Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase, a xanthophyll cycle enzyme, is regulated by membrane fluidity in model lipid bilayers

Department of Plant Physiology and Biochemistry, The Jan Zurzycki Institute of Molecular Biology and Biotechnology, Jagiellonian University, Kraków, Poland.
European Journal of Biochemistry (Impact Factor: 3.58). 10/2002; 269(18):4656-65. DOI: 10.1046/j.1432-1033.2002.03166.x
Source: PubMed

ABSTRACT This paper describes violaxanthin de-epoxidation in model lipid bilayers. Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with monogalactosyldiacylglycerol were found to be a suitable system for studying this reaction. Such a system resembles more the native thylakoid membrane and offers better possibilities for studying kinetics and factors controlling de-epoxidation of violaxanthin than a system composed only ofmonogalactosyldiacylglycerol and is commonly used in xanthophyll cycle studies. The activity of violaxanthin de-epoxidase (VDE) strongly depended on the ratio of monogalactosyldiacylglycerol to PtdCho in liposomes. The mathematical model of violaxanthin de-epoxidation was applied to calculate the probability of violaxanthin to zeaxanthin conversion at different phases of de-epoxidation reactions. Measurements of deepoxidation rate and EPR-spin label study at different temperatures revealed that dynamic properties of the membrane are important factors that might control conversion of violaxanthin to antheraxanthin. A model of the molecular mechanism of violaxanthin de-epoxidation where the reversed hexagonal structures (mainly created by monogalactosyldiacylglycerol) are assumed to be required for violaxanthin conversion to zeaxanthin is proposed. The presence of monogalactosyldiacylglycerol reversed hexagonal phase was detected in the PtdCho/monogalactosyldiacylglycerol liposomes membrane by 31P-NMR studies. The availability of violaxanthin for de-epoxidation is a diffusion-dependent process controlled by membrane fluidity. The significance of the presented results for understanding themechanism of violaxanthin de-epoxidation in native thylakoid membranes is discussed.

Download full-text


Available from: Kazimierz Strzalka, Mar 10, 2015
1 Follower
  • Source
    • "The concentration of ethanol in the reaction medium was less than 2%. Epoxidation reaction was performed for 120 minutes at room temperature and the level of three pigments (Zx, Vx, antheraxanthin (Ax)) were measured by HPLC as described earlier (Latowski et al., 2002). The reaction mixture contained: thylakoids, epoxidation medium (400 mM Sorbitol, 50 mM Hepes, 5 mM MgCl 2 , pH 7,6), albumin, sodium ascorbate, FAD, NADPH. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.
    Acta biochimica Polonica 03/2012; 59(1):105-7. · 1.39 Impact Factor
  • Source
    • "Chlorophylls concentration was determined spectrophotometrically according to Ref. [21] and carotenoids were determined by HPLC as described in Ref. [24] "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the present study, we have performed comparative analysis of different prenyllipids in Chlamydomonas reinhardtii cultures during high light stress under variety of conditions (presence of inhibitors, an uncoupler, heavy water). The obtained results indicate that plastoquinol is more active than α-tocopherol in scavenging of singlet oxygen generated in photosystem II. Besides plastoquinol, also its oxidized form, plastoquinone shows antioxidant action during the stress conditions, resulting in formation of plastoquinone-C, whose level can be regarded as an indicator of singlet oxygen oxidative stress in vivo. The pronounced stimulation of α-tocopherol consumption and α-tocopherolquinone formation by an uncoupler, FCCP, together with the results of additional model system studies, led to the suggestion that α-tocopherol can be recycled in thylakoid membranes under high light conditions from 8a-hydroperoxy-α-tocopherone, the primary oxidation product of α-tocopherol by singlet oxygen.
    Biochimica et Biophysica Acta 12/2011; 1817(3):389-94. DOI:10.1016/j.bbabio.2011.12.002 · 4.66 Impact Factor
  • Source
    • "Total chlorophyll content was estimated spectrophotometrically in 80% aqueous acetone extract as described by Arnon [26] (1949). Xanthophyll cycle pigments were estimated by high-pressure liquid chromatography (HPLC, Jasco, Japan) method as described by Latowski et al. [27]. The de-epoxidation state was calculated from the equation [% (Zx + 0.5Ax) / (Vx + Ax + Zx)]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of the present studies was analysis of the age induced changes in photochemical efficiency and xanthophylls cycle pigments of the primary cabbage(Brassica oleracea L. cv. Capitata f. alba) leaves. Photochemical efficiency of photosystem II (PS II) was studied by a pulse amplitude modulated chlorophyll fluorescence apparatus, chlorophyll concentration was analysis spectrophotometrically and xanthophyll cycle pigments were estimated by high-pressure liquid chromatography (HPLC). Leaf senescence was accompanied with a decrease both in chlorophylls concentration, the photochemical efficiency and rate constant for PS II photochemistry whereas non-photochemical parameters increased. Excitation pressure (1-qP) which is a measure of relative lumen acidification increased by 1.2× in aging leaves. The maximum quantum yield of PS II showed no significant change. The level of de-epoxidised xanthophylls increased but the concentration of mono- and di-epoxy xanthophylls decreased in aging leaves. A linear relationship between the excitation pressure and the de-epoxidation state of the xanthophyll cycle pigments and lutein, during the onset of senescence suggests that excitation pressure can be used as a sensor for monitoring the onset of senescence as well for the de-epoxidation state of the xanthophylls responsible for non-photochemical quenching in stressed leaves.