Mechanism of Initial Mn²⁺ Oxidation during Photoassembly of the Water-Oxidizing Mn₄CaO₅ Cluster in Photosystem II

The Mn₄CaO₅ cluster, the catalytic center of water oxidation in photosystem II (PSII), is assembled from free Mn²⁺ and Ca²⁺ ions through a light-driven process known as photoactivation. Despite its importance, the molecular mechanism of photoactivation remains poorly understood. Here, we investigate the mechanism underlying the initial oxidation of Mn²⁺ at its binding site in apo-PSII using time-resolved infrared spectroscopy combined with density functional theory (DFT) calculations. Two distinct kinetic phases with time constants of 100-150 μs and 1.5-2.5 ms are observed at pH 6.5-7.5. The minimal H/D isotope effect indicates that electron transfer from Mn²⁺ to Y_Z^•, rather than proton release, is the rate-limiting step. DFT calculations of the Mn³⁺/Mn²⁺ redox potential, together with ΔG° estimates derived from electron transfer rates using semiempirical equations, reveal that Mn²⁺ oxidation proceeds via a slow, endergonic electron transfer from Mn²⁺ to Y_Z^•, followed by rapid proton transfer from a water ligand to D1-H332, which provides the driving force for the reaction. The faster kinetic component is attributed to a decreased ΔG° resulting from detachment of CP43-R357 in the "open" CP43 conformation, suggesting that thermal fluctuations of the CP43 lumenal domain facilitate initial Mn²⁺ oxidation during photoactivation.