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Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few.

The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn₄Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn₄Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn₄Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism.

In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S₂-S₃ state transition of the catalytic cycle.

Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water W_s is controlled by a dynamic equilibrium between conformations in the S₂-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of W_s and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle.

To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S₃-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S₂-S₃ transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S₂ state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S₂ or the S₃ states.

Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn₄Ca-cluster to show that the briding oxygen O5  is exchanging with a near identical rate to W_s, further supporting the assignment that W_s=O5.

In conclusion, O-O bond formation most likely occurs between W2 (W_f) and O5 (W_s) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.