Photosynthetic water oxidation is catalyzed by the Mn<sub>4</sub>CaO<sub>5</sub> cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S<sub>0</sub> to S<sub>4</sub>). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S<sub>1</sub>, S<sub>2</sub>, and S<sub>3</sub> states by x-ray free-electron laser serial crystallography. No insertion of water was found in S<sub>2</sub>, but flipping of D1 Glu<sup>189</sup> upon transition to S<sub>3</sub> leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn<sub>4</sub>CaO<sub>6</sub> cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.