Origin of the 701-nm Fluorescence Emission of the Lhca2 Subunit of Higher Plant Photosystem I
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m408908200
ISSN1083-351X
AutoresRoberta Croce, Tomas Morosinotto, Janne A. Ihalainen, Agnieszka Chojnicka, Jacques Breton, Jan P. Dekker, Rienk van Grondelle, Roberto Bassi,
Tópico(s)Light effects on plants
ResumoPhotosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630–635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes. Photosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630–635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes. Photosystems I and II have a common general organization including a core complex moiety, binding Chl 1The abbreviations used are: Chl, chlorophyll; FWHM, full-width half-maximum; LD, linear dichroism; LHCI and LHCII, light-harvesting complex I and II, respectively; PSI and PSII, photosystem I and II, respectively; WT, wild type.a and β-carotene, and an outer antenna moiety, binding Chl a, Chl b, and xanthophylls. Nonetheless, their spectroscopic properties are substantially different. Both compartments of the PSI supercomplex are enriched in red-shifted chlorophyll absorption forms, whereas those of PSII are not. This property of PSI results in a preferential light harvesting in the near infrared spectral region (>700 nm) and causes the so-called "Emerson effect" that opened the way to the discovery of the two photosystems (1Evans J.R. Photobiochem. Photobiophys. 1986; 10: 135-147Google Scholar). In green plants, LHCI, the outer antenna of PSI, is the most red form-enriched compartment and is composed of four different complexes, the products of the genes lhca1 to -4 (2Haworth P. Watson J.L. Arntzen C.J. Biochim. Biophys. Acta. 1983; 724: 151-158Crossref Scopus (163) Google Scholar). These complexes are located on one side of the core complex (3Ben Shem A. Frolow F. Nelson N. Nature. 2003; 426: 630-635Crossref PubMed Scopus (662) Google Scholar, 4Boekema E.J. Jensen P.E. Schlodder E. van Breemen J.F. van Roon H. Scheller H.V. Dekker J.P. Biochemistry. 2001; 40: 1029-1036Crossref PubMed Scopus (124) Google Scholar) and can be isolated as dimers (5Croce R. Morosinotto T. Castelletti S. Breton J. Bassi R. Biochim. Biophys. Acta. 2002; 1556: 29-40Crossref PubMed Scopus (150) Google Scholar). The Lhca proteins belong to the Lhc multigenic family, and they share high sequence homology with the antenna complexes of PSII (6Jansson S. Biochim. Biophys. Acta. 1994; 1184: 1-19Crossref PubMed Scopus (590) Google Scholar). Despite these strong similarities, the spectroscopic properties of Lhca and Lhcb complexes are very different. In particular, the emission spectrum of LHCI peaks at 733 nm, more than 50 nm red-shifted as compared with the emission spectra of the antenna system of PSII (LHCII) (7Ihalainen J.A. Gobets B. Sznee K. Brazzoli M. Croce R. Bassi R. van Grondelle R. Korppi-Tommola J.E.I. Dekker J.P. Biochemistry. 2000; 39: 8625-8631Crossref PubMed Scopus (61) Google Scholar). In vitro reconstitution has shown that the red-shifted Chls are a common characteristic of Lhca complexes, although their energies differ strongly in different subunits. Emission forms at 701–702 nm have been found associated with Lhca1 and Lhca2, whereas 725- and 733-nm emissions were reported for Lhca3 and Lhca4, respectively (5Croce R. Morosinotto T. Castelletti S. Breton J. Bassi R. Biochim. Biophys. Acta. 2002; 1556: 29-40Crossref PubMed Scopus (150) Google Scholar, 8Schmid 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, 9Zhang H. Goodman H.M. Jansson S. Plant Physiol. 1997; 115: 1525-1531Crossref PubMed Scopus (49) Google Scholar, 10Schmid V.H.R. Potthast S. Wiener M. Bergauer V. Paulsen H. Storf S. J. Biol. Chem. 2002; 277: 37307-37314Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Castelletti S. Morosinotto T. Robert B. Caffarri S. Bassi R. Croce R. Biochemistry. 2003; 42: 4226-4234Crossref PubMed Scopus (85) Google Scholar). It has been shown that the red emission of Lhca3 and Lhca4 originates from the low energy absorption band of an excitonic interaction involving Chl a in sites A5 and, probably, B5 (12Morosinotto T. Breton J. Bassi R. Croce R. J. Biol. Chem. 2003; 278: 49223-49229Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), whereas the difference in energy is associated with the nature of the Chl A5 ligand, since the substitution of Asn in Lhca3 and Lhca4 by His, the usual ligand for Chl A5 in Lhc complexes, leads to the loss of the red spectral forms (12Morosinotto T. Breton J. Bassi R. Croce R. J. Biol. Chem. 2003; 278: 49223-49229Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In the present work, we analyze in detail Lhca2 with the aim of understanding the origin of the 701-nm emission that dominates the fluorescence spectrum at low temperature. It should be noticed that Lhca bears His as a ligand for Chl A5, yet its fluorescence emission is red-shifted by 20 nm with respect to the highly homologous subunit Lhcb6 (CP24) and other Lhcb proteins, thus opening the question of whether a different mechanism of modulation for the physico-chemical properties of chromophores is at work in Lhca2. By performing a detailed analysis by biochemical and spectroscopic methods on WT and mutant Lhca recombinant proteins, we conclude that the fluorescence emission band at 701 nm derives from excitonic coupling between chromophores A5 and B5, despite the presence of a His ligand for Chl A5. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (1Evans J.R. Photobiochem. Photobiophys. 1986; 10: 135-147Google Scholar, 13Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1380) Google Scholar). We suggest that in green plant LHCI, differences in interchromophore distances rather than in transition energies of the individual chromophores of the interacting pair or in the orientation of their transition vectors represent the critical factor in determining excitonic coupling. DNA Constructions and Isolation of Overexpressed Lhca Apoproteins from Bacteria—cDNAs of Lhca2 from Arabidopsis thaliana (11Castelletti S. Morosinotto T. Robert B. Caffarri S. Bassi R. Croce R. Biochemistry. 2003; 42: 4226-4234Crossref PubMed Scopus (85) Google Scholar) were mutated with the QuikChange® site-directed mutagenesis kit, by Stratagene. WT and mutant apoproteins were isolated from the BL21 strain of Escherichia coli transformed with these constructs following a protocol previously described (14Nagai K. Thøgersen H.C. Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (347) Google Scholar, 15Paulsen H. Finkenzeller B. Kuhlein N. Eur. J. Biochem. 1993; 215: 809-816Crossref PubMed Scopus (164) Google Scholar). Reconstitution and Purification of Protein-Pigment Complexes—Reconstitution and purification were performed as described in Ref. 16Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar with the following modifications. The reconstitution mixture contained 420 μg of apoprotein, 240 μg of chlorophylls, and 60 μg of carotenoids in a total of 1.1 ml. The Chl a/b ratio of the pigment mixture was 4.0. The pigments used were purified from spinach thylakoids. Protein and Pigment Concentration—High pressure liquid chromatography analysis was as in Ref. 17Gilmore A.M. Yamamoto H.Y. Plant Physiol. 1991; 96: 635-643Crossref PubMed Scopus (217) Google Scholar. The chlorophyll to carotenoid ratio and Chl a/b ratios were independently measured by fitting the spectrum of acetone extracts with the spectra of individual purified pigments in 80% acetone (18Croce R. Canino G. Ros F. Bassi R. Biochemistry. 2002; 41: 7343Crossref Scopus (161) Google Scholar). Spectroscopy—The absorption spectra at room temperature and 77 K were recorded using an SLM-Aminco DK2000 spectrophotometer, in 10 mm Hepes, pH 7.5, 20% glycerol (60% at low temperature) and 0.06% β-dodecylglucomaltoside. Wavelength sampling step was 0.4 nm, scan rate was 100 nm/min, and optical path length was 1 cm. The absorption spectra at 4 K were measured using a home-built spectrophotometer at OD 0.5 in the same buffer as described above, but with 67% glycerol. Fluorescence emission spectra at 77 K were measured using a Jasco FP-777 spectrofluorimeter and corrected for the instrumental response. The samples were excited at 440, 475, and 500 nm. The spectral bandwidth was 5 nm (excitation) and 3 nm (emission). The chlorophyll concentration was about 0.02 μg/ml in 60% glycerol and 0.03% β-dodecylglucomaltoside. The spectra between 130 and 4 K of the WT were measured with a 0.5-m imaging spectrograph (Chromex 500IS) and a CCD camera (Chromex Chromcam I). For broad band excitation, a tungsten halogen lamp (Oriel) was used with band pass filters transmitting at 420, 475, and 506 nm. For narrow band excitation, a dye laser (Coherent CR599) was used, which was pumped by an argon ion laser (Coherent Inova 310). The optical bandwidth of the laser was 0.6 nm, and the power was kept at 0.15 milliwatts/cm2. The spectra were corrected for the wavelength dependence of the detection system. CD spectra were measured at 10 °C on a Jasco 600 spectropolarimeter. The wavelength sampling step was 0.5 nm, the scan rate 100 nm/min, and spectra were recorded with eight accumulations. The OD of the samples was 1 at the maximum in the Qy transition for all complexes, and the samples were in the same solution as described for the absorption measurements. All spectra presented were normalized to the polypeptide concentration based on the Chl binding stoichiometry. LD spectra were measured as described in Ref. 19Haworth P. Tapie P. Arntzen C.J. Breton J. Biochim. Biophys. Acta. 1982; 682: 152-159Crossref Scopus (53) Google Scholar using samples oriented by the polyacrylamide gel squeezing technique. Analysis of Lhca2-WT Complex—Lhca2 WT was obtained by overexpression of the apoprotein in E. coli and reconstitution in vitro with pigments. The complex was purified from excess pigment and unfolded apoprotein by sucrose gradient ultracentrifugation, anion exchange chromatography, and glycerol gradient ultracentrifugation and was obtained in monomeric form, as previously described (11Castelletti S. Morosinotto T. Robert B. Caffarri S. Bassi R. Croce R. Biochemistry. 2003; 42: 4226-4234Crossref PubMed Scopus (85) Google Scholar). Absorption—The absorption spectrum of Lhca2 at 4 K shows its maximum in the Chl Qy region at 679.5 nm, whereas in the Chl b region two local maxima can be detected at 644 and 652 nm (Fig. 1). The second derivative analysis allows resolution of several absorption forms: three Chl b at 643, 651, and 653 nm and four Chl a at 661, 672, 680.5, and 688.5 nm. In the blue region, the minimum at 497 nm is associated to the redmost peak (S0 > S2,0 transition) of the carotenoids, whereas those at 480 and 471 nm may represent the Soret bands of two Chl b spectral forms. Fluorescence Measurements—Fluorescence emission spectra of Lhca2 were measured at different temperatures upon excitation in the blue spectral region (Fig. 2). Two emission forms at 688 and 701 nm can be detected. The latter dominates the spectrum at 4 K, but the 688-nm form is still present, indicating that the energy transfer between these two forms is not complete, thus suggesting some kind of heterogeneity in the sample. In order to obtain information on the origin of the two fluorescence emissions, fluorescence emission spectra upon selective excitation in the Qy band of the complex were measured. A selection of spectra is presented in Fig. 3A. The relative contribution of the 688- and 701-nm forms changed as a function of the excitation wavelength. In particular, the 688-nm emission reached its maximum for excitations in the 680–683-nm range, indicating that it originates from the red part of the bulk absorption. For excitation above 685 nm, the amplitude of the 688-nm emission band drops considerably, and the spectrum is dominated by the low energy emission form. In Fig. 3B, the excitation wavelength is plotted versus the maximum of the emission (squares). By increasing the excitation wavelength from 680 to 685 nm, a blue shift of the emission maximum of the redmost form to 697 nm (excitation at 685 nm) is observed. This blue shift is due to the contribution to the emission of a larger fraction of "red" pigments absorbing in the blue wing of the inhomogeneous distribution, which are selectively excited at this wavelength (20Gobets B. Van Amerongen H. Monshouwer R. Kruip J. Rögner M. van Grondelle R. Dekker J.P. Biochim. Biophys. Acta. 1994; 1188: 75-85Crossref Scopus (112) Google Scholar, 21den Hartog F.T.H. Dekker J.P. van Grondelle R. Völker S. J. Phys. Chem. B. 1998; 102: 11007-11016Crossref Google Scholar). Above 685 nm, the emission maximum undergoes a red shift, exhibiting a linear relation with the excitation wavelength, thus reaching a value of 701 nm for excitation at 690 nm. By exciting in the redmost part of the absorption spectrum, a further red shift of the emission maximum was observed. For excitations within the red tail of the absorption spectrum (around 710 nm), the emission maximum is still broad (340 cm–1), indicating large homogeneous broadening. Moreover, for excitation above 700 nm, site selection does not have a strong influence on the shape and the width of the emission, thus suggesting that the spectrum is dominated by inhomogeneous broadening. The site-selected fluorescence data allow also determining the peak wavelength of the absorption form responsible for the 701-nm emission. This value corresponds to the excitation wavelength at which the emission peak coincides with that in spectra obtained from nonselective excitation (see Ref. 20Gobets B. Van Amerongen H. Monshouwer R. Kruip J. Rögner M. van Grondelle R. Dekker J.P. Biochim. Biophys. Acta. 1994; 1188: 75-85Crossref Scopus (112) Google Scholar for more details). By following this procedure, the absorption maximum of the red band is calculated to be approximately at 690 nm, in agreement with the second derivative analysis of the absorption spectrum, which showed the redmost absorption form at 688.5 nm (see Fig. 1). Additional information on the characteristics of the red absorption tail can be obtained by fluorescence anisotropy measurements, which gives an indication of the orientation of the absorbing and emitting dipoles. In Fig. 3B (circles), the values of the fluorescence anisotropy detected at 701 nm are reported as a function of the excitation wavelength. It is clear that the values of the anisotropy also depend on the excitation wavelength. Upon excitation around 680 nm, the anisotropy value is low, and it rises to a maximum of 0.32 for excitation wavelength above 697 nm, a value very similar to that obtained in the analysis of the 702-nm emission form in a native LHCI preparation (7Ihalainen J.A. Gobets B. Sznee K. Brazzoli M. Croce R. Bassi R. van Grondelle R. Korppi-Tommola J.E.I. Dekker J.P. Biochemistry. 2000; 39: 8625-8631Crossref PubMed Scopus (61) Google Scholar). This result indicates that above this wavelength, no depolarization due to energy transfer takes place, thus suggesting that the same band is responsible for the absorption and the emission. The alternative hypothesis of energy transfer between pigments having the same dipole transition orientation is extremely unlikely. LD Spectra—The LD spectrum of Lhca2 shows strong positive contribution in the Chl a Qy absorption region, with a maximum at 679.5 nm and a shoulder around 690 nm (Fig. 4). The signal becomes slightly negative at 667 nm. In the Chl b absorption region, a positive contribution is observed at 653 nm. The reduced LD spectrum (LD/3A) shows the largest value around 690 nm, indicating that the Chl(s) responsible for this absorption forms is oriented with the largest angle with respect to the normal to the membrane plane. A second positive LD value corresponds to the maximum of the absorption spectrum (679.4 nm), whereas a local minimum is present at 683 nm. Localization of the Low Energy Absorbing Form(s) into the Lhca2 Structure—The above measurements strongly indicate that the red-shifted fluorescence signal at 701 nm is associated with an absorption form peaking at 690 nm. In order to identify which chromophore is responsible for this absorption, mutation analysis at selected Chl-binding residues was performed. Based on previous analysis with Lhca3 and Lhca4 (12Morosinotto T. Breton J. Bassi R. Croce R. J. Biol. Chem. 2003; 278: 49223-49229Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), residues coordinating Chl A5, B5, and B6, involved in the low energy absorption forms, were mutated to nonbinding residues (i.e. which cannot coordinate the central magnesium of the Chls) (see Table I for details). The apoprotein was expressed in E. coli, and the complex was reconstituted in vitro with pigments. In all cases, stable monomeric complexes were obtained. The pigment content of the complexes is shown in Table I.Table IPigment composition of the complexesSampleMutationChl a/bChl/CaroteonidChl totalLuteinViolaxanthinRLhca2-WT2.15.0101.680.32RLhca2-A5H52F2.24.581.410.35RLhca2-B5E108V/R111L4.7471.440.3RLhca2-B6E100V3.14.491.570.25 Open table in a new tab The decreased Chl/Car ratio observed in all mutants indicates loss of Chls in the Chl binding site mutants, and the higher Chl a/b ratio as compared with the WT complex implies that Chl b molecules are preferentially lost. Normalization to the carotenoid content would indicate loss of one Chl in the A5 mutant, two Chls in B5, and one Chl in B6. However, this stoichiometry may not be correct in the case of mutants A5 and B5, since the corresponding mutants in all other Lhca complexes showed a loss of carotenoid molecules. Moreover, loss of one Chl in A5 would indicate that this site has mixed occupancy, which is very unlikely, because the A5 site has been found to accommodate one Chl a molecule in all complexes analyzed so far (22Bassi R. Croce R. Cugini D. Sandona D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10056-10061Crossref PubMed Scopus (190) Google Scholar, 23Remelli R. Varotto C. Sandona D. Croce R. Bassi R. J. Biol. Chem. 1999; 274: 33510-33521Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 24Morosinotto T. Castelletti S. Breton J. Bassi R. Croce R. J. Biol. Chem. 2002; 277: 36253-36261Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). We thus suggest that the A5 mutant loses two Chls molecules, one Chl a and one Chl b, as was the case in Lhca1, whereas a small loss of carotenoids accounts for the change in Chl/Car ratio. The B5 mutant loses most probably three Chl molecules, two Chls b and one Chl a. The loss of Chl a is consistent with the analysis of the absorption spectrum (see below), which is strongly affected in the Chl a Qy absorption region. To check the influence of the mutation on the 701-nm emission form of the complex, fluorescence emission spectra at 77 K were measured (Fig. 5). Mutant A5 and B5 showed emission maximum at 686 nm, thus shifted by 15 nm to the blue as compared with the WT peak, indicating that both mutations affect the low energy emission form. The fluorescence emission of the B6 mutant was, instead, very similar to that of the WT, showing that mutation at this Chl binding site does not influence the chromophore(s) responsible for the 701-nm emission. The absorption spectra of the three mutants and the WT at 77 K are presented in Fig. 6A and the second derivative of the spectra are shown in Fig. 6B. Mutant A5 was mainly affected in the Chl a region, where the absorption form at 690 nm disappeared. A decrease in the amplitude of the spectrum in the Chl b region could also be observed, in agreement with the Chl b loss (see Table I). Mutant B5 showed dramatic changes in the absorption spectrum as compared with the WT; not only two Chl b forms (at 642 any 651 nm) were lost, but also a 688-nm absorbing Chl a form. Absorption at 680 nm was also decreased and was partially compensated by the increased signal at 670 nm, possibly due to the presence of unconnected Chls. Mutant B6 was affected mainly in the Chl b region, where the absorption at 642 nm disappeared, an indication that the Chl b accommodated in the B6 site absorbs at this wavelength. Minor changes could also be detected in the Chl a absorption region. Interestingly, the effect of the mutation at site B6 on the red-shifted absorption forms is different in Lhca2 as compared with Lhca1 (24Morosinotto T. Castelletti S. Breton J. Bassi R. Croce R. J. Biol. Chem. 2002; 277: 36253-36261Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and Lhca4. 2T. Morosinotto, R. Bassi, and R. Croce unpublished results. In these homologous proteins, loss of Chl B6 significantly affected the red-shifted emission forms. This difference may be related to the observation that the B6 mutants of Lhca1 and Lhca4 lose xanthophylls, whereas Lhca2 did not. The loss of red forms in Lhca1 and Lhca4 upon mutation at site B6 was interpreted as an indirect effect mediated by the carotenoid molecule (24Morosinotto T. Castelletti S. Breton J. Bassi R. Croce R. J. Biol. Chem. 2002; 277: 36253-36261Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The different results obtained for the B6 mutant in Lhca2 seem to indicate that the carotenoid organization in the Lhca complexes is different with respect to others Lhca subunits. The CD spectra at room temperature of WT and mutants are reported in Fig. 7. Mutant A5 is identical in the Chl b region to the WT but shows strong differences in the Chl a region. Comparison with the WT spectrum indicates that this mutant loses a negative CD signal at 687 nm and a positive one at 672 nm, thus suggesting loss of an excitonic interaction. A strong decrease in the intensity of the CD signal in the Chl b region is observed for both B6 and B5 mutants, thus implying that the Chl b molecules lost in the B5 and B6 mutants participate in pigment-pigment interactions. In particular, the comparison of the spectra of the WT with the one of mutant B6 shows that, in the latter, the loss of the negative signal in the Chl b region is connected with the loss of a positive contribution in the Chl a region (around 670 nm), thus suggesting the presence of a Chl b-Chl a interaction. The current results show that Lhca2 takes a unique position within the LHCI proteins. Unlike Lhca3 and Lhca4, it does not show strong absorption above 700 nm, but nevertheless its emission is still 22-nm red-shifted as compared with the emission maxima of all Lhcb complexes. In the case of Lhca3 and Lhca4, it was shown that the red absorption is associated with the presence of an Asn as ligand for Chl A5 (12Morosinotto T. Breton J. Bassi R. Croce R. J. Biol. Chem. 2003; 278: 49223-49229Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This is not the case of Lhca2, which has a His in this position, a feature shared with all Lhcb proteins that do not show red-shifted Chl forms. We thus proceeded with the analysis of the chromophore organization in Lhca2 and identified the absorption band responsible for the 701-nm emission band, with the aim of understanding the mechanism that allows modulation of the physicochemical properties of chlorophyll ligands. Lhca2 has been shown to have the emission maximum at 701 nm (at 4 K). A second emission form was also detected at 688 nm in the 4 K spectrum, thus suggesting heterogeneity in the sample. Several sources of spectroscopic heterogeneity have been reported for Lhc proteins, including phosphorylation-induced spectral changes in CP29 (25Croce R. Breton J. Bassi R. Biochemistry. 1996; 35: 11142-11148Crossref PubMed Scopus (58) Google Scholar), the presence of multiple gene products in the LHCII preparations with different spectral features (26Caffarri S. Croce R. Cattivelli L. Bassi R. Biochemistry. 2004; 43: 9467-9476Crossref PubMed Scopus (116) Google Scholar), and conformational changes induced by zeaxanthin binding to site L2 (27Moya I. Silvestri M. Vallon O. Cinque G. Bassi R. Biochemistry. 2001; 40: 12552-12561Crossref PubMed Scopus (196) Google Scholar, 28Formaggio E. Cinque G. Bassi R. J. Mol. Biol. 2001; 314: 1157-1166Crossref PubMed Scopus (138) Google Scholar). In the present work, a recombinant Lhca2 preparation was analyzed, which was the product of a single gene, did not contain zeaxanthin, was monomeric as detected by sucrose gradient ultracentrifugation, and was not phosphorylated. We conclude that the persistence of the two fluorescence emission bands is the product of a previously unrecognized source of molecular heterogeneity, which is reflected in the establishment or not of the molecular feature responsible for the 701-nm emission. Two hypotheses can be proposed for explanation of these data; either the Lhca2 protein is present into two conformations in which the two interacting chromophores are set at different distances and/or different orientations with respect to each other, or one of the two sites can be occupied by either Chl a or Chl b. The difference in transition energies between Chl a and Chl b and the difference in the orientation of the transition vectors (29Van Amerongen H. van Grondelle R. J. Phys. Chem. B. 2001; 105: 604-617Crossref Scopus (281) Google Scholar) would make the observable spectral shift of a Chl a/Chl b interaction much smaller with respect to that of a Chl a/Chl a interaction. The likelihood of these hypotheses will be discussed below. Site-selected fluorescence measurements indicate that the 689-nm emission originates from bulk Chls absorbing around 682 nm, whereas the 701-nm emission form is associated with a Chl absorption form at 690 nm. This result is confirmed by the mutation analysis, which showed that both mutants losing the 701-nm emission also lack the 690-nm absorption form. Spectral Characteristics of the 690-nm Absorption Form—In order to get more details on the 690-nm absorption form of Lhca2, the absorption spectrum of the WT complex was described in terms of Gaussian forms, using the values obtained by second derivative analysis as starting parameters (Fig. 8). Three Chl b bands were needed to describe the region between 640 and 660 nm, namely at 642, 649, and 655 nm. The red part of the Qy Chl a absorption region was described with two Gaussians at 686 and 690 nm, with the latter showing a broadened spectrum. An oscillation strength corresponding to the absorption of 0.85 Chl a molecules is associated with this band. This description is in good agreement with the fluorescence data; the redmost band, which is responsible for the 701-nm emission, absorbs at 690 nm, as suggested by the site-selected fluorescence measurements, and a second
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