Charilaos Goussias

University of Ioannina, Yannina, Epirus, Greece

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Publications (9)44.17 Total impact

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    Peter Faller, Charilaos Goussias, A William Rutherford, Sun Un
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    ABSTRACT: The coupling of proton chemistry with redox reactions is important in many enzymes and is central to energy transduction in biology. However, the mechanistic details are poorly understood. Here, we have studied tyrosine oxidation, a reaction in which the removal of one electron from the amino acid is linked to the release of its phenolic proton. Using the unique photochemical properties of photosystem II, it was possible to oxidize the tyrosine at 1.8 K, a temperature at which proton and protein motions are limited. The state formed was detected by high magnetic field EPR as a high-energy radical intermediate trapped in an unprecedentedly electropositive environment. Warming of the protein allows this state to convert to a relaxed, stable form of the radical. The relaxation event occurs at 77 K and seems to involve proton migration and only a very limited movement of the protein. These reactions represent a stabilization process that prevents the back-reaction and determines the reactivity of the radical.
    Proceedings of the National Academy of Sciences 08/2003; 100(15):8732-5. DOI:10.1073/pnas.1530926100 · 9.81 Impact Factor
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    ABSTRACT: The function of cytochrome b(559) in photosystem II (PSII) was investigated using a mutant created in tobacco in which the conserved phenylalanine at position 26 in the beta-subunit (PsbF) was changed to serine (Bock, R., Kössel, H., and Maliga, P. (1994) EMBO J. 13, 4623-4628). The mutant grew photoautotrophically, but the amount of PSII was reduced and the ultrastructure of the chloroplast was dramatically altered. Very few grana stacks were formed in the mutant. Although isolated PSII-enriched membrane fragments showed low PSII activity, electron paramagnetic resonance indicated the presence of functional PSII. Difference absorption spectra showed that the cytochrome b(559) contained heme. The plastoquinone pool was largely reduced in dark-adapted leaves of the mutant, based on chlorophyll fluorescence and thermoluminescence measurements. We therefore propose that cytochrome b(559) plays an important role in PSII by keeping the plastoquinone pool and thereby the acceptor side of PSII oxidized in the dark. Structural alterations as induced by the single Phe --> Ser point mutation in the transmembrane domain of PsbF evidently inhibit this function.
    Journal of Biological Chemistry 04/2003; 278(15):13554-60. DOI:10.1074/jbc.M212842200 · 4.60 Impact Factor
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    ABSTRACT: The terminal electron acceptor of Photosystem II, PSII, is a linear complex consisting of a primary quinone, a non-heme iron(II), and a secondary quinone, Q(A)Fe(2+)Q(B). The complex is a sensitive site of PSII, where electron transfer is modulated by environmental factors and notably by bicarbonate. Earlier studies showed that NO and other small molecules (CN(-), F(-), carboxylate anions) bind reversibly on the non-heme iron in competition with bicarbonate. In the present study, we report on an unusual new mode of transient binding of NO, which is favored in the light-reduced state (Q(A)(-)Fe(2+)Q(B)) of the complex. The related observations are summarized as follows: (i) Incubation with NO at -30 degrees C, following light-induced charge separation, results in the evolution of a new EPR signal at g = 2.016. The signal correlates with the reduced state Q(A)(-)Fe(2+) of the iron-quinone complex. (ii) Cyanide, at low concentrations, converts the signal to a more rhombic form with g values at 2.027 (peak) and 1.976 (valley), while at high concentrations it inhibits formation of the signals. (iii) Electron spin-echo envelope modulation (ESEEM) experiments show the existence of two protein (14)N nuclei coupled to electron spin. These two nitrogens have been detected consistently in the environment of the semiquinone Q(A)(-) in a number of PSII preparations. (iv) NO does not directly contribute to the signals, as indicated by the absence of a detectable isotopic effect ((15)NO vs (14)NO) in cw EPR. (v) A third signal with g values (2.05, 2.03, 2.01) identical to those of an Fe(NO)(2)(imidazole) synthetic complex develops slowly in the dark, or faster following illumination. (vi) In comparison with the untreated Q(A)(-)Fe(2+) complex, the present signals not only are confined to a narrow spectral region but also saturate at low microwave power. At 11 K the g = 2.016 signal saturates with a P(1/2) of 110 microW and the g = 2.027/1.976 signal with a P(1/2) of 10 microW. (vii) The spectral shape and spin concentration of these signals is successfully reproduced, assuming a weak magnetic interaction (J values in the range 0.025-0.05 cm(-)(1)) between an iron-NO complex with total spin of (1)/(2) and the spin, (1)/(2), of the semiquinone, Q(A)(-). The different modes of binding of NO to the non-heme iron are examined in the context of a molecular model. An important aspect of the model is a trans influence of Q(A) reduction on the bicarbonate ligation to the iron, transmitted via H-bonding of Q(A) with an imidazole ligand to the iron.
    Biochemistry 01/2003; 41(51):15212-23. DOI:10.1021/bi026223e · 3.19 Impact Factor
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    ABSTRACT: Myriophyllum spicatum (Haloragaceae) is a highly competitive freshwater macrophyte that produces and releases algicidal and cyanobactericidal polyphenols. Among them, beta-1,2,3-tri-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (tellimagrandin II) is the major active substance and is an effective inhibitor of microalgal exoenzymes. However, this mode of action does not fully explain the strong allelopathic activity observed in bioassays. Lipophilic extracts of M. spicatum inhibit photosynthetic oxygen evolution of intact cyanobacteria and other photoautotrophs. Fractionation of the extract provided evidence for tellimagrandin II as the active compound. Separate measurements of photosystem I and II activity with spinach (Spinacia oleracea) thylakoid membranes indicated that the site of inhibition is located at photosystem II (PSII). In thermoluminescence measurements with thylakoid membranes and PSII-enriched membrane fragments M. spicatum extracts shifted the maximum temperature of the B-band (S(2)Q(B)(-) recombination) to higher temperatures. Purified tellimagrandin II in concentrations as low as 3 microM caused a comparable shift of the B-band. This demonstrates that the target site of this inhibitor is different from the Q(B)-binding site, a common target of commercial herbicides like 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Measurements with electron paramagnetic resonance spectroscopy suggest a higher redox midpoint potential for the non-heme iron, located between the primary and the secondary quinone electron acceptors, Q(A) and Q(B). Thus, tellimagrandin II has at least two modes of action, inhibition of exoenzymes and inhibition of PSII. Multiple target sites are a common characteristic of many potent allelochemicals.
    Plant physiology 01/2003; 130(4):2011-8. DOI:10.1104/pp.011593 · 7.39 Impact Factor
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    Charilaos Goussias, Alain Boussac, A William Rutherford
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    ABSTRACT: Conceptually, photosystem II, the oxygen-evolving enzyme, can be divided into two parts: the photochemical part and the catalytic part. The photochemical part contains the ultra-fast and ultra-efficient light-induced charge separation and stabilization steps that occur when light is absorbed by chlorophyll. The catalytic part, where water is oxidized, involves a cluster of Mn ions close to a redox-active tyrosine residue. Our current understanding of the catalytic mechanism is mainly based on spectroscopic studies. Here, we present an overview of the current state of knowledge of photosystem II, attempting to delineate the open questions and the directions of current research.
    Philosophical Transactions of The Royal Society B Biological Sciences 11/2002; 357(1426):1369-81; discussion 1419-20. DOI:10.1098/rstb.2002.1134 · 6.31 Impact Factor
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    ABSTRACT: The manganese cluster of the oxygen-evolving enzyme of photosystem II is chemically reduced upon interaction with nitric oxide at -30 degrees C. The state formed gives rise to an S = 1/2 multiline EPR signal [Goussias, Ch., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261] that is attributed to a Mn(II)- Mn(III) dimer [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581]. In this work, we sought to establish whether the state could be assigned to a specific, reduced S state by using flash oxymetry, chlorophyll a fluorescence, and electron paramagnetic resonance spectroscopy. With the Joliot-type O(2) electrode, the first maximum of oxygen evolution was observed on the sixth or seventh flash. Three saturating pre-flashes were required to convert the flash pattern characteristic of NO-reduced samples to that of the untreated control (i.e., O(2) evolution maximum on the third flash). Measurements of the S state-dependent level of chlorophyll fluorescence in NO-treated PSII showed a three-flash downshift compared to untreated controls. In the EPR study, the maximum S(2) multi-line EPR signal was observed after the fourth flash. The results from all three methods are consistent with the Mn cluster being in a redox state corresponding to an S(-2) state in a majority of centers after treatment with NO. We were unable to generate the Mn(II)-Mn(III) multi-line signal using hydrazine as a reductant; it appears that the valence distribution and possibly the structure of the Mn cluster in the S(-2) state are dependent on the nature of the reductant that is used.
    Biochemistry 04/2002; 41(9):3057-64. DOI:10.1021/bi015903z · 3.19 Impact Factor
  • Charilaos Goussias, Nikolaos Ioannidis, Vasili Petrouleas
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    ABSTRACT: The spin-1/2-carrying NO molecule interacts with both the S1 and S2 states of the water oxidizing complex. The intermediates of the interaction can be resolved and trapped by NO treatment at subzero temperatures. At -30 degrees C and in the presence of approx. 500-700 microM NO, S1 loses the ability to yield by illumination an EPR active S2-state with an approximate half-time of 40-60 min. At longer incubation times (t1/2 = 4-5 h), an intense new multiline signal develops. The new signal has a hyperfine splitting similar to the S2 multiline [Dismukes, G. C., & Siderer, Y. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 274-278], but a modified shape with intense lines on the high field side. The NO modified S1 state can act as a low-temperature electron donor yielding an EPR silent state upon illumination at 200 K. NO interacts also with the S2 state of the water oxidizing complex rapidly at temperatures as low as -75 degrees C, to yield an EPR silent state. The rates of the latter interaction show analogies to the ammonia binding to the S2 state. It is possible, however, that NO, unlike ammonia, destabilizes the S2 state. On the basis of preliminary experiments with varying chloride concentrations in the range 0.1-50 mM, the S1 multiline state is attributed to binding of NO at a chloride sensitive site on the Mn cluster. The rapid interactions with the S2 state as well as the intermediate binding to the S1 state are less well understood at present, but they are tentatively assigned to the chloride-insensitive site of ammonia binding in the Mn cluster.
    Biochemistry 07/1997; 36(30):9261-6. DOI:10.1021/bi962899w · 3.19 Impact Factor
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    ABSTRACT: Incubation of photosystem II, PSII, membranes with NO for a few minutes results in the reversible elimination of the electron paramagnetic resonance (EPR) signal II from the oxidized Tyr Y(D)., presumably due to the formation of a weak Tyr Y(D).-NO complex [Petrouleas, V., & Diner, B. A. (1990) Biochim. Biophys. Acta 1015, 131-140]. Illumination of such a sample at ambient or cryogenic temperatures produces no new EPR signals. If, however, the incubation with NO is extended to the hours time range, illumination induces an EPR signal with resolved hyperfine structure in the g = 2 region. The signal shows the typical features of an immobilized iminoxyl radical (> C=NO.) with hyperfine values A(parallel) = 44 G, A(perpendicular) = 22 G, and A(iso) = 29.3 G. The following observations suggest that the iminoxyl signal is associated with PSII: (a) the signal results from an immobilized species at room temperature probably associated with a membrane-bound component, (b) the abundance of the signal is (sub)stoichiometric to PSII, (c) the signal is light-induced, (d) some of the treatments that affect PSII (Tris, Ca2+ depletion, high-salt wash) severely diminish the size of the signal, and (e) the development of the signal correlates with the release of Mn. In addition, the following observations suggest that the iminoxyl signal results from an interaction of Y(D). with NO: (a) the evolution of the signal correlates with the loss in reversibility of the Tyr Y(D).-NO interaction and (b) the size of the signal correlates with the initial amount of oxidized Tyr Y(D). It is accordingly proposed that during the incubation with NO, a weak Tyr Y(D).-NO complex is rapidly formed and is then slowly converted to a tyrosine-nitroso adduct. Light-induced oxidation of the latter produces the iminoxyl radical. The nitrosotyrosine is expected to have an oxidation potential significantly lower than the parent tyrosine and can act as an efficient electron donor in PSII even at cryogenic temperatures. It is probably this lowered redox potential of the tyrosine Y(D) that explains the release of Mn concomitant with the formation of the nitroso species.
    Biochemistry 03/1997; 36(6):1411-7. DOI:10.1021/bi9622074 · 3.19 Impact Factor
  • Charilaos Goussias, Yiannis Sanakis, Vasili Petrouleas
    Journal of Inorganic Biochemistry 08/1995; 59(2):273-273. DOI:10.1016/0162-0134(95)97376-2 · 3.27 Impact Factor

Publication Stats

422 Citations
44.17 Total Impact Points

Institutions

  • 2003
    • University of Ioannina
      • Laboratory of Physical Chemistry (Department of Chemistry)
      Yannina, Epirus, Greece
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 1997
    • National Center for Scientific Research Demokritos
      Athínai, Attica, Greece