J. Phys. Chem. B 2009, 113, 13071–13078
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Identification of the Sites of Chlorophyll Triplet Quenching in Relation to the Structure of LHC-II from Higher Plants. Evidence from EPR Spectroscopy Marilena Di Valentin, Federico Biasibetti, Stefano Ceola, and Donatella Carbonera* Dipartimento di Scienze Chimiche, UniVersita` di PadoVa, Via Marzolo 1, 35131 PadoVa, Italy ReceiVed: April 30, 2009; ReVised Manuscript ReceiVed: July 24, 2009
The Chlorophyll a (Chl a) molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric LHC-II are identified by time-resolved and pulse EPR techniques. The concept of spin angular momentum conservation during triplet-triplet energy transfer is exploited for the calculation of the spin polarization of the carotenoid triplet states. The sites with the highest probability of forming triplet states, which are quenched by the central luteins, result to be Chl603 and Chl612. “Unquenched” Chl triplet states are produced by photoexcitation in the LHC-II complex. Putative sites of these triplet states are Chl614, Chl611, Chl604, and Chl613 since they do not contribute to the formation of the observed carotenoid triplet states. Introduction In the photosystem II of higher plants, the main light harvesting function is played by the LHC-II Chl a/b-binding complex, which is the most abundant membrane protein in the chloroplasts. The crystal structure of the spinach LHC-II complex has been obtained to a resolution of 2.72 Å1 and that of pea to a resolution of 2.5 Å.2 The folding of the LHC-II protein is characterized by three trans-membrane helices, and a trimer assembly has been observed. The high-quality electron density map permitted the location of all 14 chlorophylls (Chls), eight Chl a and six Chl b, within one monomeric unit of the LHC-II complex. Three carotenoids (Cars) were also clearly identified as corresponding to two luteins and one neoxanthin. One site of mixed density was assigned to the carotenoids involved in the xanthophyll-cycle (namely, violaxanthin, antheraxanthin, zeaxanthin), which play an important role in the nonradiative dissipation of excess excitation energy formed under high light conditions.3 Luteins are bound in the center of the protein in sites L1 and L2 (see Figure 1). The binding site N1 is specific for neoxanthin and is located near the helix C. The V1 site may bind violaxanthin, lutein, or zeaxanthin depending on light conditions4 and is located at the periphery of the monomeric subunits. In high light conditions, the radiation absorbed by the light harvesting system can not be completely processed by the electron transport chain, and dissipation via other mechanisms becomes essential to avoid damage. The main photoprotective mechanism responsible for the excess energy dissipation in LHC-II is catalyzed by the nonphotochemical quenching (NPQ) mechanism based on the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin (VAZ) by violaxanthin deepoxidase, which is activated by low luminal pH3 and probably involves the V1 site.4 Carotenoids in V1 are not involved in light-harvesting and singlet energy transfer.5 Quenching in LHC-II or in the minor light-harvesting complexes is widely assumed to be caused by a conformational change that switches the complex from the active, energytransmitting state to a quenched and dissipative state.6-9 It has * Author to whom correspondence should be addressed. Tel.: +39 498275144. Fax: +39 498275161. E-mail:
[email protected].
Figure 1. Structure of the pigments associated with the basic unit of the LHC-II complex.1 Structure derived from coordinates deposited in the Brookhaven Protein Data Bank (1 RWT). The protein backbone and the Chl b molecules are in faded gray. Orange, luteins in site L1 and L2; purple, violoxanthin (Xat) in site V1; cyan, neoxanthin (Nex) in site N1; blue, Chl a603 (lower) and 612 (upper); pink, Chl a604 (right) and 613 (left); green, Chl a602 (left) and 610 (right); yellow, Chl a611; red, Chl a614.
been suggested that L1 can be a site of fast response to the qE, the energy dependent quenching component of NPQ:6,7 this site seems to undergo a conformational change as a consequence of a neoxanthin-triggered mechanism leading to the formation of a lutein-Chl a heterodimer capable of dissipating the excitation energy via a carotenoid excited singlet state. This proposed mechanism would seem to involve, in vivo, an aggregation-induced conformational change of the LHC-II proteins. However, this interpretation is still under debate in the literature. Recently, Barros et al. observed a strong emission and comparatively long fluorescence lifetime in single LHC-II crystals, indicating that the complex is unquenched and thus that the crystal structure shows the active, energy-transmitting state of LHC-II.10 Starting from this observation, they came to the conclusion that excitation energy quenching, in the lightharvesting complexes, is due to the molecular interaction with external pigments in vitro or other pigment-protein complexes such as PsbS in vivo and does not require a conformational change within the complex. Thus, at present, there is no conclusive evidence of a conformational change of LHC-II.
10.1021/jp904012j CCC: $40.75 2009 American Chemical Society Published on Web 09/03/2009
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An alternative mechanism for the qE quenching based on the formation of a Zeaxanthin radical cation and a Chl radical anion has also been proposed.11 However, the involvement of a zeaxanthin-Chl charge separated state in the dissipation of high excitation density seems to be a specific characteristic of the minor antenna complexes, while LHC-II, at least in vitro, does not show the presence of the Car cation formation up to the femtosecond time scale.12 Carotenoids in LHC-II are also known to be involved in the photoprotection process as efficient, fast, and direct quenchers of the Chl triplet states, thus preventing the formation of singlet oxygen (1O2*) and subsequent harmful oxidation of membrane and protein elements.13 This is the fastest response to the requirement of energy dissipation in the complex. The study of Car triplet states formed in LHC-II complexes has been performed in the past by means of different spectroscopic techniques (EPR, ODMR, time-resolved optical spectroscopy).14-22 Different Car triplet states have been characterized, showing specific interactions with nearby Chl molecules.16 Lampoura et al.20 assigned the Car triplets specifically to the luteins in sites L1 and L2, excluding the violaxanthin in the V1 site, since this carotenoid is not involved in singlet energy transfer and therefore cannot be involved in triplet quenching, which is a mechanism requiring closer vicinity between the donor and the acceptor. In addition, neoxanthin was excluded from participating in triplet quenching on the basis of its being surrounded by Chl b molecules which transfer the excitation to Chl a molecules so quickly that the probability of populating Chl b triplet states should be negligible. However, the recently published structures of LHC-II at high resolution1,2 have shown that both neoxanthin and violaxanthin are close to at least one Chl a molecule, thus, in principle, their involvement in triplet quenching cannot be excluded. Starting from the most recent crystallographic data, Mozzo et al.23 re-examined the problem of the carotenoid triplet assignment. They used laser-induced Triplet-minus-Singlet (TmS) spectroscopy on LHC-II preparations with different carotenoid composition and came to the conclusion that the largest part of the triplets is quenched by the lutein bound in site L1, in close proximity to the low-energy chlorophylls of the complex. The lutein in the L2 site is also active in triplet quenching, showing a longer triplet lifetime. To study the triplet quenching contribution of the carotenoids in the V1 and N1 sites, the TmS spectra of LHC-II with an empty V1 site and of LHC-II lacking both neoxanthin and violaxanthin were measured by the authors and compared with those of the wild type (WT). The shape of the spectra and the relative amplitudes of the components were only slightly different in the two complexes compared to the WT, indicating that the carotenoids in the V1 and N1 sites do not participate in triplet quenching. However, the spectral lines were too broad to exclude the presence of extra contributions with certainty, and the measurements were not accompanied by an estimate of the triplet yield in the two different samples. Moreover, small differences in the lifetimes of the spectral components were observed, in the abovementioned LHC-II complexes. Thus, although Mozzo et al.23 assigned the observed triplet states to the carotenoids present in the structure, further investigation is needed especially to identify the Chl molecules where the triplet states are initially formed and to exclude the involvement of the N1 and V1 sites. In the past, we used ODMR (optically detected magnetic resonance) to study the formation of triplet states in isolated LHC-II complexes, showing the presence of at least two distinct populations of carotenoid triplet states.16,17 More recently, in a
Di Valentin et al. time-resolved electron paramagnetic resonance (TR-EPR) and pulse-EPR work on the peridinin triplet state generated in PCP proteins from Amphidinium carterae, we exploited the concept of spin conservation during triplet-triplet energy transfer (TTET) to identify the peridinin-chlorophyll pairs directly involved in the process.24,25 In this work, we apply the advanced EPR techniques to get insight into the mechanism of formation of the carotenoid triplet states in LHC-II. We take advantage of the analysis approach previously used for PCP to identify the sites of the Chl triplet formation and quenching by carotenoids, in relation to the protein structure of LHC-II in higher plants. The EPR data are discussed in terms of energy transfer properties, specific pathways for triplet quenching, and protein structure. Materials and Methods Sample Preparation. The LHC-II complexes were isolated from spinach by using n-octyl-β-D-glucopyranoside26 after which the LHC-II trimers were purified by using n-dodecyl-β-Dmaltoside and were ultracentrifuged. For measurements, the samples were diluted in 10 mM Tricine, pH 7.8, 0.3 M sucrose, and 0.03% w/v β-dodecylmaltoside, to a final protein concentration of 3 mg/mL. Oxygen was removed from the samples by flushing argon in the EPR capillary before freezing. Glycerol, previously degassed by several cycles of freezing and pumping, was added (60% v/v) to obtain a transparent matrix. TR-EPR. TR-EPR spectra were obtained in direct detection mode using pulsed light excitation. The X-band EPR spectrometer (Bruker ECS106) was equipped with a TE102 cavity (9.4 GHz) and a nitrogen flow system. For measurements at cryogenic temperatures, an Oxford helium cryostat (ESR 200) was used. Laser excitation at 532 nm (10 mJ per pulse and repetition rate of 10 Hz) was provided by the second harmonic of a Nd:YAG laser (Quantel Brilliant). The time resolution of the TR-EPR spectrometer is about 150 ns. The microwave power used for the TR-EPR experiments was 20 mW at the cavity. No field modulation or phase-sensitive detection was used. The EPR signals were taken from the microwave preamplifier (ER047-PH Bruker, bandwidth 20 Hz-6.5 MHz) and sampled with a LeCroy LT364 oscilloscope (1 ns per point). To eliminate the laser background signal, transients were accumulated under off-resonance field conditions and subtracted from those onresonance. The spectra at different times after the laser pulses were reconstructed from kinetic traces for each field position. Pulse EPR Experiments. Experiments were performed on a Bruker Elexsys E580 pulsed EPR spectrometer. Laser excitation at 532 nm (10 mJ per pulse and repetition rate of 10 Hz) was provided by the second harmonic of a Nd:YAG laser (Quantel Brilliant) in a dielectric cavity. The temperature was controlled by means of a Helium cryostat (Oxford CF935) driven by a temperature controller (Oxford ITC503). Field-swept electron spin echo (ESE) spectra (FSE spectra) were recorded using a two-pulse ESE sequence according to the scheme: flash-DAF-π/2-τ-π-τ-echo (DAF ) delay after laser flash); the value of DAF was fixed at 50 ns, which was, in our setup, the shortest time for the detection which follows the trigger after the laser pulse. ESE-detected kinetics at the triplet canonical orientations were recorded using a two-pulse (flashDAF-π/2-τ-π-τ-echo) ESE sequence after a variable DAF between the laser flash and the first MW pulse. The π/2-pulse was of 16 ns, and the delay τ was set at 200 ns for the FSE experiment and to 120 ns for the ESE-detected kinetics.
Chl Triplet Quenching in LHC-II
Figure 2. TR-EPR spectrum of LHC-II (black line) taken 150 ns after the laser pulse at 20 K. X, Y, Z represent the ZFS canonical orientations. Reconstruction of the LHCII TR-EPR spectrum (red line), deriving from TCar (blue line) and TChl a (green line) contributions, in proportion, respectively, 1:0.3. Parameters for the reconstruction are reported in Table 1. A ) absorption, E ) emission.
EPR Spectral Analysis. Calculation of the sublevel triplet state populations of the acceptor, starting from those of the donor, was performed using a home-written program in MatLab software, following the formalism of refs 24 and 27 and utilizing the X-ray coordinates of the pigment moieties. The carotenoid zero filed splitting (ZFS) axes were determined therein using principal components analysis. The method is based upon the solution of the covariance matrix of the data, in which the eigenvalues represent the amount of variance explained by each eigenvector. The data set used in the calculations contained simply the three spatial coordinates of the atoms of each Car (only those of the conjugated chain), as reported in the PDB file 1RWT, resulting therefore in the eigenvectors being a good approximation of the ZFS axes. This is due to the particular orientation of the Car’s ZFS axes: the first solution vector is the one with the greatest eigenvalue (which explains most of the variance) and thus lies along the main axis (Z) of the Car. The second one has the second biggest eigenvalue and thus lies along the C-H bonds (X). The third vector is orthogonal to the first two (Y). The calculation was made using the MatLab function named “princomp”. The program works in the limit of a TTET which is fast, compared to the time evolution of the donor triplet spectrum, and is slow enough to allow spin alignment in the external magnetic field. Simulations of the powder spin-polarized triplet spectra were performed using a program written in MatLab with the aid of the Easyspin routine (ver. 2.6.0).28 The program is based on the full diagonalization of the triplet state spin Hamiltonian, taking into account the Zeeman and magnetic dipole-dipole interactions, assuming a powder-like distribution of molecular orientations with respect to the magnetic field direction. Input parameters are: the sublevel populations, the ZFS parameters, the line width at the canonical orientations, and the g-tensor components. Results Figure 2 shows the X-band spin-polarized TR-EPR spectrum of LHC-II, taken at 150 ns after the laser pulse, at 20 K. The spectrum reveals the presence of different species. The prevalent triplet population, having ZFS parameters, |D| ) 414 G and |E| ) 40.7 G, can be easily assigned to the carotenoid species, TCar, on the basis of the comparison with the previously reported values of the ZFS parameters determined by ODMR spectros-
J. Phys. Chem. B, Vol. 113, No. 39, 2009 13073 copy.16,17 The other species can be assigned to the Chl a triplet state (TChl a) on the basis of the ZFS parameters,24,29 as can be clearly seen by comparison with the spectrum of TChl a reported in the same figure. This spectrum has been obtained by simulation of the TR-EPR of TChl a in micelles of Triton X-100 1 mM, detected under the same experimental conditions as for LHC-II (see Supporting Information). Because of the large spectral overlap of the two triplet states, it is not easy to determine the exact polarization patterns of the two contributing triplet states by direct inspection of the TR-EPR spectrum. For this reason, we also performed pulse-ESE experiments, on the same sample, to try to better discern the different components. ESE spectroscopy is based on the principle of spin refocusing after a microwave pulse sequence. We have previously demonstrated that the TR-EPR and FSE spectra, taken at short delay after the laser flash (DAF), were identical for the PCP samples, where only carotenoid triplet states were present,30 due to the absence of fast and anisotropic relaxation processes which depend on the orientation of the carotenoid triplet states in relation to the magnetic field. On the contrary, porphyrin triplet states are known to show anisotropic relaxation processes which are reflected in their FSE spectra.31 In Figure 3, we report the FSE spectrum of LHC-II, at DAF ) 50 ns. Direct comparison of the FSE and TR-EPR spectra reveals that the central lines attributed to the Y component of the TChl are almost suppressed in the FSE spectrum, leaving the contribution of the carotenoid easily discernible. A small contribution at the Z and X canonical orientations of TChl to the FSE spectrum is however still visible. In Figure 3, we report also the FSE spectrum of TChl dissolved in Triton X-100 (1 mM), detected at the same τ and DAF used for LHC-II. It can be seen that the shape of the FSE spectrum of TChl is quite different compared to the corresponding TREPR, shown in Figure 2 (see also Supporting Information). By subtracting a proper amount of the experimental TChl a FSE spectrum from the LHC-II spectrum, the “pure” contribution of TCar is obtained (Figure 3). In this way, the correct polarization pattern of the TCar, eeaeaa, appears. As stated before, the FSE spectrum of the TCar is expected to be identical to the corresponding TR-EPR spectrum, while in the case of TChl they differ significantly. We have therefore reconstructed the TR-EPR spectrum of LHCII, using a simulation of the FSE-spectrum of TCar and a simulation of the TR-EPR spectrum of TChl obtained in micelles of Triton X-100. These two pure contributions are shown in Figure 2. A good agreement with the experimental TR-EPR spectrum of LHC-II is reached, with the relative contributions reported in Table 1. Thus, despite the large spectral overlap of the TChl and TCar, the simultaneous reconstruction of the TREPR and FSE spectra allows us to determine the spin polarization of the TCar with accuracy. This is an important starting point for further analysis. Note that a contribution from a radical species is also present in the central part of the FSE spectra. This contribution has been neglected in the reconstruction of the spectra. The kinetics of population and decay of the carotenoid triplet spin sublevels, obtained by monitoring the echo intensity as a function of DAF at the low-field canonical orientations of the ZFS tensor of the carotenoid triplet state (Z, Y, X, indicated in Figure 2), are shown in Figure 4. Inversion of the spin polarization at long delay times is expected on the basis of the anisotropy of the decay rates of the spin sublevels reported previously for other carotenoid triplet states.15,30,32 The presence of the TChl contribution affects the kinetics during the first few microseconds especially in the X canonical direction of the TCar,
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TABLE 1: Simulation Parameters for the Triplets in the Reconstruction of the TR-EPR Spectrum of LHC-IIa
carotenoid Chl a
|D|
|E|
Wx:Wy:Wz
Gauss
Gauss
Px:Py:Pz
Gauss
relative contribution
414 ( 0.5 310.3 ( 0.5
40.7 ( 0.5 43.5 ( 0.5
0.40:0.20:0.40 0.36:0.44:0.20
15 ( 1:20 ( 1:20 ( 1 25 ( 1:25 ( 1:25 ( 1
1 0.3
a Px,Py,Pz: zero-field populations of the triplet states; |D|, |E|: ZFS parameters; W ) line-width at the canonical positions in the EPR powder spectrum. Frequency of the spectrometer: ν ) 9.70 GHz. Isotropic g value: giso ) 2.0023.
Figure 3. FSE spectrum of LHC-II (black line) and of TChl a in micelles of Triton X100 (green line). Pure contribution of the carotenoid triplet state (blue line) to the field-swept ESE spectrum obtained after subtraction of the TChl a spectrum in the proportion indicated by the green spectrum. A ) absorption, E ) emission.
Figure 4. ESE-detected kinetics, at T ) 20 K, at the low-field canonical fields Z -, Y +, X + of TCar indicated in Figure 2. A ) absorption, E ) emission. For other experimental conditions, see the Materials and Methods.
while in the Z and Y directions, the TChl contribution is negligible. The kinetics show that the time evolution of the polarization is slow compared to the time resolution of the experiments. Calculation of the Spin Polarization of the Carotenoid Triplet State Based on Spin Conservation during TripletTriplet Energy Transfer. The carotenoid triplet state formation in LHC-II occurs in the nanosecond time scale,7 and it is thought to arise from TChl quenching since the direct population starting from excited singlet states, by Inter System Crossing, is a very low probability process for carotenoids. The time resolution of our TR-EPR setup is about 150 ns, while the FSE spectrum was taken at 50 ns after the laser flash. This means that, in both cases, in the hypothesis of TTET between Chl and Car, we are able to detect only the acceptor triplet state, which is the TCar. The time evolution of the EPR signal of the acceptor is slow,
3 to 40 µs depending on the zero field principal axis direction, compared to its formation so that we can consider the first detected spectra as the “initial” spin polarized spectra, maintaining the polarization inherited from the TChl donor. The TChl contribution to the LHC-II spectrum belongs to a Chl population which is unprotected and is not able to transfer the triplet energy to the carotenoids. In fact, there is no correlation between the decay rate of the observed TChl and the TCar risetime. As we have stated before, our aim is to analyze the carotenoid acceptor triplet state polarization in terms of the TTET mechanism from the TChl a donor, by exploiting the principle of spin conservation during TTET. The fast transfer time allows us to state that the initial polarization pattern of TChl a does not evolve significantly under the effect of spin relaxation before being transferred to the carotenoid. Since we are not able to detect the spectrum of the TChl a donor directly, the transfer being too fast compared to the time resolution of the setup, we have to make some assumptions in terms of TChl a sublevel populations, to be used for the calculation of the acceptor triplet populations. In the literature there are mainly two different sets of Chl triplet populations which have been reported from in vitro studies, depending on the polarity of the solvent and on the ligation state of the central Mg.24,33,34 The TChl a spectra, calculated starting from these two sets of populations, differ from each other in the initial polarization pattern (eeeaaa vs eaeaea). In the experiments performed on LHC-II, the TChl a population which is not quenched by the carotenoid and is visible in the EPR spectrum has the polarization pattern eeeaaa (see Figure 2). In the following, we present the calculations for the acceptor populating rates obtained starting from the TChl a spin populations which produce this spectral pattern, although, as the calculations demonstrate, the choice between the two polarization patterns does not influence the main conclusions that can be drawn from the results (Supporting Information). The other parameters of the calculations are the directions of the ZFS axes, in relation to the molecular frame of each of the two partners, and the relative position in space of the donor and the acceptor moieties. The TChl a ZFS axes have been chosen according to literature data35,36 and are shown in Figure 5. The ZFS axes of the carotenoid triplet state have been chosen analogously to those determined for β-carotene in crystals.37 The Z axis is along the polyene chain; the X axis is along the C-H bonds in the conjugated chain; and the Y direction is perpendicular to the conjugated XZ molecular plane (see Figure 5). Since the carotenoids in LHC-II are slightly distorted in the X-ray structure, we used a principal component analysis to derive the actual ZFS axes directions, as described in the Materials and Methods. An example of the resulting ZFS axes is shown for Lut620 in Figure 5C. Under these assumptions, we calculated the carotenoid triplet spectra for all the Chl a-Car mutual configurations reported in the X-ray structure of LHC-II. In spite of the pseudo C2 symmetry relating the two subclusters of pigments in the protein complex, differences are present in the relative position of the
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Figure 6. Calculated spin polarized triplet spectra for Lut620 (blue lines) and Lut621 (red lines) as populated by TTET from all the Chl a molecules located at a distance 8 Å are not taken into account in our calculations since TTET, being based on the Dexter mechanism, requires close vicinity of the donor-acceptor pair. For this reason, intermonomer TTET is also excluded. Although Chl a and Chl b molecules are both reported in the table, only Chls a are considered for the calculation of the TTET because the fast singlet energy transfer from Chl b to Chl a makes the possibility of forming triplet states starting from Chl b singlet states negligible. In Figure 6, the polarized triplet spectra calculated for Lut620 and Lut621, starting from each of the Chl a molecules located at a distance
