Photosynthesis Research

, Volume 98, Issue 1–3, pp 337–347 | Cite as

Directed mutagenesis of the transmembrane domain of the PsbL subunit of photosystem II in Synechocystis sp. PCC 6803

  • Hao Luo
  • Julian J. Eaton-Rye
Regular Paper


The PsbL protein is one of three low-molecular-weight subunits identified at the monomer–monomer interface of photosystem II (PSII) [Ferreira et al. (2004) Science 303:1831–1838; Loll et al. (2005) Nature 438:1040–1044]. We have employed site-directed mutagenesis to investigate the role of PsbL in Synechocystis sp. PCC 6803 cells. Truncation of the C-terminus by deleting the last four residues (Tyr-Phe-Phe-Asn) prevented association of PsbL with the CP43-less monomeric sub-complex and therefore blocked PSII assembly resulting in an obligate photoheterotrophic strain. Replacement of these residues with Ala created four photoautotrophic mutants. Compared to wild type, the F37A, F38A, and N39A strains had reduced levels of assembled PSII centers and F37A and F38A cells were readily photodamaged. In contrast, Y36A and Y36F mutants were similar to wild type. However, each of these strains had elevated levels of the CP43-less inactive monomeric complex. Mutations targeting a putative hydrogen bond between Arg-16 and sulfoquinovosyldiacylglycerol resulted in mutants that were also highly susceptible to photodamage. Similarly mutations targeting a conserved Tyr residue (Tyr-20) also destabilized PSII under high light and suggest that Tyr-20–lipid interactions or interactions of Tyr-20 with PsbT influence the ability of PSII to recover from photodamage.


Photosystem II Photodamage PsbL Site-directed mutagenesis Synechocystis sp. PCC 6803 



Blue-native polyacrylamide gel electrophoresis




n-dodecyl β-d-maltoside




Ethylenediamine tetra-acetic acid (di-sodium salt)


4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid






Optical density


Pasteur Culture Collection


Polymerase chain reaction




Photosystem II


Primary quinone electron acceptor of photosystem II


Sodium dodecyl sulfate


SDS polyacrylamide gel electrophoresis


2-[tris(hydroxymethyl)methyl]amino-1-ethanesulfonic acid





This work was supported by a grant (UOO309) from the New Zealand Marsden Fund to J.J.E.-R.


  1. Anbudurai P, Pakrasi H (1993) Mutational analysis of the PsbL protein of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Z Naturforsch 48:267–274Google Scholar
  2. Bebenek K, Kunkel T (1989) The use of native T7 DNA polymerase for site-directed mutagenesis. Nucleic Acids Res 17:5408. doi: 10.1093/nar/17.13.5408 PubMedCrossRefGoogle Scholar
  3. Bentley FK, Luo H, Dilbeck P, Burnap RL, Eaton-Rye JJ (2008) Effects of inactivating psbM and psbT on photodamage and assembly of photosystem II in Synechocystis sp. PCC 6803. Biochemistry. doi: 10.1021/bi800804h PubMedGoogle Scholar
  4. Bricker TM, Burnap RL (2005) The extrinsic proteins of photosystem II. In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase, advances in photosynthesis and respiration, vol 22. Springer, Dordrecht, pp 95–120Google Scholar
  5. Eaton-Rye JJ (2004) The construction of gene knockouts in the cyanobacterium Synechocystis sp. PCC 6803. In: Carpentier R (ed) Methods of molecular biology, photosynthesis research protocols. Humana Press, Totowa, pp 309–324CrossRefGoogle Scholar
  6. Eaton-Rye JJ, Putnam-Evans C (2005) The CP47 and CP43 core antenna components. In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase, advances in photosynthesis and respiration, vol 22. Springer, Dordrecht, pp 45–70Google Scholar
  7. Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Architecture of the photosynthetic oxygen-evolving center. Science 303:1831–1838. doi: 10.1126/science.1093087 PubMedCrossRefGoogle Scholar
  8. Güler S, Seeliger A, Hartel H, Renger C, Benning C (1996) A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyldiacylglycerol. J Biol Chem 271:7501–7507. doi: 10.1074/jbc.271.13.7501 PubMedCrossRefGoogle Scholar
  9. Hoshida H, Sugiyama R, Nakano Y, Shiina T, Toyoshima Y (1997) Electron paramagnetic resonance and mutational analysis revealed the involvement of photosystem II-L subunit in the oxidation step of Tyr-Z by P680 + to form the Tyr-Z+P680Pheo state in photosystem II. Biochemistry 36:12053–12061. doi: 10.1021/bi9710885 PubMedCrossRefGoogle Scholar
  10. Juntarajumnong W, Hirani TA, Simpson JM, Incharoensakdi A, Eaton-Rye JJ (2007) Phosphate sensing in Synechocystis sp. PCC 6803: SphU and the SphS-SphR two-component regulatory system. Arch Microbiol 188:389–402. doi: 10.1007/s00203-007-0259-0 PubMedCrossRefGoogle Scholar
  11. Kern J, Renger G (2007) Photosystem II: structure and mechanism of the water:plastoquinone oxidoreductase. Photosynth Res 94:183–202. doi: 10.1007/s11120-007-9201-1 PubMedCrossRefGoogle Scholar
  12. Kitamura K, Ozawa S, Shiina T, Toyoshima Y (1994) L protein, encoded by psbL restores normal functioning of the primary quinone acceptor, QA, in isolated D1/D2/CP47/Cytb–559/I photosystem II reaction center core complex. FEBS Lett 354:113–116. doi: 10.1016/0014-5793(94)01089-7 PubMedCrossRefGoogle Scholar
  13. Kunkel T (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492. doi: 10.1073/pnas.82.2.488 PubMedCrossRefGoogle Scholar
  14. Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685. doi: 10.1038/227680a0 CrossRefGoogle Scholar
  15. Loll B, Kern J, Saenger W, Zouni A, Biesiadka J (2005) Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438:1040–1044. doi: 10.1038/nature04224 PubMedCrossRefGoogle Scholar
  16. Luo H, Eaton-Rye J (2008) Mutations in CP47 that target putative hydrogen bonds with sulfoquinovosyl-diacylglycerol. In: Allen F, Osmond B, Golbeck J, Gant E (eds) Energy from the sun. Springer, Heidelberg, pp 737–739Google Scholar
  17. Minoda A, Sonoike K, Okada K, Sato N, Tsuzuki M (2003) Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas. FEBS Lett 553:109–112. doi: 10.1016/S0014-5793(03)00981-5 PubMedCrossRefGoogle Scholar
  18. Morgan TR, Shand JA, Clarke SM, Eaton-Rye JJ (1998) Specific requirements for cytochrome c-550 and the manganese-stabilizing protein in photoautotrophic strains of Synechocystis sp. PCC 6803 with mutations in the domain Gly-351 to Thr-436 of the chlorophyll-binding protein CP47. Biochemistry 37:14437–14449. doi: 10.1021/bi980404s PubMedCrossRefGoogle Scholar
  19. Namba O, Satoh K (1987) Isolation of a photosystem II reaction center consisting of d-1 and d-2 polypeptides and cytochrome b-559. Proc Natl Acad Sci USA 84:109–112. doi: 10.1073/pnas.84.1.109 CrossRefGoogle Scholar
  20. Nixon PJ, Sarcina M, Diner BA (2005) The CP47 and CP43 core antenna components. In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase, advances in photosynthesis and respiration, vol 22. Springer, Dordrecht, pp 71–94Google Scholar
  21. Oka A, Sugisake H, Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 295:1225–1236Google Scholar
  22. Ozawa S, Kobayashi T, Sugiyama R, Hoshida H, Shiina T, Toyoshima Y (1997) Role of PSII-L complex. 1. Over-production of wild-type and mutant versions of PSII-L protein and reconstitution into the PSII core complex. Plant Mol Biol 34:151–161. doi: 10.1023/A:1005800909495 PubMedCrossRefGoogle Scholar
  23. Rokka A, Suorsa M, Saleem A, Battchikova N, Aro E-M (2005) Synthesis and assembly of thylakoid protein complexes: multiple assembly steps of photosystem II. Biochem J 388:159–168. doi: 10.1042/BJ20042098 PubMedCrossRefGoogle Scholar
  24. Sakurai I, Mizusawa N, Ohashi S, Kobayashi M, Wada H (2007a) Effects of the lack of phosphatidylglycerol on the donor side of photosystem II. Plant Physiol 144:1336–1346. doi: 10.1104/pp.107.098731 PubMedCrossRefGoogle Scholar
  25. Sakurai I, Mizusawa N, Wada H, Sato N (2007b) Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II. Plant Physiol 145:1361–1370. doi: 10.1104/pp.107.106781 PubMedCrossRefGoogle Scholar
  26. Satoh K, Yamamoto Y (2007) The carboxyl-terminal processing of precursor D1 protein of the photosystem II reaction center. Photosynth Res 94:203–215. doi: 10.1007/s11120-007-9191-z PubMedCrossRefGoogle Scholar
  27. Schägger H (1994) Electrophoretic isolation of membrane-proteins from acrylamide gels. Appl Biochem Biotechnol 48:185–203. doi: 10.1007/BF02788741 CrossRefGoogle Scholar
  28. Sugita C, Ogata K, Shikata M, Jikuya H, Takano J, Furumichi M, Kanehisa M, Omata T, Sugiura M, Sugita M (2007) Complete nucleotide sequence of the freshwater unicellular cyanobacterium Synechococcus elongatus PCC 6301 chromosome: gene content and organization. Photosynth Res 93:55–67. doi: 10.1007/s11120-006-9122-4 PubMedCrossRefGoogle Scholar
  29. Summerfield TC, Shand JA, Bentley FK, Eaton-Rye JJ (2005) PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions. Biochemistry 44:805–815. doi: 10.1021/bi048394k PubMedCrossRefGoogle Scholar
  30. Suorsa M, Regel RE, Paakkarinen V, Battchikova N, Herrmann RG, Aro E-M (2004) Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ. Eur J Biochem 271:96–107. doi: 10.1046/j.1432-1033.2003.03906.x PubMedCrossRefGoogle Scholar
  31. Swiatek M, Regel R, Meurer J, Wanner G, Pakrasi H, Ohad I, Herrmann R (2003) Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II, as revealed by chloroplast transformation: the psbEFLJ operon in Nicotiana tabacum. Mol Genet Genomics 268:699–710PubMedGoogle Scholar
  32. Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882. doi: 10.1093/nar/25.24.4876 PubMedCrossRefGoogle Scholar
  33. Thornton LE, Roose JL, Pakrasi HB, Ikeuchi M (2005) The low molecular weight proteins of photosystem II. In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase, advances in photosynthesis and respiration, vol 22. Springer, Dordrecht, pp 121–138Google Scholar
  34. Toyoshima Y, Iwata T, Nakano Y, Hoshida H (1998) Tyr34 in PSII-L protein is essential for oxidation of Tyr-Z in PSII. In: Garab G (ed) Photosynthesis: mechanisms and effects, vol II. Kluwer Academic Publishers, Dordrecht, pp 1383–1386Google Scholar
  35. Vermaas W, Charité J, Shen G (1990) QA binding to D2 contributes to the functional and structural integrity of photosystem-II. Z Naturforsch 45:359–365Google Scholar
  36. Williams J (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol 167:766–778. doi: 10.1016/0076-6879(88)67088-1 CrossRefGoogle Scholar
  37. Whitehead TP, Kricka LJ, Carter TJN, Thorpe GHG (1979) Analytical luminescence: its potential in the clinical laboratory. Clin Chem 25:1531–1546Google Scholar
  38. Wydrzynski TJ, Satoh K (eds) (2005) Photosystem II: the light-driven water:plastoquinone oxidoreductase, advances in photosynthesis and respiration, vol 22. Springer, DordrechtGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of OtagoDunedinNew Zealand

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