Crystallographic and Computational Analysis of the Barrel Part of the PsbO Protein of Photosystem II: Carboxylate–Water Clusters as Putative Proton Transfer Relays and Structural Switches

Abstract

In all organisms that employ oxygenic photosynthesis, the membrane-extrinsic PsbO protein is a functionally important component of photosystem II. To study the previously proposed proton antenna function of carboxylate clusters at the protein-water interface, we combined crystallography and simulations of a truncated cyanobacterial (Thermosynechococcus elongatus) PsbO without peripheral loops. We expressed the PsbO β-barrel heterologously and determined crystal structures at resolutions of 1.15-1.5 Å at 100 K at various pH values and at 297 K and pH 6. (1) Approximately half of the 177 surface waters identified at 100 K are resolved at 297 K, suggesting significant occupancy of specific water sites at room temperature, and loss of resolvable occupancy for other sites. (2) Within a loop region specific to cyanobacterial PsbO, three residues and four waters coordinating a calcium ion are well ordered even at 297 K; the ligation differs for manganese. (3) The crystal structures show water-carboxylate clusters that could facilitate fast Grotthus-type proton transfer along the protein surface and/or store protons. (4) Two carboxylate side chains, which are part of a structural motif interrupting two β-strands and connecting PsbO to photosystem II, are within hydrogen bonding distance at pH 6 (100 K). Simulations indicate coupling between protein structure and carboxylate protonation. The crystal structure determined at 100 K and pH 10 indicates broken hydrogen bonding between the carboxylates and local structural change. At pH 6 and 297 K, both conformations were present in the crystal, suggesting conformational dynamics in the functionally relevant pH regime. Taken together, crystallography and molecular dynamics underline a possible mechanism for pH-dependent structural switching.

Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen

“…We therefore conclude that the pH‐induced conformational changes of PsbO occur within the properly folded β barrel of Domain I (Figs and ). The results from crystallography in combination with MD simulations of cyanobacterial PsbO (Bommer et al ) show reversible opening of the hydrogen bond between two carboxyl residues, E97 and D102. They form strong hydrogen bonds resulting in local structural change with apparent pK in the functional relevant pH range.…”

“…Several attempts to crystallize PsbO in isolated form failed to yield crystals amenable to X‐ray analysis (Anati and Adir 2000, Nowaczyk et al ). However, the crystal structure of the isolated barrel domain of a cyanobacterial PsbO protein, PsbO‐β, has been successfully resolved (Bommer et al ). The PSII crystal structures reveal that PsbO is positioned on the lumenal side of the thylakoid membrane with its β‐barrel projecting out into the lumen such that both the N‐ and C‐termini are exposed while the flexible top loops are integrated into PSII, interacting with several transmembrane PSII subunits including another PSII monomer in the PSII dimer (De Las Rivas and Barber , Bricker et al ).…”

“…PsbO therefore has both rigid and fluid parts, giving it both stability and high flexibility. This allows PsbO to respond dynamically to changes in the abundance of redox‐active thioredoxin (Roberts et al ), pH (Shutova et al , , , Bommer et al ), GTP (Lundin et al ) and the calcium ions (Heredia and De Las Rivas , Shutova et al , Murray and Barber ). The structural dynamics of PsbO have been analyzed and related to its functional role in photosynthetic water oxidation based on experiments using intra‐molecular cross‐linking (Enami et al ) and FTIR spectroscopy (Hutchison et al , Sachs et al , Offenbacher et al ).…”

“…PsbO also helps regulate PSII degradation during the PSII repair cycle (Lundin et al 2007) and calcium and chloride binding in PSII (Loll et al 2005, Popelkova et al 2011, Roose et al 2016. The binding of divalent manganese ions to PsbO proteins from higher plants and cyanobacteria in solution has also been reported (Shutova et al 2005, Bommer et al 2016. It has been suggested that PsbO contributes to the transport of protons from the water oxidation site to the thylakoid lumen via a chain of carboxylic amino acids Physiol.…”

pH‐Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes

“…A tendency of PsbO to undergo pH‐dependent structural changes was initially mentioned in conjunction with an observed hysteresis in acid–base titration experiments of isolated PsbO . Through a combination of molecular simulations and crystal structure analyses, it has been observed that upon deprotonation of the carboxylate dyad—Glu97(residue 90 in investigated construct), Asp102(95)—the Asp102 side chain moves away from Glu97 and Lys123(116) . This happens at pH values between 6 and 10.…”