Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

The conversion of violaxanthin (Vx) to zeaxanthin (Zx) in the de-epoxidation reaction of the xanthophyll cycle plays an important role in the protection of chloroplasts against photooxidative damage. Vx is bound to the antenna proteins of both photosystems. In photosystem II, the formation of Zx is essential for the pH-dependent dissipation of excess light energy as heat. The function of Zx in photosystem I is still unclear. In this work we investigated the de-epoxidation characteristics of light-harvesting complex proteins of photosystem I (LHCI) under in vivo and in vitro conditions. Recombinant LHCI (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids. All four LHCI proteins exhibited unique de-epoxidation characteristics. An almost complete Vx conversion to Zx was observed only in Lhca3, whereas Zx formation in the other LHCI proteins decreased in the order Lhca4 > Lhca1 > Lhca2. Most likely, these differences in Vx de-epoxidation were related to the different accessibility of the respective carotenoid binding sites in the distinct antenna proteins. The results indicate that Vx bound to site V1 and N1 is easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially and/or with the slower kinetics convertible to Zx. The de-epoxidation properties determined for the monomeric recombinant proteins were consistent with those obtained for isolated native LHCI-730 and LHCI-680 in the same in vitro assay and the de-epoxidation state found under in vivo conditions in native LHCIs.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

De-epoxidation of Violaxanthin in Light-harvesting Complex I Proteins

Wehner

Storf

Jahns

et al. 2004

“…Preparation of Crude VxDE Extracts-VxDE extracts were isolated from spinach essentially following the procedure described by Arvidsson et al (25). Roughly, isolated thylakoids were broken by sonification at pH 5.1, and VxDE was released from the resulting membrane fragments by increasing the pH to 7.2 for the final sonification step.…”

Section: Methodsmentioning

confidence: 99%

“…Roughly, isolated thylakoids were broken by sonification at pH 5.1, and VxDE was released from the resulting membrane fragments by increasing the pH to 7.2 for the final sonification step. After centrifugation, VxDE was precipitated from the supernatant by differential (NH 4 ) 2 SO 4 fractionation and finally collected by ultracentrifugation (25).…”

Section: Methodsmentioning

confidence: 99%

De-epoxidation of Violaxanthin after Reconstitution into Different Carotenoid Binding Sites of Light-harvesting Complex II

Jahns

Wehner

Paulsen

et al. 2001

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.

“…The activity of violaxanthin de-epoxidase was determined as in Ref. 38. All enzymatic activities were measured on freshly prepared samples (except those for violaxanthin de-epoxidase) at 25°C in the presence of saturating substrate concentrations.…”

mentioning

confidence: 99%

The Thylakoid Lumen of Chloroplasts

Kieselbach¹,

Hagman

Andersson

et al. 1998

134

The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60 -65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.The chloroplast is the photosynthetic organelle of green algae and higher plants. The chloroplast architecture comprises an envelope membrane, which encloses the soluble stroma as well as the highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the Calvin cycle, which are required for the fixation of carbon dioxide. The thylakoids, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The thylakoid membrane has a characteristic flat shape and is differentiated into appressed grana stacks and single non-appressed stroma-exposed lamellae. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, the lumen (1, 2). Electron microscopy studies of spinach thylakoids have suggested that the lumen is a densely packed space (3). No isolation method has so far been available for obtaining a high yield of pure thylakoid lumen. Thus, the present knowledge of the lumen from a compositional and functional point of view is fragmentary and is gathered from several independent approaches, addressing only single aspects of this compartment.By developing a technique for obtaining inside-out thylakoids, the investigation of the membrane surface of the lumenal side became possible (4). This work contributed to the discovery of the extrinsic proteins PsbO, PsbP, and PsbQ (5, 6) that bind to the lumenal side of photosystem II and are thought to stabilize the water oxidizing complex (7,8). More recent studies have shown that these subunits of photosystem II occur also as soluble lumenal proteins (9). This pool of unassembled PsbO, PsbQ, and PsbP was resistant to proteolytic degradation and was capa...