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Tomoko Shinomura - One of the best experts on this subject based on the ideXlab platform.

Harald Paulsen - One of the best experts on this subject based on the ideXlab platform.

  • assembly of the major light harvesting chlorophyll a b complex thermodynamics and kinetics of Neoxanthin binding
    Journal of Biological Chemistry, 2006
    Co-Authors: Stephan Hobe, Inga Trostmann, Stefan Raunser, Harald Paulsen
    Abstract:

    Abstract The major light-harvesting chlorophyll-a/b complex in most higher plants contains three carotenoids, lutein, Neoxanthin, and violaxanthin. How these pigments are assembled into the complex during its biogenesis is largely unknown. Here we show that Neoxanthin but not lutein can dissociate from the fully assembled complex. Its equilibrium binding constant in a detergent system (0.1% n-dodecyl-β-d-maltoside) was determined to be ≥ 106 m–1. Neoxanthin insertion into light-harvesting chlorophyll-a/b complex prefolded from overexpressed apoprotein (Lhcb1*2 from Pisum sativum) in the presence of chlorophylls a, b, and lutein as the sole carotenoid is kinetically controlled by an activation energy barrier of ∼120 kJ mol–1. This is the first thermodynamic and kinetic description of a binding equilibrium between a non-covalently bound pigment of the photosynthetic apparatus and its protein complex. Dissociation of Neoxanthin from the major light-harvesting chlorophyll-a/b complex upon temperature increase is discussed in terms of providing a readily available substrate pool for synthesizing abscisic acid as part of a heat and drought stress response.

  • de epoxidation of violaxanthin after reconstitution into different carotenoid binding sites of light harvesting complex ii
    Journal of Biological Chemistry, 2001
    Co-Authors: Peter Jahns, Harald Paulsen, Antje Wehner, Stephan Hobe
    Abstract:

    In higher plants, the de-epoxidation of violaxanthin (Vx) to antheraxanthin and zeaxanthin is required for the pH-dependent dissipation of excess light energy as heat and by that process plays an important role in the protection against photo-oxidative damage. The de-epoxidation reaction was investigated in an in vitro system using reconstituted light-harvesting complex II (LHCII) and a thylakoid raw extract enriched in the enzyme Vx de-epoxidase. Reconstitution of LHCII with varying carotenoids was performed to replace lutein and/or Neoxanthin, which are bound to the native complex, by Vx. Recombinant LHCII containing either 2 lutein and 1 Vx or 1.6 Vx and 1.1 Neoxanthin or 2.8 Vx per monomer were studied. Vx de-epoxidation was inducible for all complexes after the addition of Vx de-epoxidase but to different extents and with different kinetics in each complex. Analysis of the kinetics indicated that the three possible Vx binding sites have at least two, and perhaps three, specific rate constants for de-epoxidation. In particular, Vx bound to one of the two lutein binding sites of the native complex, most likely L1, was not at all or only at a slow rate convertible to Zx. In reisolated LHCII, newly formed Zx almost stoichiometrically replaced the transformed Vx, indicating that LHCII and Vx de-epoxidase stayed in close contact during the de-epoxidation reactions and that no release of carotenoids occurred.

  • effects of chlorophyll a chlorophyll b and xanthophylls on the in vitro assembly kinetics of the major light harvesting chlorophyll a b complex lhciib
    Journal of Molecular Biology, 2001
    Co-Authors: Dirk Reinsberg, Katja Ottmann, Paula J Booth, Harald Paulsen
    Abstract:

    The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio ⩽1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, Neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (Neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.

  • carotenoid binding sites in lhciib relative affinities towards major xanthophylls of higher plants
    FEBS Journal, 2000
    Co-Authors: Stephan Hobe, Hildrun Niemeier, Alexander Bender, Harald Paulsen
    Abstract:

    The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and Neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30–50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein–LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin–LHCIIb exhibits a low thermal stability like Neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein–LHCIIb and zeaxanthin–LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/Neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to Neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.

Shota Kato - One of the best experts on this subject based on the ideXlab platform.

Shinichi Takaichi - One of the best experts on this subject based on the ideXlab platform.

Stephan Hobe - One of the best experts on this subject based on the ideXlab platform.

  • assembly of the major light harvesting chlorophyll a b complex thermodynamics and kinetics of Neoxanthin binding
    Journal of Biological Chemistry, 2006
    Co-Authors: Stephan Hobe, Inga Trostmann, Stefan Raunser, Harald Paulsen
    Abstract:

    Abstract The major light-harvesting chlorophyll-a/b complex in most higher plants contains three carotenoids, lutein, Neoxanthin, and violaxanthin. How these pigments are assembled into the complex during its biogenesis is largely unknown. Here we show that Neoxanthin but not lutein can dissociate from the fully assembled complex. Its equilibrium binding constant in a detergent system (0.1% n-dodecyl-β-d-maltoside) was determined to be ≥ 106 m–1. Neoxanthin insertion into light-harvesting chlorophyll-a/b complex prefolded from overexpressed apoprotein (Lhcb1*2 from Pisum sativum) in the presence of chlorophylls a, b, and lutein as the sole carotenoid is kinetically controlled by an activation energy barrier of ∼120 kJ mol–1. This is the first thermodynamic and kinetic description of a binding equilibrium between a non-covalently bound pigment of the photosynthetic apparatus and its protein complex. Dissociation of Neoxanthin from the major light-harvesting chlorophyll-a/b complex upon temperature increase is discussed in terms of providing a readily available substrate pool for synthesizing abscisic acid as part of a heat and drought stress response.

  • de epoxidation of violaxanthin after reconstitution into different carotenoid binding sites of light harvesting complex ii
    Journal of Biological Chemistry, 2001
    Co-Authors: Peter Jahns, Harald Paulsen, Antje Wehner, Stephan Hobe
    Abstract:

    In higher plants, the de-epoxidation of violaxanthin (Vx) to antheraxanthin and zeaxanthin is required for the pH-dependent dissipation of excess light energy as heat and by that process plays an important role in the protection against photo-oxidative damage. The de-epoxidation reaction was investigated in an in vitro system using reconstituted light-harvesting complex II (LHCII) and a thylakoid raw extract enriched in the enzyme Vx de-epoxidase. Reconstitution of LHCII with varying carotenoids was performed to replace lutein and/or Neoxanthin, which are bound to the native complex, by Vx. Recombinant LHCII containing either 2 lutein and 1 Vx or 1.6 Vx and 1.1 Neoxanthin or 2.8 Vx per monomer were studied. Vx de-epoxidation was inducible for all complexes after the addition of Vx de-epoxidase but to different extents and with different kinetics in each complex. Analysis of the kinetics indicated that the three possible Vx binding sites have at least two, and perhaps three, specific rate constants for de-epoxidation. In particular, Vx bound to one of the two lutein binding sites of the native complex, most likely L1, was not at all or only at a slow rate convertible to Zx. In reisolated LHCII, newly formed Zx almost stoichiometrically replaced the transformed Vx, indicating that LHCII and Vx de-epoxidase stayed in close contact during the de-epoxidation reactions and that no release of carotenoids occurred.

  • carotenoid binding sites in lhciib relative affinities towards major xanthophylls of higher plants
    FEBS Journal, 2000
    Co-Authors: Stephan Hobe, Hildrun Niemeier, Alexander Bender, Harald Paulsen
    Abstract:

    The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and Neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30–50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein–LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin–LHCIIb exhibits a low thermal stability like Neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein–LHCIIb and zeaxanthin–LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/Neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to Neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.