Structural and functional analysis of a photosystem II mutant PsbA3-S264V.

Photosystem II (PSII) catalyzes water oxidation and oxygen evolution by a light-induced electron transfer chain, leading to the generation of electrons, protons and dioxygen. D1-S264 is a residue located close to the QB-binding site, and mutation of this residue has been shown to bring significant effects on the electron transfer and oxygen-evolving activities. Here we analyzed the structure of a Thermosynechococcus elongatus mutant PsbA3-S264V by cryo-electron microscopy at 1.96 Å resolution, which showed significant changes in the structure surrounding the bicarbonate and QB-binding region. Due to change of Ser to Val, the hydrogen-bond between the QB carbonyl oxygen and S264 is altered, which changed the protonation pathway of QB from the original route of D1-H252 through D1-S264 to QB, to a new, longer and less efficient route of D1-H252 through D1-F265 to QB. Two residues, D1-E244 and D2-E242, changed their side chain orientations significantly. Among them, D2-E242 adopted two conformations, and both are largely deviated from the original structure. All these changes led to alterations in hydrogen-bonding networks of two channels, channel A and channel B, that connect the stromal surface to QB and may function to transport protons to protonate QB. Furthermore, isothermal titration calorimetry experiments showed a diminished 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) binding affinity of the mutated PSII, which may be explained by a structural rotation of D1-F255 in the mutant based on structural analysis of DCMU-bound PSII. These findings offer valuable insights into the functions of D1-S264 in QB protonation and function, as well as in the DCMU-binding.