18710
J. Phys. Chem. B 2006, 110, 18710-18717
Probing the Electronic Structure and Conformational Flexibility of Individual Light-Harvesting 3 Complexes by Optical Single-Molecule Spectroscopy Martijn Ketelaars,† Jean-Manuel Segura,‡ Silke Oellerich,§ Ward P. F. de Ruijter, Gerhard Magis and Thijs J. Aartsma* Department of Biophysics, Leiden UniVersity, 2300 RA Leiden, The Netherlands
Michio Matsushita| and Jan Schmidt MoNOS, Leiden UniVersity, 2300 RA Leiden, The Netherlands
Richard J. Cogdell IBLS, UniVersity of Glasgow, Glasgow G12 8TA, UK
Ju1 rgen Ko1 hler Experimental Physics IV, UniVersity of Bayreuth, 95440 Bayreuth, Germany ReceiVed: February 27, 2006; In Final Form: June 22, 2006
We present fluorescence-excitation spectra of individual light-harvesting 3 (LH3 or B800-820) complexes of Rhodopseudomonas acidophila at 1.2 K. The optical single-molecule studies were employed to investigate the electronic structure as well as the conformational flexibility of the individual pigment-protein complexes. The optical spectra resemble those of individual light-harvesting 2 (LH2) complexes, in agreement with the structural similarity of both types of complexes. Although variations among the LH3 spectra are large, there is a distinct difference in the spectral features of the 800 and 820 nm region that appears in all the complexes studied. In the B800 region 4-6 narrow bands are present whereas in the B820 region a limited number of relatively broad bands are observed. These observations can generally be interpreted in terms of localized excitations in the 800 nm region and delocalized excitations in the 820 nm region. The observed heterogeneous spectral behavior, especially in the B820 band, indicates that the B820 pigments of LH3 are sensitive to light-induced local conformational changes. It is suggested that a rotation of the C3-acetyl chain of a BChl a pigment bound to the β-subunit of the light-harvesting complex is the origin of the conformational flexibility and affects the optical properties of the whole pigment-protein complex.
Introduction Pigment-protein complexes play a major role in many biological processes.1-3 Often their functional efficiency is optimized by spectral tuning of their optical properties4 by adjustment of parameters such as the site energy of the individual pigment and the interaction between pigments within one complex.5 A change of these parameters can affect the spectral features to various extents and may influence the efficiency of the biological process. This applies particularly to the primary steps of photosynthesis, when light-harvesting complexes collect sunlight and transfer this excitation energy toward the reaction center. Efficient energy transfer requires optimal spectral “tuning” of the donor and acceptor complexes. Very interesting examples of how slight conformational changes of pigments can modify the optical properties of a * To whom correspondence should be addressed. Phone: +31-(0)715275967. Fax: +31-(0)71-5275819. E-mail:
[email protected]. † Present address: Department of Radiotherapy, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands. ‡ Present address: Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fe´de´rale de Lausanne, 1015 Lausanne, Switzerland. § Present address: Experimental Physics IV, University of Bayreuth, 95440 Bayreuth, Germany. | Present address: Department of Physics, Tokyo Institute of Technology, Ookayama 2-12-1, Meguro, Tokyo 152-8551, Japan.
pigment-protein complex are the light-harvesting complexes (LH) 2 and 3 of the purple bacterium Rhodopseudomonas (Rps.) acidophila. Some strains of Rps. acidophila, such as strains 7050 and 7750, are capable of synthesizing both types of lightharvesting complexes, when grown under “stressed” conditions, for example, low-light intensities and/or low temperature.6,7 The exact response varies depending on the strain.6 Whereas in strain 7750 almost all LH2 complexes are replaced by LH3 when grown at low-temperature and low-light conditions, the 7050 strain responds to low-light conditions by increasing its LH3/ LH2 ratio. Both complexes have a major absorption band at 800 nm but differ in the position of their long-wavelength band, which is at around 850 nm for LH2 and 820 nm for LH3. Despite the spectral differences, LH2 and LH3 have almost identical structures8,9 with the bacteriochlorophyll a (BChl a) pigments arranged in two rings. One ring consists of nine pigments, which absorb at around 800 nm (B800 band) and have relatively large center-to-center distances of 21 Å. The second ring consists of 18 pigments with center-to-center distances of about 9 Å. The pigments of the latter ring absorb at around 850 nm (B850 band) in LH2, while they absorb near 820 nm (B820 band) in LH3, despite the structural similarities. On the basis of the crystal structures of LH3 and LH2 from Rps. acidophila,8,9 it was
10.1021/jp061236d CCC: $33.50 © 2006 American Chemical Society Published on Web 08/24/2006
Spectroscopy on Individual LH3 Complexes proposed that the spectral differences between LH2 and LH3 are caused by shifts of the BChl a site energies. The site energy of the BChl a molecules can be modified by various mechanisms.10 Earlier studies indicated that both the loss of the two hydrogen bonds to the C3-acetyl group of the BChl a molecule11,12 and an altered orientation of this C3-acetyl group13 could account for the blue-shifted absorption band of LH3. On the basis of the recent crystal structure it was suggested that the latter mechanism is dominant.9 Since the C3-acetyl group of a BChl a pigment bound to the β-subunits of LH3 is not hydrogen-bonded, unlike LH2, there is presumably an enhanced freedom of rotation of the C3-acetyl group of these BChl a molecules in LH3. More insight into the underlying mechanism for the observed blue-shifted absorption of LH3 can be gained by applying optical single-molecule spectroscopy. Single-complex experiments at low temperature can provide a direct insight into the parameters determining the electronic structure of these pigment-protein complexes, i.e., the intermolecular interactions and site heterogeneity.14-15 Given the high structural similarity between LH2 and LH3, it is of interest to investigate the relation between the spectral shift of the long-wavelength absorption band from 850 to 820 nm and the electronic structure of the two complexes involved. In this work we present fluorescence-excitation spectra of individual LH3 complexes of Rps. acidophila (strain 7750) at low temperature. The experimental observations reveal a large resemblance to the optical spectra of individual LH2 complexes, consistent with the high structural similarity of both complexes. Although the spectral variations among the spectra are large, the spectra can in general terms be interpreted as localized excitations in the 800 nm region and delocalized excitations in the 820 nm region. For 60% of the studied complexes the delocalized excitations are evidence for a circular exciton. The other 40% of the LH3 spectra show a large variation in terms of the number of bands and their polarization behavior in the 820 nm region. Furthermore, we address the question whether the observed heterogeneous spectral behavior of LH3 from Rps. acidophila (strain 7750) at the single-molecule level can be correlated with a conformational flexibility of the individual complexes. On the basis of the experimental results of the optical singlemolecule studies at low temperature and on numerical simulations we discuss the possibility of light-induced reversible and locally restricted conformational changes within an individual complex and the effect of these changes on the optical properties of the complex. Materials and Methods LH3 complexes of Rps. acidophila (strain 7750) were prepared as described elsewhere.16 The integrity of the sample, especially the efficiency of energy transfer from the B800 to the B820 band, was checked by comparing absorption and fluorescence-excitation spectra of a bulk sample. The LH3 complexes were diluted up to a concentration suitable for singlemolecule spectroscopy (∼5 × 10-11 M) in buffer (either 10 mM Tris, 0.1% LDAO, 1 mM EDTA, pH 8.0 or 20 mM Tris, 0.1% LDAO, pH 8) with either 1% or 1.8% (w/w) purified PVA present. A drop (about 10 µL) of the solution was spin-coated on a LiF substrate by spinning it for 15 s at 500 rpm and 60 s at 2000 rpm, producing high-quality films with a thickness of less than 1 µm. The samples were mounted in a liquid-helium cryostat and rapidly cooled to 1.2 K. The samples were illuminated with a continuous-wave tunable Ti:sapphire laser (Spectra Physics, CA) with a bandwidth of 1
J. Phys. Chem. B, Vol. 110, No. 37, 2006 18711 cm-1. A fluorescence-excitation spectrum of an individual LH3 complex was obtained in two steps. First a wide-field image of the sample was taken by exciting at 800 nm and detecting fluorescence at 845 nm with a liquid-nitrogen-cooled CCD camera (Roper Scientific, NJ). From this image a spatially wellisolated complex was selected. Next, a fluorescence-excitation spectrum of this complex was obtained by first switching to confocal illumination and then scanning the excitation wavelength while recording the fluorescence at 845 nm with an avalanche photodiode (EG&G, Canada). To reject the backscattered excitation light, interference filters (Dr. Hugo Anders, Germany) with a bandwidth of 20 nm centered at 845 nm were used for fluorescence detection. The setup used for singlemolecule spectroscopy experiments on light-harvesting complexes has been described in more detail elsewhere.17-18 To assess and minimize the effect of spectral diffusion,19 the spectra were obtained in rapid succession by repetitive scans over the whole spectral range, storing the different traces separately. With a scan speed of the laser of 4 nm per second and an acquisition time of 10 ms per data point, this yields a nominal resolution of 0.6 cm-1, ensuring that the spectral resolution is limited by the spectral bandwidth of the laser (1 cm-1). To investigate the polarization dependence of the spectra, a λ/2 plate was placed in the confocal excitation path. This plate could be rotated in steps of 1.8°. For each repetitive scan, the excitation polarization was rotated by two steps. In some experiments polarization selectivity was avoided by using circularly polarized light. A typical measurement took 1 h. The excitation intensity was typically in the range of 2-10 W/cm2. The intensity setting was a tradeoff between the need for a good signal-to-noise ratio and minimal spectral diffusion. High intensities resulted in broad, structureless spectra due to more frequent fluctuations in intensity and spectral position. Occasionally, this behavior was present even at low intensities, and no useful substructure in the spectrum could be resolved. Other than these effects, we observed little or no deterioration of the sample in terms of photobleaching. A total of 50 complexes were studied. Data analysis was performed using Labview (National Instruments, USA). The intercomplex heterogeneity was estimated from the width of the distribution of the spectral means νj calculated for every spectrum using19
νj )
∑i I(i)ν(i) ∑i I(i)
(1)
where I(i) is the fluorescence intensity at data point i, ν(i) is the spectral position corresponding to data point i, and the sums runs over all data points of the spectrum. The intracomplex heterogeneity was taken as the mean standard deviation σintra of the intensity distribution calculated for every spectrum using19
σintra ) [ν2 - νj2]1/2
(2)
where ν2 is given by
ν2 )
∑i I(i)[ν(i)]2 ∑i I(i)
(3)
18712 J. Phys. Chem. B, Vol. 110, No. 37, 2006
Ketelaars et al.
Figure 2. (a) Distribution of the spectral mean (νj) of the B800 band of 33 LH3 complexes showing the amount of intercomplex heterogeneity. The bin size is 10 cm-1. (b) Distribution of the standard deviations σintra for the spread of absorption lines in the B800 band of the individual fluorescence spectra for the same 33 LH3 complexes. The bin size is 8 cm-1.
Figure 1. Fluorescence-excitation spectra of four individual LH3 complexes (1-4) of Rps. acidophila (strain 7750). For each complex two spectra are depicted that were observed for either parallel or perpendicular polarization of the excitation light.
The numerical simulations were based on the atomic coordinates of LH3 from Rps. acidophila (strain 7050) taken from the Brookhaven Protein Data Bank (PDB code 1IJD). Results In Figure 1 some representative examples of fluorescenceexcitation spectra of individual LH3 complexes from Rps. acidophila (strain 7750) at low temperature (1.2 K) are shown, in each case for two mutually orthogonal polarizations of excitation. The fluorescence-excitation spectra of individual LH3 complexes largely resemble those of the individual LH2 complexes.15 The differences in spectral features between the 800 and 820 nm regions of the spectra, the number of bands, the bandwidths, and the typical polarization behavior confirm the similarity of the two types of antenna complexes.9,20-21 A detailed analysis of the spectra however reveals various interesting aspects, which were not or to a lesser extent observed in the LH2 experiments. First of all, 40% of the spectra show a very heterogeneous spectral behavior in the B820 region, which prohibits an unambiguous assignment of the observed absorption bands. Second, a significant structure in both the 800 and 820 nm region of the sum spectrum is observed. Finally, large spectral-diffusion effects in both the B800 band and the B820 band are observed. These observations indicate a different sensitivity and stability of the LH3 complexes compared to LH2, at least under single-molecule experimental conditions. The most notable observation in the fluorescence-excitation spectra of individual LH3 complexes is the distinction between the spectral features of the B800 band and the B820 band. B800 Band. In the B800 band the presence of multiple narrow lines indicates weak interactions between the B800 pigments, resulting in excitations that are more or less localized on individual or small groups of BChl a pigments. The line widths of 2-13 cm-1 correspond to excited-state lifetimes on the picosecond time scale, which is consistent with timeresolved21 and hole-burning20 experiments. The spectral dependence of the excited-state lifetimes in the B800 band, as was observed for both LH2 and LH3,19-22 is difficult to determine
from the single-complex spectra of LH3. Although most spectra have a narrow line around 800-805 nm, as can be seen in Figure 1, other bands are rapidly diffusing and/or strongly overlapping with others, making an unambiguous determination of their spectral widths very difficult. Nevertheless, it is possible to determine both the intra- and intercomplex heterogeneity of the B800 band using the same definitions as in van Oijen et al.;19 see also eqs 1-3 of the Materials and Methods section above. The intercomplex heterogeneity is a measure for the sample inhomogeneity resulting in a spread of the spectral means νj of the fluorescence-excitation spectra of the B800 bands of the single LH3 complexes. Figure 2a shows the distribution of νj as determined for the B800 region LH3. For this analysis only spectra of complexes were used where a clear assignment between the 800 and the 820 nm region could be made. The histogram is centered at 12 600 cm-1 with a standard deviation σinter of 43 cm-1. The latter value is an indication for the intercomplex heterogeneity of the LH3 complexes and corresponds to 102 cm-1 full width at halfmaximum (fwhm), somewhat less than the 120 cm-1 observed in LH2.19 The intracomplex heterogeneity or diagonal disorder is a measure of the variation in site energies of the BChl a molecules within a single LH3 complex. Figure 2b shows the distribution of standard deviations σintra for the spread of absorption lines in the B800 band of the individual fluorescenceexcitation spectra. The distribution is centered at 87 cm-1, which gives a value of 205 cm-1 fwhm for the intracomplex heterogeneity (130 cm-1 fwhm in LH219). Remarkably, the intracomplex heterogeneity is significantly higher in LH3 compared to LH2. The fact that the intracomplex heterogeneity is large compared to the nearest-neighbor-interaction strength Vinter (Table 1) of 18.7 cm-1 supports that the interaction between the B800 pigments is weak, resulting in more or less localized excitations. The average number of 4.6 ( 1 observed absorption lines per spectrum is less than the nine pigments in the B800 ring of LH3.9 This apparent discrepancy is most likely due to overlap of transitions in one B800 ring and to the fact that the excitations may occasionally be distributed over a small number of adjacent pigments (exciton coupling), which reduces the number of detected absorption lines. This effect has recently been observed under identical experimental conditions in LH2 complexes from Rps. molischianum.23 B820 Band. About 60% of the complexes that were studied show spectral features in the 820 nm region that strongly resemble those of the B850 band of LH2, i.e., two broad, orthogonally polarized bands. In line with the LH2 results15 we assign these two bands to the kcirc ) (1 states of a circular exciton with their degeneracy lifted by an amount δE(1 (Figure 1). The two orthogonally polarized absorption bands kcirc ) (1 and the narrow feature kcirc ) 0, which is sometimes observed
Spectroscopy on Individual LH3 Complexes
J. Phys. Chem. B, Vol. 110, No. 37, 2006 18713
TABLE 1: Comparison of the Inter- and Intrasubunit Distances, the Angles of the Qy Transition-Dipole Moments of the BChl a Molecules and Their Interaction Energies in LH2 (Strain 10050) and LH3 (Strain 7050) of Rps. acidophilaa intrasubunit intersubunit R β Vintra Vinter (Å) (Å) (deg) (deg) (cm-1) (cm-1) B800 (LH2) B800 (LH3) B850 (LH2) B850 (LH3)
9.6 9.6
21.3 21.3 8.9 8.9
-8.4 -153.6 -8.6 -155.3 -7.8 -7.4 -4.6 -7.4
254 256
-17.2 -18.7 226 210
a The nearest-neighbor interactions between the molecules were calculated using a point-dipole approximation,31 with a dipole strength of 26.8 D2 and a relative permittivity of 1. The Qy transition-dipole moment is assumed to be oriented along the axis running through the NI-NIII nitrogen atoms of the BChl a pigment with a tilt R out of the xy-plane and an angle β with the tangent of the overall circular structure in the xy-plane. The xy-plane is defined as perpendicular to the C9symmetry axis of the LH3 complex.
in the red part of the spectrum, support the contention that the excitation in this band is delocalized over most pigments in the ring. In Figure 3 the spectral properties of the two kcirc ) (1 states, i.e., the high- (kcirc ) 1high) and low-energy (kcirc ) 1low) bands, are summarized. Figure 3a shows histograms of the spectral positions of both bands, which have an average splitting of 160 cm-1. Figure 3b shows the corresponding distribution of the splitting, δE(1, which is centered at 160 cm-1 and has a standard deviation (SD) of 84 cm-1. The distribution is significantly broader than that of the LH2 complexes (Figure 3b, inset), which is centered at around 100 cm-1 with a SD of 40 cm-1.24 The distribution of the angles between the directions of the polarization of the kcirc ) (1 states, φ(1, is depicted in Figure 3c. This distribution shows a strong preference for a mutually orthogonal polarization of the two transitions. Note that this histogram has seven complexes less than those of Figures 3a and 3b, because in those cases the polarization dependence was obtained by rotating the 1/2λ plate in coarse steps of 15°. Although the obtained angles φ(1 were all close to 90°, they are less accurately determined and therefore not included in Figure 3c. A substantial part of the studied complexes (40%) however shows a spectral pattern that does not allow an unambiguous assignment of the kcirc ) (1 states. The spectra of these complexes are all quite different as shown in Figure 4. A small fraction shows spectra like complex 5 (Figure 4, top left) and lacks one of the two orthogonally polarized bands; i.e., only one broad, polarized band is observed in the B820 region. If these spectra still represent a circular exciton, then the signature might be masked either by a tilt of the complex25,26 or by an overlap of part of this spectrum with the detection window. A few complexes show spectra like that of complex 6 (Figure 4, top right) with two, broad, orthogonally polarized bands at around 810-830 nm. However, these two bands are accompanied by a third broad band that absorbs more to the red. This band, which cannot be resolved due to its partial overlap with the detection window, might be related to a rapidly diffusing kcirc ) 0 state. The last group of spectra shows spectral features as depicted for complexes 7 and 8 (Figure 4, bottom). In these spectra a conclusive assignment of the different bands in the 820 nm region is not possible, due to an overlap with the detection window and rapid spectral diffusion. The question can be raised whether the observed spectra are representative for the ensemble of LH3 complexes. Comparison of the sum spectrum of 50 individual spectra (Figure 5, solid line) and the spectrum of an ensemble of LH3 complexes (Figure
Figure 3. Spectral properties of the two kcirc ) (1 states. (a) Distribution of the spectral positions of the kcirc ) 1high and kcirc ) 1low states, representing the high-energy and low-energy exciton state, respectively. The average splitting δE(1 is 160 cm-1. The bin size is 5 nm. (b) The distribution of the splittings δE(1 is centered at 160 cm-1, with a standard deviation of 84 cm-1. The inset shows the distribution for individual LH2 complexes (Rps. acidophila), taken from Ketelaars et al.15 with slight modifications. Both histograms have a bin size of 40 cm-1. (c) Distribution of the angles φ(1 between the directions of the polarization of the kcirc ) (1 states.
Figure 4. Fluorescence-excitation spectra of four individual LH3 complexes of Rps. acidophila (strain 7750) that show spectral features completely different from the complexes presented in Figure 1. The two spectra for each complex correspond to mutually orthogonal polarizations (vertical and horizonal arrows) of the excitation light.
5, dotted line) shows that this might not be the case. The B820 band of the sum spectrum is red-shifted and shows two broad bands. A possible explanation for the observed structure is that
18714 J. Phys. Chem. B, Vol. 110, No. 37, 2006
Figure 5. Ensemble fluorescence-excitation spectra of LH3 complexes of Rps. acidophila (strain 7750) at low temperature. Spectrum of a highly concentrated sample in a PVA film (dotted line). The solid jagged line is the sum of the fluorescence-excitation spectra of 50 individual LH3 complexes.
Figure 6. Correlation between the spectral position of the low-energy kcirc ) (1 state (kcirc ) 1low) and the splitting, δE(1. (a) Individual LH3 complexes (Rps. acidophila). (b) Individual LH2 complexes (Rps. acidophila). The data in part b were taken from ref 15.
there is a preference in the experimental procedure for complexes that have a large splitting δE(1. A hint for the cause of such selectivity is given in Figure 6a where the splitting of the kcirc ) (1 states is plotted as a function of the spectral position of the kcirc ) 1low state. The figure indicates that the complexes with a large splitting δE(1 have their kcirc ) 1low state close to the detection window. The selection of complexes to be studied is apparently biased for these complexes because their emission spectrum probably has a more favorable overlap with the detection filters. This is in contrast to our previous measurements on LH2 (Figure 6b), where no correlation between the splitting and the spectral position of the kcirc ) 1low state is observed, consistent with the good agreement between the sum and bulk spectra.15 Due to a bias for observing LH3 complexes with a large splitting δE(1 (Figure 6), the histogram of Figure 3b should be considered with caution. Since the histogram may represent only a subpopulation of the ensemble it is not possible to draw definitive conclusions on a reduced symmetry of the electronic structure of the B820 band. Spectral Heterogeneity. As already mentioned, the fluorescence-excitation spectra of individual LH3 complexes exhibited much more spectral heterogeneity than those of LH2. This indicates a different sensitivity and/or stability of the LH3 complexes compared to LH2, at least under single-molecule experimental conditions. While in most cases the observed heterogeneous spectral behavior did not lead to clearly defined “substates”, for several complexes reversible spectral jumps of one absorption band could be observed. While these jumps were usually on the order of 10-40 cm-1, a very interesting case of heterogeneous spectral behavior observed for an individual LH3 complex is shown in Figure 7. The left part of the figure depicts a series of 125 fluorescence-excitation spectra of this complex (circularly polarized excitation) where each spectrum is represented by one horizontal line. For this complex, spectral jumps between two distinct configurations A and B were observed. These spectral
Ketelaars et al.
Figure 7. Fluorescence-excitation spectra of an individual LH3 complex of Rps. acidophila (strain 7750) at 1.2 K. The left part of the figure shows a stack of 125 fluorescence-excitation scans of 35 nm recorded at a scan speed of 3 nm/s. The complex was excited with circularly polarized light. The fluorescence intensity is indicated by the gray scale (white ) high intensity). The right part of the figure shows two different spectra (A and B), each corresponding to an average of the scans in the time intervals contained between the horizontal white dashed lines in the left figure (A and B). No background was subtracted, and therefore the transmission of the detection window is visible in the red edge of the spectrum.
configurations are marked on the right side of the plot. The fluorescence-excitation spectra of these two configurations are shown in the right panel of Figure 7. In configuration A two broad bands are observed, which are characteristic for a circular exciton, i.e., the kcirc ) 1high (814 nm) and kcirc ) 1low (826 nm) states, with a relatively large splitting of δE(1 ) 180 cm-1. Studying the complex with linearly polarized excitation light (data not shown) indicated that the mutual angle of polarization of these two bands is orthogonal. In the red leg of kcirc ) 1low a third, weak, narrow line at 827.1 nm is observed that is attributed to the kcirc ) 0 state. This particular feature is not distinguishable in every scan, indicating rapid spectral diffusion. In configuration B the high-energy, broad band is present at exactly the same spectral position as in configuration A (814 nm) with an identical bandwidth and intensity. At around 818 nm a second, weak, broad band is observed. However, this band overlaps with a narrow, very intense line in its red leg at 818.7 nm. The band at 814 nm and the two bands at 818 and 818.7 nm have mutually orthogonal orientations. The large difference of the two spectra in Figure 1 is very remarkable, since they originate from the same pigment-protein complex. The jumping of the complex between configurations A and B is light-induced and shows a very high spectral reproducibility as can be seen in Figure 8. In this figure, the intensity dependence for the spectral range between 770 and 835 nm is shown. The data were obtained using circularly polarized light at four different excitation intensities. At low intensities the complex is mainly in configuration A. Increasing the intensity induces a higher frequency of jumping to state B. At 2 W/cm2 this effect seems to be saturated. Note that the spectral jumping in the 820 nm region does not have any effect on the spectrum in the 800 nm band, an indication for the weak coupling between the B800 and B820 pigments. All spectra of this particular complex were obtained in two consecutive days, which emphasizes the unusually high photostability of these complexes at low temperature. Discussion The experimental observations revealed an overall resemblance to the optical spectra of individual LH2 complexes, consistent with the structural similarity of both complexes. The
Spectroscopy on Individual LH3 Complexes
Figure 8. Stack of 125 fluorescence-intensity scans obtained at a scan speed of 3 nm/s of the same complex as in Figure 7, at four different intensities between 0.5 and 4.0 W/cm2. The complex was excited with circularly polarized light.
spectra could in general be interpreted in terms of localized excitations in the 800 nm region and delocalized excitations in the 820 nm region. Interestingly, the fluorescence-excitation spectra of individual LH3 complexes exhibited much more spectral heterogeneity than those of LH2. Spectral jumps within the highly delocalized excitonic kcirc ) (1 states in the B820 band of LH3 (Figure 7) are a quite remarkable feature and were not observed in the spectra of individual LH2 complexes.14-15 What is the mechanism behind these large jumps observed in the optical spectrum of this particular LH3 complex? Since the main difference between LH2 and LH3 is the conformation of the C3-acetyl group of the β-BChl a pigments it is possible that local conformational changes of this group induce the observed spectral changes. The two spectra representing configurations A and B (Figure 7) show large differences, although they both originate from the same LH3 complex. The shift of the excitonic kcirc ) 0 state between the two configurations is very large, especially considering the average interaction strength (Vavg) between the pigments of 233 cm-1. An overall deformation of the complex or a collective change of multiple pigments seems unlikely to induce an effect of this extent at a temperature of 1.2 K. Also the fact that the band at 814 nm retains its position in both states indicates that the structure of the pigment-protein complex as a whole does not change significantly. It is more likely that the jumps are induced by a highly local conformational flexibility, i.e., a temporary, local site energy perturbation of one of the pigments in the B820 ring. Local structural fluctuations were observed to induce spectral jumps in the B800 region of LH2 from Rps. molischianum,24,27 but up to now such an effect had never been observed to occur for the highly delocalized excitonic bands at low temperature. To gain more insight into the effects of such a local energy perturbation on the optical spectrum of an individual LH3 complex we performed a series of numerical simulations, the results of which are shown in Figure 9. The electronic structure in configurations A and B is likely to be subjected to a 2-foldsymmetric perturbation; since the splitting of the two kcirc ) (1 states is large, their transitions are orthogonally polarized, and no additional high-energy exciton states are observed.15 The
J. Phys. Chem. B, Vol. 110, No. 37, 2006 18715
Figure 9. Simulation of the absorption spectrum of an individual LH3 complex assuming a local change of the site energy of pigment 6 of either -0.75Vavg or +0.50Vavg. These two cases represent configuration A or B of the complex as presented in Figure 7. The overall absorption spectra of the complexes are plotted along the x-coordinate (dashed line) and the y-coordinate (dotted line). The x-axis is chosen along the transition-dipole moment of the kcirc ) 1low state. The right part of the figure shows the Qy transition-dipole moments (arrows) of the BChl a molecules in the C2-deformed LH3 ring in the xy-plane. For both spectra the amplitude of the elliptical deformation was set to δr/r0 ) 8%.29 The dashed circles indicate the position of the local perturbation in the B820 ring. The individual transitions have homogeneous, Lorentzian line shapes with a width (fwhm) of 100 cm-1 (kcirc * 0) and 12 cm-1 (kcirc ) 0) for configuration A and 100 cm-1 (kcirc * 0) and 6 cm-1 (kcirc ) 0) for configuration B. The parameters used for the unperturbed B820 ring are ER ) 233 cm-1, Eβ ) 0 cm-1, Vintra ) 256 cm-1, and Vinter ) 210 cm-1. Interactions up to the second neighbor were included.
origin of such a 2-fold symmetric-perturbation could be either random site heterogeneity with a strong C2 component28 or a C2 modulation of the interaction energies. We assume that this 2-fold-symmetric perturbation is caused by an elliptical deformation of the LH3 complex in analogy to the model of Matsushita et al.29 In addition, we assume the presence of a local site energy perturbation of one of the pigments. The spectra were simulated assuming different degrees of site energy perturbations on the individual pigments in the B820 ring. The best match with the experimental data was obtained assuming a change for pigment 6 of -0.75Vavg in configuration A and of +0.50Vavg in configuration B, relative to the site energies of the other B820 pigments (Figure 9). Taking into account the assumed elliptical deformation of the pigment ring, pigment 6 is not a “random” choice for a modulation of the site energy, because the amplitude of the kcirc ) 1high exciton wave function is minimal at this position, while the kcirc ) 1low exciton wave function reaches its maximum amplitude.29 A local perturbation on pigment 6 will therefore shift the kcirc ) 1low and kcirc ) 0 exciton states to the red and will transfer oscillator strength from the kcirc ) 1low to the kcirc ) 0 state. Furthermore, the spectral position and oscillator strength of the blue, kcirc ) 1high exciton state will not change. As a result, the simulation of the assumed shift in site energy agrees with the main features of the experimental observations. Although the proposed model does not match the experimental observations in every detail, it gives a good qualitative explanation for the spectral jump. It shows that a local change in site energy of one of the pigments in the B820 ring can profoundly modify the collective properties of the ensemble of 18 pigments and induce large shifts of the optical spectrum. In
18716 J. Phys. Chem. B, Vol. 110, No. 37, 2006 our model, the assumed local shift in site energy of 1.25Vavg amounts to approximately 290 cm-1 with Vavg ) 233 cm-1. That the exciton model is still applicable under these conditions was previously shown for LH2 from Rps. acidophila, where the variations in site energies were distributed with a fwhm of 250 cm-1, exceeding the average interaction strength Vavg of 240 cm-1.15 Gudowska-Nowak et al.13 have shown that rotation of the C3-acetyl group of BChl a can induce changes in the site energy of the pigment of up to 25 nm (∼360 cm-1). Unlike in LH2 from Rps. acidophila, the C3-acetyl group of the BChl a pigment bound to the β-subunit of LH3 is not stabilized by a hydrogen bond9 and could be more susceptible to rotation than its counterpart of the R-subunit. Rotation of this group seems to be induced by the laser excitation, which also accounts for the intensity dependence of the effect. Depending on the extent of rotation and the time scale on which the rotation between different conformations takes place, this conformational flexibility can occasionally result in optically clearly distinguishable conformations as described here. Whether this effect is experimentally observable or not depends on different parameters. If the time scale of the conformational changes is too fast or slow to be resolved and/or if there is only a minor rotation of the C3-acetyl chain, then these changes will lead to increased broadening and heterogeneity in the optical spectra. The effect can also be masked for experimental observation when more than one pigment in the ring ensemble undergoes light-induced conformational changes. These multiple conformational changes would also result in increased broadening and spectral heterogeneity.27 These conditions are in fact far more likely to occur than the exceptional case that we discuss here. Nevertheless, this special case provides a direct insight into how the optical properties of pigment-protein complexes can be altered as a consequence of local conformational changes of just one pigment. The light-induced conformational flexibility in the pigmentprotein complex will not only change its absorption spectrum but may also induce significant changes in its emission spectrum. Large spectral jumps of the emission spectrum were recently observed for LH2 at room temperature.30 Such changes could result in a reduction of the overlap of the emission spectrum with the detection window and will lead to selectivity in the measuring procedure, which in fact was observed during the experiments. Furthermore, the conformational flexibility could also increase the susceptibility of the complex to spectral diffusion. Since the spectral diffusion is clearly light-induced and reversible (Figure 8) it is most likely caused by dissipation of excitation energy into local vibrations surrounding the BChl a pigments. If such a pigment is by chance already in a less favorable conformation, then the excess energy might induce conformational changes more easily on a very local scale, inducing heterogeneity in the individual spectra. Conclusions Fluorescence-excitation spectra of individual LH3 complexes were presented providing direct insight into the electronic structure of the complexes at the single-molecule level. The spectra confirm that the observed blue shift of the B820 absorption band in LH3 compared to the B850 absorption band in LH2 cannot be due to changes in the excitonic coupling but must be due to changes in the site energy of the BChl a pigments. In general, the spectral behavior of LH3 at the singlemolecule level highly resembles that of the previously studied LH2.14-15 In the B800 region narrow bands were observed,
Ketelaars et al. indicating that the excitations are localized on individual or small groups of BChl a pigments. In the B820 region a limited number of relatively broad bands is observed, which can be interpreted in terms of delocalized excitations. Besides the many similarities with LH2, the LH3 complexes also show some distinct differences such as larger splitting, δE(1, in the B820 band and larger spectral heterogeneity. Lightinduced, local perturbations of individual BChl a molecules in the B820 ring are thought to contribute to the heterogeneity of the optical properties of LH3. By employing optical single-molecule studies in combination with numerical simulations we have shown that LH3 complexes from Rps. acidophila (strain 7750) can exhibit highly localized conformational flexibility. This flexibility was assigned to the light-induced rotation of the C3-acetyl group of an individual BChl a pigment within the ring of the 18 excitonically coupled B820 pigments in an LH3 complex. We could show that a conformational change of an individual pigment can alter the optical properties of the whole ring ensemble. Depending on the extent and time scale of the conformational change, this will result either in optically clearly distinguishable conformational substates or in a highly heterogeneous spectral behavior. Finally, we note that the conformational stability is distinct from the functional stability, e.g., in terms of overall fluorescence intensity. Indeed, Figure 8 is taken from a series of measurements on the same single complex over a period of 2 days, illustrating the extraordinary functional stability (related to the absence of oxygen) typical of these complexes under the conditions of the experiment. In this respect, LH2 and LH3 are very comparable. Acknowledgment. This work is part of the research program of the Stichting voor Fundamenteel Onderzoek der Materie with financial aid from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) and is further supported by the “Section Earth and Life Sciences” of the NWO and by the Volkswagen-Stiftung (Germany, Hannover). S.O. acknowledges a Marie-Curie-fellowship from the European Union. R.J.C. thanks the Biotechnology and Biological Sciences Research Council (BBSRC) for financial support. M. van der Pijl, John van Egmond, and Willem Versluijs are gratefully acknowledged for excellent support in electronics and mechanics, respectively. We also thank A. van Oijen for useful discussions during the implementation of the setup. M.K. and J.-M.S. contributed equally to this work. References and Notes (1) Pettigrew, G. W.; Moore, G. R. Cytochromes c: Biological Aspects; Springer-Verlag: Berlin, 1987. (2) Ort, D. R.; Yocum, C. F. Oxygenic Photosynthesis: The Light Reactions; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1996. (3) Blankenship, R. E. Molecular Mechanisms of Photosynthesis; Blackwell Science: Oxford, U. K., 2002. (4) Zuber, H.; Cogdell, R. J. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; pp 315-348. (5) Hu, X.; Ritz, T.; Damjanovic, A.; Autenrieth, F.; Schulten, K. Q. ReV. Biophys. 2002, 35, 1. (6) Gardiner, A. T.; Cogdell, R. J.; Takaichi, S. Photosynth. Res. 1993, 38, 159. (7) Angerhofer, A.; Cogdell, R. J.; Hipkins, M. F. Biochim. Biophys. Acta 1986, 848, 333. (8) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature 1995, 374, 521. (9) McLuskey, K.; Prince, S. M.; Cogdell, R. J.; Isaacs N. W. Biochemistry 2001, 40, 8783.
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