Skip to main content
SpringerLink
Log in

Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity

  • Original article
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

X-ray crystallographic analysis (1.9-Å resolution) of the cyanobacterial photosystem II (PSII) dimer showed the presence of five phosphatidylglycerol (PG) molecules per reaction center. One of the PG molecules, PG772, is located in the vicinity of the QB-binding site. To investigate the role of PG772 in PSII, we performed site-directed mutagenesis in the cytochrome (Cyt) b559 α subunit of Synechocystis sp. PCC 6803 to change two amino acids, Thr-5 and Ser-11, which interact with PG772. The photosynthetic activity of intact cells was slightly lower in all mutants than that of cells in the control strain; however, the oxygen-evolving PSII activity was decreased markedly in cells of mutants, as measured using artificial quinones (such as p-benzoquinone). Furthermore, electron transport from QA to QB was inhibited in mutants incubated with quinones, particularly under high-intensity light conditions. Lipid analysis of purified PSII showed approximately one PG molecule per reaction center, presumably PG772, was lost in the PSII dimer from the T5A and S11A mutants compared with that in the PSII dimer from the control strain. In addition, protein analysis of monomer and dimer showed decreased levels of PsbV and PsbU extrinsic proteins in the PSII monomer purified from T5A and S11A mutants. These results suggest that site-directed mutagenesis of Thr-5 and Ser-11, which presumably causes the loss of PG772, induces quinone-dependent inhibition of PSII activity under high-intensity light conditions and destabilizes the binding of extrinsic proteins to PSII.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

BQ:

p-Benzoquinone

BN:

Blue native

Chl:

Chlorophyll

Cyt:

Cytochrome

DCBQ:

2,6-Dichloro-p-benzoquinone

DCMU:

3-(3,4-Dichlorophenyl)-1,1-dimethylurea

DGDG:

Digalactosyldiacylglycerol

DM:

n-Dodecyl-β-d-maltoside

DMBQ:

2,6-Dimethyl-p-benzoquinone

Em:

Erythromycin

EmR :

Erythromycin-resistant gene cassette

Fecy:

Potassium ferricyanide

HP:

High potential

IP:

Intermediate potential

Km:

Kanamycin

LP:

Low potential

MGDG:

Monogalactosyldiacylglycerol

PG:

Phosphatidylglycerol

PQ:

Plastoquinone

PSI:

Photosystem I

PSII:

Photosystem II

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SQDG:

Sulfoquinovosyldiacylglycerol

References

  • Barbato R, Friso G, Ponticos M, Barber J (1995) Characterization of the light-induced cross-linking of the α-subunit of cytochrome b 559 and the D1 protein in isolated photosystem II reaction centers. J Biol Chem 270:24032–24037

    Article  CAS  PubMed  Google Scholar 

  • Bligh EG, Dyer WJ (1961) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  Google Scholar 

  • Chiu YF, Lin WC, Wu CM, Chen YH, Hung CH, Ke SC, Chu HA (2009) Identification and characterization of a cytochrome b 559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions. Biochim Biophys Acta 1787:1179–1188

    Article  CAS  PubMed  Google Scholar 

  • Chiu YF, Chen YH, Roncel M, Dilbeck PL, Huang JY, Ke SC, Ortega JM, Burnap RL, Chu HA (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 1827:507–519

    Article  CAS  PubMed  Google Scholar 

  • Chu HA, Chiu YF (2016) The roles of cytochrome b 559 in assembly and photoprotection of photosystem II revealed by site-directed mutagenesis studies. Front Plant Sci 6:1–7

    Article  CAS  Google Scholar 

  • Endo K, Mizusawa N, Shen JR, Yamada M, Tomo T, Komatsu H, Kobayashi M, Kobayashi K, Wada H (2015) Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II. Photosynth Res 126:385–397

    Article  CAS  PubMed  Google Scholar 

  • Endo K, Kobayashi K, Wada H (2016) Sulfoquinovosyldiacylglycerol has an essential role in Thermosynechococcus elongatus BP-1 under phosphate-deficient conditions. Plant Cell Physiol 57:2461–2471

    Article  CAS  PubMed  Google Scholar 

  • Gombos Z, Wada H, Murata N (1991) Direct evaluation of effects of fatty-acid unsaturation on the thermal-properties of photosynthetic activities, as studied by mutation and transformation of Synechocystis PCC 6803. Plant Cell Physiol 32:205–211

    Article  CAS  Google Scholar 

  • Gombos Z, Várkonyi Z, Hagio M, Iwaki M, Kovács L, Masamoto K, Itoh S, Wada H (2002) Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone QB in the photosystem II reaction center. Biochemistry 41:3796–3802

    Article  CAS  PubMed  Google Scholar 

  • Guskov A, Kern J, Gabdulkhakov A, Broser M, Zouni A, Saenger W (2009) Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride. Nat Struct Mol Biol 16:334–342

    Article  CAS  PubMed  Google Scholar 

  • Hagio M, Gombos Z, Várkonyi Z, Masamoto K, Sato N, Tsuzuki M, Wada H (2000) Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis. Plant Physiol 124:795–804

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hung CH, Huang JY, Chiu YF, Chu HA (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 1767:686–693

    Article  CAS  PubMed  Google Scholar 

  • Hung CH, Hwang HJ, Chen YH, Chiu YF, Ke SC, Burnap RL, Chu HA (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 285:5653–5663

    Article  CAS  PubMed  Google Scholar 

  • Itoh S, Kozuki T, Nishida K, Fukushima Y, Yamakawa H, Domonkos I, Laczkó-Dobos H, Kis M, Ughy B, Gombos Z (2012) Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II. Biochim Biophys Acta 1817:287–297

    Article  CAS  PubMed  Google Scholar 

  • Kaminskaya O, Shuvalov VA, Renger G (2007a) Evidence for a novel quinone-binding site in the photosystem II (PS II) complex that regulates the redox potential of cytochrome b 559. Biochemistry 46:1091–1105

    Article  CAS  PubMed  Google Scholar 

  • Kaminskaya O, Shuvalov VA, Renger G (2007b) Two reaction pathways for transformation of high potential cytochrome b 559 of PS II into the intermediate potential form. Biochim Biophys Acta 1767:550–558

    Article  CAS  PubMed  Google Scholar 

  • Kashino Y, Lauber WM, Carroll JA, Wang Q, Whitmarsh J, Satoh K, Pakrasi HB (2002) Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium Synechocystis sp. PCC 6803 reveals the presence of novel polypeptides. Biochemistry 41:8004–8012

    Article  CAS  PubMed  Google Scholar 

  • Kern J, Renger G (2007) Photosystem II: structure and mechanism of the water: plastoquinone oxidoreductase. Photosynth Res 94:183–202

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi K, Endo K, Wada H (2016) Roles of lipids in photosynthesis. In: Nakamura Y, Li-Beisson Y (eds) Lipids in plant and algae development. Springer International Publishing, Cham, pp. 21–49

    Chapter  Google Scholar 

  • Kopečná J, Pilný J, Krynická V, Tomčala A, Kis M, Gombos Z, Komenda J, Sobotka R (2015) Lack of phosphatidylglycerol inhibits chlorophyll biosynthesis at multiple sites and limits chlorophyllide reutilization in Synechocystis sp. strain PCC 6803. Plant Physiol 169:1307–1317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krieger-Liszkay A, Rutherford AW (1998) Influence of herbicide binding on the redox potential of the quinone acceptor in photosystem II: Relevance to photodamage and phytotoxicity. Biochemistry 37:17339–17344

    Article  CAS  PubMed  Google Scholar 

  • Lupínková L, Metz JG, Diner BA, Vass I, Komenda J (2002) Histidine residue 252 of the photosystem II D1 polypeptide is involved in a light-induced cross-linking of the polypeptide with the α subunit of cytochrome b-559: study of a site-directed mutant of Synechocystis PCC 6803. Biochim Biophys Acta 1554:192–201

    Article  PubMed  Google Scholar 

  • Mizusawa N, Wada H (2012) The role of lipids in photosystem II. Biochim Biophys Acta 1817:194–208

    Article  CAS  PubMed  Google Scholar 

  • Morita SY, Terada T (2015) Enzymatic measurement of phosphatidylglycerol and cardiolipin in cultured cells and mitochondria. Sci Rep 5:1–15

    Article  Google Scholar 

  • Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI (2007) Photoinhibition of photosystem II under environmental stress. Biochim Biophys Acta 1767:414–421

    Article  CAS  PubMed  Google Scholar 

  • Nishiyama Y, Allakhverdiev SI, Murata N (2006) A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. Biochim Biophys Acta 1757:742–749

    Article  CAS  PubMed  Google Scholar 

  • Pagliano C, Saracco G, Barber J (2013) Structural, functional and auxiliary proteins of photosystem II. Photosynth Res 116:16–188

    Article  CAS  Google Scholar 

  • Pakrasi HB, Williams JG, Arntzen CJ (1988) Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b 559 in photosystem II. EMBO J 7:325–332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394

    Article  CAS  Google Scholar 

  • Renger G, Renger T (2008) Photosystem II: the machinery of photosynthetic water splitting. Photosynth Res 98:53–80

    Article  CAS  PubMed  Google Scholar 

  • Roncel M, Ortega JM, Losada M (2001) Factors determining the special redox properties of photosynthetic cytochrome b 559. Eur J Biochem 268:4961–4968

    Article  CAS  PubMed  Google Scholar 

  • Sakurai I, Hagio M, Gombos Z, Tyystjarvi T, Paakkarinen V, Aro EM, Wada H (2003) Requirement of phosphatidylglycerol for maintenance of photosynthetic machinery. Plant Physiol 133:1376–1384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sato N, Hagio M, Wada H, Tsuzuki M (2000) Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes. Proc Natl Acad Sci USA 97:10655–10660

    Article  CAS  PubMed  Google Scholar 

  • Satoh K, Koike H, Ichimura T, Katoh S (1992) Binding affinities of benzoquinones to the QB site of photosystem II in Synechococcus oxygen-evolving preparation. Biochim Biophys Acta 1102:45–52

    Article  CAS  Google Scholar 

  • Satoh K, Oh-hashi M, Kashino Y, Koike H (1995) Mechanism of electron flow through the QB site in photosystem II. 1. Kinetics of the reduction of electron acceptors at the QB and plastoquinone sites in photosystem II particles from the cyanobacterium Synechococcus vulcanus. Plant Cell Physiol 36:597–605

    Article  CAS  Google Scholar 

  • Shi LX, Hall M, Funk C, Schröder WP (2012) Photosystem II, a growing complex: updates on newly discovered components and low molecular mass proteins. Biochim Biophys Acta 1817:13–25

    Article  CAS  PubMed  Google Scholar 

  • Shinopoulos KE, Brudvig GW (2012) Cytochrome b 559 and cyclic electron transfer within photosystem II. Biochim Biophys Acta 1817:66–75

    Article  CAS  PubMed  Google Scholar 

  • Stewart DH, Brudvig GW (1998) Cytochrome b 559 of photosystem II. Biochim Biophys Acta 1367:63–87

    Article  CAS  PubMed  Google Scholar 

  • Tyystjärvi E (2008) Photoinhibition of photosystem II and photodamage of the oxygen evolving manganese cluster. Coord Chem Rev 252:361–376

    Article  CAS  Google Scholar 

  • 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 473:55–60

    Article  CAS  PubMed  Google Scholar 

  • Van Eerden FJ, Melo MN, Frederix PWJM, Periole X, Marrink SJ (2017) Exchange pathways of plastoquinone and plastoquinol in the photosystem II complex. Nat Commun 8:15214

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (16J10129), and by CREST, Japan Science and Technology Agency.

Author information

Authors and Affiliations

  1. Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, 153-8902, Japan

    Kaichiro Endo, Koichi Kobayashi & Hajime Wada

  2. Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, Republic of China

    Hsing-Ting Wang & Hsiu-An Chu

  3. Research Institute for Interdisciplinary Science, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama, 700-8530, Japan

    Jian-Ren Shen

Authors
  1. Kaichiro Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Koichi Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hsing-Ting Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hsiu-An Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jian-Ren Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hajime Wada
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hajime Wada.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 13 KB)

Supplementary Fig. 1

BN-PAGE/SDS-PAGE analyses of PSII complexes purified from cells of the control strain and mutants. PSII complexes corresponding to 3 μg Chl were analyzed by BN-PAGE in the first dimension, and then by SDS-PAGE in the second dimension. Proteins in the gels of SDS-PAGE were visualized with silver staining. (TIF 6142 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Endo, K., Kobayashi, K., Wang, HT. et al. Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity. Photosynth Res 139, 267–279 (2019). https://doi.org/10.1007/s11120-018-0555-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11120-018-0555-3

Keywords

Search

Navigation