Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S-0 to S-4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S-1, S-2, and S-3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S-2, but flipping of D1 Glu(189) upon transition to S-3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 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.