Photosystem II (PSII) is a membrane protein complex that catalyzes the light-driven oxidation of water, forming the molecular oxygen indispensable to life on Earth. The goal of this thesis is to elucidate how PSII orchestrates water delivery at its oxygen-evolving complex (OEC) through finely tuned protein–cofactor–water networks. Five interconnected projects employ a range of structural and spectroscopic techniques—including serial femtosecond crystallography (SFX), high-resolution cryo-electron microscopy (cryo-EM), time-resolved membrane inlet mass spectrometry (TR-MIMS), EPR spectroscopy, and in-vivo variable fluorescence—to reveal key mechanistic steps in water oxidation.

Project I captures the final S3→[S4]→S0 transition of the Kok cycle using time-resolved SFX, unveiling a two-step Mn₄CaO₅–O_x cluster reduction and a potential peroxidic intermediate. Project II uses cryo-EM to resolve a 1.71 Å resolution light-activated structure of Thermosynechococcus vestitus PSII, revealing crucial proton and water positions, clarifying the mechanism of two-step Q_B reduction, and reinforcing the role of the O1-channel as a primary substrate route. Project III reevaluates a two-site two-conformation exchange model to reconcile O5 as the slowly exchanging substrate, emphasizing how the conformational equilibrium of the Mn₄CaO₅ cluster dictates kinetics. Project IV settles the debate over O1-channel accessibility in Synechocystis sp. PCC 6803 PSII by showing that site-directed mutations in channel bottleneck residues diminish substrate water exchange efficiency. Finally, Project V presents a unifying framework for multi-step substrate exchange, exemplified by the D1-N298A mutation’s differential impact on S₂- and S₃-state kinetics; this mutation disrupts N298 hydrogen-bond network in the O1-terminal cavity, impairing Y_Z oxidation and specifically slowing substrate exchange in the S₃ state.

Together, these studies demonstrate how well-defined channels, protein hydrogen-bonding motifs, and water clusters collectively govern PSII’s water oxidation. By integrating diverse methodological approaches, the thesis reveals the centrality of protein–water dynamics in regulating the substrate water molecule management in the OEC. The findings refine current mechanistic models of O–O bond formation, laying a foundation for future research into the design of bioinspired catalysts and further explorations of nature’s remarkable water-splitting machinery.