An additional water is introduced into the manganese cluster during the formation of the S₃ state of photosystem II.

Abstract

During photosynthetic water oxidation, the Mn₄Ca cluster in Photosystem II progresses through five intermediate S_i (i = 0-4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S₃ state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S₃ state or in the subsequent S₃ to S₀ transition. Here we report conventional high-resolution data for the S₁, S₂, and S₃ states to a resolution of ~1.9 Å, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, O_X (or O6), in the S₃ state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The O_X-O5 distance is ~2.1 Å, supporting no strong interaction between them in the S₃ state, suggesting that if this is the O-O bond formation site, it is formed during the S₃ to S₀ transition initiated by the final oxidation of the cluster.