Probing substrate water access through the O1 channel of Photosystem II by single site mutations and membrane inlet mass spectrometry

Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO₂ to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn₄CaO₅ cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated from Thermosynechococcus vestitus (T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn₄CaO₅ cluster during its S₂→S₃ and S₃→S₄→S₀ state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacterium Synechocystis sp. PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitus numbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn₄CaO₅ cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn₄CaO₅ cluster that includes the pentameric, near planar 'water wheel' of the O1 channel.