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J. Biol. Chem., Vol. 279, Issue 47, 48543-48549, November 19, 2004
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From the
Istituto di Biofisica, CNR, Trento, c/o ITC via Sommarive 18, Povo, Trento 38100, Italy, ¶Dipartimento Scientifico e Tecnologico, Università di Verona, 15 Strada Le Grazie, Verona 37134, Italy, ||Université Aix-Marseille II, LGBP-Faculté des Sciences de Luminy, Département de Biologie, Case 901, 163 Avenue de Luminy, Marseille 13288, France, **Faculty of Science, Division of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands, and Service de Bioenergetique, Bat. 532, Commissariat a l'Energie Atomique, Saclay, Gif-sur-Yvette Cedex 91191, France
Received for publication, August 4, 2004 , and in revised form, August 24, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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 (1). 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 (2). These complexes are located on one side of the core complex (3, 4) and can be isolated as dimers (5). The Lhca proteins belong to the Lhc multigenic family, and they share high sequence homology with the antenna complexes of PSII (6). 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) (7). 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 (5, 8–11). 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 (12), 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 (12). 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 (1, 13). 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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Reconstitution and Purification of Protein-Pigment Complexes—Reconstitution and purification were performed as described in Ref. 16 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. 17. 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 (18).
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% -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% -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. 19 using samples oriented by the polyacrylamide gel squeezing technique.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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.
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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. 20 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 (7). 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.
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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.
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (25), the presence of multiple gene products in the LHCII preparations with different spectral features (26), and conformational changes induced by zeaxanthin binding to site L2 (27, 28). 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 (29) 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.
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The energetic separation between the absorption maximum and the emission peak represents the Stokes shift. For the 690-nm absorption form, which emits at 701 nm, the Stokes shift is 11 nm (240 cm–1). This is larger than observed in Lhcb complexes (where it is around 2 nm). From the Stokes shift, a value of the optical reorganization energy (S
) can be obtained, which corresponds to 120 cm–1 (Stokes shift 
2S.).
The characteristics of the absorption band broadening can yield important information on the chromophore protein environment. The analysis of the WT minus mutant difference spectra indicates that at 77 K the FWHM of the band is around 400 cm–1. At this temperature, the homogeneous broadening of the band can be described as follows,
 | (Eq. 1) |
obtained from the Stokes shift, the value for the FWHMhom is 270 cm–1, which gives a value of the inhomogeneous broadening of around 300 cm–1. Such a large value for the inhomogeneous broadening is consistent with data from site-selected fluorescence measurements, since the excitation in the red tail did not affect the shape and width of the emission band. The 690-nm absorption form of Lhca2 is thus characterized by a larger value of both homogeneous and inhomogeneous broadening as compared with the bulk Chl absorption form, for which these two values are both around 100 cm–1 (30). In this respect, the 690-nm band of Lhca2 shows characteristics very similar to the red absorption forms observed in several organisms (20), although its energy is significantly higher.
The Origin of the 701-nm Emission—Large values of optical reorganization energy are usually associated with the presence of pigment-pigment interactions. It has already been suggested that the red forms of Lhca3 and Lhca4 are the low energy term of an interaction between two Chl a molecules. Mutational analysis shows that these two chromophores are bound to sites Chl A5 and Chl B5, since mutation at these sites completely abolishes the red emission forms. This conclusion is in agreement with the finding that site A5 binds Chl a in Lhca as well as in any other Lhc protein (22–24), whereas site B5 either binds Chl a or has a mixed Chl a-Chl b occupancy as derived from pigment analysis of WT and mutant protein as well as their spectroscopic analysis. We conclude that the low energy spectral forms originate from the same protein domain in all Lhca complexes and involve Chls A5 and B5. Nevertheless, the energy level of these red-shifted transitions is modulated to a different extent in each Lhca subunit.
The presence of pigment-pigment interaction inducing the absorption at 688–690 nm of Lhca2 is supported by the comparison of the CD spectra of WT and A5 mutant, which lack a negative contribution around 687 nm and a positive one at 672 nm (values at room temperature). Similar components can be detected in the WT minus A5 absorption and LD difference spectra (Fig. 9). In both cases, two absorptions, at 688–689 nm and at 672–674 nm, can be detected, representing Chl forms lost by the effect of the mutation at site A5 and having positive LD spectra. These data can be interpreted as the loss of an excitonic interaction having the low energy band at 688–690 nm and the high energy band at 672–674 nm, as the effect of A5 mutation. To describe both difference spectra, a negative band peaking at 679 nm is required. This band is present in the mutant but not in the WT and shows a positive LD signal (Fig. 6). Considering that all spectroscopic techniques consistently indicate that the interaction leading to the 690-nm absorption involves two Chl a molecules and that in the A5 mutant only one Chl a molecule is lost, the gain in the absorption can be interpreted as the contribution of the noninteracting monomer, which is left alone in the A5 mutant. Based on the mutation analysis, which suggested that the two interacting Chls are accommodated in sites A5 and B5, we conclude that the "new" 679-nm form detected in WT minus A5 difference spectra represents the absorption of the monomeric Chl a in site B5. This is in agreement with the analysis of the homologous protein CP29 (22), showing that a noninteracting Chl a absorbing at 679 nm is bound to site B5.
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Once it is concluded that in all Lhca complexes the red forms originate from interaction between Chls a in sites A5 and B5, it is interesting to investigate the origin of the large difference in interaction energy between the complexes, which is reflected in a difference of around 30 nm between the emission maxima of Lhca2 and Lhca4. Two major factors control the amplitude of the interaction energy: the distance between the chromophores and the relative orientation of their dipole moment transition (the geometric factor k). (V1,2 = 5.5kµ1 µ2/R3, where R is the center to center distance between the chromophores and µ represents the dipole moment of the Chls). The analysis of the LD spectra of the complexes indicates that the redmost absorption form of both Lhca4 and Lhca2 forms the largest angle with the normal to the membrane plane (12) (this work). Although small differences in the orientation between the Chls in the two complexes cannot be excluded, we can assume, in a first approximation, a similar geometric factor in the two complexes. On this basis, the difference in interaction energy can be explained by considering that the distance between the two interacting Chls is around 2 Å larger in Lhca2 compared with Lhca4 by applying the equation, V = 90k/R3. Recently, the structure of the PSI·LHCI complex has been elucidated at 4.4-Å resolution, and the position of the bound Chl has been defined. The center to center distance between Chl A5 and B5 in Lhca4 has been shown to be 7.9 Å, whereas in Lhca2 the value is 10.4 Å (3). This is consistent with the proposal that in Lhca4, the presence of the Asn as a ligand for Chl A5 allows a shorter distance between the two interacting Chls as compared with the complexes that have His for Chl A5 ligation (12).
Comparison with LHCII—Previous work with Lhca3 and Lhca4 has highlighted the importance of the presence of an Asn as ligand for Chl A5 for the generation of the red-shifted 730-nm fluorescence in Lhca3 and Lhca4, the only members of the Lhc superfamily holding Asn as a Chl A5 ligand. The present work shows that a significant, although lower, level of excitonic interaction can be obtained without the intervention of Asn ligation, and yet this interaction is sufficient for yielding the 701-nm emission form of Lhca2. This finding implies that additional structural signatures differentiate the Lhca from the Lhcb subfamily as a requirement for the chromophore-chromophore interactions in the helix C domain. This problem can be addressed thanks to the recent elucidation of LHCII structure at high resolution, since in Lhcb 1–3, the components of trimeric LHCII, the ligand of Chl A5 is His, as in Lhca2, and yet no 701-nm fluorescence emission can be detected. Moreover, the two structures in this domain are also very similar (3, 13), thus allowing detailed comparison. Why does LHCII not have red forms? Both x-ray structure (13) and mutation analysis (23, 31) consistently identify the ligand at site B5 as Chl b. Although the distance and mutual orientation between Chl a A5 and Chl b B5 allow for interaction in LHCII (13), the large energy gap between Chl a and Chl b makes the shift induced in the absorption form very small. In the case of Lhca2, biochemical and spectral analysis are consistent with site B5 having low selectivity and thus allowing for binding of both Chl a and Chl b. This mixed occupancy is crucial to explain the presence of two emission forms in the emission spectrum of Lhca2. We suggest that the blue form (emission at 689 nm) originates from the population of the ensemble having Chl b in site B5, whereas the 701-nm emission is originating from the population in which site B5 accommodates a Chl a molecule. The crucial question is thus how the selectivity of each Chl binding site is determined in Lhca proteins. The structure of LHCII shows that all but one Chl b present have the formyl group hydrogen-bonded with protein residues of water molecules. It can thus be speculated that the possibility to form this hydrogen bond affects the affinity of a site for Chl b versus Chl a, through an energy minimization effect. In LHCII, Chl b in site B5 is stabilized by hydrogen bond with the NH of Gln131 (13). In the Lhca2 complex, as well as in all Lhca complexes, this Gln is substituted by a Glu residue. The Glu cannot act as hydrogen donor, and it can be expected that in this case Chl B5 loses its high affinity for Chl b and thus can also accommodate Chl a.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 390461405360; Fax: 390461405372; E-mail: croce{at}itc.it.
1 The 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.
2 T. Morosinotto, R. Bassi, and R. Croce unpublished results.
3 R. Croce, T. Morosinotto, J. A. Ihalainen, A. Chojnicka, J. P. Dekker, R. van Grondelle, and R. Bassi, manuscript in preparation.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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