Abstract
The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA(1) or the psbA(3) gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491-1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b(559) (Cyt b(559)) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b(559) properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b(559) likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.
Highlights
Photosystem II (PSII) is often exposed to photo-oxidative stress
For the assembly and synthesis of the de novo D1 protein in PSII complex, the old D1 polypeptide is digested by specific proteases such as FtsH and Deg/HtrA [14], and the newly synthetized D1 is assembled into the PSII complex
Cyanobacterial species have with a His6 tag on the C terminus of CP43; WT*, His6 tag T. elongatus strain in which the psbA1 and psbA2 genes were deleted; WT*/⌬PsbJ, His6-tag T. elongatus strain in which the psbA1, psbA2, and psbJ genes were deleted; 43H/⌬PsbJ, His6 tag T. elongatus strain in which the psbJ gene was deleted; HP, high potential; LP, low potential
Summary
Construction of ⌬PsbJ Strains—DNA fragments of Ϸ930 bp of the 5Ј-flanking region of psbJ (gene number tsr1544) were cloned from T. elongatus wild-type genomic DNA by PCR amplification and subcloned into a plasmid vector pBluescript II SKϩ between KpnI and XbaI sites. The 43H/⌬PsbJ transformants were selected as single colonies on DTN agar plates containing 25 g of spectinomycin mlϪ1, 10 g of streptomycin mlϪ1, and 40 g of kanamycin mlϪ1 as previously described in Refs. Gel Permeation Chromatography—For size separation, the PSII complex was further treated with 1.5% n-dodecyl--Dmaltoside at a Chl concentration of 1.5 mg mlϪ1 for 30 min at 4 °C in the dark. SDS-Polyacrylamide Gel Electrophoresis—PSII complexes suspended in 40 mM MES/NaOH (pH 6.5), 10 mM NaCl, 10 mM CaCl2, 10 mM MgCl2, 0.03% n-dodecyl--D-maltoside were solubilized with 2% lithium lauryl sulfate and analyzed by SDS-polyacrylamide gel electrophoresis with a 16 –22% gradient gel containing 7.5 M urea as described in Ref. 40. Samples were illuminated for ϳ2 min, long enough to induce the oxidation of Cyt b559 in a proportion of the centers but short enough to avoid the warming of the sample (even at 77 K) and, to prevent the relaxation of the non-relaxed into the relaxed state of the light-induced oxidized Cyt b559
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
