Abstract
Due to the highly oxidative chemistry required for water oxidation, individual subunits of photosystem II (PS II) are frequently damaged and replaced by a sophisticated repair machinery. Binding of a bicarbonate ligand to the non-heme iron center (NHI) is inhibited in pre-complexes to avoid the generation of singlet oxygen. The NHI mediates electron transfer to a mobile plastoquinone (PQ) electron acceptor in the QB site that shuttles protons and electrons to the next complex of the photosynthetic electron transport chain. Binding of the assembly factor Psb28 at the acceptor side has been correlated with conformational changes of the QB site, inhibiting binding of PQ and HCO3 −. Two homologs of Psb28 exist in cyanobacteria. Their evolution was reconstructed by phylogenetic inference, revealing conservation of Psb28-2 in fewer than half of selected cyanobacterial species. Horizontal gene transfer (HGT) could be excluded to explain the sporadic conservation. De novo structure prediction of Psb28-2 confirmed structural homology to Psb28-1. Using molecular dynamics (MD) simulations, a conserved insertion in a loop region of Psb28-2 was deemed responsible for destabilization of a PS II pre-complex. During purification of Synechocystis Psb28 proteins from recombinant E. coli, concentrated Psb28-2 precipitated while Psb28-1 remained soluble. Psb28 proteins were knocked out in mutant strains of Synechocystis substituting either of the symmetrical residues Tyr246 and Tyr244 from the D1 and D2 reaction core subunits of PS II, respectively, to alanine. These tyrosines coordinate the HCO3 − ligand to the NHI and are involved in protonation of QB. The assembly factors were also knocked out in D2-Y244F and D2-K264A strains. Deletion of Psb28-1 from D2-K264A and D1-Y246A strains increased the number of active PS II centers, although without improving overall photosynthetic activity. Surprisingly, interruption of psb28-2 in the D1-Y246A mutant consistently resulted in the introduction of frameshift mutations in the genes encoding for D1 and D2 proteins, inhibiting PS II assembly. The D1 residues Ala251, His252 and Ser268 thought to be involved in protonation of QB were substituted to their counterparts from the purple bacterial reaction center. The D1-His252Asp mutation on its own did not affect PS II structure but destabilized the semiquinone state of QB. The D1-Ala251Glu mutation on the other hand severely disrupted the structural integrity of PS II.