Photosynthesis Research

, Volume 139, Issue 1–3, pp 475–486 | Cite as

Consequences of structural modifications in cytochrome b559 on the electron acceptor side of Photosystem II

  • Makoto Nakamura
  • Alain Boussac
  • Miwa SugiuraEmail author
Original Article


Cytb559 in Photosystem II is a heterodimeric b-type cytochrome. The subunits, PsbE and PsbF, consist each in a membrane α-helix. Mutants were previously designed and studied in Thermosynechococcus elongatus (Sugiura et al., Biochim Biophys Acta 1847:276–285, 2015) either in which an axial histidine ligand of the haem-iron was substituted for a methionine, the PsbE/H23M mutant in which the haem was lacking, or in which the haem environment was modified, the PsbE/Y19F and PsbE/T26P mutants. All these mutants remained active showing that the haem has no structural role provided that PsbE and PsbF subunits are present. Here, we have carried on the characterization of these mutants. The following results were obtained: (i) the Y19F mutation hardly affect the Em of Cytb559, whereas the T26P mutation converts the haem into a form with a Em much below 0 mV (so low that it is likely not reducible by QB) even in an active enzyme; (ii) in the PsbE/H23M mutant, and to a less extent in PsbE/T26P mutant, the electron transfer efficiency from QA to QB is decreased; (iii) the lower Em of the QA/QA couple in the PsbE/H23M mutant correlates with a higher production of singlet oxygen; (iv) the superoxide and/or hydroperoxide formation was not increased in the PsbE/H23M mutant lacking the haem, whereas it was significantly larger in the PsbE/T26P. These data are discussed in view of the literature to discriminate between structural and redox roles for the haem of Cytb559 in the production of reactive oxygen species.


Photosystem II Cytb559 Redox Haem axial ligand Acceptor side 





Accessory Chl on the D1 or D2 side, respectively








Midpoint redox potential versus SHE, the standard hydrogen electrode

HP, IP, LP forms

High-potential, intermediate-potential and low-potential forms of Cytb559


2–(N–morpholino) ethanesulfonic acid


Primary electron donor

PD1 and PD2

Chl monomer of P680 on the D1 or D2 side, respectively




Photosystem II


Chlorophyll dimer acting as the electron donor


α-(4-Pyridyl N-oxide)-N-tert-butylnitrone


Primary quinone acceptor


Secondary quinone acceptor

T. elongatus

Thermosynechococcus elongatus


Reactive oxygen species




T. elongatus mutant strain containing only the psbA3 gene



We would like to thank Anja Krieger-Liszkay (CEA Saclay) for discussions and technical suggestions for the trapping of ROS. This work was supported by JSPS-KAKENHI grant in Scientific Research on Innovative Areas “Innovations for Light-Energy Conversion (I4LEC)” (17H064351 for M. S.) and a JSPS-KAKENHI grant (17K07367 for M.S.). AB was supported in part by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05.


  1. Askerka M, Brudvig GW, Batista VS (2017) The O2-evolving complex of photosystem II: recent insights from quantum mechanics/molecular mechanics (QM/MM), extended X-ray absorption fine structure (EXAFS), and femtosecond X-ray crystallography data. Acc Chem Res 50:41–48CrossRefGoogle Scholar
  2. Bondarava N, Gross CM, Mubarakshina M, Golecki JR, Johnson GN, Krieger-Liszkay A (2010) Putative function of cytochrome b 559 as a plastoquinol oxidase. Physiol Plant 138:463–473CrossRefGoogle Scholar
  3. Buchta J, Grabolle M, Dau H (2007) Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements - Method, rationale, and results on the activation energy of dioxygen formation. Biochim Biophys Acta 1767:565–574CrossRefGoogle Scholar
  4. Chiu Y-F, Lin W-C, Wu C-M, Chen Y-H, Hung C-H, Ke S-C, Chu H-A (2009) Identification and characterization of a cytochrome b 559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions. Biochim Biophys Acta 1787:1179–1188CrossRefGoogle Scholar
  5. Chiu Y-F, Chen Y-H, Roncel M, Dilbeck PL, Huang J-Y, Ke SC, Ortega JM, Burnap RL, Chu H-A (2013) Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b 559 in photosystem II. Biochim Biophys Acta 1827:507–519CrossRefGoogle Scholar
  6. Cox N, Messinger J (2013) Reflections on substrate water and dioxygen formation. Biochim Biophys Acta 1827:1020–1030CrossRefGoogle Scholar
  7. Cser K, Vass I (2007) Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803. Biochim Biophys Acta 1767:233–243CrossRefGoogle Scholar
  8. Dau H, Zaharieva I, Haumann M (2012) Recent developments in research on water oxidation by photosystem II. Curr Op Chem Biol 16:3–10CrossRefGoogle Scholar
  9. Diner BA, Rappaport F (2002) Structure, dynamics, and energetic of the primary photochemistry of photosystem II of oxygenic photosynthesis. Annu Rev Plant Biol 53:551–580CrossRefGoogle Scholar
  10. Ducruet JM (2003) Chlorophyll thermoluminescence of leaf discs: simple instruments and progress in signal interpretation open the way to new ecophysiological indicators. J Exp Bot 54:2419–2430CrossRefGoogle Scholar
  11. Ducruet JM, Vass I (2009) Thermoluminescence: experimental. Photosynth Res 101:195–204CrossRefGoogle Scholar
  12. Guerrero F, Zurita JL, Roncel M, Kirilovsky D, Ortega JM (2014) The role of the high potential form of the cytochrome b 559: study of Thermosynechococcus elongatus mutants. Biochim Biophys Acta 1837:908–919CrossRefGoogle Scholar
  13. Hamilton ML, Franco E, Deák Z, Schlodder E, Vass I, Nixon PJ (2014) Investigating the photoprotective role of cytochrome b-559 in photosystem II in a mutant with altered ligation of the haem. Plant Cell Physiol 55:1276–1285CrossRefGoogle Scholar
  14. Hung C-H, Huang J-Y, Chiu Y-F, Chu H-A (2007) Site-directed mutagenesis on the heme axial-ligands of cytochrome b 559 in photosystem II by using cyanobacteria Synechocystis PCC 6803. Biochim Biophys Acta 1767:686–693CrossRefGoogle Scholar
  15. Hung C-H, Hwang H-J, Chen Y-H, Chiu Y-F, Ke S-C, Burnap RL, Chu H-A (2010) Spectroscopic and functional characterizations of cyanobacterium Synechocystis PCC 6803 mutants on and near the heme axial ligand of cytochrome b 559 in photosystem II. J Biol Chem 285:5653–5663CrossRefGoogle Scholar
  16. Ikeuchi M, Inoue Y (1988) A new 4.8-kDa polypeptide intrinsic to the PS II reaction center, as revealed by modified SDS-PAGE with improved resolution of low-molecular-weight proteins. Plant Cell Physiol 29:1233–1239Google Scholar
  17. Joliot P, Kok B (1975) Oxygen evolution in photosynthesis. In: Govindjee (ed) Bioenergetics of photosynthesis. Academic Press, New York, pp 387–412CrossRefGoogle Scholar
  18. Kaminskaya OP, Shuvalov VA (2013a) Biphasic reduction of cytochrome b 559 by plastoquinol in photosystem II membrane fragments. Evidence for two types of cytochrome b 559/plastoquinone redox equilibria. Biochim Biophys Acta 1827:471–483CrossRefGoogle Scholar
  19. Kaminskaya OP, Shuvalov VA (2013b) Towards an understanding of the nature of the redox forms of cytochrome b559 in photosystem II. Dokl Biochem Biophys 450:151–154CrossRefGoogle Scholar
  20. Kern J, Loll B, Lüneberg C, DiFiore D, Biesiadka J, Irrgang K-D, Zouni A (2005) Purification, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor. Biochim Biophys Acta 1706:147–157CrossRefGoogle Scholar
  21. Kok B, Forbush B, McGloin M (1970) Cooperation of charges in photosynthetic O2 evolution–I. A linear four step mechanism. Photochem Photobiol 11:457–475CrossRefGoogle Scholar
  22. Ma J-J, Li L-B, Jing Y-X, Kuang T-Y (2007a) Mutation of residue arginine18 of cytochrome b 559 α-subunit and its effects on photosystem II activities in Chlamydomonas reinhardtii. J Integr Plant Biol 49:1054–1061CrossRefGoogle Scholar
  23. Ma J-J, Li L-B, Jing Y-X, Kuang T-Y (2007b) Mutagenesis of Ser(24) of cytochrome b 559 alpha subunit affects PSII activities in Chlamydomonas reinhardtii. Chin Sci Bull 52:896–902CrossRefGoogle Scholar
  24. Mayhew SG (1978) The redox potential of dithionite and SO2 from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase. Eur J Biochem 85:535–547CrossRefGoogle Scholar
  25. Michelet L, Roach T, Fischer BB, Bedhomme M, Lemaire SD, Krieger-Liszkay A (2013) Down-regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii. Plant Cell Environ 36:1204–1213CrossRefGoogle Scholar
  26. Morais F, Kühn K, Stewart DH, Barber J, Brudvig GW, Nixon PJ (2001) Photosynthetic water oxidation in cytochrome b 559 mutants containing a disrupted heme-binding pocket. J Biol Chem 276:31986–31993CrossRefGoogle Scholar
  27. Müh F, Glöckner C, Hellmich J, Zouni A (2012) Light-induced quinone reduction in photosystem II. Biochim Biophys Acta 1817:44–65CrossRefGoogle Scholar
  28. Müh F, Plöckinger M, Renger T (2017) Electrostatic asymmetry in the reaction center of photosystem II. J Phys Chem Lett 8:850–858CrossRefGoogle Scholar
  29. Pakrasi HB, De Ciechi P, Whitmarsh J (1991) Site directed mutagenesis of the heme axial ligands of cytochrome b 559 affects the stability of the photosystem II complex. EMBO J 10:1619–1627CrossRefGoogle Scholar
  30. Pospíšil P (2011) Enzymatic function of cytochrome b 559 in photosystem II. J Photochem Photobiol B 104:341–347CrossRefGoogle Scholar
  31. Pospíšil P (2012) Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II. Biochim Biophys Acta 1817:218–231CrossRefGoogle Scholar
  32. Pospsil P, Snyrychova I, Kruk J, Strzalka K, Naus J (2006) Evidence that cytochrome b 559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b 559 tobacco mutant. Biochem J 397:321–327CrossRefGoogle Scholar
  33. Rappaport F, Lavergne J (2009) Thermoluminescence: theory. Photosynth Res 101:205–216CrossRefGoogle Scholar
  34. Rehman AU, Cser K, Sass L, Vass I (2013) Characterization of singlet oxygen production and its involvement in photodamage of photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping. Biochem Biophs Acta 1827:689–698Google Scholar
  35. Renger G (2011) Light–induced oxidative water splitting in photosynthesis: energetics, kinetics, and mechanism. J Photochem Photobiol B 104:35–43CrossRefGoogle Scholar
  36. Roncel M, Ortega JM, Losada M (2001) Factors determining the special redox properties of photosynthetic cytochrome b 559. Eur J Biochem 268:4961–4968CrossRefGoogle Scholar
  37. Roncel M, Boussac A, Zurita JL, Bottin H, Sugiura M, Kirilovsky D, Ortega JM (2003) Redox properties of the photosystem II cytochromes b 559 and c 550 in the cyanobacterium Thermosynechococcus elongatus. J Biol Inorg Chem 8:206–216CrossRefGoogle Scholar
  38. Roncel M, Kirilovsky D, Guerrero F, Serrano A, Ortega JM (2012) Photosynthetic cytochrome c 550. Biochim Biophys Acta 1817:1152–1163CrossRefGoogle Scholar
  39. Rutherford AW, Krieger-Liszkay A (2001) Herbicide-induced oxidative stress in photosystem II. TIBS 26:648–653Google Scholar
  40. Sedoud A, Kastner L, Cox N, El-Alaoui S, Kirilovsky D, Rutherford AW (2011) Effects of formate binding on the quinone–iron electron acceptor complex of photosystem II. Biochim Biophys Acta 1807:216–226CrossRefGoogle Scholar
  41. Shen J-R (2015) The structure of photosystem II and the mechanism of water oxidation in photosynthesis. Annu Rev Plant Biol 66:23–48CrossRefGoogle Scholar
  42. Shinopoulos KE, Brudvig GW (2012) Cytochrome b 559 and cyclic electron transfer within photosystem II. Biochim Biophys Acta 1817:66–75CrossRefGoogle Scholar
  43. Stewart DH, Brudvig GW (1998) Cytochrome b 559 of photosystem II. Biochim Biophys Acta 1367:63–68CrossRefGoogle Scholar
  44. Suga M, Akita F, Hirata K, Ueno G, Murakami H, Nakajima Y, Shimizu T, Yamashita K, Yamamoto M, Ago H, Shen J-R (2015) Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 517:99–103CrossRefGoogle Scholar
  45. Sugiura M, Inoue Y (1999) Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43. Plant Cell Physiol 40:1219–1231CrossRefGoogle Scholar
  46. Sugiura M, Boussac A, Noguchi T, Rappaport F (2008) Influence of histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus. Biochim Biophys Acta 1777:331–342CrossRefGoogle Scholar
  47. Sugiura M, Kato Y, Takahashi R, Suzuki H, Watanabe T, Noguchi T, Rappaport F, Boussac A (2010) Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA 1 or psbA 3 gene. Biochim Biophys Acta 1797:1491–1499CrossRefGoogle Scholar
  48. Sugiura M, Nakamura M, Koyama K, Boussac A (2015) Assembly of oxygen-evolving photosystem II efficiently occurs with the apo-Cytb 559 but the holo-Cytb 559 accelerates the recovery of functional enzyme upon photoinhibition. Biochim Biophys Acta 1847:276–285CrossRefGoogle Scholar
  49. Telfer A (2014) Singlet oxygen production by PSII under light stress: mechanism, detection and the protective role of β-Carotene. Plant cell Physiol 55:1216–1223CrossRefGoogle Scholar
  50. Telfer A, Steven MB, Phillips D, Barber J (1994) Isolated photosynthetic reaction center of photosystem II as a sensitizer for the formation of singlet oxygen. J Biol Chem 269(18):13244–13253Google Scholar
  51. Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473:55–60CrossRefGoogle Scholar
  52. Yano J, Yachandra V (2014) Mn4Ca cluster in photosynthesis: where and how water is oxidized to dioxygen. Chem Rev 114:4175–4205CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Graduate School of Science and TechnologyEhime UniversityMatsuyamaJapan
  2. 2.I2BC, CNRS UMR 9198, CEA SaclayGif-sur-YvetteFrance
  3. 3.Proteo-Science Research CenterEhime UniversityMatsuyamaJapan

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