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Inhomogeneous spectral broadening of the B820 subunit form of LH1
238
Biochimica et Biophysica Acta, 1141 (1993) 238-244 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00
BBABIO 43762
Inhomogeneous spectral broadening of the B820 subunit form of LH1 Ronald W. Visschers, Frank van Mourik, Ren6 Monshouwer and Rienk van Grondelle Department of Biophysics, Faculty of Physics and Astronomy, Free University, Amsterdam (The Netherlands) (Received 7 May 1992)
Key words: Light-harvesting; Antenna complex; LH1; Fluorescence spectroscopy; Photosynthesis; Energy transfer; (Rs. rubrum)
Site-selected fluorescence spectra of the B820 subunit of LH1 from Rhodospirillum rubrum G9 were measured at temperatures between 160 K and 4.2 K. A linear correlation between excitation wavelength and maximal emission wavelength was observed across the whole Qy absorption band of the B820 subunits, which persists at higher temperatures. This demonstrates that (a) the Qr absorbance band of the B820 subunit is inhomogeneously broadened; (b) the subunit consists of a strongly interacting dimer of BChi a; (c) no energy transfer occurs among these dimers. The temperature dependence of the shape of the emission spectrum confirms that the protein forms a 'glass-like' environment for the bacteriochlorophyll pigments. After reassociating the octyl-glucoside-solubilized B820 subunits into the reassociated B873 form, the emission wavelength is independent of the wavelength of excitation at 4.2 K. This implies that the reassociated B873 form consists of a large number of interacting pigment molecules. The nature and origin of the spectral inhomogeneity in the B820 subunits is discussed.
Introduction
There is now accumulating evidence that the main near-infrared (NIR) absorbance bands due to bacteriochlorophyll in the light-harvesting complexes of purple bacteria are spectrally heterogeneous. Such behavior has been described both for the core antenna complexes (LH1) from several purple bacteria [1,2] and for the B800 band of the peripheral light-harvesting complex (LH2) from Rb. sphaeriodes [3]. For isolated LH1 complexes from Rb. sphaeroides and Rs. rubrum it has been shown that the fluorescence polarization is not constant upon excitation across the absorption band at 77 K, but increases when excitation occurs on the red side of the absorbance band. This observations led to a model in which excitations are trapped on a red-shifted BChl species (B896) that constitutes 10-20% of the total oscillator strength of this band. Time-resolved measurements probing energy-transfer within the core antenna complex provided additional evidence for the presence of red-shifted components [4,5]. However, this view was recently challenged by Timpmann et al. [6], who observed that for the LH1 antenna of Rs.
Correspondence to: R.W. Visschers, Department of Biophysics, Faculty of Physics and Astronomy, Free University, De Boelelaan 1081, 1081 HV Amsterdam.
rubrum at 4 K the fluorescence life-time depends on the emission wavelength, being shorter on the blue side of the fluorescence spectrum, and progressively increasing if the emission is detected at longer wavelengths. Therefore, they proposed that at least three, but probably many more, spectrally distinct pools of bacteriochlorophylls must exist in the LH1 antenna complex. Hole-burning studies on the B800 band of LH2 complexes from Rb. sphaeriodes demonstrated that narrow holes could be permanently burnt into this absorbance band at temperatures between 1.2 and 30 K [3], thus providing that the B800 band is inhomogeneously broadened. From the width of the holes the homogeneous linewidth (Fho m) was estimated 70 +_ 10 GHz, independent of burning wavelength between 791 and 804 nm. This homogeneous width corresponds to an energy transfer time of 2.45 ps at 4.2 K, in good agreement with time-resolved measurements of this process [7]. No fluorescence line-narrowing was observed in these experiments for the direct emission of the B800 bacteriochlorophylls. Hole-burning experiments on intact chromatophores from the Rb. sphaeroides mutant strain NF57 [8] yielded similar results for the B800 band [9]. They showed on the other hand that the B850 band of LH2 in these chromatophores and the B880 of the LH1 complex behave like homogeneous bands, since no narrow holes could be burnt in these bands [10].
239 In a recent study [11] we have reported that for isolated LH1 complexes of the LHl-only mutant strain Rb. sphaeroides M2192 [12] the position of the fluorescence band depends on the wavelengths of excitation at 4.2 K. Site-selection studies using a narrow-band, continuous wave laser showed that the emission spectrum of these pigment-protein complexes becomes significantly red-shifted upon excitation on the red side of the absorption band. These results were interpreted in terms of a model that treats separate light-harvesting complexes as clusters of a finite number of pigments among which fast energy transfer occurs. It was shown that the most significant parameter determining the exact wavelength of the onset of the red-shifted emission is the number of interacting pigments within a cluster. To gain further insight into the physical parameters that determine the position and shape of the long wavelength absorption band of photosynthetic pigment-protein complexes we have studied the simplest photosynthetic pigment-protein complex currently available, the B820(OG) subunit. This subunit can be obtained by reversible dissociation of core antenna complexes from several purple bacteria by addition of the detergents octyl glucoside [13-16] and n-octyldipropyl sulfoxide (ODPS) [17]. The biochemical and spectroscopic properties of the B820 subunit have been thoroughly characterized [14,17-21]. It was proposed to be an excitonically interacting BChl a dimer, associated with a single (a/3)polypeptide pair. To account for the high fluorescence polarization as well as the relatively low intensity of the high-energy exciton component, the Qy transitions of both BChl a molecules in the dimer have to be nearly parallel to each other in a head to tail or head to head arrangement. The subunits have been shown to reaggregate into the B873(reassoc) that resembles the original LH1 structure, upon lowering the detergent concentration [13,14]. In this work we report the polarized emission spectra of the B820(OG) subunit as a function of excitation wavelength and temperature between 4.2 and 160 K. In addition, the ground-state absorbance spectrum of B820(OG) was measured as a function of temperature between 4.2 K and 300 K. The results show that the Qy absorption band of B820(OG) is inhomogeneously broadened and are fully consistent with the view that B820(OG) is a strongly coupled dimer of BChl a. Materials and Methods
B820(OG) subunits were prepared according to the method of Miller et al. [14] with the following modifications; purified chromatophores of the carotenoid-less Rs. rubrum G9 were prepared by sonication followed by sucrose density centrifugation. Purified chromatophores were diluted to Asm ----20 and solubilized with
OGP until a nearly complete conversion into B820(OG) was observed. Aliquots of 0.2-0.5 ml were subsequently applied to a Superose 6 (Pharmacia) gelfiltration FPLC column equilibrated with buffer containing 1% OG. The complexes eluted with an apparent molecular mass of 60 kDa. The peak fraction containing purified B820(OG) subunits was stored at -20°C until later use. Low-temperature samples were prepared by dilution of thawed B820(OG) complexes into a buffer containing 70% (v/v) glycerol, 50 mM potassium phosphate (pH 7.5) and 1.15% OGP. To minimize re-absorbance the A820 was kept below 0.2 and the laser passed through the sample in such a way that the fluorescence only travelled through the sample for a very short distance (1-2 mm). Re-aggregation of the B820(OG) subunits into B873(reassoc) was achieved by diluting B820(OG) subunits into 70% (v/v) glycerol buffer containing no detergent. Fluorescence spectra were measured as described in Ref. 11. Briefly, a cw dye laser (Coherent model 590, Styryl 9) pumped by a 3W Ar + laser (SpectraPhysics, model 164) was used as an excitation source. This combination produced 30-100 mW of continuous light, tunable between 810 and 910 nm with a nominal bandwidth of > 30 GHz. To avoid local heating of the sample the beam was expanded to a diameter of 2 mm before striking it. Samples, contained in acrylic fluorescence cuvettes with 1 cm pathlength (Sarstedt) were cooled in a liquid helium flow cryostat (Oxford instruments, CF1204). Fluorescence was detected through a double 1/8 m monochromator (bandwidth 1.2 nm) with a cooled S1 (EMI 9684) photomuitiplier. Absorbance spectra were measured on a Cary 219 spectrophotometer with a resolution of 1 nm. Results
Fig. 1 shows the absorbance spectrum of B820(OG) subunits between 77 K and 300 K. Although the position of the absorbance bands is not strongly dependent on temperature [20], they are prominently broadened at room temperature; the major effect that is observed upon cooling from room temperature to about 200 K is a decrease of the width of the absorbance band. The sharpening of the spectrum in this temperature range is accompanied by a shift of the absorbance maximum from 820 nm at room temperature to 826 nm at 200 K. In addition, a minor contribution of the 777 form becomes visible. Cooling from 77 K to 4.2 K induces only a very minor additional decrease of the width of the absorbance band, and does not shift the absorbance maximum further to the red (see Fig. 3 also for the 4.2 K spectrum). No fine structure is observed in the low-temperature absorbance spectra nor in the second and fourth derivative thereof (not shown).
240 AbsoFDance I
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Wavelength ( nm ) Fig. 1. Absorbance spectra of the B820(OG) subunit of the core light-havesting complex from Rs. rubrum. The absorbance spectra were taken at 77 K (solid) 150 K (dash), 200 K (dot), and 300 K (chain-dash). (Bandwidth 1 nm.) The complexes were contained in buffer containing 1.15% (w/v) OG, 50 mM potassium-phosphate buffer (pH 7.5) and 80% v/v glycerol to obtain glassy, transparent samples at low temperatures.
The emission spectra of B820(OG) upon narrowband excitation near the absorbance maximum (Aexc = 823 nm) at four different temperatures are given in Fig. 2. The sharp peak at 823 nm is partly due to direct scattering of laser light. The scattering prevents direct observation of the resonant zero-phonon line. The emission is broad and featureless. No vibronic finestructure is observed in the emission band at 4.2 K. Furthermore, the shape of this band does not change drastically either upon exciting more to the red, or
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upon increasing the temperature. Two major effects can be observed when the temperature of the sample is increased from 4.2 K: at 4.2 K no emission is discernible on the blue side of the excitation wavelength, whereas a progressive increase of emission on the blue side of the excitation wavelength is observed upon increasing the temperature; and secondly, the maximum of the emission shifts to the blue by 4 nm independent of the excitation wavelength, (see also Fig. 3), due mainly to the disappearance of the small trough on the red side of the excitation wavelength. In Fig. 3 the correlation between excitation wavelength and the position of the emission maximum is shown for several temperatures, together with the absorbance spectrum of B820(OG) at 4.2 K. The shape and position of the B820(OG) NIR absorption band in this spectrum is nearly identical to the 77 K spectrum shown in Fig. 1, demonstrating that no additional sharpening of the absorbance band takes place. From Fig. 3 it is clear that a linear correlation between excitation wavelength and emission maximum exists, when the excitation wavelength is tuned across the whole absorbance band. The slope is the same (0.88 _+ 0.08) within the margin of error at different temperatures. The polarization of the fluorescence (not shown) is independent of temperature and higher than 0.4 (Acxc = 823 nm). It is also independent of the detection wavelength, which indicates the absence of energy transfer and a nearly parallel orientation of the Qy dipoles [20]. Note that for intact LH1 complexes [11] and for the B850 band of the accessory light-harvesting complex (unpublished data) the emission wavelength dependence of the polarization can be ascribed to intra-cluster energy transfer. A more detailed analysis of the shape of the emission that is observed on the blue side of the of the
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Wavelength (nm) Fig. 2. Fluorescence spectra from the B820(OG) subunit. Sample conditions as given in Fig. 1 but Aszo =, 0.2. The excitation wavelength of the dye laser was 823 nm. 1"he sharp peak at 823 nm is due to direct scattering of laser light. The emission spectra were measured with the polarizer set to the magic angle (54 deg) to obtain isotropic emission spectra. The traces shown were taken at 4.2 K (solid), 40 K (dashed), 80 K (dotted), and 160 K (chain-dash).
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Wavelength (nm) Fig. 3. Dependence of the maximum of the emission wavelength on excitation wavelength at different temperatures; squares 4.2 K, circles 40 K, triangles 77 K. 2"he solid curve shows the 4.2 K absorbance spectrum of the B820(OG) subunit.
241
Ant,i-Stokes fluorescence i
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Fig. 4. (A) Linear least-square fit of the anti-Stokes fluorescence as a function of energy difference between excitation and detection wavelength at three different temperatures (A~x¢ = 827): squares, 40 K; circles, 77 K; triangles, 160 K. Only 1 / 3 of the datapoints used for the fit are actually shown. T h e first 2 nm were not used in the fit since these points contain a scattering contribution. T h e data was fitted to an exponential function given by: c *exp (-aE/kbr) (where zlE is the energy difference between excitation and emission wavelength, K b is Boitzmann's constant). T h e temperature p a r a m e t e r T and constant c were varied to obtain satisfactory fits. The fits shown by the solid lines correspond to the following effective temperatures: 47.6 K, 75.4 K and 115 K (for m e a s u r e m e n t s at 40 K, 77 K and 160 K, respectively). (B) Same fit as in (A). T h e data were obtained by excitation in the red wing of the B820(OG) (Ae~c = 837 nm, T = 77 K) Only 1 / 2 of the actual datapoints are shown. For the exponential fit the first 20 points were not used. There is now a clear deviation of the exponential distribution in the vicinity of the scattering peak.
excitation wavelength at various temperatures is presented in Fig. 4. Photons emitted with higher energy than the excitation light derive from thermally popu-
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excitation wavelength Fig. 5. Absorbance spectrum of B873(OG, reassoc) at 4.2 K. T h e position of the fluorescence m a x i m u m at several excitation wavelengths are indicated by squares. For comparison, the same data obtained for purified LH1 complexes from Rb. sphaeroides M2192 are also shown (circles) [11]. To allow a direct comparison, the latter
have been blue-shifted by 8 nm. This is done since the absorbance maximum of the 13873 is blue shifted by 8-9 nm with respect to the LH1 complexes of Rb. sphaeroidp_s M2192, and in order to obtain cluster-sizes, the red-shift of the emission spectra has to be compared relative to the position of the absorbance maximum. The red shift of the emission maximum, observed for the purified complexes agrees with a cluster of 8 dimers [11]. Since no increase is observed for the B873(reassoc) it must comprise more than 32 dimers among which energy transfer takes place.
lated vibronic states in the system, which, in view of the small energies involved, probably not arise from intramolecular vibrations but from coupling of the electronic transitions with the surrounding protein medium. Thus, the shape of the anti-Stokes fluorescence contains information regarding the distribution of these states. A direct comparison of the population of levels at a given energy difference is prevented by the fact that the normalization of the emission spectra at different temperatures is rather awkward. To circumvent this problem we have fitted the complete blue emission with an exponential function. Fig. 4 shows that the observed anti-Stokes fluorescence indeed decays exponentially with the energy difference between excitation and detection wavelength. For the 40 K and 77 K data the fits corresponded to a Boltzmann activation temperature of 47.6 K and 75.4 K, respectively. For the 160 K data the activation t e m p e r a t u r e determined from the 'blue' fluorescence is 115 K. When the excitation wavelength is chosen on the red side of the absorbance maximum a deviation of the exponential behavior can be observed. Fig. 4B shows that in this case the observed fluorescence falls below the exponential curve in the vicinity of the excitation wavelength. Fig. 5 shows the absorbance spectrum of the B873(reassoc) at 4.2 K. T h e absence of significant absorbance in the 820 nm region demonstrates that a nearly complete reassociation of the B820(OG) has taken place [14]. The width and shape of the band are again very similar to the 77 K absorbance spectra [20]. Fig. 5 further shows the relation between excitation
242 and emission wavelengths for the B873(reassoc) at 4.2 K. There is a clear difference from the linear relation that is found for the B820(OG) subunit (Fig. 3). The wavelength at which the emission maximum occurs remains constant upon excitation across the absorbance band and no shift of the emission spectrum to the red could be detected when the excitation wavelength is chosen in the red wing of the absorbance band. The polarization of the fluorescence has dropped to a value of 0.15 (Aexc=873 nm), indicative of energy-transfer among pigments with different orientation. For comparison, we have added the data for purified LH1 complexes from Rhodobacter sphaeroides M2192. The original data from van Mourik et al. [11] have been shifted 8 nm to the blue to allow a direct comparison with our data. This is done since the Qr absorbance band of B873(reassoc) is also 8-9 nm blue-shifted compared to the absorption band of LH1 complexes due to the absence of carotenoids [13]. For the purified LH1 complexes it was found that the emission wavelength depends on the excitation wavelength only in the low energy wing ()rexc > 877 nm in Fig. 5). From these measurements it was concluded that the purified LH1 is a cluster of 16 BChl a pigments (8 dimers) randomly sampled from the totally inhomogeneously broadened pigment tool. It is clear that the B873(reassoc) behaves like a more extreme case and that the cluster size is probably significantly larger than of the purified LH1.
Discussion Recently we have developed a model that describes the effect of inhomogeneous broadening of the absorption bands on the fluorescence properties of cluster of pigments in photosynthetic light-harvesting complexes [11]. Based on the assumption that at 4.2 K the absorbance band is inhomogeneously broadened, and that efficient energy transfer only takes place following a downhill energy gradient within a cluster, the dependence of the emission spectrum on the excitation wavelength could be qualitatively and quantitatively modelled. The most important parameter in this statistical description is the number of pigments within a cluster between which efficient energy transfer takes place. The number of coupled pigments in a cluster was shown to directly determine the excitation wavelength at which the onset of the gradually increasing red shift of the emission maximum can be observed. A comparison between theoretical curves and the measurements of the correlation between excitation wavelength and the position of the emission maximum for the purified LH1 complex from Rb. sphaeroides M2192 showed that
approximately 8 closely interacting BChl a dimers are energetically coupled within such a complex.
Inhomogeneous broadening of B820 To further asses the validity of this 'cluster-model' and to determine the physical properties that define the absorbance properties of the photosynthetic antenna pigments we now present a similar study for the B820(OG) subunit of LH1 from Rs. rubrum G9. This detergent-solubilized subunit of the core antenna is assumed to be a single BChl a bound to an a/3 polypeptide pair. For clusters comprising one pigment (or one closely interacting dimer as is the case for the B820 subunit [20]) with an inhomogeneously broadened absorption band, we expect a linear correlation between the observed emission maximum and the excitation wavelength under the condition that the homogeneous bandwidth is much smaller than the actual width of the absorption band. The data presented in Fig. 3 shows that for the B820(OG) subunit such a relation is indeed found. We therefore conclude that the strongly coupled dimer behaves like a single transition in these experiments and that the absorbance band of the B820(OG) subunit is inhomogeneously broadened. Furthermore, the absence of energy transfer cannot be due to a temperature dependence of the F6rster transfer rates, since the linear relation is also found at 77 K. These results show that the origin of the inhomogeneity is present within the smallest subunit of the lightharvesting complexes and does not originate from large-scale aggregations of the pigments involved. To understand the broad and featureless site selection fluorescence spectra of the B820(OG) subunit we compare our measurements to those of Van der Laan et al. [22]. Their site selection fluorescence spectra of monomeric BChl a in triethylamine glasses show many well resolved vibronic fine structures. However, when dissolved in L D A O / w at er mixtures, these features gradually disappear when the LDAO concentration is decreased. Thus at low LDAO concentrations the emission spectra become broad and featureless as a result of increased electron-phonon coupling. In the case of BChl a in L D A O / w at er mixtures these effects probably originate from self aggregation of the pigment molecules, which is a well documented process for BChl a [23]. In the case of the B820(OG) subunit the source for the spectral inhomogeneity originates from within the protein binding pocket that surrounds the BChl a molecules. It is interesting to note that it has been previously shown that the interaction with the protein environment influences the position of the absorbance bands [24,25] and more recently it has been shown that replacing single tyrosine residues in the accessory antenna concurs with large shifts of the absorbance in LH2 [26]. Also in site selection spectroscopy of the biliprotein c-phycocyanine, the ob-
243 served emission is broad and featureless, which is ascribed to extensive electron-phonon coupling [27].
Temperature effects To evaluate the temperature dependence of the inhomogeneous broadening we have measured the NIR absorbance spectrum of the B820 subunits at several temperatures between 300 K and 4.2 K (Fig. 1). The overall changes are rather small and occur predominantly in the 300-200 K range. A comparable temperature dependence has been observed for the absorbance spectrum of Photosystem I from plants, where narrowing of the absorbance band stops at approx. 30 K [28]. The temperature dependence of the absorbance of the B820(OG) band shows that the total width of the absorbance band is only slightly determined by the decrease of the homogeneous bandwidth in the region between 100 K and 4.2 K. The relatively small changes of the bandwidth of the B820(OG) subunits in the temperature region between 100 and 4.2 K can be attributed to the larger width of the B820 absorbance band due to site-inhomogeneous broadening as compared to the PS I main absorbance band. A pronounced influence of the temperature on the emission spectrum is observed at all temperatures between 4.2 K and 160 K. At 4.2 K the emission spectrum is relatively red-shifted (for a given excitation wavelength) and shows no emission on the high energy side of the excitation wavelength. Thus, at 4.2 K fluorescence predominantly occurs from the lowest energy level of a particular B820(OG) subunit, to which the system rapidly relaxes after excitation. Increasing the temperature to 40 K or 77 K results in a small blue shift of the emission maximum and a clear increase of fluorescence on the blue side of the excitation wavelength. This-emission must derive from a thermally increased population of low frequency modes of the $1 level. The effective temperature that can be extracted from the exponential dependence of this 'blue' emission on the difference between emission and excitation energy is rather close to the actual temperature of the system, indicative for the population of a continuous set of low frequency phonon modes of the surrounding protein pocket. This is in good agreement with the observation that hole-burning spectra of BChl a in glassy environments display broad side-holes close to the burn wavelength (20 cm -1) that are ascribed to coupling to a specific low frequency mode of the host [22]. At 160 K we find that the effective temperature that is obtained from the anti-Stokes fluorescence is lower than the actual temperature. This is probably due to the fact that at 160 K the thermal energy corresponds to a substantial part of the total width of the B820(OG) Qy absorption band. If the excitation wavelength is chosen in the red wing of the B820(OG) absorption band the exponential
shape of the anti-Stokes is somewhat perturbed (Fig. 4B). This may indicate that in fact a distinct energy difference of 20-50 cm-1 exists between the 0-0 transition (which is selectively excited in the red wing) and the most abundant protein modes that are coupled to the electronic transition.
B873(reassoc) In Fig. 5 the results of site selection fluorescence spectroscopy for the reassociated subunits is shown. The reassociated subunits, in which the absorbance maximum is shifted to 873 nm, do not display a detectable change in the maximum of the emission upon excitation across the absorption band. We estimate that a red shift would probably have been detected if the cluster comprised less than 32 coupled dimers. The absence of a red-shift indicates that the reassociated form is even larger, and thus is different from highly purified carotenoid-containing LH1 complexes which appear to consist of 8 interacting dimers [11]. In fact, we find that in this aspect the B873(reassoc) behaves rather similar to chromatophores of the LH1 only mutant Rb. sphaeroides M2192 for which we could only observe a small red-shift of the emission maximum at 4.2 K (Visschers et al., unpublished data). We note that in recent study Braun and Scherz [29] have discussed how the aggregation state of the peripheral antenna may be accounted for using strongly coupled dimers as basic building blocks. Currently, we work on a similar characterization of the aggregation properties and low-temperature energy transfer of purified LH2 and LH1 mutants of Rb. sphaeroides.
Acknowledgments The authors would like to thank Ing. F. Calkoen for isolating the B820 subunits, Mr. H.A. van der Stroom for preparing chromatophore membranes, Dr. L. Valkunas (present adress: Institute of Physics, Lithuanian Academy of Sciences, Lithuania) for helpful discussions, and Prof. S. V61ker (Center for the study of excited state of Molecules, University of Leiden, Leiden, The Netherlands) for critically reading the manuscript. This research was financially supported by EEC research grant SC1 0004-C, and the Netherlands Organization for Scientific Research (NWO) through The Dutch Foundation for Biophysics.
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244 4 Freibcrg, A., God/k, V.I., Timpmann, K., (1987) in Progress in Photosynthesis Research, Vol. I (Biggens J., ed), Nijhoff, Dordrecht. 5 Hunter, C.N., Bergstr6m, H., Van Grondelle, R. and Sundstr6m, V. (1990) Biochemistry 29, 391-3203-3207. 6 Timpmann, K., Freiberg, A. and God/k, V.I. (1991) Chem. Phys. Lett. 182, 617-622. 7 Bergstr6m, H., Westerhuis, W.H.J., Sundstr6m, V., Van Grondelle, R., Niederman, R.A. and Gillbro, T. (1988) FEBS Lett. 233, 12-16. 8 Ashby, M.K., Coomber, S.A. and Hunter, C.N. (1987) FEBS Lett. 213, 245-248. 9 Reddy, N.R.S., Small, G.J., Seibert, M. and Picorel, R. (1991) Chem. Phys. Lett. 181,391-399. 10 Reddy, N.R.S., Picorel, R. and Small, G.J., (1992) J. Phys. Chem. 94, 7545-7546. 11 Van Mourik, F., Visschers, R.W. and Van Grondelle, R. (1992) Chem. Phys. Lett. 193, 1-7. 12 Hunter, C.N., Van Grondelle, R. and Van Dorssen, R. (1989) Biochim. Biophys. Acta 973, 383-389. 13 Loach, P.A., Parkes, P.S., Miller, J.F., Hinchigeri, S. and Callahan, P.M. (1985) Molecular biology of the photosynthetic apparatus (Steinback, K., Bonitz, S., Arntzen, C. and Bogorad, L., eds. pp. 197-209), Cold Spring Harbor Laboratories, New York. 14 Miller, J.F., Hinchigeri, S.B., Parkes-Loach, P.S., Callahan, P.M., Sprinkle, J.R. and Loach, P.A. (1987) Biochemistry 26, 5055-5062. 15 Chang, M.C., Meyer, L. Loach and P.A. (1990) Photochem. Photobiol. 52, 873-881. 16 Heller, B.A., Loach, P.A. (1990) Photochem. Photobiol. 51,621627.
17 Visschers, R.W., Nunn, R., Calkoen, F.. Van Mourik, F., Hunter, C.N., Rice, D.W. and Van Grondelle, R., (1992) Biochim. Biophys. Acta 1100, 259-266. 18 Parkes-Loach, P.S., Sprinkle, J.R. and Loach, P.A. (1988) Biochemistry 27, 2718-2727. 19 Chang, M.C., Callahan, P.M., Parkes-Loach, P.S., Cotton, T.M. and Loach, P.A. (1990) Biochemistry 29, 421-429. 20 Visschers, R.W., Chang, C.M., Van Mourik, F., Parkes-Loach, P.S., Heller, B.A., Loach, P.A. and Van Grondelle, R. (1991) Biochemistry 30, 5734-5742. 21 Van Mourik, F., van tier Oord, C.J.R., Visscher, K.J., ParkesLoach, P.S., Loach, P.A., Visschers, R.W. and Van Grondelle, R. (1991) Biochim. Biophys. Acta 1059, 111-119. 22 Van der Laan, H., Smorenburg, H.E., Schmidt Th. and V61ker, S. (1992) J. Opt. Soc. Am. B 9, 931-940. 23 Scherz, A., Rosenbach-Belkin, V. and Fisher, J.R.E. (1990) Proc. Natl. Acad. Sci. USA 87, 5430-5434. 24 Eccles, J. and Honig, B. (1983) Proc. Natl. Acad. Sci. USA 80, 4959-4962. 25 Gudowska-Novak, E., Newton, M.D. and Fajer, J. (1990) J. Phys. Chem. 94, 5795-5802. 26 Fowler, G.J.S., Visschers, R.W., Grief, G.G., Van Grondelle, R. and Hunter, C.N. (1992) Nature 355, 848-850. 27 K6hler, W., Friedrich, J., Fischer, R. and Scheer, H. (1998) Chem. Phys. Lett. 143, 169-173. 28 Jia, Y., Jean, J.M., Werst, M.M., Chan, C.K., Fleming, G.R. (1992) Biophys. J. 63, 259-273. 29 Braun, P. and Scherz, A. (1991) Biochemistry 30, 5177-5184.
Biochimica et Biophysica Acta, 1141 (1993) 238-244 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00
BBABIO 43762
Inhomogeneous spectral broadening of the B820 subunit form of LH1 Ronald W. Visschers, Frank van Mourik, Ren6 Monshouwer and Rienk van Grondelle Department of Biophysics, Faculty of Physics and Astronomy, Free University, Amsterdam (The Netherlands) (Received 7 May 1992)
Key words: Light-harvesting; Antenna complex; LH1; Fluorescence spectroscopy; Photosynthesis; Energy transfer; (Rs. rubrum)
Site-selected fluorescence spectra of the B820 subunit of LH1 from Rhodospirillum rubrum G9 were measured at temperatures between 160 K and 4.2 K. A linear correlation between excitation wavelength and maximal emission wavelength was observed across the whole Qy absorption band of the B820 subunits, which persists at higher temperatures. This demonstrates that (a) the Qr absorbance band of the B820 subunit is inhomogeneously broadened; (b) the subunit consists of a strongly interacting dimer of BChi a; (c) no energy transfer occurs among these dimers. The temperature dependence of the shape of the emission spectrum confirms that the protein forms a 'glass-like' environment for the bacteriochlorophyll pigments. After reassociating the octyl-glucoside-solubilized B820 subunits into the reassociated B873 form, the emission wavelength is independent of the wavelength of excitation at 4.2 K. This implies that the reassociated B873 form consists of a large number of interacting pigment molecules. The nature and origin of the spectral inhomogeneity in the B820 subunits is discussed.
Introduction
There is now accumulating evidence that the main near-infrared (NIR) absorbance bands due to bacteriochlorophyll in the light-harvesting complexes of purple bacteria are spectrally heterogeneous. Such behavior has been described both for the core antenna complexes (LH1) from several purple bacteria [1,2] and for the B800 band of the peripheral light-harvesting complex (LH2) from Rb. sphaeriodes [3]. For isolated LH1 complexes from Rb. sphaeroides and Rs. rubrum it has been shown that the fluorescence polarization is not constant upon excitation across the absorption band at 77 K, but increases when excitation occurs on the red side of the absorbance band. This observations led to a model in which excitations are trapped on a red-shifted BChl species (B896) that constitutes 10-20% of the total oscillator strength of this band. Time-resolved measurements probing energy-transfer within the core antenna complex provided additional evidence for the presence of red-shifted components [4,5]. However, this view was recently challenged by Timpmann et al. [6], who observed that for the LH1 antenna of Rs.
Correspondence to: R.W. Visschers, Department of Biophysics, Faculty of Physics and Astronomy, Free University, De Boelelaan 1081, 1081 HV Amsterdam.
rubrum at 4 K the fluorescence life-time depends on the emission wavelength, being shorter on the blue side of the fluorescence spectrum, and progressively increasing if the emission is detected at longer wavelengths. Therefore, they proposed that at least three, but probably many more, spectrally distinct pools of bacteriochlorophylls must exist in the LH1 antenna complex. Hole-burning studies on the B800 band of LH2 complexes from Rb. sphaeriodes demonstrated that narrow holes could be permanently burnt into this absorbance band at temperatures between 1.2 and 30 K [3], thus providing that the B800 band is inhomogeneously broadened. From the width of the holes the homogeneous linewidth (Fho m) was estimated 70 +_ 10 GHz, independent of burning wavelength between 791 and 804 nm. This homogeneous width corresponds to an energy transfer time of 2.45 ps at 4.2 K, in good agreement with time-resolved measurements of this process [7]. No fluorescence line-narrowing was observed in these experiments for the direct emission of the B800 bacteriochlorophylls. Hole-burning experiments on intact chromatophores from the Rb. sphaeroides mutant strain NF57 [8] yielded similar results for the B800 band [9]. They showed on the other hand that the B850 band of LH2 in these chromatophores and the B880 of the LH1 complex behave like homogeneous bands, since no narrow holes could be burnt in these bands [10].
239 In a recent study [11] we have reported that for isolated LH1 complexes of the LHl-only mutant strain Rb. sphaeroides M2192 [12] the position of the fluorescence band depends on the wavelengths of excitation at 4.2 K. Site-selection studies using a narrow-band, continuous wave laser showed that the emission spectrum of these pigment-protein complexes becomes significantly red-shifted upon excitation on the red side of the absorption band. These results were interpreted in terms of a model that treats separate light-harvesting complexes as clusters of a finite number of pigments among which fast energy transfer occurs. It was shown that the most significant parameter determining the exact wavelength of the onset of the red-shifted emission is the number of interacting pigments within a cluster. To gain further insight into the physical parameters that determine the position and shape of the long wavelength absorption band of photosynthetic pigment-protein complexes we have studied the simplest photosynthetic pigment-protein complex currently available, the B820(OG) subunit. This subunit can be obtained by reversible dissociation of core antenna complexes from several purple bacteria by addition of the detergents octyl glucoside [13-16] and n-octyldipropyl sulfoxide (ODPS) [17]. The biochemical and spectroscopic properties of the B820 subunit have been thoroughly characterized [14,17-21]. It was proposed to be an excitonically interacting BChl a dimer, associated with a single (a/3)polypeptide pair. To account for the high fluorescence polarization as well as the relatively low intensity of the high-energy exciton component, the Qy transitions of both BChl a molecules in the dimer have to be nearly parallel to each other in a head to tail or head to head arrangement. The subunits have been shown to reaggregate into the B873(reassoc) that resembles the original LH1 structure, upon lowering the detergent concentration [13,14]. In this work we report the polarized emission spectra of the B820(OG) subunit as a function of excitation wavelength and temperature between 4.2 and 160 K. In addition, the ground-state absorbance spectrum of B820(OG) was measured as a function of temperature between 4.2 K and 300 K. The results show that the Qy absorption band of B820(OG) is inhomogeneously broadened and are fully consistent with the view that B820(OG) is a strongly coupled dimer of BChl a. Materials and Methods
B820(OG) subunits were prepared according to the method of Miller et al. [14] with the following modifications; purified chromatophores of the carotenoid-less Rs. rubrum G9 were prepared by sonication followed by sucrose density centrifugation. Purified chromatophores were diluted to Asm ----20 and solubilized with
OGP until a nearly complete conversion into B820(OG) was observed. Aliquots of 0.2-0.5 ml were subsequently applied to a Superose 6 (Pharmacia) gelfiltration FPLC column equilibrated with buffer containing 1% OG. The complexes eluted with an apparent molecular mass of 60 kDa. The peak fraction containing purified B820(OG) subunits was stored at -20°C until later use. Low-temperature samples were prepared by dilution of thawed B820(OG) complexes into a buffer containing 70% (v/v) glycerol, 50 mM potassium phosphate (pH 7.5) and 1.15% OGP. To minimize re-absorbance the A820 was kept below 0.2 and the laser passed through the sample in such a way that the fluorescence only travelled through the sample for a very short distance (1-2 mm). Re-aggregation of the B820(OG) subunits into B873(reassoc) was achieved by diluting B820(OG) subunits into 70% (v/v) glycerol buffer containing no detergent. Fluorescence spectra were measured as described in Ref. 11. Briefly, a cw dye laser (Coherent model 590, Styryl 9) pumped by a 3W Ar + laser (SpectraPhysics, model 164) was used as an excitation source. This combination produced 30-100 mW of continuous light, tunable between 810 and 910 nm with a nominal bandwidth of > 30 GHz. To avoid local heating of the sample the beam was expanded to a diameter of 2 mm before striking it. Samples, contained in acrylic fluorescence cuvettes with 1 cm pathlength (Sarstedt) were cooled in a liquid helium flow cryostat (Oxford instruments, CF1204). Fluorescence was detected through a double 1/8 m monochromator (bandwidth 1.2 nm) with a cooled S1 (EMI 9684) photomuitiplier. Absorbance spectra were measured on a Cary 219 spectrophotometer with a resolution of 1 nm. Results
Fig. 1 shows the absorbance spectrum of B820(OG) subunits between 77 K and 300 K. Although the position of the absorbance bands is not strongly dependent on temperature [20], they are prominently broadened at room temperature; the major effect that is observed upon cooling from room temperature to about 200 K is a decrease of the width of the absorbance band. The sharpening of the spectrum in this temperature range is accompanied by a shift of the absorbance maximum from 820 nm at room temperature to 826 nm at 200 K. In addition, a minor contribution of the 777 form becomes visible. Cooling from 77 K to 4.2 K induces only a very minor additional decrease of the width of the absorbance band, and does not shift the absorbance maximum further to the red (see Fig. 3 also for the 4.2 K spectrum). No fine structure is observed in the low-temperature absorbance spectra nor in the second and fourth derivative thereof (not shown).
240 AbsoFDance I
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The emission spectra of B820(OG) upon narrowband excitation near the absorbance maximum (Aexc = 823 nm) at four different temperatures are given in Fig. 2. The sharp peak at 823 nm is partly due to direct scattering of laser light. The scattering prevents direct observation of the resonant zero-phonon line. The emission is broad and featureless. No vibronic finestructure is observed in the emission band at 4.2 K. Furthermore, the shape of this band does not change drastically either upon exciting more to the red, or
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upon increasing the temperature. Two major effects can be observed when the temperature of the sample is increased from 4.2 K: at 4.2 K no emission is discernible on the blue side of the excitation wavelength, whereas a progressive increase of emission on the blue side of the excitation wavelength is observed upon increasing the temperature; and secondly, the maximum of the emission shifts to the blue by 4 nm independent of the excitation wavelength, (see also Fig. 3), due mainly to the disappearance of the small trough on the red side of the excitation wavelength. In Fig. 3 the correlation between excitation wavelength and the position of the emission maximum is shown for several temperatures, together with the absorbance spectrum of B820(OG) at 4.2 K. The shape and position of the B820(OG) NIR absorption band in this spectrum is nearly identical to the 77 K spectrum shown in Fig. 1, demonstrating that no additional sharpening of the absorbance band takes place. From Fig. 3 it is clear that a linear correlation between excitation wavelength and emission maximum exists, when the excitation wavelength is tuned across the whole absorbance band. The slope is the same (0.88 _+ 0.08) within the margin of error at different temperatures. The polarization of the fluorescence (not shown) is independent of temperature and higher than 0.4 (Acxc = 823 nm). It is also independent of the detection wavelength, which indicates the absence of energy transfer and a nearly parallel orientation of the Qy dipoles [20]. Note that for intact LH1 complexes [11] and for the B850 band of the accessory light-harvesting complex (unpublished data) the emission wavelength dependence of the polarization can be ascribed to intra-cluster energy transfer. A more detailed analysis of the shape of the emission that is observed on the blue side of the of the
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Wavelength (nm) Fig. 2. Fluorescence spectra from the B820(OG) subunit. Sample conditions as given in Fig. 1 but Aszo =, 0.2. The excitation wavelength of the dye laser was 823 nm. 1"he sharp peak at 823 nm is due to direct scattering of laser light. The emission spectra were measured with the polarizer set to the magic angle (54 deg) to obtain isotropic emission spectra. The traces shown were taken at 4.2 K (solid), 40 K (dashed), 80 K (dotted), and 160 K (chain-dash).
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Wavelength (nm) Fig. 3. Dependence of the maximum of the emission wavelength on excitation wavelength at different temperatures; squares 4.2 K, circles 40 K, triangles 77 K. 2"he solid curve shows the 4.2 K absorbance spectrum of the B820(OG) subunit.
241
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Fig. 4. (A) Linear least-square fit of the anti-Stokes fluorescence as a function of energy difference between excitation and detection wavelength at three different temperatures (A~x¢ = 827): squares, 40 K; circles, 77 K; triangles, 160 K. Only 1 / 3 of the datapoints used for the fit are actually shown. T h e first 2 nm were not used in the fit since these points contain a scattering contribution. T h e data was fitted to an exponential function given by: c *exp (-aE/kbr) (where zlE is the energy difference between excitation and emission wavelength, K b is Boitzmann's constant). T h e temperature p a r a m e t e r T and constant c were varied to obtain satisfactory fits. The fits shown by the solid lines correspond to the following effective temperatures: 47.6 K, 75.4 K and 115 K (for m e a s u r e m e n t s at 40 K, 77 K and 160 K, respectively). (B) Same fit as in (A). T h e data were obtained by excitation in the red wing of the B820(OG) (Ae~c = 837 nm, T = 77 K) Only 1 / 2 of the actual datapoints are shown. For the exponential fit the first 20 points were not used. There is now a clear deviation of the exponential distribution in the vicinity of the scattering peak.
excitation wavelength at various temperatures is presented in Fig. 4. Photons emitted with higher energy than the excitation light derive from thermally popu-
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excitation wavelength Fig. 5. Absorbance spectrum of B873(OG, reassoc) at 4.2 K. T h e position of the fluorescence m a x i m u m at several excitation wavelengths are indicated by squares. For comparison, the same data obtained for purified LH1 complexes from Rb. sphaeroides M2192 are also shown (circles) [11]. To allow a direct comparison, the latter
have been blue-shifted by 8 nm. This is done since the absorbance maximum of the 13873 is blue shifted by 8-9 nm with respect to the LH1 complexes of Rb. sphaeroidp_s M2192, and in order to obtain cluster-sizes, the red-shift of the emission spectra has to be compared relative to the position of the absorbance maximum. The red shift of the emission maximum, observed for the purified complexes agrees with a cluster of 8 dimers [11]. Since no increase is observed for the B873(reassoc) it must comprise more than 32 dimers among which energy transfer takes place.
lated vibronic states in the system, which, in view of the small energies involved, probably not arise from intramolecular vibrations but from coupling of the electronic transitions with the surrounding protein medium. Thus, the shape of the anti-Stokes fluorescence contains information regarding the distribution of these states. A direct comparison of the population of levels at a given energy difference is prevented by the fact that the normalization of the emission spectra at different temperatures is rather awkward. To circumvent this problem we have fitted the complete blue emission with an exponential function. Fig. 4 shows that the observed anti-Stokes fluorescence indeed decays exponentially with the energy difference between excitation and detection wavelength. For the 40 K and 77 K data the fits corresponded to a Boltzmann activation temperature of 47.6 K and 75.4 K, respectively. For the 160 K data the activation t e m p e r a t u r e determined from the 'blue' fluorescence is 115 K. When the excitation wavelength is chosen on the red side of the absorbance maximum a deviation of the exponential behavior can be observed. Fig. 4B shows that in this case the observed fluorescence falls below the exponential curve in the vicinity of the excitation wavelength. Fig. 5 shows the absorbance spectrum of the B873(reassoc) at 4.2 K. T h e absence of significant absorbance in the 820 nm region demonstrates that a nearly complete reassociation of the B820(OG) has taken place [14]. The width and shape of the band are again very similar to the 77 K absorbance spectra [20]. Fig. 5 further shows the relation between excitation
242 and emission wavelengths for the B873(reassoc) at 4.2 K. There is a clear difference from the linear relation that is found for the B820(OG) subunit (Fig. 3). The wavelength at which the emission maximum occurs remains constant upon excitation across the absorbance band and no shift of the emission spectrum to the red could be detected when the excitation wavelength is chosen in the red wing of the absorbance band. The polarization of the fluorescence has dropped to a value of 0.15 (Aexc=873 nm), indicative of energy-transfer among pigments with different orientation. For comparison, we have added the data for purified LH1 complexes from Rhodobacter sphaeroides M2192. The original data from van Mourik et al. [11] have been shifted 8 nm to the blue to allow a direct comparison with our data. This is done since the Qr absorbance band of B873(reassoc) is also 8-9 nm blue-shifted compared to the absorption band of LH1 complexes due to the absence of carotenoids [13]. For the purified LH1 complexes it was found that the emission wavelength depends on the excitation wavelength only in the low energy wing ()rexc > 877 nm in Fig. 5). From these measurements it was concluded that the purified LH1 is a cluster of 16 BChl a pigments (8 dimers) randomly sampled from the totally inhomogeneously broadened pigment tool. It is clear that the B873(reassoc) behaves like a more extreme case and that the cluster size is probably significantly larger than of the purified LH1.
Discussion Recently we have developed a model that describes the effect of inhomogeneous broadening of the absorption bands on the fluorescence properties of cluster of pigments in photosynthetic light-harvesting complexes [11]. Based on the assumption that at 4.2 K the absorbance band is inhomogeneously broadened, and that efficient energy transfer only takes place following a downhill energy gradient within a cluster, the dependence of the emission spectrum on the excitation wavelength could be qualitatively and quantitatively modelled. The most important parameter in this statistical description is the number of pigments within a cluster between which efficient energy transfer takes place. The number of coupled pigments in a cluster was shown to directly determine the excitation wavelength at which the onset of the gradually increasing red shift of the emission maximum can be observed. A comparison between theoretical curves and the measurements of the correlation between excitation wavelength and the position of the emission maximum for the purified LH1 complex from Rb. sphaeroides M2192 showed that
approximately 8 closely interacting BChl a dimers are energetically coupled within such a complex.
Inhomogeneous broadening of B820 To further asses the validity of this 'cluster-model' and to determine the physical properties that define the absorbance properties of the photosynthetic antenna pigments we now present a similar study for the B820(OG) subunit of LH1 from Rs. rubrum G9. This detergent-solubilized subunit of the core antenna is assumed to be a single BChl a bound to an a/3 polypeptide pair. For clusters comprising one pigment (or one closely interacting dimer as is the case for the B820 subunit [20]) with an inhomogeneously broadened absorption band, we expect a linear correlation between the observed emission maximum and the excitation wavelength under the condition that the homogeneous bandwidth is much smaller than the actual width of the absorption band. The data presented in Fig. 3 shows that for the B820(OG) subunit such a relation is indeed found. We therefore conclude that the strongly coupled dimer behaves like a single transition in these experiments and that the absorbance band of the B820(OG) subunit is inhomogeneously broadened. Furthermore, the absence of energy transfer cannot be due to a temperature dependence of the F6rster transfer rates, since the linear relation is also found at 77 K. These results show that the origin of the inhomogeneity is present within the smallest subunit of the lightharvesting complexes and does not originate from large-scale aggregations of the pigments involved. To understand the broad and featureless site selection fluorescence spectra of the B820(OG) subunit we compare our measurements to those of Van der Laan et al. [22]. Their site selection fluorescence spectra of monomeric BChl a in triethylamine glasses show many well resolved vibronic fine structures. However, when dissolved in L D A O / w at er mixtures, these features gradually disappear when the LDAO concentration is decreased. Thus at low LDAO concentrations the emission spectra become broad and featureless as a result of increased electron-phonon coupling. In the case of BChl a in L D A O / w at er mixtures these effects probably originate from self aggregation of the pigment molecules, which is a well documented process for BChl a [23]. In the case of the B820(OG) subunit the source for the spectral inhomogeneity originates from within the protein binding pocket that surrounds the BChl a molecules. It is interesting to note that it has been previously shown that the interaction with the protein environment influences the position of the absorbance bands [24,25] and more recently it has been shown that replacing single tyrosine residues in the accessory antenna concurs with large shifts of the absorbance in LH2 [26]. Also in site selection spectroscopy of the biliprotein c-phycocyanine, the ob-
243 served emission is broad and featureless, which is ascribed to extensive electron-phonon coupling [27].
Temperature effects To evaluate the temperature dependence of the inhomogeneous broadening we have measured the NIR absorbance spectrum of the B820 subunits at several temperatures between 300 K and 4.2 K (Fig. 1). The overall changes are rather small and occur predominantly in the 300-200 K range. A comparable temperature dependence has been observed for the absorbance spectrum of Photosystem I from plants, where narrowing of the absorbance band stops at approx. 30 K [28]. The temperature dependence of the absorbance of the B820(OG) band shows that the total width of the absorbance band is only slightly determined by the decrease of the homogeneous bandwidth in the region between 100 K and 4.2 K. The relatively small changes of the bandwidth of the B820(OG) subunits in the temperature region between 100 and 4.2 K can be attributed to the larger width of the B820 absorbance band due to site-inhomogeneous broadening as compared to the PS I main absorbance band. A pronounced influence of the temperature on the emission spectrum is observed at all temperatures between 4.2 K and 160 K. At 4.2 K the emission spectrum is relatively red-shifted (for a given excitation wavelength) and shows no emission on the high energy side of the excitation wavelength. Thus, at 4.2 K fluorescence predominantly occurs from the lowest energy level of a particular B820(OG) subunit, to which the system rapidly relaxes after excitation. Increasing the temperature to 40 K or 77 K results in a small blue shift of the emission maximum and a clear increase of fluorescence on the blue side of the excitation wavelength. This-emission must derive from a thermally increased population of low frequency modes of the $1 level. The effective temperature that can be extracted from the exponential dependence of this 'blue' emission on the difference between emission and excitation energy is rather close to the actual temperature of the system, indicative for the population of a continuous set of low frequency phonon modes of the surrounding protein pocket. This is in good agreement with the observation that hole-burning spectra of BChl a in glassy environments display broad side-holes close to the burn wavelength (20 cm -1) that are ascribed to coupling to a specific low frequency mode of the host [22]. At 160 K we find that the effective temperature that is obtained from the anti-Stokes fluorescence is lower than the actual temperature. This is probably due to the fact that at 160 K the thermal energy corresponds to a substantial part of the total width of the B820(OG) Qy absorption band. If the excitation wavelength is chosen in the red wing of the B820(OG) absorption band the exponential
shape of the anti-Stokes is somewhat perturbed (Fig. 4B). This may indicate that in fact a distinct energy difference of 20-50 cm-1 exists between the 0-0 transition (which is selectively excited in the red wing) and the most abundant protein modes that are coupled to the electronic transition.
B873(reassoc) In Fig. 5 the results of site selection fluorescence spectroscopy for the reassociated subunits is shown. The reassociated subunits, in which the absorbance maximum is shifted to 873 nm, do not display a detectable change in the maximum of the emission upon excitation across the absorption band. We estimate that a red shift would probably have been detected if the cluster comprised less than 32 coupled dimers. The absence of a red-shift indicates that the reassociated form is even larger, and thus is different from highly purified carotenoid-containing LH1 complexes which appear to consist of 8 interacting dimers [11]. In fact, we find that in this aspect the B873(reassoc) behaves rather similar to chromatophores of the LH1 only mutant Rb. sphaeroides M2192 for which we could only observe a small red-shift of the emission maximum at 4.2 K (Visschers et al., unpublished data). We note that in recent study Braun and Scherz [29] have discussed how the aggregation state of the peripheral antenna may be accounted for using strongly coupled dimers as basic building blocks. Currently, we work on a similar characterization of the aggregation properties and low-temperature energy transfer of purified LH2 and LH1 mutants of Rb. sphaeroides.
Acknowledgments The authors would like to thank Ing. F. Calkoen for isolating the B820 subunits, Mr. H.A. van der Stroom for preparing chromatophore membranes, Dr. L. Valkunas (present adress: Institute of Physics, Lithuanian Academy of Sciences, Lithuania) for helpful discussions, and Prof. S. V61ker (Center for the study of excited state of Molecules, University of Leiden, Leiden, The Netherlands) for critically reading the manuscript. This research was financially supported by EEC research grant SC1 0004-C, and the Netherlands Organization for Scientific Research (NWO) through The Dutch Foundation for Biophysics.
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