J. Phys. Chem. B 2009, 113, 12565–12574
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Carotenoid Structures and Environments in Trimeric and Oligomeric Fucoxanthin Chlorophyll a/c2 Proteins from Resonance Raman Spectroscopy Lavanya Premvardhan,*,† Luc Bordes,† Anja Beer,‡ Claudia Bu¨chel,‡ and Bruno Robert† CEA, Institut de Biologie et Technologie de Saclay, and CNRS, 91191 Gif-sur-YVette Cedex, France, and Institute of Molecular Biosciences, UniVersity of Frankfurt, Frankfurt, Germany ReceiVed: April 2, 2009; ReVised Manuscript ReceiVed: July 6, 2009
Resonance Raman (RR) spectroscopy is used to characterize the structures and environments of the carotenoid fucoxanthin (Fx), the primary light harvester in the membrane-intrinsic fucoxanthin chlorophyll a/c2 proteins (FCP) from the diatom Cyclotella meneghiniana, thereby building on the findings from Stark spectroscopy and calculations (J. Phys. Chem. B 2008, 112 (37), 11838-11853). Solvent-dependent effects on the RR bands of isolated Fx and the xanthophyll-cycle carotenoid, diadinoxanthin (Ddx), are studied to better characterize the protein-specific environmental factors that affect their geometry and spectral signatures. In addition, excitation-wavelength-dependent (441.6-570 nm) changes in the RR bands of the ν1 and ν3 modes, as well as the conjugated C8 carbonyl stretch, allow the identification of 5–6 in both the trimeric (FCPatrim) and oligomeric (FCPbolig) forms of FCP. These Fx’s are broadly classified into two each of high (Fxblue) and low (Fxred) energy, and 1–2 of intermediate (Fxgreen) energy that are allied to their location and function in the protein. The CdC stretching frequencies (ν1), which indicate conjugation over at least 7 double bonds, and the low intensity of the ν4 C-H bending modes attest to their planar all-trans conformations both in the protein and in solution, with the protein-bound Fxred’s exhibiting signs of nonlinearity. Additionally, rededge excitation of Fx in solution, and in the FCPs, exhibits the effect of mixing between the two lowestenergy, 21Ag*--like and 11Bu*+-like, excited states, which underpins the high light-harvesting and energytransfer efficiency of the Fxred’s. RR spectra also reveal differences between FCPatrim and FCPbolig complexes, such as the greater prevalence of Ddx in FCPbolig. Importantly, the identification of 5–6 Fx’s per FCP monomer suggests that there may be more than the four Fx’s previously assumed per FCP monomer, or else there is definite heterogeneity in Fx structures and/or binding sites. Introduction Diatoms are unicellular eukaryotes that are responsible for about 25% of the primary biomass production on Earth1 but have been much less studied than higher eukaryotic thylakoid systems, which have a similar photosynthetic cycle.2,3 In diatoms and brown algae the light-harvesting system is based on fucoxanthin chlorophyll a/c2 proteins (FCPs). The solar energy absorbed by these proteins is transferred, namely, excited-state energy transfer (ET), to Chl-a in the lightharvesting complex (LHC) and then onto the reaction center of photosystems I and II, to be eventually stored as potential chemical energy. Although FCP complexes from brown algae and diatoms have been characterized by steady-state and ultrafast spectroscopic techniques,4-11 the lack of a crystal structure for these proteins hinders the ability to accurately attribute properties and functions to the individual pigments. The paucity of structural information about FCP thus results in the recourse to the LHC structure of higher plants (LHCII) as a reference, because of their partly homologous primary sequence.2,3,9 The macroscopic similarity in the structure and function of the LHCs of these unicellular organisms to that of higher plants, however, does not imply functional similarity at a molecular level, particularly if their pigment structures * Corresponding author. Current address: Synchrotron SOLEIL, L’Ormes des Merisiers, Saint Aubin BP 48, 91192 Gif-sur-Yvette Cedex, France. E-mail:
[email protected]. Tel: +33-(0)1-693597935. † CEA, Institut de Biologie et Technologie de Saclay, and CNRS. ‡ University of Frankfurt.
and organization differ. To this end, the resonance Raman (RR) study aims to provide information about the structure and composition of the carotenoids (Cars) in two different FCP complexes by selectively probing fucoxanthin (Fx in Figure 1), the Car in FCP, with 12 discrete excitation wavelengths between 413.7 and 570 nm. These FCP complexes, isolated from the centric diatom Cyclotella meneghiniana, include a fraction composed of trimers (FCPatrim) and another of oligomers (FCPbolig). Although both FCP and LHCII are composed of Cars and chlorophylls (Chls), the pigment composition of FCP differs in a way that reflects the adaptation of diatoms to their marine environment. First of all, not only is the Car Fx found in greater amounts in FCP relative to lutein (Lut) in LHCII but its lightabsorbing capability extends into the blue-green range and it functions as the primary light harvester, in contrast to LHCII (or LH2), where (bacterio)chlorophylls assume this function. In addition, chlorophyll c2 (Chl-c2) facilitates light harvesting at higher energy in FCP, instead of Chl-b. Previously, differences in the photophysical and dipolar properties of the functionally different Fx’s in FCP led to the identification of two red-absorbing species, Fxred, as well as a higher energy Fxblue and an Fxgreen.11 Structural information obtained thus far, about FCP, from circular and linear dichroism5,7-9 is achieved in more detail in the current RR study. Because RR is a vibronic technique, small differences among electronically similar Fx’s may be uniquely identified on the basis of even small deformations of a specific bond. Furthermore, RR permits differentiation
10.1021/jp903029g CCC: $40.75 2009 American Chemical Society Published on Web 08/21/2009
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Premvardhan et al. efficient light harvesting, as well as ET from S2 (∼40%).10 Calculations on Fx have shown that the S1/ICT state is a 1 *+ combination of the “covalent” 21A*g -like and the “polar” 1 Bu like states, and the mixing of these states, which is dependent on the environment, influences the spectroscopic properties of both S1 and S2.11 In FCP, the red-most-absorbing Fx’s are expected to be most prone to this phenomenon, explaining their high light-harvesting and ET efficiency10,18 relative to the higherenergy Fx’s, which must also function as photoprotectors and structural stabilizers. In spite of the complexity of the electronic properties of Fx, the RR spectra, obtained with excitations between 413.7 and 570 nm, ably identify and provide a set of structural markers for these Fx’s in FCPatrim and FCPbolig. The other Car in FCP, diadinoxanthin (Ddx in Figure 1), is similar to violaxanthin in LHCII19,20 and associated with regulation of the excitation energy in FCP on its deepoxidation to diatoxanthin (Dtx).21,22 Although present in trace amounts in FCPatrim (0.016 Ddx/Chl-a) and FCPbolig (0.025 Ddx/Chl-a),12 the RR spectra suggest the presence of Ddx in FCPbolig and are briefly discussed. Material and Methods
Figure 1. (a) Absorption spectra of the trimeric (FCPatrim: light solid line) and oligomeric (FCPbolig: dark solid line) FCP complexes in glycerol-buffer solutions shown at 77 K and RT on an arbitrary y-axis scale with the Qy band areas normalized (in cm-1 units) for FCPatrim and FCPbolig, separately. The spectra are shown in linear energy units of wavenumbers, cm-1, with a break at 600 nm. The spectra on the right, up to 725 nm, are stretched for better visualization. In parts b and c are shown the fits to the RT absorption spectra of FCPatrim and FCPbolig, respectively, using the pigment absorptions in solution (see Materials and Methods). Dashed vertical lines indicate laser wavelengths (413.7-570 nm) used to obtain RR spectra.
of identical pigments bound at different protein sites by the selective enhancement of vibronic modes coupled to the electronic transition probed by the excitation.13 The electronic properties of the carbonyl-containing Cars such as Fx, as well as peridinin and siphonoxanthin, which function as the primary light harvesters in marine organisms,14,15 have a typical polyene spectrum with an S0 f S2 transition that is allowed and a forbidden S0 f S1 transition that confers specific photophysical properties and controls their function.16 In Fx, the S1 (21Ag*--like) state plays an important role in transferring the solar energy, absorbed by S2 (11Bu*+-like), to Chl-a. More precisely, it is an intramolecular charge-transfer (ICT) state, strongly coupled to the S1 state, the so-called S1/ICT state, that has been identified as the principal ET conduit, in these Cars,14 and is evident in both Fx- and peridinin-containing LHCs.10,15,17 Stark (electroabsorption) studies showed that photoinduced charge transfer (CT) undergone by Fx11 is implicated in the
Sample Preparation. FCPs were prepared from the diatom C. meneghiniana grown under low-light conditions (40 µmol m-2 s-1 of white light for 8 h per day) according to Beer et al.12 In brief, thylakoids were isolated after French press breakage of the cells by differential centrifugation. After solubilization with 15 mM β-dodecyl maltoside, FCPs were separated using ion-exchange chromatography. Both complexes were checked for purity and oligomeric states using SDS-PAGE and gel filtration chromatography.12 Excitation ET from Chl-c2 and Fx to Chl-a was assessed by fluorescence excitation spectra at room temperature (RT). Concentrated solutions of Fx, isolated from thylakoids using acetone,9 in distilled acetonitrile (Acn), and HPLC-grade (>99% purity, Sigma Aldrich) toluene and methanol (MeOH), were used to measure the RR spectra. Acn may contain trace amounts of water because it was not transferred under nitrogen. Ddx in ethanol (EtOH), purchased from DHI Water & Environment (Denmark), was used neat. Instrumentation and Experimental Method. RT absorption spectra of FCP were obtained in a buffer (10 mM Mes, pH 6.5, 2 mM KCl, 0.03% β-DDM) and glycerol/buffer mixtures (55/ 45, v/v) in a Perkin-Elmer spectrometer. Low-temperature (77 K) spectra were obtained using glycerol/buffer solutions of FCPatrim and FCPbolig in 1-cm-path-length, 1-mm-wide cuvettes. (Clear glasses could not be obtained in 1-cm-wide cuvettes.) 25-100 µL of FCP in buffer sans glycerol, or Fx in solvent, was dropped on a 1.2-cm-Ø round microscope slide fit onto a holder and immediately immersed into a trough with liquid nitrogen (LN2) and then into a LN2 flow cryostat (Air Liquide). Laser excitations at 413.7 nm; 441.6 nm; 457.9, 476.5, 488.0, 496.5, 514.5, and 528.7 nm were obtained with Kr+ (Innova 90, Coherent), He-Cd (Linconix), and Ar+ (Innova 100, Coherent) lasers, respectively. The broad-band output (∼6 W) of the Ar+ laser pumped a Spectra Physics dye laser to produce 540, 550, 560, and 570 nm excitations. Raman scattered photons, from the sample oriented at 45° to the incident beam, were focused into a U1000 Raman spectrometer (Jobin-Yvon) equipped with a blue-sensitive back-illuminated LN2-cooled CCD camera.23 The RR spectra of the isolated Car in solution (Figure 2), or bound to FCP (Figures 3 and 4), were obtained with a 1200 g mm-1 grating (resolution ∼ 1.9 cm-1 at 441.6 nm to 0.8 cm-1 at 528.7 nm). The high concentration of the samples and the
Resonance Raman of Fucoxanthin and FCP
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Figure 2. RR spectra for excitation between 441.6 and 527.9 nm, of Fx in toluene (FxTol as a solid line), acetonitrile (FxAcn as a dashed line), and methanol (FxMeOH as a dotted line) and diadinoxanthin in ethanol (DdxEtOH as a dot-dashed line) shown from 940 to 1800 cm-1, for excitation between 441.6 and 528 nm. The spectra are normalized to the ν1 band (1500-1570 cm-1) at a value of 100. The ν2, ν3, and ν4 bands at ∼1160, 1010, and 960 cm-1, respectively, are shown in the left panel and the carbonyl stretching region (1550-1700 cm-1) in the right panel. Note the different y-axis axis scales: ν3 and ν4 at 0-60 (left); ν1 and ν2 at 0-120 (middle); and carbonyl at 0-17 (magnified ∼20 times relative to ν1).
absence of glycerol, standard in our RR measurements, significantly decrease light transmission, and sample absorption spectra cannot be obtained before and after measurement of the Raman spectra. Sample stability and integrity were assumed based on the similarity between the first and final Raman spectra. Output laser powers of 20-30 mW are attenuated at the sample to
