Title: Photoprotection in the Antenna Complexes of Photosystem II
Abstract: In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O2 to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O2. As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins. In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O2 to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O2. As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins. Under normal light conditions, the photosynthetic apparatus is very efficient in harvesting light energy and transferring excitation energy to the reaction center, where it is used to induce charge separation. In high light, when the amount of energy harvested by the system exceeds the capacity for electron transport to available sinks, other de-excitation mechanisms become important (1Niyogi K.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1597) Google Scholar). Part of the excitations decay via intersystem crossing leading to the production of chlorophyll (Chl) 2The abbreviations used are:ChlchlorophyllLhclight-harvesting complexAbsabsorptionTmSTriplet-minus-SingletWTwild type.2The abbreviations used are:ChlchlorophyllLhclight-harvesting complexAbsabsorptionTmSTriplet-minus-SingletWTwild type. triplets, which live rather long (τ = 600–1100 μs in different solvents (2Chauvet J.-P. Bazin M. Santus R. Photochem. Photobiol. 1985; 41: 83-90Crossref Scopus (14) Google Scholar, 3Fujimori E. Livingston R. Nature. 1957; 180: 1036-1038Crossref Scopus (69) Google Scholar)) and can react with molecular oxygen producing singlet oxygen, a very reactive and harmful species (4Knox J.P. Dodge A.D. Planta. 1985; 164: 30-34Crossref PubMed Scopus (23) Google Scholar). The damage includes oxidation of lipids (5Tardy F. Havaux M. J. Photochem. Photobiol. B. 1996; 34: 87-94Crossref PubMed Scopus (75) Google Scholar), protein, and pigments (6Formaggio E. Cinque G. Bassi R. J. Mol. Biol. 2001; 314: 1157-1166Crossref PubMed Scopus (135) Google Scholar), leading to photoinhibition of the photosynthesis machinery and photobleaching. Light-harvesting complexes are protected against singlet-oxygen formation by carotenoids (7Koyama Y. J. Photochem. Photobiol. B. 1991; 9: 265-280Crossref Scopus (199) Google Scholar). These isoprenoids can act in two ways: (i) by quenching of Chl triplets (τ = 500 ps (8Schödel R. Irrgang K.D. Voigt J. Renger G. Biophys. J. 1999; 76: 2238-2248Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar)) and (ii) by directly scavenging singlet oxygen (τ = 0.7 ns in benzene (9Farmilo A. Wilkinson F. Photochem. Photobiol. 1973; 18: 447-450Crossref PubMed Scopus (148) Google Scholar)); in both cases carotenoid triplets are formed that decay to the ground state, producing heat (τ = 9 μs in benzene (10Land E.J. Sykes A. Truscott P.G. Photochem. Photobiol. 1971; 13: 311-320Crossref Scopus (46) Google Scholar)). For these processes to occur, Chls and carotenoids need to be in close contact because triplets are transferred from Chls to carotenoids via the Dexter exchange mechanism (11Siefermann-Harms D. Physiol. Plant.. 1987; 69: 561-568Crossref Scopus (527) Google Scholar). The close distances are maintained by the proteins, which coordinate the pigments, thereby allowing fast energy transfer and efficient photoprotection.The carotenoid composition of higher plants is highly conserved; the chloroplast-encoded subunits of Photosystem I and Photosystem II core complexes bind β-carotene, whereas the outer antennae, composed of nucleus-encoded light-harvesting complexes (Lhc), accommodate lutein, neoxanthin, and violaxanthin in moderate light, whereas zeaxanthin is produced (12Demmig-Adams B. Winter K. Kruger A. Czygan F.-C. Briggs W.R. Photosynthesis. Plant Biology. 8. Alan R. Liss, New York1989Google Scholar) via de-epoxidation of violaxanthin under light stress conditions. The structure of LHCII (13Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1350) Google Scholar), the major antenna complex of photosystem II, shows the location of four carotenoids. Two xanthophylls are bound in the center of the molecule in sites L1 and L2 which accommodate mainly lutein (14Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). A third carotenoid binding site (N1), highly specific for neoxanthin, is located near helix C (15Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar). The fourth site (V1) is at the periphery of the monomeric subunits, and it accommodates violaxanthin, lutein, or zeaxanthin depending on light conditions (16Ruban A.V. Lee P.J. Wentworth M. Young A.J. Horton P. J. Biol. Chem. 1999; 274: 10458-10465Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). All carotenoids but the one in the V1 site (17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) are involved in light-harvesting and singlet energy transfer (18Peterman E.J.G. Gradinaru C.C. Calkoen F. Borst J.C. Grondelle van R. Amerongen van H. Biochemistry. 1997; 36: 12208-12215Crossref PubMed Scopus (102) Google Scholar, 19Gradinaru C.C. Stokkum van I.H.M. Pascal A.A. Grondelle van R. Amerongen van H. J. Phys. Chem. B. 2000; 104: 9330-9342Crossref Scopus (181) Google Scholar, 20Croce R. Müller M.G. Bassi R. Holzwarth A.R. Biophys. J. 2001; 80: 901-915Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar).Three carotenoid binding sites are present in Lhcb4 and Lhcb5, although these complexes seem to coordinate less than three xanthophylls, possibly due to some loss during purification. As in LHCII, site L1 binds lutein, N1 neoxanthin, and L2 violaxanthin (in Lhcb4) or lutein (in Lhcb5) (16Ruban A.V. Lee P.J. Wentworth M. Young A.J. Horton P. J. Biol. Chem. 1999; 274: 10458-10465Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 21Caffarri S. Passarini F. Bassi R. Croce R. FEBS Lett. 2007; 581: 4704-4710Crossref PubMed Scopus (69) Google Scholar). Singlet energy transfer was observed from all three xanthophylls (22Frank H.A. Das S.K. Bautista J.A. Bruce D. Vasil'ev S. Crimi M. Croce R. Bassi R. Biochemistry. 2001; 40: 1220-1225Crossref PubMed Scopus (42) Google Scholar, 23Croce R. Müller M.G. Caffarri S. Bassi R. Holzwarth A.R. Biophys. 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Acta. 1994; 1188: 243-250Crossref Scopus (26) Google Scholar). Although there is general agreement on the lifetime values under anaerobic conditions, differences exist with respect to the values measured in the presence of oxygen. Peterman et al. (18Peterman E.J.G. Gradinaru C.C. Calkoen F. Borst J.C. Grondelle van R. Amerongen van H. Biochemistry. 1997; 36: 12208-12215Crossref PubMed Scopus (102) Google Scholar) found a biexponential decay of the carotenoid triplets with components of 2 and 4 μs, associated, respectively, to the TmS spectra peaking at 505 and 525 nm; Schödel et al. (30Schödel R. Irrgang K.D. Voigt J. Renger G. Biophys. J. 1998; 75: 3143-3153Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) described the decay kinetics with a monoexponential decay of 2 μs, whereas a longer lifetime, around 7 μs, was found by Siefermann-Harms and Angerhofer (27Siefermann-Harms D. Angerhofer A. Photosynth. Res. 1998; 55: 83-94Crossref Scopus (38) Google Scholar). Because the presence of oxygen enhances the intersystem crossing (34Mathis P. Galmiche J.M. Comptes Rendus Hebdomadaires des Seances de l'Academie des Sciences Serie D 264. 1967; 15: 1903-1967Google Scholar), it was suggested that this difference is due to the sample preparation procedure; it was assumed that the faster lifetimes are associated to LHCII complexes that have partly lost their structural integrity, which is required to prevent oxygen from diffusing into the complexes (27Siefermann-Harms D. Angerhofer A. Photosynth. Res. 1998; 55: 83-94Crossref Scopus (38) Google Scholar). Because most of the xanthophylls have similar absorption spectra, it has not been possible to unequivocally determine how many and which carotenoids contribute to the TmS spectrum. However, due to the very fast energy transfer from Chl b to Chl a (35Visser H.M. Kleima F.J. Stokkum van I.H.M. Grondelle van R. Grondelle van R. Chem. Phys. 1996; 210: 297-312Crossref Scopus (93) Google Scholar), it is expected that triplets are mainly formed on Chls a, and thus, carotenoids should be located in their close proximity (36Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1529) Google Scholar). At that time the available structure of LHCII (36Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1529) Google Scholar) showed only the location of the two central xanthophylls, and carotenoid to Chl singlet energy transfer measurements have shown that these xanthophylls transfer their excitation energy primarily to Chl a molecules, supporting the hypothesis that Chls a are in close proximity of luteins L1 and L2 (18Peterman E.J.G. Gradinaru C.C. Calkoen F. Borst J.C. Grondelle van R. Amerongen van H. Biochemistry. 1997; 36: 12208-12215Crossref PubMed Scopus (102) Google Scholar, 19Gradinaru C.C. Stokkum van I.H.M. Pascal A.A. Grondelle van R. Amerongen van H. J. Phys. Chem. B. 2000; 104: 9330-9342Crossref Scopus (181) Google Scholar, 20Croce R. Müller M.G. Bassi R. Holzwarth A.R. Biophys. J. 2001; 80: 901-915Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Later, Lampoura et al. (32Lampoura S.S. Barzda V. Owen G.M. Hoff A.J. Grondelle van R. Biochemistry. 2002; 41: 9139-9144Crossref PubMed Scopus (65) Google Scholar) speculated that the violaxanthin in the V1 site could not be involved in triplet quenching since it was not active in singlet energy transfer (17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and that also the neoxanthin was not involved since it was surrounded by Chl b molecules (15Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar). However, from the 2.72-Å resolution structure of LHCII (13Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1350) Google Scholar) it is now known that at least one Chl a molecule is in close proximity of each xanthophyll; L1 is the carotenoid closest to Chls 610, 612, 613, and 614 (all Chl a according to the structure). L2 is the nearest neighbor of Chls 602 (a), 603 (a), 607 (b), and 609 (b), N1 of Chls 604 (a), 605 (b), 606 (b), and 608 (b), and V1 of Chls 601 (b) and 611 (a). Moreover, in the equilibrated system part of the energy, although small, is still located on Chls b (37Novoderezhkin V.I. Palacios M.A. Grondelle van R. Grondelle van R. J. Phys. Chem. B. 2005; 109: 10493-10504Crossref PubMed Scopus (234) Google Scholar), which thus, might also require triplet quenching. In this work we have investigated the role of all individual xanthophylls in photoprotection by analyzing native LHCII preparations with different carotenoid composition. We also report for the first time on the carotenoid triplet properties of the minor antenna complexes Lhcb4 (CP29) and Lhcb5 (CP26). By combining spectroscopic measurements with structural data on the LHCII holocomplex, we derive detailed information about the functional role of the individual carotenoids in the Lhc antenna complexes.EXPERIMENTAL PROCEDURESPlant Material—The wild type (WT) and mutants of Arabidopsis thaliana (ecotype Col-0) npq2 (38Dall'Osto L. Caffarri S. Bassi R. Plant Cell. 2005; 17: 1217-1232Crossref PubMed Scopus (199) Google Scholar) and chy1chy2lut5 (39Fiore A. Dall'Osto L. Fraser P.D. Bassi R. Giuliano G. FEBS Lett. 2006; 580: 4718-4722Crossref PubMed Scopus (54) Google Scholar) were grown under controlled light conditions (photoperiod of 8 h of light and 16 h of dark; 100 μmol of photons m–2 s–1 for WT and npq2; 30 μmol of photons m–2 s–1 for chy1chy2lut5 plants, because of their higher photosensitivity), temperature (23 °C/20 °C, day/night) m and relative air humidity (60–70%).Thylakoid Preparation; Solubilization and Sample Preparation—Unstacked thylakoids were isolated from leaves, as described previously (40Bassi R. Rigoni F. Barbato R. Giacometti G.M. Biochim. Biophys. Acta. 1988; 936: 29-38Crossref Scopus (76) Google Scholar). LHCII trimers from WT and mutants were purified by sucrose gradient as reported (17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Trimeric WT LHCII was further fractionated by flatbed isoelectrofocusing at 4 °C as described previously (41Dainese P. Hoyer-Hansen G. Bassi R. Photochem. Photobiol. 1990; 51: 693-703Crossref PubMed Google Scholar). Green bands were harvested and eluted from a small column with 10 mm HEPES, pH 7.5, and 0.06%, n-dodecyl-α-d-maltoside (α-DM) and further fractionated on a 0.1–1 m sucrose gradient containing 0.06% α-DM and 10 mm HEPES, pH 7.5, for 24 h at 280,000 × g at 4 °C.Reconstituted Complexes—The apoproteins of Lhcb1, Lhcb4 from Zea mays (14Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 42Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar), and Lhcb5 from A. thaliana (38Dall'Osto L. Caffarri S. Bassi R. Plant Cell. 2005; 17: 1217-1232Crossref PubMed Scopus (199) Google Scholar) were overexpressed in the SG13009 Escherichia coli strain transformed with constructs following a protocol described previously (43Nagai K. Thøgersen H.C. Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (347) Google Scholar, 44Paulsen H. Finkenzeller B. Kühlein N. Eur. J. Biochem. 1993; 215: 809-816Crossref PubMed Scopus (164) Google Scholar). Reconstitution and purification of pigment-protein complexes were performed as described in Giuffra et al. (42Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar) using a Chl a/b mixture with ratio 2.4 for Lhcb1 and 3.0 for Lhcb4 and Lhcb5.Pigment Analysis—The pigments were extracted with acetone (80%) and separated and quantified by high performance liquid chromatography, as described in Gilmore and Yamamoto (45Gilmore A.M. Yamamoto H.Y. Plant Physiol. 1991; 96: 635-643Crossref PubMed Scopus (216) Google Scholar) and by fitting the spectra of the acetone extracts with the spectra of individual pigments (46Croce R. Cinque G. Holzwarth A.R. Bassi R. Photosynth. Res. 2000; 64: 221-231Crossref PubMed Scopus (82) Google Scholar).Spectroscopy—The absorption spectra at room temperature in 10 mm Hepes, pH 7.5, 0.2 m sucrose, and 0.06% d-maltoside were recorded using a SLM-Aminco DK2000 spectrophotometer. The wavelength sampling step was 0.4 nm, the scan rate was 100 nm/min, and the optical path length was 1 cm. Fluorescence emission spectra were measured using a Fluorolog (Jobin Yvon) spectrofluorimeter and were corrected for the instrument response. The samples were excited at 440 and 475 nm. The spectral bandwidth was 5 nm (excitation) and 3 nm (emission). The chlorophyll concentration was about 0.02 μg/ml in 10 mm HEPES and 0.03% d-maltoside.Light-induced absorbance changes were recorded with a home-built high sensitivity laser-based spectrophotometer as described in Croce et al. (47Croce R. Mozzo M. Morosinotto T. Romeo A. Hienerwadel R. Bassi R. Biochemistry. 2007; 46: 3846-3855Crossref PubMed Scopus (36) Google Scholar) in the presence and absence of oxygen. The anaerobic conditions were obtained incubating the sample with 20 μg/ml glucose oxidase, 40 μg/ml catalase, and 0.02 mm/ml of glucose for 15 min.During the measurements under aerobic conditions, the signal intensity at 510 nm was checked at regular time intervals to detect possible loss of intensity due to bleaching of the sample. This value was used to build a curve of the intensity variation as a function of time to correct the data. The correction was particularly important for LHCII-L and LHCII-LZ, which showed amplitude reduction during the experiment.For a given wavelength the kinetics of the absorbance change were recorded with variable delay times between the actinic and the detection light pulses, ranging from 5 ns to 9 ms. The delay time was obtained by setting the electronic trigger for each laser light pulse. The delay of the light pulses was determined with fast silicon detectors (Thorlabs-Det210). For each kinetic trace, a set of measurements with increasing delay times was performed. The time between excitations was 300 ms. For aerobic conditions, 29 different delay times were taken and 48 delay times for anaerobic conditions. The kinetics were measured 5–10 times and averaged. The GraphPad PRISM program (GraphPad Software) was used for globally analyzing the kinetics between 70 ns and 9 ms over the 420–580-nm wavelength range.RESULTSLHCII trimers were purified from WT plants and mutant plants affected in the carotenoid biosynthesis; that is, npq2, which is blocked at the level of the zeaxanthin epoxidase and accumulates only lutein and zeaxanthin (LHCII-LZ), and chy1chl2lut5, which lacks all the xantophylls in the β-β branch and contains only lutein (LHCII-L). The trimers from WT plants were purified under mild and more aggressive conditions, which led to complexes that differ in the carotenoid/protein ratio (17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Monomeric LHCII (Lhcb1), Lhcb4, and Lhcb5 were obtained by refolding in vitro.Pigment ContentThe pigment composition of the complexes is reported in Table 1. The two different preparations of LHCII WT, WT4 and WT3, bind 4 and 3 carotenoids, respectively. They differ in the amount of lutein and violaxanthin, in agreement with the absence of the xanthophyll in the V1 site in the complex purified by flatbed isoelectrofocusing (LHCII-WT3), as was shown previously (17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). LHCII-LZ binds 3.5 carotenoids. The absence of neoxanthin and violaxanthin is partially compensated by the accumulation of zeaxanthin, whereas the amount of lutein is unchanged. The presence of zeaxanthin in LHCII trimers obtained by flatbed isoelectrofocusing (48Connelly J.P. Müller M.G. Bassi R. Croce R. Holzwarth A.R. Biochemistry. 1997; 36: 281-287Crossref PubMed Scopus (116) Google Scholar) indicates that this xanthophyll also binds to the internal sites (L1 and L2), although it is not possible to discriminate between them. LHCII-L coordinates 2.7 luteins (39Fiore A. Dall'Osto L. Fraser P.D. Bassi R. Giuliano G. FEBS Lett. 2006; 580: 4718-4722Crossref PubMed Scopus (54) Google Scholar), which are likely to be accommodated in sites L1, L2, and V1, whereas site N1 is empty (49Dall'Osto L. Cazzaniga S. North H. Marion-Poll A. Bassi R. Plant Cell. 2007; 19: 1048-1064Crossref PubMed Scopus (150) Google Scholar). Monomeric Lhcb1 coordinates three carotenoids lacking the xanthophylls in the V1 site (50Remelli R. Varotto C. Sandona D. Croce R. Bassi R. J. Biol. Chem. 1999; 274: 33510-33521Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Lhcb4 coordinates lutein, violaxanthin, and neoxanthin, which are accommodated in sites L1, L2, and N1, respectively (Table 1). The L1 and N1 sites of Lhcb5 have similar occupancy as those of Lhcb4, whereas the L2 site binds lutein (16Ruban A.V. Lee P.J. Wentworth M. Young A.J. Horton P. J. Biol. Chem. 1999; 274: 10458-10465Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 21Caffarri S. Passarini F. Bassi R. Croce R. FEBS Lett. 2007; 581: 4704-4710Crossref PubMed Scopus (69) Google Scholar).TABLE 1Pigment compositionSample preparationChl a/Chl bChl totalCarsNeoViolaLuteZeaLHCII-WT4LHCII WT trimer native1.4144.01.00.42.6LHCII-WT3LHCII WT trimer IEF1.3133.01.00.21.8LHCII-LZLHCII npq2 trimer1.4143.52.51LHCII-LLHCII chy1chy2lut5 trimer1.5142.72.7Lhcb1rLhcb1 WT monomer1.3123.01.00.31.7Lhcb4rLhcb4 WT2.781.90.50.50.9Lhcb5rLhcb5 WT1.992.40.80.11.5 Open table in a new tab Absorption SpectraThe absorption spectra of the trimers with altered carotenoid composition are reported in Fig. 1, where they are compared with the WT spectrum (WT4). The WT minus LHCII-L absorption difference spectrum (Fig. 1B) is identical both in the carotenoid and in the Chl absorption region to the difference spectrum (WT minus Lhcb1-L) obtained for Lhcb1 monomers reconstituted in vitro in which the N1 site is empty (14Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Note the decrease in the absorption of Chl b at 650 nm, which is typical for complexes lacking neoxanthin, and the lack of a 488 nm band, which is the lowest energy absorption maximum of the neoxanthin (15Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar). It can be concluded that the environment of the neoxanthin does not change upon trimerization.In the difference spectrum WT minus LHCII-LZ (Fig. 1A) a positive band around 490 nm and a negative around 504 nm were detected, corresponding, respectively, to the neoxanthin, which is absent in LHCII-LZ, and the zeaxanthin, which absorbs mainly around 504 nm, in agreement with previous results on recombinant complexes (15Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar). This difference spectrum is very similar to that of LHCII-WT minus LHCII-L (the sample without neoxanthin), suggesting that the N1 site is at least partially empty, also in LHCII-LZ. No decrease in the absorption at 510 nm is observed, thus suggesting that site L2 still accommodates lutein. Moreover, the main absorption band of zeaxanthin was found at around 504 nm, 18-nm red-shifted as compared with the absorption in solution. This shift is compatible with the binding of zeaxanthin in sites L1 and N1, which induces a similar shift in lutein and neoxanthin absorption in the WT complex (15Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar).The WT4-WT3 difference spectrum (not shown) is identical to the one reported by Caffarri et al. (17Caffarri S. Croce R. Breton J. Bassi R. J. Biol. Chem. 2001; 276: 35924-35933Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) showing the loss of a xanthophyll band around 486 nm.Triplet Minus Singlet SpectraTriplet formation in native and recombinant Lhcb complexes was studied by flash-induced transient absorption under aerobic and anaerobic conditions; 5-ns flashes excited Chl b at 640 nm, and absorption changes were detected in the 420–580-nm range.LHCII Trimers—The decay of the carotenoid triplets in LHCII-WT4 under aerobic conditions could be best fitted by a biexponential decay, the first component having a lifetime of 2.30 μs, a maximum at 507 nm, and a bleaching at 490 nm, and the second component having a lifetime of 3.6 μs, maximum at 522.5 nm, and bleaching at 505 nm (Fig. 2A, Table 2). This indicates that one or more carotenoids absorbing around 490 nm is responsible for the fast decay, whereas a red-shifted xanthophyll (absorption around 505 nm) is responsible for the slower decay. These results are in agreement with those of Peterman et al. (28Peterman E.J.G. Dukker F.M. Grondelle van R. Amerongen van H. Biophys. J. 1995; 69: 2670-2678Abstract Full Text PDF PubMed Scopus (153) Google Scholar), but in disagreement with other TmS experiments which showed a single decay component (8Schödel R. Irrgang K.D. Voigt J. Renger G. Biophys. J. 1999; 76: 2238-2248Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 27Siefermann-Harms D. Angerhofer A. Photosynth. Res. 1998; 55: 83-94Crossref Scopus (38) Google Scholar). The amplitudes of the two components differ; 68% of the total spectrum is associated to the fast decay and 32% to the slow decay, as shown in Fig. 2A. Under anaerobic conditions, the triplet decay is also best fitted with two exponentials. The fast component has a lifetime of 9 μs, maximum at 510 nm, and a shoulder at 522 nm, and it corresponds to Car triplet decay (Fig. 2B, Table 2). The difference in the carotenoid triplet lifetime for aerobic and anaerobic conditions has been observed before (28Peterman E.J.G. Dukker F.M. Grondelle van R. Amerongen van H. Biophys. J. 1995; 69: 2670-2678Abstract Full Text PDF PubMed Scopus (153) Google Scholar), and it is due to the presence of oxygen, which enhances intersystem crossing (34Mathis P. Galmiche J.M. Comptes Rendus Hebdomadaires des Seances de l'Academie des Scien
Publication Year: 2008
Publication Date: 2008-03-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 187
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