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

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 of monogalactosyldiacylglycerol 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 31 P-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 the mechanism of violaxanthin de-epoxidation in native thylakoid membranes is discussed.Keywords: xanthophyll cycle; de-epoxidation; liposomes; violaxanthin; zeaxanthin Xanthophyll cycle is a photoprotective mechanism widespread in nature operating in the thylakoid membranes of all higher plants, ferns, mosses and several algal groups [1]. This cycle involves two reversible reactions, light-dependent de-epoxidation of violaxanthin to zeaxanthin via antheraxanthin as an intermediate and light-independent epoxidation of zeaxanthin to anteraxanthin and violaxanthin [2]. The conversion of violaxanthin to zeaxanthin is catalysed by violaxanthin de-epoxidase (VDE) and the reverse reaction of violaxanthin formation from zeaxanthin is catalysed by another enzyme, zeaxanthin epoxidase. VDE has been isolated from spinach and lettuce chloroplasts and the molecular mass of the native enzyme was estimated as 43 kDa [3][4][5]. The gene encoding VDE has been already isolated and cloned [6]. VDE is located on the lumenal side of the thylakoid membrane, shows an optimum activity at pH 4.8 when present in chloroplasts and at 5.2 for the isolated enzyme [7] and requires ascorbate as a reductant [8]. In the dark, when the pH in thylakoid lumen is neutral or alkaline, VDE is inactive, whereas under strong light conditions, pH in the thylakoid lumen decreases, the enzyme binds to the membrane, becomes active and converts...

Maturation Process of Photosynthetic Membranes Observed by Proton Magnetic Relaxation and Sorption Isotherm

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Rehydration of Digalactosyldiacylglycerol Model Membrane Lyophilizates Observed by NMR and Sorption Isotherm

“…The structure of this kind consists of rod-like aggregates which are formed by the polar lipid headgroups located at the center of the rods along with some water, while the fatty acid chains point outward so that the surfaces of the rod are hydrophobic. It is worth mentioning that the presence of H II phase, has been detected in the native photosynthetic membranes [5,23] as well as in phosphatidylcholine liposomes containing MGDG at the amount ranging 50% [24].…”

The Effect of Water Accessible Paramagnetic Ions on Subcellular Structures Formed in Developing Wheat Photosynthetic Membranes as Observed by NMR and by Sorption Isotherm

“…A good candidate for water-encapsulating lipid structures could be the tubules of inverted hexagonal phase formed by monogalactosyldiacylglycerol (MGDG), a dominating lipid species of the photosynthetic membranes. Domains enriched in inverted hexagonal phases have been detected both in the native photosynthetic membranes [30] as well as in model phosphatidylcholine liposomes containing MGDG [31]. However, the question if such structures present in fully hydrated membranes also occur in lyophilized thylakoids, remains to be found.…”