes. Monitoring the isotope-edited FTIR signals of a Mes buffer estimated that nearly two protons are released upon the Mn²⁺ oxidation. Quantum chemical calculations for models of the Mn binding site suggested that the proton of a water ligand is transferred to D1-H332 through a hydrogen bond upon the Mn³⁺ formation and then released to the bulk as the Mn³⁺ shifts to bind to this histidine. Another Mn²⁺ ion may be inserted to form a binuclear Mn³⁺Mn²⁺ complex, whose structure was calculated to be stabilized by a μ-hydroxo bridge hydrogen-bonded with deprotonated D1-H337. Nearly one additional proton can thus be released from this histidine, assuming that it is mostly protonated before illumination. Alternatively, a proton could be released by further insertion of Ca²⁺, forming a Mn³⁺Mn²⁺Ca²⁺ complex with another hydroxo ligand connecting Ca²⁺ to the Mn³⁺Mn²⁺ complex.