The role of D2 and CP43 proteins in the bicarbonate-binding environment of Photosystem II

In Photosystem II (PS II), absorbed light energy is used to transfer electrons from water to a primary plastoquinone electron acceptor (QA) and then to the secondary plastoquinone acceptor (QB). A non-heme iron (NHI) is located between QA and QB and ligated by His residues from the D1 and D2 reaction centre protein; in addition, a bicarbonate ion forms a bidentate ligand to the NHI. Stabilisation of bicarbonate is provided by D2-Tyr244, D1-Tyr246 and the two water molecules, W622 and W582, through hydrogen bonds (PDB 4UB6). These two water molecules and amino acid residues around the bicarbonate-binding environment have been hypothesised to participate in stabilisation of a hydrogen-bond network for delivering protons via the bound HCO3- to the QB2-(H+) form of the secondary plastoquinone electron acceptor of PS II. The main purpose of this study was to investigate the importance of D2 and CP43 amino acid residues in maintaining the hydrogen-bond network to provide the stabilisation of bicarbonate and their roles in the structure, electron transfer and protonation steps in the QA-NHI-QB complex of PS II. Mutations targeting four amino acid residues from D2 protein (D2-Glu242, D2-Thr243, D2-Tyr244 and D2-Lys264) and one amino acid residue from CP43 protein (CP43-Arg448) were selected. Nearly all mutants showed impaired oxygen evolution and were highly susceptible to photodamage in the presence of PS II-specific electron acceptors; however, they were able to acclimate to high light when HCO3– was added. In addition, acceptor side electron transfer was altered in these strains, with a decrease in the forward electron transfer between QA and QB, as well as the back reaction from QA, in the presence of the PS II-specific inhibitor 3,4-dichloro-1,1-dimethyl urea (DCMU). Furthermore, all mutants except the D2-Glu242 mutant exhibited the accumulation of unassembled complexes containing the CP43 subunit and an ineffective repair mechanism was also observed in these mutants that showed delayed protein synthesis following the high-light treatment based on 35S-Met protein labelling experiments. This study indicated that the disruption of the hydrogen-bond network associated with the bicarbonate-binding environment impairs PS II assembly, electron transfer and also the repair mechanism of the photosystem following high-light-induced damage