On B800-B800 energy transfer in the LH2 complexof purple bacteria

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1 Journal of Luminescence 98 (2002) On B800-B800 energy transfer in the LH2 complexof purple bacteria Valter Zazubovich, Ryszard Jankowiak, Gerald J. Small* Ames Laboratory USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA Abstract The B800 Q y -states of the light harvesting 2 (LH2) photosynthetic complex of purple bacteria exhibit two relaxation channels. One is B800-B850 excitation energy transfer that occurs in B2 ps in low-temperature limit. This channel is dominant for excitation wavelengths located to lower energy of the B800 absorption band maximum. The second additional channel sets in for excitation on the high-energy side of the maximum. The mechanism for the additional channel has been controversial. That it is due to relaxation of Bacteriochlorophyll a (Bchl a) vibrational modes has already been excluded. This left intra-band B800-B800 energy transfer and relaxation of mixed B800 B850 exciton levels as possible mechanisms. High-pressure hole burning results for LH2 of Rhodopseudomonas acidophila are presented, which, together with excitonic calculations, indicate that the latter mechanism is relatively unimportant. Intra-band B800-B800 excitation energy transfer is considered from a probabilistic viewpoint. It appears that the B800 zero-phonon hole action spectrum corresponds to those of Bchl a sites on the C 9 ring that cannot undergo downward energy transfer to a nearest-neighbor site. One-third of the sites fall into this category. It is concluded that the additional decay channel is most likely due to above intra-band transfer. r 2002 Published by Elsevier Science B.V. Keywords: Light-harvesting complex; Non-photochemical hole burning; High pressure; Energy transfer 1. Introduction Some years ago our group began using nonphotochemical hole burning (NPHB) spectroscopy combined with high hydrostatic pressure at low temperatures to probe the excitonic structure and intermolecular interactions between chlorophyll molecules in photosynthetic antenna complexes [1]. Examples of systems studied since then are the light-harvesting complexii of photosystem II of green plants [2], reaction center of photosystem II *Corresponding author. Tel.: ; fax: address: gsmall@ameslab.gov (G.J. Small). [3], the LH1 [4] and LH2 [5 7] of purple bacteria, photosystem I of cyanobacteria [8,9] and chlorosome antenna of green sulfur bacterium Chlorobium tepidum [10]. Taken as a whole, the results show that when the linear pressure shift rates for an exciton level in chlorophyll aggregate is higher than B 0.25 cm 1 /MPa, electron-exchange coupling between nearest-neighbor Chls is important. Such coupling, in contrast to electrostatic coupling, can induce significant charge transfer character to the exciton states, which, unless symmetry dictates otherwise, leads to large permanent dipole moment. Stark hole burning spectroscopy is a powerful tool for measuring the dipole moment change associated with the S 0 -S /02/$ - see front matter r 2002 Published by Elsevier Science B.V. PII: S (02)

2 124 V. Zazubovich et al. / Journal of Luminescence 98 (2002) (Q y ) transitions of Chl aggregates [11]. Importantly, there is a strong positive correlation between the linear pressure shift rate, permanent dipole moment change and linear electron phonon coupling strength associated with the spectral transition (see Ref. [8] and references therein). We note that utilization of pressure improves the resolution of NPHB spectroscopy [8,9]. Very recently, we carried out a detailed study of how pressure affects bacteriochlorophyll (Bchl) Bchl excitonic couplings and the site energy distribution function (SDF) of Bchl molecules in the LH2 complexof Rps. acidophila [12]. In this paper, the results from that paper and new results are used to address the problem of the mechanism of excitation energy transfer between B800 molecules of that complex. Before defining the problem a brief description of the LH2 structure is in order. The X-ray structure revealed that LH2 carries cyclic C 9 symmetry with the B800 and B850 Bchl molecules located on the opposite sides of the membrane [13]. (B800 and B850 indicating absorption at 800 and 850 nm at room temperature.) The B850 ring is a 9 mer of Bchl a dimers with strong excitonic coupling between nearest neighbors, B300 cm 1, see Ref [14] for a review. The B800 ring is a 9 mer of monomers with weak nearest-neighbor couplings (B25 cm 1 ) due to large B21 A center to center distances between them. The magnitude of energy disorder (diagonal and/or off-diagonal) relative to the coupling determines the extent to which the B800 and B850 excited states are delocalized. (Temperature is also a factor due to the exciton phonon couplings.) It is generally agreed that the former are highly localized while the latter are delocalized, although the extent of delocalization is still controversial [15]. Fig. 1 shows the 4.2 K absorption of LH2. The vertical lines below locate the exciton levels of the B850 ring. C 9 symmetry dictates that two levels are non-degenerate (A) while the others are doubly degenerate (E), see Ref. [16]. The subscripts l and u indicate the levels that stem mainly from the lower and upper Q y levels of the basic dimer. In the absence of disorder, all levels except E 1l level are, for all intent and purposes, forbidden in absorption. Note that there are several u-levels in the near vicinity of the B800 band. (b) (a) B The exciton level structures in Fig. 1 were calculated using the theory of Wu et al. [16,17], in which the nearest dimer dimer coupling approximation is used. In the absence of energy disorder: E j l ¼ E l þ 2V l cosð2pj=nþ ð1þ for lower manifold and Eu j ¼ E u þ 2V u cosð2pj=nþ WAVENUMBER (cm -1 ) B850 A u E 1u E 4l E 2u E 3u x E 2l E 1l A l A u E 1u * E 3u x E 2l E 1l A l WAVELENGTH (nm) Fig. 1. Absorption spectrum of LH2 at 5 K and normal pressure and B850 excitonic level structures in the absence of disorder for (a) V l ¼ 200 cm 1, V u ¼ 100 cm 1, V ul ¼ 130 cm 1 and E u E l ¼ 600 cm 1 [16] and (b) V l ¼ 300 cm 1, V u ¼ 100 cm 1, V ul ¼ 130 cm 1 and E u E l ¼ 600 cm 1. For level structure (b), V l was increased to account for experimentally observed gap between B850 and lowest-energy band B870 [18]. The asterisk denotes closely spaced E 2u and E 4l levels. X denotes closely spaced E 4u and E 3l levels. Both level structures are shifted to have strongly absorbing E 1l level coincident with the B850 band maximum. ð2þ for upper manifold. E l and E u are the energies of the lower and upper levels of the basic dimer. The

3 V. Zazubovich et al. / Journal of Luminescence 98 (2002) coupling between the upper and lower levels is given by H j int ¼ 2V ul cosð2pj=nþ: ð3þ In the above equations n ¼ 9 and j runs from 4 to4. The degenerate E 1l level corresponds to j ¼ 71: The parameter values used to calculate the level structure (a) in Fig. 1 are from Ref [16] (based on room temperature data [14]) and are given in the figure caption. For level structure (b), V l was increased to account for the gap between the B850 band and lowest-energy band B870 experimentally observed at liquid helium temperature [18]. We note that other calculations also place upper (u) exciton levels in the near vicinity of B800 band [19]. With the above background material in mind, we return to the relaxation dynamics of the B800 ring that follows excitation at different wavelengths within B800 band. At low temperatures, both hole burning [5,20] and fs pump probe [5] experiments have shown, for both Rps. Acidophila and Rb. Sphaeroides, that excitation of the B800 band at wavelengths located at the B800 band maximum and to lower energies yielded a constant total dephasing time. The pump probe experiments proved that the dephasing time at liquid helium temperatures is due to B800-B850 energy transfer that occurs in B2 ps. However, the hole burning results as well as those from spectroscopic studies of single LH2 complexes [21] showed that the dephasing time shortens, approximately linearly, as the excitation wavelength is tuned to the blue of the B800 band maximum. This has been referred to as the additional decay channel of the B800 molecules. It was proven that this channel is not due to vibrational relaxation of modes that build on the origin band of B800 and B850 molecules [5]. In the same work it was also shown that it is not due to direct excitation of the upper exciton levels of B850 ring. This left only two explanations for the additional decay channel. One is that it is due to intra-band B800-B800 transfer involving only B800 molecules. The other is that it is a manifestation of coupling between the B800 states and the upper exciton levels of B850 ring [5,22]. This mixing could facilitate relaxation of the mixed states to lower exciton levels of B850 Table 1 Excitonic couplings, their pressure dependencies and monomer energy shift rate from [12] Parameter V l (0.1 MPa) V u (0.1 MPa) V ul (0.1 MPa) E u E l (0.1 MPa) dv l =dp dðe u E l Þ=dp dv u =dp a dv ul =dp a Bchl a monomer energy shift rate ring or a faster energy transfer within the B800 band. It was suggested in Ref. [22] that this mechanism is more likely. In this paper we use results from Ref. [12] pertaining to the pressure dependencies of the parameters in Eqs. (1) (3) (Table 1), new highpressure hole-burning data and statistical arguments to argue that the possible mixing between the B800 and B850 states is of no importance for explaining the additional decay channel. 2. Experimental Value 260 cm cm cm cm cm 1 /MPa 0.40 cm 1 /MPa 0.15 cm 1 /MPa 0.15 cm 1 /MPa cm 1 /MPa a Estimated assuming that dv u =dp¼ dv ul =dp¼ dv l =dp: Purified LH2 complexes from Rps. Acidophila were prepared according to van Oijen et al. [22]. Samples were dissolved in 20 mm Tris HCl buffer (ph=8) containing 0.1% of LDAO detergent and mixed with glycerol at a ratio of 1:2. Gelatin capsules containing the sample were placed into Unipress high-pressure cell with optical windows, connected to a U11 three-stage hydraulic compressor through a flexible alloy capillary with inner diameter of 0.3 mm. Helium gas was used as the pressure-transmitting medium. For details of the high-pressure apparatus see Ref. [1]. The highpressure cell was cooled in a Janis 11DT helium cryostat. Temperature was measured and stabilized with a Lakeshore Cryotronic model 330 temperature controller. Unless otherwise mentioned the sample temperature was B7 K. The laser used for spectral hole burning was a CW

4 126 V. Zazubovich et al. / Journal of Luminescence 98 (2002) Coherent 899 Ti-Sapphire tunable laser (linewidth of 2 GHz) pumped by Coherent Innova 200 Ar-ion laser. A Bruker HR 120 Fourier transform spectrometer was used for recording pre- and post-burn absorption spectra with resolution of 0.5 cm 1. Holes were burned into the B800 absorption band at 7 11 different wavelengths at pressures of 0.1, 290, 610, 875 and 1015 MPa. The burn fluences used are given in the figure captions. 3. Results The solid and dashed curves in Fig. 2 are the B800 absorption profiles at ambient pressure and 875 MPa, respectively. The solid squares are zerophonon hole (ZPH) widths produced by excitation at wavelengths located across the B800 band. The solid circles are the ZPH widths for a pressure of 875 MPa. The solid vertical arrows mark the onset of the broadening of the ZPH. For all pressures used, the offset of the broadening to the blue from the B800 maximum is 2775cm 1. Thus, the HOLE WIDTH (cm -1 ) cm cm WAVENUMBER (cm -1 ) Fig. 2. Absorption spectra and wavelength dependencies of the hole width for ambient pressure (solid curve, squares) and for 875 MPa (dashed curve, circles). Lower-energy holes (width B4cm 1 ) were burned with 25 mw/cm 2 for 100 s (2.5 J/cm 2 ). Burn fluences of up to 20 J/cm 2 were required to burn observable holes one the high-energy side of B800 band. offsets of 25 and 32 cm 1 shown in the Fig. 2 are equal within the experimental uncertainty. The relevance of the independence of the offset on pressure to the additional B800 decay channel is discussed in the following section. The constant hole widths (for shallow burns, fractional depth o0.03) on the low-energy side of B800 band were found to be essentially independent of pressure between 0.1 and 1015 MPa. Widths ranged between 4.0 and 4.5 cm 1, corresponding to B800-B850 transfer times of ps. However, the uncertainty in the hole width is 70.5 cm 1. It should be noted that the energy gap (DE) between the B800 and B850 bands increases from B960 cm 1 at 0.1 MPa to B1420 cm 1 at 1015 MPa. As pointed out in earlier work [5,6], the resilience of the transfer rate to pressure-induced changes in DE is due to the homogeneous width of the spectral density being very large. It is contributed by B850 exciton levels and Bchl vibrations and protein phonons that build on them. Relevant for what follows is that the linear pressure shift rate of ZPH burned across the B800 band is almost constant, 0.13 cm 1 /MPa on average (results not shown). The solid data points in Fig. 3 are the depths of ZPH burned under constant burn fluence conditions (burn intensity of 25 mw/cm 2 and time of 100 s) at ambient pressure. The maximal fractional hole depth is The dashed curve is a Gaussian fit normalized to account for 30% of integrated intensity of the B800 band (solid curve), see Discussion. 4. Discussion We begin by considering whether or not the observation that the displacement between the onset of the additional decay channel and the B800 band maximum (the offset referred to earlier) is independent on pressure (see Fig. 2) is consistent with the hypothesis that mixed B800 B850 states are responsible for the additional decay channel. (It has been suggested that these states, located predominantly on the high-energy side of the B800 band, might lead to increased rates of energy transfer within the B800 manifold and

5 V. Zazubovich et al. / Journal of Luminescence 98 (2002) Table 2 Pressure shift rates of the levels of B850 in the vicinity of B800 band 0.4 Level Pressure shift rate a (cm 1 /MPa) Pressure shift rate b (cm 1 /MPa) ABSORBANCE A u E 1u E 4l E 2u E 3u a Calculated assuming that dv u =dp¼ dv ul =dp¼ dv l =dp: b Calculated assuming that dv u =dp : dv ul =dp : dv l =dp ¼ V u : V ul : V l : WAVENUMBER (cm -1 ) Fig. 3. B800 absorption spectrum (solid line) and the wavelength dependence of the depth of spectral holes burned with constant burn fluence of 2.5 J/cm 2 (ZPH action spectrum) at ambient pressure (diamonds). The dashed curve is Gaussian fit to action spectrum and its intensity is adjusted to account for 30% of B800 band integral absorption, see text. The dotted spectrum is the difference between the absorption spectrum and the dashed curve. It represents the absorption profile of Bchl a molecules capable of downward energy transfer to nearestneighbor Bchl a molecules. B800-B850 transfer [5].) To this end we performed excitonic calculations to determine the pressure dependencies of the energies of upper exciton levels of the B850 ring, the objective being to compare their linear pressure shift rates with the shift rate of B800 Q y -states, vide supra. The results from Ref. [12] given in Table 1 were used, where E u ; E l ; V u ; V l and V ul are defined by Eqs. (1) (3). The values of dv l =dp ¼ 0:15 cm 1 /MPa and dðe u E l Þ=dpÞ ¼0:40 cm 1 /MPa were determined using high-pressure data on the E 1l and A l levels. Note that the latter rate results in de u =dp ¼ 0:20 cm 1 /MPa. The pressure dependencies of V u and V ul were determined as described in the footnote a. The shift rate for monomer Bchl a was set equal to the shift rate observed for spectral holes burned at the low-energy side of the B800 band, cm 1 / MPa [12]. The results are given in the second column of Table 2. The key finding is that all of the upper exciton levels of B850 ring exhibit positive shift rates. Thus, they cannot follow the pressureinduced shifting of the B800 band that occurs with a rate of cm 1 /MPa (and of ZPH burned within that band). The absence of tracking is inconsistent with mixed B800 B850 states being mainly responsible for the additional decay channel since the aforementioned offset (from the B800 band maximum) where broadening of ZPH begins is independent of pressure. The rates given in the third column were calculated assuming that dv u =dp : dv ul =dp : dv l =dp ¼ V u : V ul : V l : Again, they are positive. Additional support for the mixed states being unimportant comes from the observation that the pressure shift rates of ZPH burned across the B800 band are weakly dependent of wavelength (data not shown). We turn next to the question of whether the additional decay channel can be understood in terms of B800-B800 energy transfer involving only B800 molecules. In earlier work from our group [5,22] it was suggested that this is unlikely. In Ref. [22] the function Fðo ex Þ¼ Z oex N do gðoþdðo ex oþ ð4þ upon which the F.orster-type energy transfer rate (with energy disorder taken into account) is proportional to, was considered. gðoþ is the site distribution function (SDF) and Dðo ex oþ is the spectral density in the rate expression for sites excited at o ex that transfer energy to lower-energy

6 128 V. Zazubovich et al. / Journal of Luminescence 98 (2002) sites. It was found that whether Dðo ex oþ is smooth and slowly varying function or a window-type function, hole width data shown in Fig. 2 cannot be explained. However, it was implied in Ref. [22] that except for the lowestenergy site of each B800 ring, all sites (excited) have a nearest neighbor that lies lower in energy. (The structure of the B800 ring and the inverse 6th power dependence of F.orster transfer rate on the distance between donor and acceptor molecules results in the energy transfer rate between nearest neighbors being B40 times higher than between next-nearest neighbors.) Here, we show, using combinatorics, that this assumption is invalid. Given energy disorder due to structural heterogeneity, it is highly improbable that any of the nine Q y excitation energies in a B800 ring could be equal. Thus, we assume the absence of accidental degeneracies in what follows. (We also make the reasonable assumption that energy transfer between nearest neighbors dominates the energy transfer kinetics.) We denote the sites by L n ; n ¼ 1; y; 9; where n ¼ 1 for the lowest-energy site in any given ring. Obviously, and independent of any model, 1/9 of the sites in an ensemble (L 1 -sites) are incapable of downward energy transfer in the B800 manifold. Thus, their excitation decay is determined by B800-B850 energy transfer. Concerning L 2 ; the probability that L 1 is its nearest neighbor is 2/8, thus the probability of L 2 sites incapable of downward energy transfer is 3/4. It is not difficult to show that the probability of site L n being incapable of downward transfer is (for n ¼ 1; y; 7) given by 9 * 2 * 6! * C 2;9 n =9!; where C 2;9 n ¼ð9 nþ!=2!ð9 n 2Þ!: Thus, for example, this probability is 1/28 for n ¼ 7 (third highest energy). Note that the probability for n ¼ 8 and 9 is zero. Totally, 1/3 of all B800 molecules are incapable of B800-B800 transfer, which explains why the intensity of the ZPH spectrum (dashed curve) in Fig. 3 was adjusted to account for 30% of the intensity of the B800 band. The majority of these molecules will absorb to the red of the B800 maximum. Note that the fit to the low-energy tail of the B800 band is good. The dotted curve in Fig. 3 is the distribution of the sites capable of undergoing downward transfer to the nearest neighbors. Since this curve extends into the region of dashed curve, the question arises as to whether it is consistent with the hole width data presented in Fig. 2, which show that the hole width is constant at lower energies. There is no inconsistency, since the measured holes were shallow and the hole-burning efficiency of the sites associated with the additional decay channel is about a factor of 8 lower than for sites that are not. That is, a broad contribution to the hole profiles from the former sites would not have been observable. The results in Fig. 3 qualitatively explain the hole width data in Fig. 2. Detailed calculations of the Q y electronic structure and intra-band energy transfer rates of B800 ring, that take into account energy disorder, together with data from further single complexspectroscopic studies, are required to determine if B800-B800 intra-band transfer mechanism can quantitatively account for the results in Fig. 2. Nevertheless, we believe that results presented here indicate that this mechanism is of primary importance and that the mixing between the B800 and B850 states is of secondary importance. Acknowledgements Research at the Ames Laboratory was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, US Department of Energy. Ames Laboratory is operated for USDOE by Iowa State University under Contract W-7405-Eng-82. We thank S. Matsuzaki for help in setting up the high-pressure experiments and Professor R.J. Cogdell, University of Glasgow, for providing LH2 samples. References [1] N.R.S. Reddy, G.J. Small, in: Biophysical Techniques in Photosynthesis, Kluwer Academic Publishers, Dordrecht, 1995, pp [2] J. Peiper, M. R.atsep, R. Jankowiak, K.-D. Irrgang, J. Voigt, G. Renger, G.J. Small, J. Phys. Chem. A 103 (1999) [3] H.-C. Chang, R. Jankowiak, N.R.S. Reddy, G.J. Small, Chem. Phys. 197 (1995) 307.

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