Photosynthetic water oxidation is catalyzed by the Mn CaO cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S to S ). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S , S , and S states by x-ray free-electron laser serial crystallography. No insertion of water was found in S , but flipping of D1 Glu upon transition to S 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 CaO 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.