Comment on <i>Photosystem II: light-dependent oscillation of ligand composition at its active site</i> : existence of O6 in S ₃ -state photosystem II revealed by omit maps

Photosystem II (PSII) catalyzes the light-driven oxidation of water to molecular oxygen through a series of redox states (S 0 –S 4 ) in the Kok cycle. This reaction is catalyzed by a Mn 4 CaO 5 cluster bound to PSII in the dark-stable S 1 state, and insertion of a sixth oxygen ligand (O6) in the S 3 state has been shown to be a key step in O—O bond formation by multiple pump–probe X-ray free-electron laser (XFEL) studies. However, a recent reanalysis of the experimental XFEL data by Wang [(2024), Acta Cryst. D 80 , 850–861] challenged the existence of O6, attributing the observed difference electron density to an artifact arising from a two-conformer model of O5 instead of the insertion of a new oxygen O6. Here, we reanalyzed our 2F dataset using improved omit-map strategies, including conformation-specific analyses, and clearly resolved electron density corresponding to a partially occupied O6 site. Moreover, omit maps from time-resolved serial femtosecond X-ray crystallography datasets at room temperature revealed a transient intermediate (O6*) near the Ca site shortly after the second flash, which subsequently migrated to the O6 position. These findings provide structural evidence that supports the assignment of O6 as a state-specific ligand in the S 3 state, and are inconsistent with the interpretation proposed by Wang.