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J. Phys. Chem. 1996, 100, 2438-2442
Energy Transfer and Exciton Coupling in Isolated B800-850 Complexes of the Photosynthetic Purple Sulfur Bacterium Chromatium tepidum. The Effect of Structural Symmetry on Bacteriochlorophyll Excited States John T. M. Kennis,*,† Alexander M. Streltsov,†,‡ Thijs J. Aartsma,† Tsunenori Nozawa,§ and Jan Amesz† Department of Biophysics, Huygens Laboratory of the UniVersity of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands, and Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku UniVersity, Sendai 980, Japan ReceiVed: August 24, 1995; In Final Form: NoVember 2, 1995X
Energy transfer and exciton coupling in isolated B800-850 complexes from the purple sulfur bacterium Chromatium tepidum were studied by means of spectrally resolved absorbance difference spectroscopy with a time resolution of 200 fs. Energy transfer from bacteriochlorophyll (BChl) 800 to BChl 850 was found to occur with a time constant of 0.8-0.9 ps. Remarkably, the amplitude of the absorbance changes of BChl 850 was 4 times larger than that of BChl 800. By relating this result to the crystal structure of B800-850 complexes of Rhodopseudomonas acidophila (MacDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs; N. W. Nature 1995, 374, 517), it was concluded that the spectral properties of BChl 850 are mainly determined by strong exciton interactions between BChl 850 molecules in a circular symmetric arrangement, which lead to concentration of the oscillator strength in a few optically allowed transitions, corresponding to delocalized eigenstates. In BChl 850, a rapid red shift of the bleaching was observed. This relaxation process may be ascribed either to vibrational relaxation or exciton scattering. A similar red shift appears to occur in BChl 800.
Introduction During the last years it has become clear that energy transfer in photosynthetic antenna systems is very fast and occurs on a time scale of 1 ps or less. An example is given by the B800850 complex of the purple non-sulfur bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris, where the decay time of excited bacteriochlorophyll (BChl) 800 was found to be slightly less than 1 ps by time resolved absorbance spectroscopy.1,2 However, these time constants, as far as we know, have not been confirmed by measurements of the rise time of BChl 850. Hole-burning experiments have indicated a somewhat longer lifetime of about 2 ps at low temperature.3,4 Time-resolved studies of energy trapping by the reaction center have pointed to an interesting phenomenon. It appeared that the value of absorption changes induced by exciting the light harvesting (LH) 1 antenna exceeded by far those by photooxidation of the primary donor.5-7 Moreover, it was found that the absolute value of absorption changes upon excitation of the LH1 antenna corresponded to the bleaching of several monomeric BChls.8 These results were explained by a model in which the BChl molecules in LH1 are arranged as a circular oligomer, with strong dipolar interactions between the BChls.9 The recent X-ray structure of the B800-850 complex of Rhodopseudomonas acidophila10 has shown that in B800-850 complexes, such a model may be applicable to BChl 850 as well, whereas the BChl 800 molecules can be considered as monomers. Thus, time-resolved measurements of energy transfer from BChl 800 to BChl 850 give a possibility to directly check the points mentioned above. * Corresponding author. Telephone 31-71-5275982, FAX 31-71-5275819, e-mail:
[email protected]. † Huygens Laboratory of the University of Leiden. ‡ Permanent address: Institute for Physical and Chemical Biology, Moscow State University, 119899 Moscow, Russia. § Tohoku University. X Abstract published in AdVance ACS Abstracts, January 1, 1996.
0022-3654/96/20100-2438$12.00/0
In this paper we describe the results of a femtosecond study on the isolated B800-850 complex of the purple sulfur bacterium Chromatium tepidum. Energy transfer from BChl 800 to BChl 850 was found to occur in 0.8-0.9 ps. From the relative magnitudes of the absorbance difference signals of excited BChl 800 and BChl 850 we conclude that the spectral properties of BChl 850 are indeed largely determined by exciton interactions between BChl molecules in a symmetric arrangement. Materials and Methods Cells of Chr. tepidum were grown, chromatophores were prepared and B800-850 complexes were isolated as described earlier.11 The B800-850 complexes were diluted in a TrisTGA buffer (pH 8.4) to an absorbance of about 0.7 at the maximum of the BChl 800 band. Time-resolved absorbance difference spectroscopy was performed by means of a home-built amplified femtosecond laser system with optical multichannel analyzer (OMA) detection. Ultrashort pulses were supplied by a hybridly mode-locked dye laser, which was synchronously pumped by the frequencydoubled output of an actively mode-locked Nd:YAG laser (Spectron Laser Systems). It produced pulses with a length of about 100 fs at a central wavelength of 620 nm at a frequency of 76.6 MHz. Amplification of the ultrashort pulses took place in two dye amplifiers, pumped by a home-built Q-switched Nd: YAG laser operating at a frequency of 10 Hz. In the first stage, an amplification of 104 was achieved in a multipass amplifier. Subsequent amplification by a factor of 20 took place in a Bethune cell with a length of 2 cm. The beam was subsequently focused in a glass cuvette of 1 cm path length containing a low concentration of rhodamine 640 in methanol, in order to generate a continuum, which was sent into a pump-probe setup. Part of the continuum was amplified in a glass flow cuvette with a length of 1 cm containing the dye LDS 821, transversely © 1996 American Chemical Society
Energy Transfer in Isolated B800-850 Complexes
J. Phys. Chem., Vol. 100, No. 6, 1996 2439
Figure 1. Absorption spectrum of B800-850 complexes of Chr. tepidum (solid line) and the spectrum of the excitation pulse (broken line).
pumped by the Q-switched Nd:YAG laser. Then it was passed through an interference filter to produce an excitation pulse centered around the desired pump wavelength. This was sent into a variable delay line and focused in the sample. The other part of the continuum was split into a probe beam and a reference beam. The probe beam was focused and overlapped with the excitation beam in the sample and sent into an OMA (EG&G PAR). The reference beam did not pass through the sample and was led directly into the OMA. To determine the exact pump wavelength, the sample was replaced by a 1 mm cuvette containing a scattering medium, and the light of the pump beam scattered along the path of the probe beam was recorded by the OMA. The cross correlation of the system in the infrared after continuum generation and amplification had a width of 200 fs. During the experiments, about 1.5% of the BChl molecules were excited with each excitation pulse, and a few thousand spectra per delay were collected. All measurements were performed at room temperature.
Figure 2. Absorbance difference spectra upon excitation at 797 nm with pump and probe polarized under the magic angle at delays (a, top) between -0.33 and 0.6 ps and (b, bottom) between 0.6 and 3.0 ps.
Results Figure 1 shows the absorption spectrum of the B800-850 complex of Chr. tepidum, along with the spectrum of the pump pulse. The absorption spectrum of the B800-850 complex shows the characteristic Qy transitions peaking at 800 and 855 nm. The spectrum of the pump pulse, with a maximum at 797 nm, showed little overlap with the BChl 850 band, which means that BChl 800 was selectively excited. Figure 2a shows the time-resolved spectra upon excitation at 797 nm with pump and probe beam polarized at the magic angle at increasing delays between -0.33 and 0.6 ps. Around zero delay, a bleaching near 800 nm developed, which is due to formation of excited BChl 800. The bleaching is due to a decrease of absorption, caused by ground state depletion and to stimulated emission from the excited level to the ground state. At 0.13 ps the bleaching reached its maximum value. In Figure 2b, the time-resolved spectra recorded at delays from 0.6 to 3 ps are shown. At increasing delays, the amplitude of the bleaching near 800 nm progressively decreased, whereas a bleaching appeared around 860 nm, indicating energy transfer from BChl 800 to BChl 850. This bleaching was accompanied by an absorbance increase at wavelengths shorter than 842 nm. After a few ps, energy transfer from BChl 800 to BChl 850 was complete, and the absorbance difference spectrum was now entirely due to excited BChl 850. The bleaching, with a maximum at the long-wavelength side of the absorption band, can be attributed to ground state depletion of BChl 850 combined with stimulated emission. The absorbance increase
Figure 3. Kinetics of absorbance changes upon excitation at 797 nm with pump and probe polarized under the magic angle at (a, top) 802 nm and (b, bottom) 860 nm. The kinetic trace at 802 nm is fitted with a single decay of 0.8 ps. The kinetic trace at 860 nm is fitted with a rise time of 0.8 ps during the first 3 ps after the excitation pulse. A convolution with a 200 fs wide (sech)2 function was used for all fits.
at wavelengths shorter than 842 nm can be assigned to excited state absorption by BChl 850. The spectrum at 3 ps is similar to that obtained at picosecond time resolution with membrane fragments of Chr. tepidum.12 Its shape is typical for the excited state of various spectral forms of BChl a in antennae of purple bacteria6,7,12-14 and has been explained in various ways.9,14 It is obvious that the final bleaching near 860 nm was considerably larger than the maximum bleaching near 800 nm. We shall return to this point in the Discussion section. Figure 3a shows the kinetics at 802 nm derived from the timeresolved spectra. It displays a bleaching which decayed with a
2440 J. Phys. Chem., Vol. 100, No. 6, 1996
Figure 4. Absorbance difference spectra upon excitation at 797 nm with pump and probe polarized parallel at delays (a, top) between -0.08 and 0.58 ps and (b, bottom) between 0.58 and 5.25 ps.
single time constant of 0.8 ps into an absorbance increase. The kinetics at 861 nm showed a rise time of 0.8 ps (Figure 3b). After about 3 ps, the signal decreased somewhat, which is presumably a consequence of singlet-singlet annihilation within the complex or between aggregated complexes. These results show that energy transfer from BChl 800 to BChl 850 occurs with a time constant of 0.8 ps, which is essentially the same value as those obtained for the lifetime of BChl 800 in the purple non-sulfur bacteria Rb. sphaeroides and Rps. palustris.1,2 This suggests that the distances between BChl 800 and BChl 850 in LH2 complexes from Rb. sphaeroides and Chr. tepidum are not much different. At increasing delays, the maximum of the bleaching around 860 nm shifted to the red by 5 nm with a time constant of about 1 ps. This time constant may be limited by that of energy transfer to BChl 850. However, the intrinsic time constant for this dynamic process in BChl 850 cannot be very much less than 1 ps, because otherwise the effect would not be observed. There are several mechanisms that may underlie the red shift, such as intramolecular vibrational relaxation or exciton scattering to lower lying exciton levels. We believe that at present it is hard to favor one of the mechanisms. One might consider that the shift is due to energy transfer between energetically inequivalent BChl 850 molecules within a B800-850 complex, but we reject this possibility as will be argued in the Discussion section. We also performed measurements with pump and probe polarized parallel under otherwise identical conditions. Figure 4a,b shows the time-resolved spectra at increasing delays between -0.08 and 5.25 ps. The results were very similar to those obtained with magic angle polarization, with the same relative magnitudes of the signals around 800 and 850 nm. A red shift of the spectra around 860 nm by 5 nm with increasing delay was also seen. A similar red shift appears to occur in BChl 800, as can be seen from the difference spectra of Figure 4a. Figure 5 shows the kinetics at 802 and 860 nm. The kinetics at 802 nm displayed an initial bleaching due to excitation of
Kennis et al.
Figure 5. Kinetics of absorbance changes upon excitation at 797 nm with pump and probe polarized parallel at (a, top) 802 nm and (b, bottom) 860 nm. The kinetic trace at 802 nm is fitted with a single decay of 0.9 ps. The kinetic trace at 860 nm is fitted with a rise time of 0.9 ps. A convolution with a 200 fs wide (sech)2 function was used for all fits.
BChl 800 and a decay with a time constant of 0.9 ps. The signal at 860 nm showed a rise time with exactly the same time constant. In contrast to the experiments performed under magic angle, there was no evidence for singlet-singlet annihilation occurring in this measurement. This is partly explained by the somewhat lower excitation pulse energies used. Moreover, fluctuations in excitation pulse energy were larger during the magic angle measurements, so the most intense shots in this series could give rise to the annihilation effect. Discussion Our results unambiguously indicate that energy transfer from BChl 800 to BChl 850 in isolated B800-850 complexes of Chr. tepidum occurs with a time constant of 0.8-0.9 ps. This is reflected both by the decay of excited BChl 800 and by the rise of excited BChl 850. It may be noted that in previous timeresolved studies of B800-850 complexes from Rb. sphaeroides either only the decay of excited BChl 800 was monitored during one-color measurements2 or the rise time of excited BChl 850 did not match the decay of excited BChl 800.1 The most peculiar aspect of the data presented here is the relative magnitude of the absorbance difference signal measured for excited BChl 850 as compared to that of excited BChl 800. In Figure 2a, the maximum bleaching for BChl 800 is given by the time-resolved absorbance difference spectrum at a delay of 0.13 ps. In Figure 2b, the fifth spectrum is taken at a delay of 3 ps, when all excitations have been transferred to BChl 850. Obviously, the absorbance difference signal induced by excitation of BChl 850 is much larger than that by excitation of BChl 800; the integrated bleaching of BChl 850 is 4 times larger than that of BChl 800 (corrected for the convolution with the excitation pulse). The same observations can be made for the measurements with pump and probe polarized parallel, as can be seen in Figure 4a,b. In the crystalline B800-850 complex from Rps. acidophila the center-to-center distance of the BChl 800 molecules is 21 Å,10 and one may regard them as monomers. The arrangement of BChl 800 in Chr. tepidum is probably different. Close inspection of the BChl 800 band shows that it is composed of
Energy Transfer in Isolated B800-850 Complexes two bands, as is also indicated by the second derivative of the absorption spectrum (not shown). At low temperature the BChl 800 band splits in two, with peaks at 797 and 809 nm, which have about the same amplitude.15 The circular dichroism spectrum shows a conservative double feature at the corresponding wavelengths.11 These observations suggest that, unlike the situation in Rps. acidophila, the BChl 800 molecules in Chr. tepidum are organized as excitonically coupled dimers, giving rise to a splitting of the absorption band.11 The equal intensities of the two absorption bands around 800 nm indicate that the oscillator strength is evenly distributed between two exciton components of the coupled dimer. Hence, the absorbance difference signal associated with exciting a pair of molecules in such an arrangement will again be equal to that of a monomeric bleaching. It thus appears that the bleaching observed upon excitation of BChl 850 is 4 times larger than for monomeric BChl. This factor is underestimated because the bleaching of the BChl 850 band is partly compensated for by excited state absorption. As mentioned in the Introduction, similar observations concerning the LH1 antennae in purple bacteria have already been reported.5-8 Our results may qualitatively be understood as follows. Let us assume that the arrangement of BChl 850 molecules in the B800-850 complex of Chr. tepidum is similar to that in the corresponding complex of Rps. acidophila. The crystal structure of the B800-850 complex of the latter organism indicates a highly symmetric ring structure, and the distances between BChl 850 molecules, about 9 Å, lead to strong dipolar intermolecular couplings. We thus are dealing with a circular aggregate of strongly interacting molecules. In the case of perfect symmetry, strict selection rules for optical transitions apply for such aggregates, and the oscillator strength of the constituent monomers is concentrated in a few optically allowed transitions, corresponding to delocalized eigenstates.9,16-20 Absorption of a photon or energy transfer from adjacent molecules to the aggregate then leads to ground state depletion of a transition carrying the oscillator strength of several monomers. The cross section for stimulated emission from the excited state to the ground state is enhanced as well, proportionally to the oscillator strength of the transition. In a series of papers, Novoderezhkin and Razjivin have performed calculations on circular aggregates to account for absorption difference measurements in LH1.9,19,20 According to these calculations, the absorption from the singly excited exciton level to the doubly excited exciton level is blue-shifted with respect to the main absorption band, which in our case explains the observed absorbance increase at wavelengths shorter than 842 nm. They have estimated that, upon excitation, an aggregate size of about 20 gives rise to an absorbance change which is increased by a factor of 4 relative to that of a monomer,9 in agreement with our experiments. This would imply that the excitations in LH2 are delocalized over the entire BChl 850 aggregate. However, the effect of energetic disorder on this result of the calculations remains to be investigated. The B800-850 complex of Rps. acidophila is a nonamer of Rβ-subunits, with 18 BChl 850 molecules and 9 BChl 800 molecules.10 Electron microscopic and sedimentation studies indicate a hexameric and an octameric structure for the B800850 complexes of Rb. sphaeroides and Rhodospirillum molischianum, respectively.21,22 We suppose that a similar number applies to the B800-850 complex of Chr. tepidum. However, if the BChl 800 molecules are arranged as dimers, a circular symmetric structure cannot be 9-fold for Chr. tepidum, but must be related to an even number.
J. Phys. Chem., Vol. 100, No. 6, 1996 2441 The mutual orientation of the BChls 850 and the strength of the dipolar interaction have a significant influence on the wavelength of the dipole-allowed transitions. In the crystal structure of Rps. acidophila, the BChls 850 are about 9 Å apart and the Qy transition moments are inclined by about 20° with respect to the tangent of the ring. One can calculate that the dipole-allowed transition then is red-shifted by about 40 nm with respect to monomeric BChl (using a dipolar strength of 40 D2 for BChl a23). This partly explains the red shift of BChl 850 with respect to BChl 800. Hydrogen bonds between the chromophores and the protein environment presumably also contribute to the red shift of the absorption.24 From our measurements, it becomes clear that the width of the BChl 850 bleaching is larger than that of BChl 800. This may be due to a dispersion in exciton interactions because of variations in site energies of the individual BChls or to a significant lifetime broadening associated with ultrafast exciton scattering to nearby energy levels. Strictly speaking, these contributions cannot be separated at room temperature since both are determined by thermally induced fluctuations in the environment. Assuming that the exciton scattering mechanism dominates, we estimate that the characteristic time constant for this process is a few tens of femtoseconds. In fact, such a time constant, attributed to exciton relaxation, has recently been observed in LH2 complexes of Rb. sphaeroides.25 At this point it is useful to consider the origin of the dynamic red shift of the absorbance difference around 860 nm. A similar shift observed in LH1 has been assigned to localized excitations at energetically inequivalent BChl dimers.14 Energy transfer will then lead to a rapid thermalization of the excited state. In view of the structure of LH2,10 it appears that the exciton model should be favored: the strong dipolar interactions between the BChls are difficult to reconcile with localized excitations. This is consistent with our results which indicate that the excitations are delocalized over more than two BChls. The dynamic red shift may be attributed to thermalization of the excited state by means of exciton scattering to lower lying energy levels. Considering the Huang-Rhys factor for the electron-phonon coupling in bacterial antenna systems, typically about 0.5,18,26 intramolecular vibrational relaxation may also contribute to the band shift. In conclusion, our results indicate that the spectral properties of BChl 850 in the B800-850 complex of Chr. tepidum are mainly determined by a symmetric arrangement and strong dipolar interactions between BChl 850 molecules. This leads to concentration of oscillator strength in a few optically allowed eigenstates that are strongly delocalized, possibly over the entire aggregate. Acknowledgment. The investigation was supported by the Life Science Foundation (SLW), which was subsidized by the Netherlands Organisation for Scientific Research (NWO), and was also supported by the European Union (Contract No. SCI*CT92-0796). References and Notes (1) Shreve, A. P.; Trautman, J. K.; Frank, H. A.; Owens, T. G.; Albrecht, A. C. Biochim. Biophys. Acta 1991, 1058, 280. (2) Hess, S.; Feldchtein, F.; Babin, A.; Nurgaleev, I.; Pullerits, T.; Sergeev, A.; Sundstro¨m, V. Chem. Phys. Lett. 1993, 216, 247. (3) De Caro, C.; Visschers, R. W.; Van Grondelle, R.; Vo¨lker, S. J. Phys. Chem. 1994, 98, 10584 (4) Reddy, N. R. S.; Small, G. J.; Seibert, M. ; Picorel, R. Chem. Phys. Lett. 1991, 181, 391. (5) Xiao, W.; Lin, S.; Taguchi, A. K. W. ; Woodbury, N. W. Biochemistry 1994, 33, 8312.
2442 J. Phys. Chem., Vol. 100, No. 6, 1996 (6) Kennis, J. T. M.; Aartsma, T. J.; Amesz, J. Biochim. Biophys. Acta 1994, 1188, 278. (7) Abdourakhmanov, I. A.; Danielius, R. V.; Razjivin, A. P. FEBS Lett. 1989, 245, 47. (8) Abdourakhmanov, I. A.; Danielius, R.; Razjivin, A. P.; Rotomskis, R. In Poster Abstracts of the 7th International Congress on Photosynthesis, ProVidence, Aug 10-15, 1986; Biggins, J., Ed.; 1986; p 303. (9) Novoderezhkin, V. I.; Razjivin, A. P. FEBS Lett. 1993, 245, 47. (10) MacDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs; N. W. Nature 1995, 374, 517. (11) Nozawa, T.; Fukada, T.; Hatano, M.; Madigan, M. T. Biochim. Biophys. Acta 1986, 852, 191. (12) Kennis, J. T. M.; Aartsma, T. J.; Amesz, J. Chem. Phys. 1995, 194, 285. (13) Nuijs, A. M.; Van Grondelle, R.; Joppe, H. L. P.; Van Bochove, A. C.; Duysens, L. N. M. Biochim. Biophys. Acta 1985, 810, 94. (14) Visser, H. M.; Somsen, O. J. G.; Van Mourik, F.; Lin, S.; Van Stokkum, I. H. M.; Van Grondelle, R. Biophys. J. 1995, 69, 1083. (15) Garcia, D.; Parot, P; Verme´glio, A. ; Madigan, M. T. Biochim. Biophys. Acta 1986, 850, 390. (16) Kasha, M. In Spectroscopy of the Excited State; Di Bartolo, Ed.; Plenum Press: New York, 1976; p 337.
Kennis et al. (17) Pearlstein R. M.; Zuber, H. In Antennas and Reaction Centers of Photosynthetic Bacteria; Michel-Beyerle, M. E., Ed.; Springer Series in Chemical Physics 42; Springer Verlag: Berlin, 1985; p 53. (18) Reddy, N. R. S.; Picorel, R.; Small, G. J. J. Phys. Chem 1992, 96, 6458. (19) Novoderezhkin, V. I.; Razjivin, A. P. Biophys. J. 1995, 68, 1089. (20) Novoderezhkin, V. I.; Razjivin, A. P. Photosynth. Res. 1994, 42, 9. (21) Boonstra, A. J.; Visschers, R. W.; Calkoen, F.; Van Grondelle, R.; Van Bruggen, E. F. J.; Boekema, E. J. Biochim. Biophys. Acta 1993, 1142, 181. (22) Kleinekofort, W.; Germeroth, L.; Van den Broek, J. A.; Schubert, D.; Michel, H. Biochim. Biophys. Acta 1992, 1140, 102. (23) Pearlstein R. M. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991; p 1047. (24) Olsen, J. D.; Sockalingum, G. D.; Robert, B.; Hunter, C. N. Proc. Natl Acad. Sci U.S.A. 1994, 91, 11050. (25) Pullerits, T.; Chachisvilis, M.; Jones, M. R.; Hunter, C. N.; Sundstrom, V. Chem. Phys. Lett. 1994, 224, 355. (26) Pullerits, T.; Monshouwer, R.; Van Mourik, F.; Van Grondelle, R. Chem. Phys. 1995, 194, 395.
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