J. Phys. Chem. 1994,98, 10307-10312
10307
Singlet-Triplet Spectroscopy of the Light-Harvesting BChl a Complex of Prosthecochloris aestuarii. The Nature of the Low-Energy 825 nm Transition? F. van Mourik; R. R. Verwijst, J. M. Mulder, and R. van Grondelle Department of Biophysics, Faculty of Physics and Astronomy, Free University of Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands Received: April 1, 1994; In Final Form: July 14, 1994@
The process of energy transfer in the trimeric water-solule BChl a complex of Prosthecochloris aestuarii was studied (as a function of temperature) using singlet-triplet annihilation. At 77 K the presence of one triplet state per trimer (21 BChl a molecules) is sufficient to quench the fluorescence of the trimer completely. Therefore, it was concluded that the aggregation state of the complex in solution is trimeric. At 4 K the efficiency of quenching by triplets is reduced; a mechanism for energy transfer between subunits is proposed to explain this observation. Laser-flash-induced (polarized) triplet-singlet difference spectra show that all Qy absorption bands of the complex are coupled by (strong) exciton interactions. The polarized T-S difference spectrum indicates that the 825 nm band of the timer is split into three transitions, two degenerate transitions, which are oriented in the plane of the trimer, and one slightly red-shifted transition, which is oriented parallel to the trimer C3 axes. The nondegenerate transition was found to have a negligibly small contribution to the ground-state absorption spectrum. A model for the organization of the 825 nm transition dipoles within the trimer is presented.
Introduction The efficient transfer of excitation energy among photosynthetic (antenna) pigments is mandatory for the unique (high) quantum yield of photosyn3esis. For each pigment-protein complex (the reaction center or RC) capable of converting light energy into chemical free energy there are numerous antenna pigment-protein complexes, which absorb (sun)light and transfer their excitations to the RC. In most cases more than 90% of the photosynthetic pigments are associated with this antenna function. For efficient energy transport to the RC the antenna pigments need to be closely spaced since the transfer rate between pigments is inversely proportional to the distance to the sixth power.3 On the other hand, a close spacing of the pigments can also lead to perturbations of the spectral properties of the pigments. All light-harvestingantennae show clear signs of exciton interaction. In the case of the antennae of purple bacteria spectroscopic evidence indicates that the basic “pigment species” is a dimer of BChl.1,2 However, the packing of these dimers in the antenna is rather dense, and the interactions between dimers is probably of the same order of magnitude as the interactions within the dimer. Thus, the question arises at which level of organization of the antenna the process of energy transfer should be described in terms of Forster tran~fer,~ or hopping between (clusters of) pigments, and at which level the exciton formalism is more appropriate. The best subject for the study of exciton coupling in BChlprotein complexes is undoubtedly the water-soluble BChl a light-harvesting complex of the green sulfur bacterium Prosthecochloris a e ~ t u a r i i(hereafter ~,~ Fh40 complex). This is the only BChl a containing light-harvesting complex for which the 3-D structure has been resolved. The FMO complex consists of a trimer of subunits of ca. 47 kDa, each binding seven BChl a’s. The nearest neighbor Mg-Mg distances of the chroA preliminary version of part of this work was published: Van Mourik, F.; Verwijst, R. R.; Mulder, J. M.; Van Grondelle, R. Excitation transfer dynamics and spectroscopic properties of the light-harvesting BChl a complex of Prosthecochloris aestuarii. J . Lumin. 1992, 53, 499-502. Abstract published in Advance ACS Abstracts, September 1, 1994. @
0022-365419412098-10307$04.5010
mophores within a subunit vary between 11.3 and 14.4 A, while the edge to edge distances between subunits are about 25 A. Singlet-singlet annihilation measurements have shown that at room temperature efficient energy transfer takes place within a trimer.8 The absorption, circular dichroism, and linear dichroism spectra are indicative of strong exciton interaction between the pigment^.^^^ Stark spectroscopy has revealed that the absorption spectrum of the FMO complex is free of charge transfer bands.16 Magnetic CD measurements have shown no evidence for major perturbation of the directions of the BChl a transition dipoles.20 Thus, we could expect to be able to describe the spectroscopic properties of the FMO complex using relatively simple exciton models. However, even with the available crystal structure it has turned out to be a difficult task to relate the spectroscopic properties of the FMO complex to the coupling between the pigment^.^,^^^^ The reason for this difficulty originates from the fact that there are at least seven fit parameters: the absorption wavelengths of the individual (monomeric) pigments. Calculations of the effects of conformation and surrounding of the pigments on their spectral properties have indicated that the seven individual pigments in the complex could differ in energy by as much as 500 ~ m - ’ . This ~ is about 3 times larger than the strongest coupling between any pair of pigments of the complex.l 4 Therefore, exciton calculations of the spectrum of the FMO complex must include the site energies of the seven pigments. As was shown by Pearlstein, the absorption spectrum of the FMO complex can be simulated reasonably well using exciton calculations in combination with the assignment of reasonable site energies to the pigments.14 In order to reproduce the longwavelength absorption band (825 nm) of the complex, pigment 7 (using the numbering scheme of Matthews and Fenna4) must be placed at 822 nm. This makes pigment 7 special, since it is by far the most red-shifted BChl of the complex. Pigment 7 is also special in another sense, since it is the pigment with the largest intersubunit interaction (with the corresponding pigments 7 of the two other subunit^!).^^^^^ Hole-burning have indicated the presence of more than seven exciton levels. In
0 1994 American Chemical Society
Van Mourik et al.
10308 J. Phys. Chem., Vol. 98, No. 40, 1994
9, 790
1
1
I
I
I
eo0
810
820
830
840
Wavelength (nm) Figure 1. Upper trace (offset by 0.5): 4 K absorption spectrum of the FMO complex. Lower trace: T-S difference spectrum induced by a 6 ns 10 mJ/cm2590 nm laser flash (normalized, typical AODg25 = 0.1). The bandwidth of the transient absorption measurements was 1 nm.
particular, the 825 nm band was found to be composed of two contributions peaking at 824 and 827 nm. These are ascribed to inter-subunit exciton splitting. In this work we have specifically looked at the nature of this 825 nm band using polarized T-S spectroscopy and linear dichroism. A second question, related to the first, that we have tried to answer concerns the “size” of the complex. First of all, the fact that the complex crystallizes in a trimeric form is no proof that this is also the structure in solution. Stacking of Fh40 trimer^'^,^^ could certainly introduce additional interactions between pigments on different trimers, and there have been indications that this could play a role.I8 Here we measured the effective size of the complex (number of pigments coupled by energy transfer) using singlet-triplet annihilation measurements at 77 and 4 K. Materials and Methods The FMO complex (a kind gift of Dr. S. Otte, State University of Leiden) was prepared following the procedure of Olson.5 On Superose-6 FPLC the complex was homogeneous with a molecular weight of 140 000, characteristic for the trimeric s t r u c t ~ r e . ~Absorption .~ and LD spectra were recorded on a home-built spectrophotometer. Polarized laser-flash-induced T-S absorption difference spectra were measured with the transient absorption spectrophotometer described in ref 2. Samples of the FMO complex were mixed with a buffered (10 mM Tris/HCl at pH 8.0) glass-forming solution of 50% (v/v) glycerol/water. For LD measurements the samples were oriented by embedding them in a buffered (10 mM Tris/HCl at pH 8.0) polyacrylamide gel, 14% w/v, acrylamide:N,N-methylenebisacrylamide 29: 1 with 50% (v/v) glycerol. The gels were compressed by a factor of 1.25, in both x and y directions. The LD is measured as the difference in absorption between z- and y-polarized light (incident along the x-axis of the sample). Samples were cooled in an Oxford Instruments CF1204 helium flow cryostat. Results and Discussion
1. The T-S Spectrum: Exciton Coupling within the FMO Subunit. Figure 1 (upper trace) shows the 4 K absorption spectrum of the FMO complex. The spectrum is very similar to that reported earlie$ and shows characteristic bands at 825, 812, 804, 800, and 792 nm. Figure 1 (lower trace) shows the
4 K T-S difference spectrum obtained with nonpolarized laser flashes in the Qx region of the spectrum. At all wavelengths the flash-induced bleaching or induced absorption signal decayed single-exponentially with a lifetime of 55 ps. The spectrum shows maximal bleaching at 825 nm, conciding with the 825 nm absorption band. At shorter wavelengths both positive and negative features are observed. Note that the “first-derivative” shape of the spectrum around 812 nm appears to be due to a bandshift of the 812 nm band, which dominates the groundstate absorption spectrum. The relatively sharp features around 810-820 nm were less pronounced in the 77 K T-S spectrum (results not shown), but apart from this difference, which correlates well with the slightly broader absorption bands at 77 K, the 77 K T-S spectrum was identical to the spectrum in Figure 1. We interpret the T-S spectrum as indicative of strong exciton coupling in the FMO complex. The formation of a triple state in the complex appears to affect all the absorption bands. The fact that the 825 nm band appears as the major feature of the T-S spectrum can be understood using the results of the exciton calculations of Pear1~tein.l~At 4 K we expect the formation of triplet states to take place from the lowest exciton level of the system, the 825 nm band, since relaxation to this level is fast1’,l3and since it is the only energy level that is populated at thermal equilibrium (4 K). In the simulations of Pearlstein the 825 nm band is mainly due to pigment 7, which is by far (120 cm-’) the pigment with the lowest singlet excited state energy. Consequently, it is safe to assume that pigment 7 is also the pigment with the lowest energy triplet state, and we can expect the triplets to be localized on this pigment. This enables us to simulate the T-S spectrum by calculating the ground-state absorption spectrum with and without pigment 7, thereby assuming that in the triplet state pigment 7 has little or no effect on the spectrum of the remaining six pigments. Since the BChl aT has no absorption bands in the Qy region of the spectrum,2 this is a reasonable assumption. We calculated the spectrum of just one subunit (seven pigments), since the calculated absorption spectra of the monomeric and trimeric subunits do not differ ~ignificantly.’~Figure 2a,b presents the results of this simulation. Figure 2a shows the stick spectrum for the Pearlstein (single subunit) Hamiltonian with (dashed, upside-down since it is the bleaching component of the T-S spectrum) and without pigment 7. In Figure 2b the stimulated difference spectrum is shown in which the stick spectra of Figure 2a have been convoluted with an arbitrary Gaussian bandwidth of 100 cm-’. There is a striking similarity between Figure 2b and the T-S spectrum of Figure 1. Since there are no adjustable parameters in the simulation, this should be taken as evidence that the assignment of a strong red shift to the monomer absorption of pigment 7 could be correct. Moreover, the success of the simulation indicates the spectroscopic properties of the FMO complex are largely defined by the properties of the (monomeric) subunit. 2. Singlet-Triplet Annihilation in Relation to the Couping of the Pigments in the FMO Complex. Above, we have treated the spectroscopy of the FMO complex as if it were due to a single subunit only, and obtained a good agreement between theory and experimental data using this assumption. Nevertheless, it remains a matter of controversy to which extent the spectroscopic properties of the FMO trimer should be viewed as resulting from “strong” interactions between all the BChl a molecules in the trimer. Moreover, the aggregation state of the complex in solution is not u n d i ~ p u t e d ; ~ .that ’ ~ ~is, ’ ~interactions could occur between BChl a molecules located on different FMO trimers. This degree of coupling (or “size”) can be
J. Phys. Chem., Vol. 98, No. 40, 1994 10309
Spectroscopy of the Light-Harvesting BChl a Complex
QJ
4
0
a
z
‘r 7kO
,
770
7iO
1
1
810
830
Si0
Wavelength (nm)
‘1
2: ‘780
I
I
I
I
I
800
810
820
830
840
Wavelength (nm) Figure 2. (a) Calculated stick spectra using the subunit Hamiltonian from Pearl~tein,’~ with (dashed, upside-down) and without the 822 nm pigment. A dipole strength of 1 corresponds to the dipole strength of 1 monomeric pigment (50-60 Dz). (b) Difference spectrum of Figure 2a in which the stick spectra have been convoluted with Gaussians with fwhm = 100 cm-’. The simulated spectrum is normalized on the 825 nm bleaching band.
investigated by measuring the annihilation of multiple excitations following a laser flash.*J* Here we have applied singlet(S)-triplet(T) annihilation12to the FMO complex at 77 and 4 K. The relative concentration of triplets (fraction of bleached pigments) produced by the laser flash was calculated by taking the ratio between the integrated difference spectrum and the integrated ground-state absorption spectrum over the range 790840 nm. This ratio reflects the loss of dipole strength of the FMO complex due to triplet formation. Typically, the maximally attainable (limited by annihilation and saturation effects during the laser flash) integrated bleaching amounted to 2% of the integrated dipole strength over the same spectral range. The degree of quenching of the fluorescence by the triplet, S-T annihilation,’* was measured using a weak xenon flash, fired with a variable delay after the laser flash. Figure 3 shows the 77 and 4 K results. Within the error of measurement a linear relation was obtained between the amount of triplets and the fluorescence yield at both temperatures. The slope of the curve is clearly steeper at 77 K than at 4 K, showing that on the average a triplet state is a more efficient quencher at 77 K than at 4 K. Extrapolation of the curves to zero fluorescence yield shows that 1 triplet per 23 f 2 BChl a’s at 77 K and 1 triplet
10310 J. Phys. Chem., Vol. 98, No. 40, 1994
Van Mourik et al.
7
..
0
21
’ 790
,., ...
.”. : ’, . .. . .. . ... ...
‘I 1
I
I
1
I
800
810
820
830
840
..................................
‘ 7;O
0
.
........
,,
:.. . .... . ..,.
,
I
I
I
I
1
770
790
810
830
850
Wavelength (nm) Figure 4. Polarization of the bleaching of the 825 nm band (squares, measured at 825 nm, with a bandwidth of 1 nm, 4 K) as a function of the excitation wavelength. The solid curve is the 4 K ground-state absorption spectrum. Excitation with a 6 ns 1 rd/cm2 laser flash.
Figure 5. 4 K LD and LD/A (dashed) spectra of the FMO complex. The dotted curve is the absorption spectrum of the LD sample, offset by 0.1. The bandwidth of the measurements was 0.5 nm.
band, we have measured the polarization of the 825 nm bleaching upon excitation over the Qy region. The result is shown in Figure 4. Upon excitation and detection in the peak of the 825 nm band the polarization is weakly positive (P x 0.05-O.l), which is in good agreement with the polarizedfluorescence excitation spectrum of the 825 nm band, which also has a polarization of 0.1 (W. H. J. Westerhuis, private communication). Surprisingly, the polarization of the 825 nm bleaching dropped to P < -0.2 upon red-edge excitation. The absolute values of the polarization should be taken as lower limits, since the experiments are performed with slightly saturating pulses.2 These results cannot be understood on the basis of the assumption that a single transition contributes to the 825 nm band. There must be an additional absorption band on the red side of the 825 nm band, which cannot be resolved in the absorption spectrum. The most probable explanation for the observed polarization behavior is that the 825 nm band is split into three trimer transitions which arise from the interactions between the “subunit transitions”. A trimer with C3 symmetry gives rise to three energy levels/transitions, two of which are degenerate and are oriented in the plane of the trimer, whereas the third transition is perpendicular to the plane of the trimer, along the C3 symmetry axis.23 In this interpretation the negative 825 nm polarization upon excitation at wavelengths longer than 825 nm is due to excitation of a transition perpendicular to the plane of the trimer. The perpendicular transition should also have a significantly different LD compared to that of the two degenerate bands, which are oriented in the plane of the trimer. Therefore, we specifically looked for spectral inhomogeneity of the LD signal of the 825 nm band, as shown in Figure 5. Since the complexes are disc-like particles, our orientation technique produces negative LD signals for transitions oriented perpendicular to the plane of the disc/trimer, whereas transitions that lie in the plane of the disc give rise to a positive LD signal. The absolute value of the LDIA of the 825 nm band corresponds with an angle of approximately 25” between the transition dipole moment and the plane of the disc.22 However, this should be taken as a maximal value, since it is based on the assumption of perfect orientation, whereas small proteins generally give a lower degree of orientation. The LD/A signal in the 825 nm region is constant over the band (the small drop of the LD/A signal at the red side of the band is not significant). Consequently, the LDIA spectrum gives no indication for the presence
of the long-wavelength absorption band seen in the polarized T-S spectrum. Apparently, the two degenerate high-energy transitions consitute by far the dominant contribution to the 825 nm absorption band. This puts an upper limit on the intensity of the perpendicular transition, since it should exhibit a strongly negative LD signal. Due to the necessary base-line corrections of the 4 K LD and OD spectra, the error in the signal in the red tail of the spectra can amount to about 5% of the 825 nm signals. Therefore, the dipole strength of the red band is maximally about 5% of the main 825 nm band. This distribution of the dipole strength over the three exciton components corresponds to an angle between the plane of the trimer and the “subunit 825 nm transition dipole” of less than 13”. A lower limit for the dipole strength of the 825 nm transition is harder to get. The polarized T-S spectra, which gave evidence for this transition, were recorded with rather intense laser pulses (1-2 mJ/cm2). This corresponds to a photon flux that is about 10 times the total number of FMO trimers in the sample; that is, the laser pulse is several orders of magnitude too intense for a linear response. The fact that the 825 nm band is inhomogeneously combined with the narrow bandwidth of the laser (0.2 cm-’) makes things even worse, since this implies that only a fraction of the inhomogeneously broadened pool of FMO trimers are excited by the pulse. Consequently, excitation in the red wing results in hole-buming of the red wing of the inhomogeneously broadened 825 nm band. On the other hand, the presumed weak (forbidden) band has the same density of states (but a much smaller absorption cross section) as each of the two transitions that are responsible for the main 825 nm band. Consequently, a saturating pulse exciting this “forbidden” band will result in (almost) the same number of excited states as a saturating pulse exciting the allowed 825 nm transitions! Since the produced triplet states will be localized (in “thermal equilibrium”) on one of the redshifted pigments (pigment 7), regardless of the initially produced singlet state, excitation of the forbidden state will yield the same bleaching spectrum per absorbed photon as excitation of the main 825 nm band. This explains how the forbidden transition can dominate the red part of the 825 nm band in the polarized T-S spectrum, whereas it does not show up in the LD/A spectrum. The polarized T-S spectrum and the LD signal of the 825 nm band(s) are consistent with the predictions for an excitoncoupled trimer of subunit 825 nm transitions with C3 symmetry.
Wavelength (nm)
J. Phys. Chem., Vol. 98, No. 40, 1994 10311
Spectroscopy of the Light-Harvesting BChl a Complex
Figure 6. Schematic representation of the geometry of the trimer of “825 nm subunit transition dipoles” and the energy splitting of the band. The transition dipoles are presumed to be oriented in the plane of the trimer. For angles 0 less than 40.2”the “forbidden” transition is lower in energy than the two degenerate allowed transitions; for angles > 40.2” the order is reversed.
The sign of the LD signal and the absence of a strong out of plane transition dictate that the 825 nm subunit transition dipoles are oriented in the plane of the trimer. The degree of polarization of both the fluorescence and the bleaching upon 825 nm excitation is fully consistent with the assignment of the 825 nm band to a pair of mutually perpendicular transitions in the plane of the trimer. Figure 6 shows the geometry of such a trimer. The angle 8 determines the sign and magnitude of the coupling between the subunit transitions and thereby the relative energies of the exciton transitions. For 8 < 40.2” (cos2 8 =- 7/12) the forbidden transition is located at the red side of the two degenerate transitions. For larger angles the order of the energies of the transitions is reversed. In our experiments the forbidden transition appears to be at the long-wavelength side of the allowed transition, corresponding to an angle 8 < 40.2”. The trimer calculations of Pearlstein gave the opposite r e s ~ 1 t . l The ~ reason for this discrepancy is not clear, although it must be due to a different orientation of the subunit 825 nm transition dipoles than that obtained from the crystal structure. This could also explain the failure of the simulation of the CD spectrum of the trimer.14 The calculated trimer CD spectrum has a positive CD band at about 830 nm, which is completely absent in the experimental (77 K) CD spectrum. If the subunit 825 nm transitions are indeed oriented close to perfectly in the plane of the trimer, as our measurements suggest, then the trimer interactions between these transitions will not induce a CD spectrum. In this case the CD signal of the 825 nm band will be entirely due to the interactions within the subunits. The low dipole strength that we find for the long-wavelength transition is in good agreement with the burning-power dependence of the FMO hole-burning spectra.” The “exciton band structure” of the hole-burned spectrum obtained with nonsaturated burning powers differs significantly from that obtained with high-power burning in Figure 3 of ref 11. In the saturated spectrum the intensity of the 814 nm band is higher. This correlates well with the fact that this transition has an out of plane orientation in our LD measurements and therefore gives rise to a highly polarized signal upon photoselection via the (also out of plane oriented) 827 nm transition. The ZPH features obtained with low burning power in the 827 nm region are probably mostly due to the 825 nm transitions. The low dipole strength of the 827 nm band should also result in a low fluorescence quantum yield at low temperatures (
