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Site-directed mutagenesis of the basic residues 321K to321 G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803

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Abstract

CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 µM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting.

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References

  1. Aro E-M, Virgin I, Andersson B: Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochim Biophys Acta 1143: 113-134 (1993).

    PubMed  Google Scholar 

  2. Boussac A, Setif P, Rutherford AW: Inhibition of tyrosine Z photooxidation after formation of the s3 state in calciumdepleted and chloride-depleted photosystem II. Biochemistry 31: 1224-1234 (1992).

    PubMed  Google Scholar 

  3. Bricker TM, Frankel LK: Use of a monoclonal antibody in structural investigations of the 49 kDa polypeptide of photosystem II. Arch Biochem Biophys 256: 295-301 (1987).

    PubMed  Google Scholar 

  4. Bricker TM, Odom WR, Quierolo CB: Close association of the 33 kDa extrinsic protein with the apoprotein of Cpa-1 in photosystem II. FEBS Lett 231: 111-117 (1988).

    Article  Google Scholar 

  5. Bricker TM: The structure and function of CPa-1 and CPa-2 in photosystem II. Photosyn Res 24: 1-13 (1990).

    Google Scholar 

  6. Burnap RL, Sherman LA: Deletion mutagenesis in Synechocystis6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for oxygen evolution. Biochemistry 30: 440-446 (1991).

    PubMed  Google Scholar 

  7. Debus R: The manganese and calcium ions of photosynthetic oxygen evolution. Biochim Biophys Acta 1102: 269-352 (1992).

    PubMed  Google Scholar 

  8. DeRose VJ, Latimer MJ, Zimmermann JL, Mukerji I, Yachandra VK, Sauer K, Klein MP: Fluoride substitution in the mn cluster from photosystem II-EPR and x-ray absorption spectroscopy studies. Chem Phys 194: 443-459 (1995).

    Article  Google Scholar 

  9. Dismukes GC, Zheng M, Hutchins R, Philo JS: The inorganic biochemistry of photosynthetic water oxidation. Biochem Soc Trans 22: 323-327 (1994).

    PubMed  Google Scholar 

  10. Eaton-Rye JJ, Vermaas WFJ: Oligonucleotide-directed mutagenesis of psbB, the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis6803. Plant Mol Biol 17: 1165-1177 (1991).

    PubMed  Google Scholar 

  11. Enami I, Kaneko M, Kitamura N, Koike H, Sinoike K, Inoue Y, Katoh S: Total immobilization of the extrinsic 33 kDa protein in spinach photosystem II membrane preparations. Proteins stiochiometry and stabilization of oxygen evolution. Biochim Biophys Acta 1060: 224-232 (1991).

    Google Scholar 

  12. Frankel LK, Bricker TM: Monoclonal antibodies directed against the 33, 24 and 17 kDa extrinsic proteins of spinach photosystem II. In: Batcheffsky M (ed) Current Research in Photosynthesis, vol. 1, pp. 825-828. Kluwer Academic Publishers, Dordrecht, Netherlands (1990).

    Google Scholar 

  13. Frankel LK, Bricker TM: Interaction of CPa-1 with the manganese stabilizing protein of photosystem II: Identification of domains on CPa-1 which are shielded from Nhydroxysuccinimide biotinylation. Biochemistry 31: 11059- 11064 (1992).

    PubMed  Google Scholar 

  14. Gleiter HM, Haag E, Shen J-R, Eaton-Rye JJ, Inoue Y, Vermaas WFJ, Renger G: Functional characterization of mutant strains of the cyanobacterium Synechocystissp. PCC 6803 lacking short domains within the large, lumen-exposed loop of the chlorophyll protein CP47 in photosystem II. Biochemistry 33: 12063-12070 (1994).

    PubMed  Google Scholar 

  15. Gleiter HM, Haag E, Shen J-R, Eaton-Rye JJ, Seeliger AG, Inoue Y, Vermaas WFJ, Renger G: Involvement of the CP 47 protein in stabilization and photoactivation of a functional water oxidizing complex in the cyanobacterium Synechocystissp. PCC 6803. Biochemistry 34: 6847-6856 (1995).

    PubMed  Google Scholar 

  16. Haag E, Eaton-Rye JJ, Renger G, Vermaas WFJ: Functionally important domains of the large hydrophilic loop of CP 47 as probed by oligonucleotide-directed mutagenesis in Synechocystissp. PCC 6803. Biochemistry 32: 4444-4454 (1993).

    PubMed  Google Scholar 

  17. Homann PH: The relation between the chloride, calcium and polypeptide requirements of photosynthetic water oxidation. J Bioenerg Biomembr 19: 105-123 (1987).

    PubMed  Google Scholar 

  18. Itoh S, Yerkes CT, Koike H, Robinson HH, Crofts AR: Effects of chloride depletion on electron donation from the water oxidizing complex to the photosystem reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts. Biochim Biophys Acta 766: 612-622 (1984).

    Google Scholar 

  19. Kelly PM, Izawa S: The role of chloride ion in photosystem II: Effects of chloride ion on photosystem II electron transport and hydroxylamine inhibition. Biochim Biophys Acta 502: 198- 210 (1978).

    PubMed  Google Scholar 

  20. Kunkle TA: Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci 82: 488- 492 (1985).

    PubMed  Google Scholar 

  21. Lindberg K, Vanngard T, Andreasson L-E: Studies of the slowly exchanging chloride in PSII of higher plants. Photosyn Res 8: 401-408 (1993).

    Google Scholar 

  22. Lubbers K, Drevenstedt W, June W: Chloride depletion of photosyntheticwater oxidase. FEBS Lett 336: 304-308 (1994).

    Article  Google Scholar 

  23. Nixon P, Diner B: Aspartate 170 of the photosystem II reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster. Biochemistry 31: 942- 948 (1992).

    PubMed  Google Scholar 

  24. Nixon P, Diner B: Analysis of water-oxidation mutants constructed in the cyanobacterium Synechocystis6803. Biochem Soc Trans 22: 338-343 (1994).

    PubMed  Google Scholar 

  25. Odom WR, Bricker TM: Interaction of Cpa-1 with the manganese-stabilizing protein of photosystem II: Identification of domains cross-linked by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Biochemistry 31: 5616-5620 (1992).

    PubMed  Google Scholar 

  26. Ono T, Inoue Y: Ca2+-dependent restoration of oxygenevolving activity in CaCl2-washed photosystem II particles depleted of 33 kDa, 24 kDa and 16 kDa proteins. FEBS Lett 168: 281-286 (1984).

    Article  Google Scholar 

  27. Ono T, Zimmermann JL, Inoue Y, Rutherford AW: EPR evidence for a modified S-state transition in chloride-depleted photosystem II. Biochim Biophys Acta 851: 193-201 (1986).

    Google Scholar 

  28. Ono T, Noguchi T, Inoue Y, Kusonoki M, Yanaguchi H, Oyaragi H: XANES spectroscopy formonitoring intermediate reaction states of chloride depleted Mn cluster in photosynthetic water oxidation. J Am Chem Soc 117: 6383-6387 (1995).

    Google Scholar 

  29. Putnam-Evans C, Bricker TM: Site-directed mutagenesis of the CP47 protein of photosystem II: alteration of the basic residue pair 384;384R to 384;385G leads to a defect associated with the oxygen-evolving complex. Biochemistry 31: 11482- 11488 (1992).

    PubMed  Google Scholar 

  30. Putnam-Evans C, Bricker TM: Site-directedmutagenesis of the CP 47 protein of photosystem II: Alteration of the basic residue 448R to 448G prevents the assembly of functional photosystem II centers under chloride-limiting conditions. Biochemistry 33: 10770-10776 (1994).

    PubMed  Google Scholar 

  31. Putnam-Evans C, Burnap R, Wu Jituo, Whitmarsh John, Bricker TM: Site-directed mutagenesis of the CP 47 protein of photosystem II:Alteration of conserved charged residues in the domain 364E-440D. Biochemistry 35: 4046-4053 (1996).

    Article  PubMed  Google Scholar 

  32. Rippka RA, Derulles J, Waterbury JB, Herdman M, Stanier RY: Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1-61 (1979).

    Google Scholar 

  33. Rutherford AW, Boussac A: The oxygen-evolving enzyme: effects of calcium and chloride ions. Biochem Soc Trans 22: 352-357 (1994).

    PubMed  Google Scholar 

  34. Sandusky PO, Yocum CF: The chloride requirement of photosynthetic oxygen evolution: Factors affecting nucleophilic displacement of chloride from the oxygen evolving complex. Biochim Biophys Acta 849: 85-93 (1982).

    Google Scholar 

  35. Sandusky PO, Yocum CF: The mechanism of amine inhibition of the photosynthetic oxygen-evolving complex. FEBS Lett 162: 339-343 (1983).

    Article  Google Scholar 

  36. Sinclair J: The influence of anions on oxygen evolution by isolated spinach chloroplasts. Biochim Biophys Acta 764: 247- 252 (1984).

    Google Scholar 

  37. Tamura N, Cheniae G: Photoactivation of the water oxidizing complex in photosystem II membranes depleted of manganese and extrinsic proteins. I. Biochemical and kinetic characterization. Biochim Biophys Acta 890: 179-194 (1987).

    Google Scholar 

  38. Theg SM, Jursinic PA, Homann PH: Studies on the mechanism of chloride action on photosynthetic water oxidation. Biochim Biophys Acta 766: 636-646 (1984).

    Google Scholar 

  39. van der Bolt F, Vermaas WFJ: Photoinactivation of photosystem II as studiedwith site-directed D2 mutants of the cyanobacterium Synechocystissp. pcc 6803. Biochim Biophys Acta 1098: 247-254 (1992).

    Google Scholar 

  40. van Vliet P, Rutherford AW: Properties of the chloride-depleted oxygen-evolving complex of photosystem II studied by electron paramagnetic resonance. Biochemistry 35: 1829-1839 (1996).

    Article  PubMed  Google Scholar 

  41. van Vliet P, Boussac A, Rutherford AW: Chloride-depletion effects in the calcium-deficient oxygen-evolving complex of photosystem II. Biochemistry 33: 12990-13004 (1994).

    PubMed  Google Scholar 

  42. Vermaas WFJ, Williams JGK, Arntzen CJ: Sequencing and modification of psbB, the gene encoding the CP 47 protein of photosystem II in the cyanobacterium Synechocystis6803. Plant Mol Biol 8: 317-326 (1987).

    Google Scholar 

  43. Williams JGK: Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis6803. Meth Enzymol 167: 766-778 (1988).

    Google Scholar 

  44. Yachandra VK, DeRose VJ, Latimer MJ, Mukerji I, Sauer K, Klein MP: Where plants make oxygen: A structural model for the photosynthetic oxygen-evolving manganese cluster. Science 260: 675-679 (1993).

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Biology, East Carolina University, Greenville, NC, 27858, USA

    Cindy Putnam-Evans

  2. Department of Microbiology, Louisiana State University, Baton Rouge, LA, 70803, USA

    Terry M. Bricker

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  1. Cindy Putnam-Evans
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  2. Terry M. Bricker
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Putnam-Evans, C., Bricker, T.M. Site-directed mutagenesis of the basic residues 321K to321 G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803. Plant Mol Biol 34, 455–463 (1997). https://doi.org/10.1023/A:1005826411702

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