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

Peter JahnsDagger §, Antje WehnerDagger , Harald Paulsen, and Stephan Hobe

From the Dagger  Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany and  Institut für Allgemeine Botanik, Johannes-Gutenberg-Universität Mainz, Müllerweg 6, D-55128 Mainz, Germany

Received for publication, March 9, 2001, and in revised form, March 30, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antenna proteins serve as light-harvesting systems in all photosynthetic organisms that collect energy for the primary light reactions in the reaction centers. In higher plants, the antenna proteins of both photosystems constitute the large family of light-harvesting chlorophyll (Chl)1 a/b-binding (LHC) proteins (1). All LHC proteins are encoded by nuclear Lhc genes, show high sequence similarity among themselves (1-3), and have presumably similar structures (1). The structure of the major LHC of photosystem II (PS II), the so-called LHCII or Lhcb1/2 protein, has been determined at 3.4 Å by electron crystallography (5). According to biochemical analysis, LHCII binds two lutein (Lu), 1 neoxanthin (Nx), and substoichiometric amounts of violaxanthin (Vx)/monomer (6-8). Two xanthophylls, which are associated with the two central membrane-spanning alpha -helices, have been identified in the crystal structure and tentatively assigned to Lu (4, 5). Data obtained from site-directed mutagenesis of LHCII indicated that the Nx binding site is most likely associated with the more peripheral transmembrane helix (9). In CP29 (or Lhcb4), one of the minor Chl a/b binding proteins of PSII, only the two Lu binding sites, called L1 and L2, seem to be occupied, one by Lu and the other by Vx or Nx (10).

The selectivity of the three carotenoid binding sites in LHCII has been studied using recombinant protein (11, 12). It was shown that Vx could bind to all binding sites of the native complex, the two Lu (L1 and L2) as well as the Nx binding site, albeit with lower affinity than Lu and Nx. In the absence of other carotenoids during reconstitution, 2-3 Vx were found to bind to LHCII (11, 12). In the presence of equal amounts of Lu and Vx, roughly 2 Lu and 1 Vx bound to the complexes (11, 12).

The dissipation of excess light energy in the antenna of PSII, frequently measured as non-photochemical quenching (NPQ) of Chl fluorescence, is supposed to be important for the protection of plants against photo-oxidative damage (13-15). The de-epoxidation of Vx to zeaxanthin (Zx) via the intermediate antheraxanthin (Ax) in the xanthophyll cycle plays a crucial role in the Delta pH-dependent qE-component of NPQ (16), with the additional requirement of the psbS protein in this process (17). The xanthophyll cycle pigments are bound to the different antenna subcomplexes of both photosystems with different stoichiometries varying from about 0.1 to 1.5/monomer (8, 18-22). Under in vivo conditions, xanthophyll conversion occurs in various antenna subcomplexes with different kinetics and to a different extent, which seems to be related to distinct functions of single subcomplexes in different components of NPQ (18, 20). It is unclear, however, whether the differential xanthophyll conversion in single antenna complexes is determined by specific carotenoid binding sites or simply by a different accessibility of the Vx de-epoxidase (VxDE) to distinct antenna complexes. In this work, we investigated the possible role of different carotenoid binding sites for Vx convertibility using the most abundant Chl a/b protein, LHCII, as a model system for all LHC proteins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Pigments, Protein, and Reconstitution-- Pigments and overexpressed Lhcb1*2 were isolated as described (12). Reconstitution was performed by detergent exchange as described (23) at a Chl a/b ratio of 3 and a Chl and xanthophyll concentration of 0.8 and 0.1 mg/ml, respectively. Xanthophylls were added either as single species or as 1:1 mixtures. Vx/Zx ratios for reconstitution of Nx·Vx·Zx complexes were as depicted in Fig. 2. Because reconstituted LHCII was separated from unbound pigments by sucrose density centrifugation, sucrose was omitted from the solubilization buffer. 200 µl of reconstitution sample were applied to 11 ml of sucrose gradients, generated by one freeze-thaw cycle (0.4 M sucrose, 5 mM Tricine-NaOH, pH 7.8, 0.05% (w/v) dodecylmaltoside), and centrifuged at 280,000 × g for 17 h at 4 °C.

Pigment Analysis-- Pigments were extracted from all samples following the method of (24) and separated by reversed-phase HPLC as described in (18).

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. After centrifugation, VxDE was precipitated from the supernatant by differential (NH4)2SO4 fractionation and finally collected by ultracentrifugation (25).

In Vitro De-epoxidation-- For de-epoxidation, VxDE extracts were diluted 15-fold with 0.4 M citrate-NaOH, pH 5.1. Recombinant LHCII or isolated Vx was mixed with monogalactosyl diacylglycerol (MGDG) at a molar Vx/MGDG ratio of 1:30 and added to the assay yielding a final Vx concentration of about 100 nM. De-epoxidation, performed at 28 °C, was started by the addition of 30 mM ascorbate. For kinetic analysis, de-epoxidation was stopped by mixing the sample with 2-butanol at indicated time points. The pigment stoichiometry of complexes upon incubation with VxDE was determined after concentrating the sample in 30-kDa Centricon tubes (Amicon), resolubilization in 0.1% n-dodecyl-beta -D-maltoside and subsequent purification of monomeric complexes on sucrose gradients.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The pigment composition of LHCII reconstituted with different carotenoids is summarized in Table I. 2.8-3.0 xanthophylls/monomer were determined in all complexes. In LHCII reconstituted in the presence of all major xanthophylls the data were compatible with a stoichiometry of 2 Lu and 1 Nx/monomer, whereas reconstitution with Vx and Nx in the absence of Lu yielded complexes containing 1.6 Vx and 1.1 Nx/monomer. Reconstitution of LHCII with Lu and Vx in the absence of Nx resulted in binding of 2 Lu and about 1 Vx/monomer, and with Vx as the single carotenoid species about 2.8 Vx were bound per LHCII protein.

                              
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Table I
Pigment stoichiometries of different LHCII complexes
Pigments were extracted with 2-butanol from bands of monomeric LHCII obtained after sucrose density gradient centrifugation. Pigment composition was analysed by HPLC. Data were normalized to 13 Chl (a + b). Mean values ± S.D. of 4-5 measurements are shown.

We investigated the convertibility of Vx in the reconstituted complexes. Under our experimental conditions, free Vx was fully converted to Zx by the VxDE within 10 min (Fig. 1A), with a transient accumulation of the intermediate Ax peaking at 2 min of incubation. A very similar result was obtained with Lu·Vx monomers containing 2 Lu and 1 Vx, although the kinetics was retarded by about a factor of 2 (Fig. 1B) when compared with nonbound Vx. In LHCII containing 1.5 Vx and 1 Nx/monomer, de-epoxidation was even more strongly retarded. Moreover, the convertibility was restricted to about 50% of the total Vx, indicating a partial accessibility of Vx for de-epoxidation (Fig. 1C). Replacement of all carotenoids by Vx led to a multiphasic kinetics of de-epoxidation; a fraction of about 60% was convertible to Zx within 30 min as before (Fig. 1, A and B), whereas the remaining portion showed a very slow turnover (Fig. 1D).


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Fig. 1.   Time course of Vx de-epoxidation in LHCII monomers with different carotenoid composition. Recombinant LHCII was reconstituted in the presence of Chl a, Chl b, and varying carotenoids. Pigmented monomeric complexes with different xanthophyll composition were purified by sucrose density gradient centrifugation. De-epoxidation was then performed at 28 °C after the addition of MGDG and partially purified VxDE prepared from spinach thylakoids. The reaction was started by adding ascorbate and stopped by rapidly mixing the samples with 2-butanol. The VxDE content and Vx concentration were in a similar range in all experiments. Experiments are shown with: A, non-protein bound Vx isolated from spinach thylakoids; B, LHCII containing 2 Lu and 1.1 Vx/monomer; C, LHCII containing 1.6 Vx and 1.1 Nx/monomer; and D, LHCII containing 2.8 Vx/monomer. Mean values of 2-3 independent experiments are shown. Error bars indicate upper and lower limits.

We determined the rate constants for both steps, Vx right-arrow Ax and Ax right-arrow Zx, of the de-epoxidation reactions in all experiments. Because the de-epoxidation follows a first-order reaction (26-28) and Vx bound to different sites may exhibit different kinetics, we fitted the data with 1, 2, or 3 exponentials according to the following scheme.
<UP>Vx</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>11</SUB>, k<SUB>12</SUB>, k<SUB>13</SUB></UL></LIM> <UP>Ax</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>  k<SUB>2</SUB>  </UL></LIM> <UP>Zx</UP>

<UP><SC>Scheme</SC> 1</UP>
A single exponential term was sufficient to fit the data for free Vx, Vx·Nx monomers, and Lu·Vx monomers (Table II), revealing rate constants in the range of 0.1 to 0.5 min-1. The time-dependent decay of Vx in Vx monomers (Fig. 1D), however, could only be modeled with at least two exponentials. For the rapidly convertible fraction (55-65%) of Vx, similar rate constants were determined as before, whereas the remaining fraction (35-45%) was characterized by a 10-20 times lower rate constant of about 0.01 min-1. Because of the few measuring points, several sets of exponentials yielded reasonable fits. Therefore, column B (under "Vx monomers") in Table II also depicts a set of three exponentials with equivalent amplitudes representing three molecules of Vx. Obviously, three possible Vx binding sites have at least two and perhaps three specific rate constants for de-epoxidation. It is worth noting that the rate constant for the second step of de-epoxidation, Ax right-arrow Zx, was very similar in all complexes and was comparable with the conversion of unbound Vx to Ax.

                              
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Table II
Kinetics of Vx to Zx conversion
Rate constants were determined for the Vx to Zx conversion shown in Figs. 1 and 2. Assuming a first-order kinetics for this reaction, rate constants were derived for both steps of de-epoxidation, Vx right-arrow Ax (k11) and Ax right-arrow Zx (k2). For Vx monomers, the data points could be fitted with 2 or 3 exponentials with a range of rate constants (A, B). Amplitudes are given in parentheses.

We further investigated whether the reaction product Zx was re-bound (or stayed bound) to the LHCII complexes. For this purpose, samples were incubated for 70 min under conditions of de-epoxidation. Subsequently, pigmented protein complexes were separated from free pigment by sucrose density gradient centrifugation, and the pigment content of the resulting bands was analyzed by HPLC (summarized in Table III). To distinguish between VxDE-dependent shifts in pigment stoichiometry and potential pigment loss due to incubation temperature (28 °C) and the subsequent reconcentrating step, control samples in the absence of VxDE were also analyzed.

                              
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Table III
Pigment stoichiometries of LHCII complexes after de-epoxidation
Monomeric complexes were obtained by sucrose density gradient centrifugation and used for de-epoxidation experiments. For de-epoxidation, samples were incubated for 70 min at 28 °C in the presence (monomer + VxDE) or absence (Control) of VxDE. After de-epoxidation, samples were concentrated and reloaded on a sucrose density gradient, and fractions containing monomeric LHCII and unbound pigments were analysed. Data were normalized to 12 or 13 Chl (a + b). Mean values of two experiments are shown with deviations no larger than ±12%. Xan, xanthophylls; DEPS, de-epoxidation state (Zx/(Zx + Vx)).

The de-epoxidation state (DEPS), defined as Zx/(Zx + Vx), of total pigment samples (i.e. the value shown for free pigment + monomers in Table III) of Nx·Vx-LHCII (0.45) and Vx-LHCII (0.8) after a 70-min VxDE treatment corresponded to the values obtained with the kinetic assays (Fig. 1, C and D). Only Vx·Lu-LHCII (0.9) exhibited a slightly decreased value when compared with the DEPS of 1.0 in the former experiment (Fig. 1B).

Comparing total pigment samples with reisolated monomers, a partial loss of xanthophylls and also of Chl was observed in all samples, including controls, which can be related to the incubation at 28 °C for 70 min. In comparison with untreated monomers (Table I), we observed a loss of about 0.5 Vx in Vx monomers and Vx·Lu complexes leading to 2.3 and 0.5 Vx, respectively. In Nx·Vx complexes, the Vx content remained unchanged whereas Nx was slightly reduced (Table III).

Newly formed Zx was bound to all LHCII complexes with different stoichiometries (Table III). In Vx-LHCII, 1.4 Zx were bound to the monomers while 0.7 Vx withstood the VxDE incubation. The fraction of VxDE-resistant Vx was even greater in Nx·Vx-LHCII, where 1.2 Vx and only 0.3 Zx were bound. The remaining 0.5 Vx in the control samples of Vx·Lu monomers was almost completely converted to 0.4 Zx with only minor amounts of Vx persisting VxDE treatment. Thus, somewhat reduced if similar DEPS values were found in the reisolated monomers as noted before in the kinetic analysis, indicating that the newly formed Zx molecules were rebound by or stayed bound to the complexes.

De-epoxidation with Vx·Nx-LHCII containing 1.6 Vx and nearly 1 Nx/monomer (Fig. 1C) resulted in a maximum DEPS of about 0.5, indicating that about 1 Vx/monomer was inaccessible for de-epoxidation. This partial conversion to Zx could result from two different restrictions. Assuming that the Vx molecules are bound to the two Lu binding sites, L1 and L2, of the native complex, de-epoxidation could be generally suppressed if one of the two Lu binding sites is occupied by Zx independently of the binding site. Alternatively, one specific binding site, either L1 or L2, could be unavailable for de-epoxidation, e.g. for structural reasons.

We designed the following experimental approach to decide between these two possibilities. LHCII complexes were reconstituted with a mixture of Nx, Vx, and Zx yielding stoichiometries of 1 Nx/momomer in all cases but with Vx/Zx ratios varying between 1.9:0.5 and 0.03:1.9/monomer (Fig. 2A). Fig. 2B indicates an equivalent relative binding affinity KVx/Zx of 0.24 in L1 and L2. The absence of any biphasic behavior of the competition curve with a slope of 0.24 strongly suggests that L1 and L2 preferentially bind Zx with respect to Vx but do not differ in their relative affinities toward these carotenoids.


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Fig. 2.   Vx/Zx/Nx competition. Reconstitutions were carried out at constant Chl and Nx concentrations with Vx/Zx ratios as indicated. Complexes were isolated on sucrose gradients and analyzed for pigment stoichiometry. A, number of xanthophylls bound, normalized to a total of 12 Chl. The Chl a/b ratio was 1.7 in the reconstituted complexes. B, Vx/Zx ratio in reconstituted complexes. A linear regression of data points is given with a slope of 0.24. r2 = 0.99 (coefficient of determination).

We expected that no de-epoxidation would occur if the presence of Zx in one of the two Vx binding sites generally restricts de-epoxidation of the Vx present in these complexes. This should become particularly obvious in complex preparations with a Zx/Vx ratio >=  1. On the other hand, de-epoxidation of about 50% of the Vx should be observed again, if one specific binding site is inaccessible for de-epoxidation and if both binding sites have a similar affinity toward Vx and Zx. In fact, at all Vx/Zx ratios we found that de-epoxidation was limited to about 50% of the respective Vx pool. This is shown as an example for a Vx/Zx ratio of 1:1 in Fig. 3. The rate constants for the reactions were in a similar range as in the respective experiments with Vx·Nx monomers (Table II). The observation that only 50% of Vx is convertible to Zx, irrespective of the Zx/Vx ratio in the reconstituted complexes, indicates that Vx is accessible to the VxDE only in one of the two Lu binding sites (L1 or L2).


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Fig. 3.   Time course of Vx de-epoxidation in LHCII monomers with a carotenoid composition of 1 Vx, 1 Zx, and 1 Nx. All other conditions were as given in the legend of Fig. 1. Mean values of 2 experiments are shown. Error bars indicate upper and lower limits.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown for the first time that Vx bound to solubilized LHCII can be de-epoxidized in vitro by partially purified VxDE prepared from spinach thylakoids. It has been found2 that purified VxDE did not de-epoxidize Vx bound to pigment protein fractions of LHCII even when the latter was supplemented with MGDG (19). In these experiments, it is most likely trimeric LHCII isolated from thylakoid membranes was used, whereas our data were obtained with recombinant monomeric LHCII. In experiments with the recombinant trimeric forms of the different complexes, however, we have also observed de-epoxidation, but to a lower extent (maximum 50% conversion) and at much slower rates (a factor of about 10; data not documented). Thus, it is reasonable to assume that Vx bound to trimeric LHCII is not readily accessible for in vitro de-epoxidation. Because our experiments were performed at 28 °C, Vx might have become accessible by thermally induced destabilization of the trimers.

Incubation of complexes under de-epoxidation conditions and subsequent re-purification leads to a preferential loss of Chl a (compare Table I and control monomers in Table III). The normalization of initially isolated versions of LHCIIb to 13 Chls and reisolated monomers to 12 Chls is justified by this loss. Otherwise, if a number of 12 Chls would be assumed in the original samples, the insertion of up to 1 Chl b after treatment for de-epoxidation had to be considered, which is very unlikely.

Our pigment data indicate binding of almost 3 carotenoids/monomer in all complexes, in rough agreement with previous studies (11, 12). For LHCII containing either Vx as its single carotenoid species or Lu plus Vx, lower stoichiometries of 2.2-2.3 have also been reported (11, 12). These differences are most likely based on varying purification conditions and reflect a low affinity of Vx to the Nx binding site of the native protein.

According to previous work (11, 12), we assume that in complexes containing 2 Lu and 1 Vx, the two Lu binding sites of the native complex, L1 and L2, are both occupied by Lu and that Vx is bound either to the Nx binding site of the native complex or to a peripheral site. The notion that Vx is most rapidly converted to Zx in complexes containing 2 Lu and 1 Vx could then be explained by assuming that Vx is easily accessible for VxDE at these sites.

In complexes reconstituted with Vx and Nx, 1.6 Vx and 1 Nx were determined per monomer. Because Nx has a much higher affinity to the Nx binding site of the native complex in comparison with Vx (11), it is likely that in these complexes the Vx molecules are bound to the L1 and L2 site of LHCII. Our experiments have shown that only a portion of this Vx can be converted into Zx (Fig. 1C) and that Vx bound to one specific site, either L1 or L2, is not available for de-epoxidation (Fig. 3). This may indicate that one of the two Lu binding sites essentially requires binding of a carotenoid, whereas the other one could, at least transiently, be empty. A similar conclusion has been drawn from recent studies with recombinant Lhcb4 (CP29) (10). In this monomeric antenna protein of PSII, the L1 site is occupied by Lu, whereas the L2 site can bind either Vx or Nx (10). Vx bound to Lhcb4 is known to be de-epoxidized in vivo under high light stress (18, 20). Thus the proposed structural similarity among all Chl a/b-binding proteins would support the assumption that Vx bound to the site L2 of LHCII can be de-epoxidized to Zx, whereas the Vx bound to site L1 cannot. This is further in accordance with the observation that the L1 site is obligatory occupied by Lu in all Chl a/b-binding proteins analyzed so far, whereas the L2 site can be occupied by Vx to various extents in the different complexes (8).

In LHCII complexes containing Vx as the single carotenoid species, all three binding sites are occupied by Vx. In contrast to the Vx·Nx monomers, Vx bound to both the L1 and L2 site was convertible to Zx. Obviously, the unexpected accessibility of Vx bound to L1 was related to the absence of Nx in these complexes. This interpretation would suggest a stabilizing function of Nx for the structure of LHCII. Because we did not observe a pronounced dissociation of these complexes, the lack of Nx may simply result in a slightly changed overall conformation providing an increased accessibility of Vx bound to L1. Two of the three Vx were rapidly convertible to Zx in these complexes with similar kinetics as found in the Lu·Vx and Vx·Nx monomers (Fig. 1, Table II). This finding confirms the conclusion that Vx bound to the L2 site and the Nx binding site of the native complex is easily accessible for de-epoxidation.

Our analyses of the reaction kinetics indicated that Vx is de-epoxidized at different carotenoid binding sites at specific rates (Table II). It cannot be decided from our data whether this specificity is determined by different binding affinities of Vx to each site or by a different accessibility of VxDE to the three carotenoid binding sites. With the exception of the slowest phase, determined in the experiment with Vx monomers (about 0.01 min -1, Table II), the rate constants for de-epoxidation are in agreement with the kinetics found in earlier studies at saturating light intensities in intact leaves (26, 27) or, under optimum conditions, in isolated thylakoids (28, 29). Thus, it is reasonable to assume that the kinetics determined with recombinant LHCII and VxDE extracts can be applied also to membrane bound antenna proteins.

It is known that most of Vx present in thylakoid membranes is bound to PSI antenna proteins and to the minor PSII antenna proteins Lhcb4-6 but not to LHCII (8, 18-20). According to the structural similarity among the different antenna proteins, the characteristics of Vx de-epoxidation at recombinant LHCII may be used as a model for all Chl a/b-binding proteins in higher plants. Furthermore, the analysis of Lu-deficient mutants of Arabidopsis have shown that Lu can presumably be replaced even under in vivo conditions by other xanthophylls, in particular Vx and Ax, without affecting the viability of the plants (30, 31).

The kinetic analyses and the fact that the reaction product Zx is (re)bound to the LHC II complexes further provide new insights into the mechanism of de-epoxidation. The kinetic analyses have shown that, in contrast to the first step (Vx right-arrow Ax) of de-epoxidation, the kinetics of the second Ax right-arrow Zx step are nearly identical in all experiments and thus independent of the Vx binding site. This leads to ratios of k2/k1(1,2,3) varying from 2 (in Vx·Lu monomers) to 80 (slowest phase in Vx monomers) indicating that both reactions are rate-limited by different factors. According to our data, the first step (Vx right-arrow Ax) seems to be kinetically controlled by the binding strength of Vx to the LHCII complex (or the accessibility of the VxDE to the binding site), whereas the Ax right-arrow Zx step may rather be rate-limited by processes related to the VxDE itself. It is tempting to speculate that the dynamic interaction of the intermediate Ax with VxDE (which is required to make the second epoxy group accessible to the active center of the enzyme) determines the kinetics of the second step. Because the Ax right-arrow Zx conversion follows similar kinetics in the presence and absence of LHCII complexes, the movement of Ax must be independent of antenna proteins. This lets us speculate that the intermediate Ax is not bound to the LHC II complexes but rather is associated with the VxDE. After de-epoxidation of the second epoxy group, the product Zx can be rebound by the LHCII complexes.

It is unknown to what extent xanthophylls are released into the lipid phase after de-epoxidation under in vivo conditions. At least for LHCII and Lhcb4 it has been reported that occupation of some of the binding sites depends on the de-epoxidation state of the xanthophyll cycle pigments (20, 21). Both a redistribution of xanthophyll cycle pigments among chlorophyll-binding proteins (20) as well as a partial release into the lipid phase (21) can be assumed on the basis of these studies. Moreover, recent analyses of xanthophyll cycle mutants from Arabidopsis thaliana indicated that xanthophylls may serve important functions not only in energy dissipation but also as membrane stabilizers and antioxidants in the lipid phase (32, 33). The latter characteristics resemble the proposed functions of tocopherols in the membrane (34). Thus, at least a partial release of Zx into the lipid phase would make sense as judged from the physiological functions of xanthophylls.

In our experiments with recombinant LHCII, however, the xanthophyll/Chl ratios of the de-epoxidized and control LHCII monomers were only slightly different in Vx complexes (0.18 and 0.19, respectively) and were identical for Nx·Vx-LHCII and Vx·Lu-LHCII, although Zx was also present in the free pigment zone (Table III). Obviously, the partial loss of xanthophylls was independent of the DEPS and was caused simply by the incubation at 28 °C. We conclude from this result that under our experimental conditions accessible Vx is converted to Zx and, provided that complexes do not fall apart, all newly formed Zx is rebound by the LHCII complexes. Considering the low concentrations of LHCII complexes and carotenoids in our assay, it can be assumed that LHCII and VxDE stay in close contact during the de-epoxidation reactions and that no release of carotenoids into the lipid phase occurs.

In conclusion, our data support the view that the kinetics and the extent of Vx de-epoxidation is controlled by the carotenoid binding site of a distinct antenna protein. Thus, it is very likely that the differential xanthophyll conversion in single antenna complexes observed under in vivo conditions (18, 20) is determined by the characteristics of the Vx binding sites and not by the accessibility of VxDE to the antenna proteins. Further work is now needed to characterize the Vx de-epoxidation in other antenna proteins and to explore the influence of the xanthophyll conversion of protein modifications that occur under different light conditions (binding of protons, phosphorylation) and that are likely to be central to the regulation of light harvesting.

    ACKNOWLEDGEMENTS

The help of S. Raunser with the preparation of some of the reconstituted samples is gratefully acknowledged. Zx was a kind gift from Roche Diagnostics (Switzerland).

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft SFB 189, TP B13, and Ja 665/2-1 (to P. J.) and Pa-324/5-3 (to H. P.) and a grant from the Stiftung Rheinland Pfalz für Innovation (to H. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: +49-211-81-13862; Fax: +49-211-81-13706; E-mail: pjahns@uni-duesseldorf.de.

Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M102147200

2 K. Hindehoffer, A. Lee, P. Thornber, and H. Y. Yamamoto, unpublished work.

    ABBREVIATIONS

The abbreviations used are: Chl, chlorophyll; Ax, antheraxanthin; DEPS, de-epoxidation state; HPLC, high pressure liquid chromatography; LHC, light-harvesting complex; Lu, lutein; MGDG, monogalactosyl diacylglycerol; NPQ, non-photochemical quenching of chlorophyll fluorescence; Nx, neoxanthin; PSI, photosystem I; PSII, photosystem II; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Vx, violaxanthin; VxDE, violaxanthin de-epoxidase; Zx, zeaxanthin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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