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Characterisation of a H2O2-oxidisable cytochrome b-559 in intact chloroplasts with a new type of LED Array Spectrophotometer

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Abstract

Cytochrome (cyt) b-559 absorbance changes in intact chloroplasts were deconvoluted using a previously described LED-Array-Spectrophotometer (Klughammer et al. (1990), Photosynth Res 25: 317–327). When intact chloroplasts were isolated in the presence of ascorbate, approx. 15% of the total cyt b-559 could be transiently oxidised by 200 μM H2O2 in the dark. This fraction displays low-potential properties, as it can be also oxidised by menadione in the presence of 5 mM ascorbate. Heat pretreatment increased the size of this fraction by a factor of 3–4. Low concentrations of cyanide (in the μM range) prolonged the oxidation time while high concentrations suppressed the oxidation (I50=1.5 mM KCN). The former KCN-effect relates to inhibition of ascorbate dependent H2O2-reduction which is catalysed by ascorbate peroxidase, whereas the latter effect reflects competition between H2O2 and CN− for the same binding site at the cytochrome heme. In the light, much lower concentrations of H2O2 were required to obtain oxidation, the amplitude depending on light intensity and on the concentration of the added H2O2, but never exceeding approx. 15% of the total cyt b-559. In the light, but not in the dark, H2O2 also induced the transient oxidation of a cyt f fraction similar in size to the H2O2-oxidisable cyt b-559 fraction. In this case, H2O2 serves as an acceptor of Photosystem I in conjunction with the ascorbate peroxidase detoxification system. Light can also induce oxidation of a 15% cyt b-559 fraction without H2O2-addition, if nitrite is present as electron acceptor and the chloroplasts are depleted of ascorbate. It is concluded that light-induced cyt b-559 oxidation in vivo is likely to be restricted to the H2O2-oxidisable cyt b-559 LP fraction and is normally counteracted by ascorbate.

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Abbreviations

APX:

ascorbate peroxidase

chl:

chlorophyll

cyt:

cytochrome

HP:

high potential

LP:

low potential

MDA:

monodehydroascorbate

PQ:

plastoquinone

PS I and PS II:

Photosystems I and II

References

  • Ananyev G, Renger G, Wecker U and Klimov V (1994) The photoproduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b-559. Photosynth Res 41: 327–338

    Google Scholar 

  • Anderson JM and Boardman NK (1973) Localization of low potential cytochrome b-559 in Photosystem I. FEBS Lett 32: 157–160

    Article  PubMed  Google Scholar 

  • Arnon AI and Tang G (1988) Cyt b-559 and proton conductance in oxygenic photosynthesis. Proc Natl Acad Sci USA 85: 9524–9528

    Google Scholar 

  • Asada K and Takahashi M (1987) Production and scavenging of active oxygen in photosynthesis. In: Kyle et al. (eds) Photoinhibition, pp 227–287. Elsevier, Amsterdam

    Google Scholar 

  • Asada K, Kiso K and Yoshikawa K (1974) Univalent reduction of molecular oxygen by spinach chloroplasts on illumination. J Biol Chem 249: 2175–2181

    PubMed  Google Scholar 

  • Barabas K, Kravcova T and Garab G (1993) Flash-induced reduction of cyt b-559 by QB − in chloroplasts in the presence of protonophores. Photosynth Res 6: 59–64

    Google Scholar 

  • Bendall DS (1982) Photosynthetic cytochromes of oxygenic organisms. Biochim Biophys Acta 683: 119–151

    Google Scholar 

  • Buser CA, Diner BA and Brudvig GW (1992) Photooxidation of cytochrome b-559 in oxygen-evolving Photosystem II. Biochem 31: 11449–11459

    Google Scholar 

  • Canaani O and Havaux M (1990) Evidence for a biological role in photosynthesis for cytochrome b-559-a component of Photosystem II reaction centre. Proc Natl Acad Sci USA 87: 9295–9299

    PubMed  Google Scholar 

  • Cheniae GM and Martin IF (1970) Sites of function of manganese within Photosystem II. Roles in O2 evolution and system II. Biochim Biophys Acta 197: 219–239

    PubMed  Google Scholar 

  • Cramer WA, Fan HN and Böhme H (1971) High and low potential states of chloroplast cytochrome b-559 and thermodynamic control of non-cyclic electron transport. Bioenerg 2: 289–303

    Google Scholar 

  • Cramer WA, Tae GS, Furbacher PN and Böttger M (1993) Minireview: The enigmatic cytochrome b-559 of oxygenic photosynthesis. Physiol Plant 88: 705–711

    Article  Google Scholar 

  • Erixon K, Lozier R and Butler W (1972) The redox state of cytochrome c/plastocyanin oxidoreductases. Biochim Biophys Acta 267: 375–382

    PubMed  Google Scholar 

  • Grace S, Pace R and Wydrzynski T (1995) Formation and decay of monodehydroascorbate radicals in illuminated thylakoids as determined by EPR spectroscopy. Biochim Biophys Acta 1229: 155–165

    Google Scholar 

  • Groden D and Beck E (1979) H2O2 destruction by ascorbate-dependent systems from chloroplasts. Biochim Biophys Acta 546: 426–435

    PubMed  Google Scholar 

  • Heber U and Santarius KA (1970) Direct and indirect transport of ATP and ADP across the chloroplast envelope. Z Naturforsch 25b: 718–728

    Google Scholar 

  • Heber U, Kirk MR and Boardman NK (1979) Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts. Biochim Biophys Acta 546: 292–306

    PubMed  Google Scholar 

  • Heimann S (1995) Charakterisierung der Rolle des Cytochrom b-559 beim Photosynthese-Prozeß in Chloroplasten mit Hilfe optischer Meßmethoden. Master Thesis, Bayerische-Julius-Maximilians-Universität Würzburg, Germany

  • Hiller RG, Anderson JM and Boardman NK (1971) Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature. Biochim Biophys Acta 245: 439–452

    PubMed  Google Scholar 

  • Klughammer C (1992) Entwicklung und Anwendung neuer absorptions-spektroskopischer Methoden zur Charakterisierung des photosynthetischen Elektronentransportes in isolierten Chloroplasten und intakten Blättern. Doctoral Thesis, Bayerische-Julius-Maximilians-Universität Würzburg, Germany

  • Klughammer C and Schreiber U (1993) Selective interaction of valinomycin/K+ with the cytochrome b/f complex of chloroplasts. FEBS Letters 336: 491–495

    Article  PubMed  Google Scholar 

  • Klughammer C, Kolbowski J and Schreiber U (1990) LED Array Spectrophotometer for measurement of time-resolved difference spectra in the 530–600 nm wavelength region. Photosynth Res 25: 317–327

    Google Scholar 

  • Krieger A and Weis E (1993) The role of calcium in the pH-dependent control of Photosystem II. Photosynth Res 37: 117–130

    Google Scholar 

  • Krishtalik LI, Tae G-S, Cherepanov DA and Cramer WA (1993) The redox properties of cytochrome b imposed by the membrane electrostatic environment. Biophys J 65, 184–195

    PubMed  Google Scholar 

  • Melis A (1991) Review: Dynamics of photosynthetic membrane composition and function. Biochim Biophys Acta 1058: 87–106

    Google Scholar 

  • Miyake C and Asada K (1992) Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiol 33: 541–553

    Google Scholar 

  • Miyake C and Asada K (1994) Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiol 35: 539–549

    Google Scholar 

  • Neubauer C and Schreiber U (1989) Photochemical and nonphotochemical quenching of chlorophyll fluorescence induced by hydrogen peroxide. Z Naturforsch 44c: 262–270

    Google Scholar 

  • Njus D and Kelley PM (1993) The secretory-vesicle ascorbate-regenerating system: A chain of concerted H+/e−-transfer reactions. Biochim Biophys Acta 1144: 235–248

    PubMed  Google Scholar 

  • Ortega JM, Hervas M and Losada M (1989) Location of cytochrome b-559 between Photosystem II and Photosystem I in noncyclic electron transport. Biochim Biophys Acta 975: 244–251

    Google Scholar 

  • Pakrasi HB, Nyhus KJ and Granok H (1990) Targeted deletion mutagenesis of the β-subunit of cytochrome b-559 protein destabilizes the reaction centre of Photosystem II. Z Naturforsch 45c: 423–429

    Google Scholar 

  • Poulson M, Samson G and Whitmarsh J (1995) Evidence that cytochrome b-559 protects Photosystem II against photoinhibition. Biochem 34: 10932–10938

    Google Scholar 

  • Rich PR (1988) A critical examination of the supposed variable proton stoichiometry of the chloroplast cytochrome b/f complex. Biochim Biophys Acta 932: 33–42

    Google Scholar 

  • Samson G and Fork DC (1991) Enhanced susceptibility of the oxidized and unprotonated forms of high potential cytochrome b-559 toward DCMU. Photosynth Res 27: 179–187

    Google Scholar 

  • Schreiber U and Berry JA (1977) Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus. Planta 136: 233–238

    Google Scholar 

  • Schreiber U and Neubauer C (1987) The polyphasic rise of chlorophyll fluorescence upon onset of strong continuous illumination: II. Partial control by the Photosystem II donor side and possible ways of interpretation. Z Naturforsch 42c: 1255–1264

    Google Scholar 

  • Schreiber U and Neubauer C (1990) O2-dependent electron flow, membrane energization and the mechanisms of nonphotochemical quenching of chlorophyll fluorescence. Photosynth Res 25: 279–293

    Google Scholar 

  • Schreiber U, Reising H and Neubauer C (1991) Contrasting pH-optima of light-driven O2- and H2O2-reduction in spinach chloroplasts as measured via chlorophyll fluorescence quenching. Z Naturforsch 46c: 635–643

    Google Scholar 

  • Schreiber U, Hormann H, Asada K and Neubauer C (1995) O2-dependent electron flow in intact spinach chloroplasts: Properties and possible regulation of the Mehler-Ascorbate Peroxidase Cycle. In: Mathis P (ed) Photosynthesis: From Light to Biosphere, Vol II, pp 813–818 Kluwer Academic Publishers, Dordrecht

    Google Scholar 

  • Shuvalov VA and Krasnovsky AA (1975) Investigation of the photoreduction of oxygen in chloroplasts measured as chemiluminescence of luminol and chlorophyll. Biokhimia USSR 40: 358–367

    Google Scholar 

  • Sundby C, Melis A, Hideg E, Mäenpää P and Andersson B (1986) Temperature-dependent changes in the antenna size of Photosystem II. Reversible conversion of Photosystem IIα to Photosystem IIβ. Biochim Biophys Acta 891: 475–483

    Google Scholar 

  • Thompson JK and Brudvig GW (1988) Cytochrome b-559 may function to protect Photosystem II from photoinhibition. Biochem 27: 6653–6658

    Google Scholar 

  • Weis E (1985) Light- and temperature-induced changes in the distribution of excitation energy between Photosystem I and Photosystem II in spinach leaves. Biochim Biophys Acta 807: 118–126

    Google Scholar 

  • Weis E and Berry JA (1987) Quantum efficiency of Photosystem II in relation to energy dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–208

    Google Scholar 

  • Whitmarsh J, Samson G and Poulson M (1994) Photoprotection in Photosystem II-the role of cytochrome b-559. In: Baker NR and Bower J (eds) Photoinhibition of Photosynthesis-From Molecular Mechanisms to the Field, pp 75–93. BIOS Scientific, Oxford

    Google Scholar 

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  1. Lehrstuhl Botanik I, Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082, Würzburg, Germany

    Sebastian Heimann & Ulrich Schreiber

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  1. Sebastian Heimann
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  2. Ulrich Schreiber
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Heimann, S., Schreiber, U. Characterisation of a H2O2-oxidisable cytochrome b-559 in intact chloroplasts with a new type of LED Array Spectrophotometer. Photosynth Res 47, 187–197 (1996). https://doi.org/10.1007/BF00016181

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  • DOI: https://doi.org/10.1007/BF00016181

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