De-epoxidation of Violaxanthin in the Minor Antenna Proteins of Photosystem II, LHCB4, LHCB5, and LHCB6
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m602915200
ISSN1083-351X
AutoresAntje Wehner, Thomas Graßes, Peter Jahns,
Tópico(s)Plant Diversity and Evolution
ResumoThe conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4-6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein. The conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4-6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein. Light-harvesting chlorophyll (Chl) 2The abbreviations used are: Chl, chlorophyll; Ax, antheraxanthin; Car, carotenoid; DEPS, de-epoxidation state; HPLC, high performance liquid chromatography; LHC, light-harvesting complex; Lut, lutein; Nx, neoxanthin; PSI, photosystem I; PSII, photosystem II; Vx, violaxanthin; VxDE, violaxanthin de-epoxidase; Zx, zeaxanthin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. a/b-binding (LHC) proteins serve as an efficient antenna system that captures solar energy for the primary light reactions in plant photosynthesis (1Green B.R. Anderson J.M. Parson W.W. Green B.R. Parson W.W. Light-harvesting Antennas in Photosynthesis. Kluwer Academic Publishers Group, Dordrecht, Netherlands2003: 1-28Google Scholar). In higher plants, the LHC family is composed of at least 11 different antenna proteins of both photosystems, Lhca1-5 in photosystem I (PSI) and Lhcb1-6 in photosystem II (PSII) (2Jansson S. Biochim. Biophys. 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Plant. 2003; 119: 347-354Crossref Scopus (97) Google Scholar). Recent analysis of the x-ray structure of trimeric LHCII from spinach (7Liu Z.F. Yan H.C. Wang K.B. Kuang T.Y. Zhang J.P. Gui L.L. An X.M. Chang W.R. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1380) Google Scholar) and pea (8Standfuss J. van Scheltinga A.C.T. Lamborghini M. Kuhlbrandt W. EMBO J. 2005; 24: 919-928Crossref PubMed Scopus (642) Google Scholar), which consists of Lhcb1-3 proteins, identified the four different xanthophyll binding sites in these proteins. Two luteins (Luts) are bound to the central L1 and L2 sites associated with the two central transmembrane helices A and B, and neoxanthin (Nx) is bound to the more peripheral helix B and protrudes into the lipid phase, whereas violaxanthin (Vx) is located at the monomer interface (8Standfuss J. van Scheltinga A.C.T. Lamborghini M. Kuhlbrandt W. EMBO J. 2005; 24: 919-928Crossref PubMed Scopus (642) Google Scholar). 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Plant. 1997; 100: 806-816Crossref Google Scholar), is bound to each of the LHC proteins in different stoichiometries and at variable binding sites, either L2, N1, or V1 (6Morosinotto T. Caffarri S. Dall'Osto L. Bassi R. Physiol. Plant. 2003; 119: 347-354Crossref Scopus (97) Google Scholar). Zx has at least two different photoprotective functions; it is essentially required for thermal dissipation of excess light energy (so-called qE-quenching) in the antenna of PSII (18Demmig B. Winter K. Krüger A. Czygan F.-C. Plant Physiol. 1987; 84: 218-224Crossref PubMed Google Scholar, 19Niyogi K.K. Grossman A.R. Bjorkman O. Plant Cell. 1998; 10: 1121-1134Crossref PubMed Scopus (757) Google Scholar, 20Horton P. Wentworth M. Ruban A. FEBS Lett. 2005; 579: 4201-4206Crossref PubMed Scopus (267) Google Scholar) and additionally acts as an antioxidant in the lipid phase of the thylakoid membrane (21Havaux M. 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Based on spectroscopic analysis, a direct involvement of Zx in the quenching process due to the presence of a Chl-Zx heterodimer and the formation of a carotenoid (Car) cation has been proposed as the molecular mechanism of energy quenching in PSII (26Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science. 2005; 307: 433-436Crossref PubMed Scopus (640) Google Scholar, 27Ma Y.Z. Holt N.E. Li X.P. Niyogi K.K. Fleming G.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4377-4382Crossref PubMed Scopus (170) Google Scholar). However, the requirement of additional antenna proteins for qE has been shown in different studies (28Falk S. Krol M. Maxwell D.P. Rezansoff D.A. Gray G.R. Huner N.P.A. Physiol. Plant. 1994; 91: 551-558Crossref Scopus (22) Google Scholar, 29Härtel H. Lokstein H. Biochim. Biophys. Acta. 1995; 1228: 91-94Crossref Scopus (50) Google Scholar, 30Jahns P. Krause G.H. Planta. 1994; 192: 176-182Crossref Scopus (70) Google Scholar, 31Chow W.S. Funk C. Hope A.B. 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In vivo analysis of the dynamics of xanthophyll conversion demonstrated that the extent and kinetics of Zx formation differ in single antenna subcomplexes (36Färber A. Young A.J. Ruban A.V. Horton P. Jahns P. Plant Physiol. 1997; 115: 1609-1618Crossref PubMed Scopus (176) Google Scholar), most likely reflecting different affinities of Vx binding sites in different antenna proteins. It is reasonable to assume that these characteristics of Vx conversion are related to different functions of the single antenna proteins in the dissipation of excess light energy. To understand the contribution of each of the single antenna proteins and/or specific xanthophyll binding sites to the formation of Zx (and, thus, to energy dissipation), the convertibility of Vx to Zx has been investigated in recombinant antenna proteins. Recent studies on recombinant Lhcb1 (37Jahns P. Wehner A. Paulsen H. Hobe S. J. Biol. Chem. 2001; 276: 22154-22159Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and Lhca1-4 (38Wehner A. Storf S. Jahns P. Schmid V.H.R. J. Biol. Chem. 2004; 279: 26823-26829Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) brought evidence that Vx bound to V1 and N1 is, like non protein-bound Vx, easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially, and/or with slower kinetics, convertible to Zx. These characteristics are likely to reflect the different affinities of Vx to the respective xanthophyll binding sites in the distinct antenna proteins. In this work we investigated the de-epoxidation characteristics of the three minor PSII antenna proteins, Lhcb4 - 6, which are supposed to bind Vx at the L2 site under in vivo conditions (6Morosinotto T. Caffarri S. Dall'Osto L. Bassi R. Physiol. Plant. 2003; 119: 347-354Crossref Scopus (97) Google Scholar). Recombinant proteins were reconstituted with different xanthophyll mixtures to gain information about the characteristics of Vx conversion at the different Car binding sites in these proteins. Clearly different de-epoxidation characteristics were found for each of the minor PSII antenna proteins, confirming the importance of protein-specific properties for the regulation of Vx conversion to Zx. Together with former studies on the other LHC proteins our data allow us to relate the multiphasic kinetics of Vx de-epoxidation found under in vivo conditions and isolated thylakoids to different xanthophyll binding properties of the single antenna proteins. Isolation and Cloning of lhcb4, lhcb5, and lhcb6 Genes—The cDNA fragments of a tobacco cDNA library (Nicotiana tabacum) were cloned into the EcoRI site of the Lambda ZAP II vector system (Stratagene). For screening of the cDNA library for the minor antenna proteins, heterologous sequence tag clones from Solanum lycopersicum (accession numbers X61287 and M32606 for lhcb5 and lhcb6, respectively) and Arabidopsis thaliana (accession number X71878 for lhcb4) were used (kindly provided by Bernhard Grimm). Samples were [32P]dCTP-labeled using the Random Primers DNA labeling system (Invitrogen) according to the manufacturer's manual. Plaque hybridization was performed overnight at 65 °C (for lhcb5 and lhcb6) or 56 °C (for lhcb4) in hybridization buffer (0.75 m NaCl, 75 mm sodium citrate, pH 7.0, 0,1% SDS, 0,1 mg/ml salmon sperm, 1 g/liter Ficoll 400, 1 g/liter polyvinylpyrrolidone, 1 g/liter bovine serum albumin), and hybridized filters were exposed to a radiographic film (Eastman Kodak Co.). In vivo excision of the phagemid was achieved by using the ExAssist™/SOLR™ system (Stratagene). The cDNA sequences were analyzed with the PCGENE program (Intelligenetics, Mountain View, CA). The sequences have been submitted to the GenBank™ data base with accession numbers DQ676843 (for Lhcb4), DQ676844 (for Lhcb6), and DQ676845 (for Lhcb5). Cloning of the Overexpression Plasmids—The coding sequences of the respective mature antenna proteins of Lhcb6, Lhcb5, and Lhcb4 were cloned into the overexpression vector pET21a (Novagene) using the BamHI and Hin-dIII restriction sites. For DNA amplification, the following primer sets were used: Lhcb6, 5′-ATAATTGGATCCGCGGCAGCAGCTGCTCCCAAG-3′ and 5′-GCGCCGAAGCTTCAAACCAAGAGCTCCAAGGGGTAT-3′, Lhcb5, 5′-ATAATTGGATCCAAGAAGGCTGCTGCTGCCCCT-3′ and 5′-GCGCAGAAGCTTCAAAGTGGGGGCTCTTTCAGC-3′; Lhcb4, 5′-GAGAGGGGATCCCGATTCGGATTCGGAAAAAA-3′ and 5′-GCGGCGAAGCTTAGAGAAGAAGCCGAATGTGTC-3′. Overexpression and Reconstitution of LHC Apoproteins—For overexpression, the plasmids were transformed into the Escherichia coli strain BL21. Overexpression and purification of the proteins as inclusion bodies were carried out following the protocol established for recombinant Lhcb1 protein (39Paulsen H. Rümler U. Rüdiger W. Planta. 1990; 181: 204-211Crossref PubMed Scopus (227) Google Scholar). Protein quantification was performed using the Bio-Rad dye binding assay according to the manufacturer's manual. Pigments used for reconstitution were isolated from pea thylakoids as described in Paulsen and Schmid (40Paulsen H. Schmid V.H.R. Smith A.G. Witty M. Heme, Chlorophyll and Bilins: Methods and Protocols. Humana Press Totowa, NJ2002: 235-253Google Scholar). Reconstitution was performed using the detergent exchange method (41Schmid V.H.R. Cammarata K.V. Bruns B.U. Schmidt G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7667-7672Crossref PubMed Scopus (133) Google Scholar). The reconstitution mixture for Lhcb6 complexes contained 180 μg of the inclusion body protein, 300 μg of Chl (a/b = 1), and 45 μg of xanthophylls. For Lhcb4 complexes the same assay was used, but the Chl a/b concentration was raised to 3. Lhcb5 complexes were reconstituted with a mixture of 240 μg of protein, 300 μg of Chl (a/b = 3), and 45 μg of xanthophylls. Xanthophylls were added as single species or in 1:1 ratios. In Vitro De-epoxidation—In vitro de-epoxidation was carried out with VxDE extracts from spinach chloroplasts as described before (37Jahns P. Wehner A. Paulsen H. Hobe S. J. Biol. Chem. 2001; 276: 22154-22159Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For repurification of the de-epoxidized complexes, samples were concentrated with Centricon YM-50 tubes after 2 h of de-epoxidation. The concentrated sample was loaded on a sucrose gradient (0.1-0.8 m sucrose, 5 mm Tricine, pH 7.6, and 0.06% β-dodecyl maltoside) and centrifuged for 18 h at 300,000 × g and 4 °C. The resulting bands were harvested with a syringe, and the pigment content was analyzed by high performance liquid chromatography (HPLC). Pigment Analysis—Pigments were extracted from all samples with 2-butanol (42Martinson, T. A., and Plumley, F. G. (21995) Anal. Biochem. 228, 123-130Google Scholar) and analyzed by reversed-phase HPLC as described earlier (36Färber A. Young A.J. Ruban A.V. Horton P. Jahns P. Plant Physiol. 1997; 115: 1609-1618Crossref PubMed Scopus (176) Google Scholar). Spectroscopy—Fluorescence emission spectra (PerkinElmer Life Sciences luminescence spectrometer LS 55) were recorded at room temperature. The samples were diluted to a Chl concentration of 1 μg/ml in 10 mm HEPES, pH 7.6, 0.06% β-dodecyl maltoside, 20% glycerol, 200 μg/ml glucose oxidase, and 40 μg/ml katalase. Apoproteins of Lhcb4 - 6 were overexpressed in E. coli and reconstituted with pigment extracts isolated from spinach thylakoids. The xanthophyll mixture used for reconstitution was varied to yield either complexes with the native xanthophyll composition (Lhcb4 and Lhcb5, Lut + Vx + Nx; Lhcb6, Lut + Vx) or complexes, in which either Lut or Nx were replaced by Vx. The pigment composition of the different complexes is summarized in Table 1. The pigment stoichiometry per monomer of Lhcb4 and Lhcb5 complexes with the native xanthophyll composition (Lhcb4-LVN and Lhcb5-LVN) was in agreement with the data published for recombinant proteins of Zea mays (43Crimi M. Dorra D. Bosinger C.S. Giuffra E. Holzwarth A.R. Bassi R. Eur. J. Biochem. 2001; 268: 260-267Crossref PubMed Scopus (52) Google Scholar, 44Croce R. Canino G. Ros F. Bassi R. Biochemistry. 2002; 41: 7334-7343Crossref PubMed Scopus (161) Google Scholar). In accordance with the literature, two xanthophylls per monomer are bound by these complexes. The slightly higher stoichiometries obtained in Lhcb5 complexes in presence of Nx (Lhcb5-LVN and Lhcb5-NV) may indicate that a third xanthophyll binding site might be occupied under these reconstitution conditions. In Lhcb6, significantly less than two xanthophylls were found per monomer when the data are normalized to 10 Chls per monomer (Table 1). This is in contrast to the stoichiometry of two xanthophylls per monomer (or 10 Chls) reported in other studies (45Pagano A. Cinque G. Bassi R. J. Biol. Chem. 1998; 273: 17154-17165Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 46Moya I. Silvestri M. Vallon O. Cinque G. Bassi R. Biochemistry. 2001; 40: 12552-12561Crossref PubMed Scopus (196) Google Scholar). Thus, either a fraction of recombinant Lhcb6 binds only one xanthophyll per monomer under our reconstitution conditions, or the number of coordinated Chl molecules is lower than 10. The latter possibility is supported by the fact that Lhcb6 is known to be rather unstable and tends to loose Chl molecules in its isolated form (45Pagano A. Cinque G. Bassi R. J. Biol. Chem. 1998; 273: 17154-17165Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). If the data are normalized to 8 Chl per monomer, a stoichiometry of two xanthophylls per monomer would also be given in Lhcb6.TABLE 1Pigment stoichiometries of reconstituted Lhcb4-6 Apoproteins of Lhcb4-6 were reconstituted with different pigment mixtures as described under "Experimental Procedures." After reconstitution, complexes were concentrated and separated from free pigments by sucrose density gradient centrifugation. For quantification, pigments were extracted with 2-butanol from the respective gradient bands and analyzed by HPLC. Data are normalized to yield the stoichiometries per monomer assuming that 8, 9, and 10 Chls (a + b) are coordinated per monomer of Lhcb4, -5, and -6, respectively. Mean values (±S.D.) of 6-10 independent experiments are shown. Ζ Car, sum of all carotenoids (Nx + Vx + Lut); L, lutein; V, violaxanthin; N, neoxanthin.Pigments per monomerProteinNxVxLutΣ CarChl a/bLhcb4-LVN0.53 ± 0.030.51 ± 0.050.86 ± 0.131.90 ± 0.113.37 ± 0.42Lhcb4-LV00.70 ± 0.040.81 ± 0.081.50 ± 0.052.56 ± 0.23Lhcb4-NV0.56 ± 0.021.06 ± 0.0601.62 ± 0.083.47 ± 0.18Lhcb5-LVN0.73 ± 0.040.23 ± 0.041.28 ± 0.052.25 ± 0.052.51 ± 0.27Lhcb5-LV00.37 ± 0.061.53 ± 0.081.90 ± 0.091.98 ± 0.15Lhcb5-NV0.99 ± 0.101.31 ± 0.0202.30 ± 0.121.75 ± 0.12Lhcb6-LV00.84 ± 0.090.81 ± 0.051.64 ± 0.051.09 ± 0.04Lhcb6-V011.52 ± 0.0201.52 ± 0.021.09 ± 0.03 Open table in a new tab Reconstitution of Lhcb6 in absence of Lut (Lhcb6-V) was possible without changes of the Chl a/b ratio or the Car/Chl stoichiometry. It can, thus, be assumed that Lut was stoichiometrically replaced by Vx in Lhcb6-V. Reconstitution of Lhcb5 in the absence of Nx (Lhcb5-LV) or Lut (Lhcb5-NV), however, resulted in a reduction of the Chl a/b ratio, reflecting the variability of this complex in binding either Chl a or Chl b without affecting the xanthophyll binding properties (44Croce R. Canino G. Ros F. Bassi R. Biochemistry. 2002; 41: 7334-7343Crossref PubMed Scopus (161) Google Scholar). In Lhcb5, it was therefore possible to replace both Nx and Lut by Vx. Only in Lhcb4, reconstitution in the absence of Nx (Lhcb4-LV) or Lut (Lhcb4-NV) led to a reduction of the xanthophyll binding capacity. It has to be noted, however, that the stoichiometries of Nx and Lut per monomer are not altered in the presence and absence of Lut and Nx, respectively. It can, thus, be assumed that the binding affinity of Vx to the Nx and Lut binding site is reduced in comparison with Nx and Lut, respectively. As a consequence, a fraction of Lhcb4 complexes bound only one xanthophyll per monomer when either Nx or Lut was absent during reconstitution. The functionality of the recombinant proteins was tested by recording the fluorescence emission spectra at excitation wavelengths that preferentially excite Chl a (440 nm) or Chl b (475 nm) (Fig. 1). Independent of the excitation wavelength, fluorescence emission was found to peak at about 680 nm in Lhcb4, supporting the functional coupling of the chlorophylls in these complexes (Fig. 1, A-C). Only in Lhcb4-LV complexes (Fig. 1B), a very faint shoulder at about 660 nm was visible upon excitation of Chl b at 475 nm, indicating that a small fraction of the excitation energy is not completely transferred from Chl b to Chl a. A similar picture was found for Lhcb5 complexes reconstituted with the full complement of xanthophylls (Lhcb5-LVN, Fig. 1D). Reconstitution in the absence of Nx (Lhcb5-LV) or Lut (Lhcb5-NV) resulted in a more pronounced shoulder of the emission spectra at 660 nm (Fig. 1, E and F), indicating again a less efficient energy transfer from Chl b to Chl a in a small fraction of complexes. It should be noted that the appearance of the shoulder at 660 nm in Lhcb4-LV, Lhcb5-LV, and Lhcb5-NV correlates with the reduction of the Chl a/b ratio in comparison with the complexes with the native xanthophyll composition, Lhcb4-LVN, and Lhcb5-LVN, respectively (see Table 1). The markedly increased peak at 660 nm upon excitation at 475 nm in Lhcb6 under both reconstitution conditions (Fig. 1, G and H) is in agreement with former studies on recombinant Lhcb6 from maize (45Pagano A. Cinque G. Bassi R. J. Biol. Chem. 1998; 273: 17154-17165Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Thus, even in Lhcb6 complexes with the native xanthophyll composition (Lhcb6-LV), a pronounced fraction of the excitation energy is not efficiently transferred from Chl b to Chl a. This property of recombinant Lhcb6 might be related to the fraction of complexes that possibly binds only one xanthophyll per monomer (see above). The enzymatic conversion of Vx to Zx in the reconstituted LHCs was studied during 2 h of incubation at 20 °C with a VxDE extract isolated from spinach thylakoids. Non-protein-bound Vx, which was used as the control substrate to test the VxDE activity in each assay was nearly completely de-epoxidized within 10 min (Fig. 2A). In recombinant Lhcb6 complexes with the native xanthophyll composition (Lhcb6-LV, Fig. 2B), ∼35% of the Vx was convertible to Zx with somewhat slower kinetics as non-protein bound Vx, whereas the remaining fraction of Vx was not accessible for de-epoxidation (Table 2). We ensured that the activity of the enzyme did not limit the reaction by testing the conversion of freshly added Vx at the end of the experiment (not shown). Thus, two different populations of Lhcb6-LV complexes must exist that differ in the accessibility of Vx for de-epoxidation. After replacement of Lut by Vx (Lhcb6-V, Fig. 2C), about 40% of the Vx was converted to Zx with even more retarded kinetics than before (Table 2). Obviously, the replacement of Lut by Vx induced only a partial increase of the Vx convertibility. This indicates that Vx is not accessible for de-epoxidation at the (former) Lut binding sites in Lhcb6.TABLE 2Kinetics of Vx conversion Under assumption of an irreversible first order reaction, rate constants were determined for the Vx conversion shown in Figs. 1, 2, 3. For simplification, analyses were restricted to the first step of de-epoxidation (Vx → Ax). For all Lhcb5 complexes (Fig. 3) and non-protein-bound Vx (Fig. 2A), data points were fitted with two exponentials, in all other cases with a single exponential (k1). Amplitudes (A1 and A2) and rate constants (k1 and k2) represent mean values ± S.D. of three independent experiments.SampleAmplitudeRate constantA1A2k1k2%10−3 min−1Non protein-bound Vx85 ± 415 ± 4310 ± 3010 ± 6Lhcb4-LVN15 ± 1270 ± 40Lhcb4-LV31 ± 2290 ± 60Lhcb4-NV17 ± 1230 ± 40Lhcb5-LVN10 ± 190 ± 1290 ± 803 ± 0.2Lhcb5-LV76 ± 424 ± 3250 ± 2010 ± 5Lhcb5-NV27 ± 273 ± 1460 ± 104 ± 0.3Lhcb6-LV36 ± 190 ± 6Lhcb6-V38 ± 130 ± 3 Open table in a new tab In Lhcb5, the de-epoxidation characteristics were different (Fig. 3). In all complexes a biphasic conversion was found (Table 2). The majority of complexes with the native xanthophyll composition (Lhcb5-LVN, Fig. 3B) showed slower conversion kinetics than the corresponding complexes of Lhcb6, and the reaction was not completed within the time frame of the experiment. Fitting of the data indicated that all of the Vx bound to Lhcb-LVN was convertible to Zx but with strongly retarded kinetics in comparison with non-protein-bound Vx (Table 2). It can, thus, be assumed that Vx is generally accessible for de-epoxidation in Lhcb5, but Vx conversion occurs only with very slow kinetics. After reconstitution of Lhcb5 in the absence of Nx (Lhcb5-LV, Fig. 3B), the Vx convertibility was dramatically accelerated (Table 2). The rapid and complete deepoxidation of most of the Vx (about 75%) under these conditions let us conclude that the presence of Nx is responsible for the restriction of the Vx accessibility in Lhcb5-LVN complexes. On the other hand, when Lut was omitted from the reconstitution assay (in Lhcb5-NV, Fig. 3D), the fraction of rapidly convertible Vx was increased in comparison with Lhcb5-LVN complexes. About 30% of the Vx pool was convertible with fast kinetics (like the majority of Lhcb5-LV complexes), and about 70% was convertible with slow kinetics (like the majority of Lhcb5-LVN complexes) (Table 2). According to the increased fraction of rapidly convertible Vx in Lhcb5-LV complexes, we assume that this portion of Vx is related to complexes that did not bind Nx. Vx de-epoxidation in recombina
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