J. Phys. Chem. 1991, 95, 471-479 under our experimental conditions, the net ovalbumin charge is approximately -4.7,35 and the Debye screening length is on the order of 2 nm. The analysis of Russel and Glendinning? in which the electrostatic repulsion is approximated by an excluded shell model, indicates that, under our experimental conditions, QES should indeed measure an accurate mutual diffusion coefficient. Their analysis, however, because of its approximate nature, does not preclude the possibility that long-range electrostatic interactions may be responsible, at least in part, for the comparatively low values of the QES diffusivity. The independence of observed diffusivity upon scattering angle, and thus upon small changes in k , in our experiments is not particularly disturbing since the observed QES diffusivities very nearly approximate the measured intradiffusivities. 2. Oualbumin Scattering Power Polydispersity. This polydispersity might arise for example from the presence of ovalbumin aggregates in solution or the charge heterogeneity associated with the different phosphorus contents of various ovalbumin species. Polydispersity fluctuations in solution would then relax with a diffusion Coefficient closely related to the ovalbumin intradiffusivity since the relaxation would involve the motion of slightly different ovalbumin species relative to one another. Such an effect has been demonstrated for intentionally polydisperse systems4' and appears
471
to be the most likely explanation for the observed discrepancy between the boundary-relaxation and QES diffusivities. Both of the above phenomena, however, would probably manifest themselves in a multiexponential autocorrelation function, as measured by QES, and/or wave vector dependent decay constants. As mentioned previously, the autocorrelation functions derived from the QES experiments are well represented by single exponentials, and second-order cumulant analysis does not yield significantly different estimates of the ovalbumin diffusivity. It is thus apparent that significant ambiguities exist in the interpretation of either the boundary-relaxation or the QES experimental data. It is thought, however, that the PFGNMR technique provides relatively unambiguous measures of the intradiffusion coefficient and that use of this technique may aid future investigations of protein diffusion. Acknowledgment. We are grateful to Professor H. Yu for the use of his light-scattering apparatus. S.J.G. received financial support from the Wisconsin Alumni Research Foundation and the National Science Foundation in the form of Graduate Fellowships. The N M R equipment was obtained with the help of a major gift from the Shell Foundation. We are grateful to a reviewer for pointing out helpful references.
Excited-State Structure and Energy-Transfer Dynamics of the Bacteriochlorophyll a Antenna Complex from Prosthecochloris aestuarii Stephen G. Johnson and Gerald J. Small* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 5001 I (Received: April 30, 1990)
Persistent nonphotochemicalhole-burning results at 4.2 K are reported for an antenna complex of the photosynthetic bacterium Prosthecochloris aestuarii which consists of a trimer of subunits with each subunit containing seven bacteriochlorophyll a (BChl a) molecules. The data for the Q, (SI)region are consistent with an excitonically coupled system and indicate the existence of more than seven exciton levels, with two contributing to the lowest energy absorption band at 825 nm. State assignments are discussed in terms of various models, including one that invokes interaction between BChl a belonging to different subunits of the same trimer. Exciton level decay times are reported and discussed in terms of exciton-phonon scattering involving phonon emission. These times are compared to and discussed in terms of the ultrafast (SI00fs) decay times for the accessory pigment Qystates of the bacterial RC. The hole spectra also allow for the determination of the linear exciton-phonon coupling strength (optical reorganization energy) and the contribution of complex heterogeneity to the line widths of the absorption profiles.
1. Introduction Electronic energy relaxation and transfer within the lightharvesting complex (LHC) of the photosynthetic unit and subsequent trapping by the reaction center (RC) complex have long attracted very considerable attention.' The problem is considerably more complex than that of exciton transport and trapping by a dopant molecule in a host (donor) crystal because of the heterogeneous nature of the LHC. Generally, the LHC is comprised of several protein complexes and, furthermore, the diagonal energy disorder for pigments within a given complex can be signifi~ant.~"Compounding the difficulty of the problem for most
systems is the unavailability of an X-ray structure even for a subunit, e.g., photosystems I and I1 of green plants. Structure together with data on the kinetics and pathways of energy transfer, excited-state assignments, diagonal energy disorder, and optical nuclear reorganization energies are important for the testing of energy-transfer theories. Time domain spectroscopies have been the principal tools used to study energy transfer which occurs typically on a picosecond time scale.4 However, spectral hole burning has recently been shown to be an important complementary t e c h n i q ~ e . ~ qIn ~ -addition ~ to the kinetics, spectral hole (3) Fetisova, 2.G.;Freiberg, A. M.; Timpmann, K. E. Nature (London) 1988, 334, 633.
( I ) van Grondelle, R. Biochim. Biophys. Acta 1985,811, 147. Geacintov, N. E.; Breton, J. In CRC Critical Reviews in Plant Science; CRC Press: Boca Raton, FL, 1987; Vol. 5, p I . Govindjee, Ed.Bioenergetics ofPhotasynthesis; Academic Press: New York, 1975. Michel-Beyerle, M. E., Ed. Antennas and Reaction Centers of Photosynthetic Bacteria; Springer-Verlag: West Berlin, 1985. Jortner, J.; Pullman, B., Eds. Perspectives in Photosynthesis; Kluwer Academic: Dordrecht, 1990. Clayton, R. K.; Sistrom, W. R., Eds. The Photosynthetic Bacteria; Plenum Press: New York, 1978. Breton, J.; Vcrmeglio, A.; Eds. The Photosynthetic Bacterial Reaction Center, Plenum Press: New York, 1988. (2) Gillic, J. K.; Small, G . J.: Golbeck, J. H. J . Phys Chem. 1989, 93, 1620.
(4) Holzwarth, A. R. Q.Reu. Biophys. 1989, 22, 239. (5) Kohler, W.; Friedrich, J.; Fischer, R.; Scheer, H. Chem. Phys. Lett. 1988, 143, 169. (6) Kohler, W.; Friedrich, J.; Fischer, R.; Scheer, H. J . Chem. Phys. 1988, 89, 871. (7) Johnson, S. G.; Tang, D.; Jankowiak, R.; Hayes, J . M.; Small, G. J.; Tiede, D. M. J . Phys. Chem. 1989, 93, 5953. (8) Tang, D.; Johnson, S. G.; Jankowiak, R.; Hayes, J. M.; Small, G. J.; Tiede, D. M. In Perspectives in Photosynthesis; Jortner, J.; Pullman, B., Eds.; Kluwer Academic: Dordrecht, 1990; p 99. (9) Johnson, S. G.; Tang, D.; Jankowiak, R.; Hayes, J. M.; Small, G. J.; Tiede, D. M. J . Phys. Chem. 1990, 94, 5849.
0022-3654/91/2095-0471.$02.50/00 1991 American Chemical Society
412
The Journal of Physical Chemistry, Vol. 95, No. I , 1991 WAMLENGTH (nm)
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.1
ti IO
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Figure I . 4.2 K absorption of the antenna complex from Prosthecochloris aestuarii in buffered glass forming solvent. Solid arrows indicate burn wavelengths used in this study. Resolution 4 cm-l.
burning can provide information on diagonal energy disorder, heterogeneity, and the linear electron-phonon coupling. The watcr-soluble BChl a complex from Prosthecochloris aestuarii, the Fenna-Matthews-Olson (FMO) complex, is one for which structural information is available.'0*" The crystal structure revealed that the basic structural unit is a trimer of subunits, cach containing seven BChl a molecules which are not symmetry equivalent. Nearest-neighbor M g M g distances within a subunit vary between 11.3 and 14.4 A, while the edge-to-ed e distances between subunits (within a given trimer) are =25 . The complex is devoid of the RC. Furthermore, the relative orientations of the BChl a molecules within the subunit are not appropriate for the formation of "dark" intermolecular chargetransfer states which might mediate energy transfer between the "light" Qy states.9," Thus, the FMO complex is an interesting and, perhaps, relatively simple system for the study of excited-state structure and relaxation dynamics. Pairwise dipole-dipole matrix elements within the subunit are as large as ~ 2 0 cm-1.12J3 0 The weakest interactions between BChl monomers in a given subunit are ;=IO cm-I, which is comparable to the strongest interaction between monomers of different subunits of the trimer. The 4.2 K absorption spectrum of the FMO complex in a glass is shown in Figure 1 and exhibits five discernible components (none of which can be assigned to vibrations because the required Franck-Condon factors are far too large).2 Although this structure and the CD spectra cannot be understood in the absence of excitonic interactions, attempts to do so in terms of excitonic interactions within a single subunit have not been successful.13J4 One difficulty has been the prediction of the absorption band at 825 nm. (See refs 12, 15, and 16 for a discussion of other difficulties.) It has been suggested that this difficulty cannot be resolved by allowing for diagonal energy disorder of the seven BChl a.15 Such disorder is mandated by the X-ray structure which shows conformational variations between the BChl a monomers as well as significant differences in axial ligand binding to Mg. Indeed, recent quantum chemical calculations by GudowskaNowak et a1.I6 indicate that the diagonal energy disorder for the
1
(10) Matthews, B. W.; Fenna, R. E. Arc. Chem. Res. 1980, 13, 309. ( I I ) Tronrud, D. E.; Schmid, M. F.;Matthews, B. W. J . Mol. Eiol. 1986, 188. 443.
( I 2) Pearlstein, R. M. In Photosynthetic Light-Harvesting Systems; Scheer, H.; Schneider, S., Eds.; De Gruyter: Berlin, 1988; p 555. (13) Matthews, B. W.; Fenna, R. E.; Bolognesi, M. C.; Schmid, M. F.; Olson,J . M. J . Mol. Biol. 1979, 131, 259. (14) Pearlstein, R. M.; Hemenger, R. D. froc. Narl. Aeod. Sei. U.S.A. 1978, 75, 4920.
( I 5 ) Pearlstein, R. M. Private communication. (16) Gudowska-Nowak, E.; Newton, M. D.; Fajer, J. J. Phys. Chem. 1990, 94, 5795.
Johnson and Small
Q,, states is greater than the observed width of the Q,, absorption 0 Although these calculations overestimate region ( ~ 5 0 cm-I). the diagonal energy disorder, they indicate that this disorder should be treated on an equal footing with the excitonic interactions. An additional complication is that the structure of the complex in solution may be different from that in the crystal, because of aggregation effects. (Neither absorption nor CD spectra are available for the crystal.) This possibility has been considered by Pearlstein, who has suggested that in solution two subunits belonging to different trimers are able to strongly interact across the /3 sheet (the protein portion of the ~ u b u n i t ) .With ~ ~ ~this ~~ model his calculations show, for example, that these additional strong interactions are important for formation of an absorption band at 825 nm. Thus, what began as a seven BChl a aggregate problem for the Qy state structure is augmented to one with 14 BChl a. We note, however, that with the adoption of this model the neglect of the interactions between subunits of the same trimer is questionable. In this paper we report the results of nonphotochemical holeburning (NPHB) studies designed to provide additional insight on the Qy state structure and excitonic interactions and the first determination of the inhomogeneous, linear electron-phonon coupling and dynamical contributions to the widths of the absorption features of Figure 1. An additional motivation was to compare the Q,, state energy-transfer decay times with those of the "accessory" pigment Q,, states of the RC of Rhodopseudomonas viridis and Rhodobacter sphaeroides. Aside from the interaction between the monomers of the special pair (primary donor state), the range of pairwise excitonic interactions in these RC is comparable to that of the BChl a complex of P. aestaurii." Very recently, spectral line-narrowing experiments on the bacterial RC have shown that the accessory BChl and bacteriopheophytin Qy states decay in the remarkably short time of 30-50 fs at 4.2 K? Earlier time domain studies had shown that these states decay and the primary donor state is populated in 5100 fs at 10 K.18 Energy-transfer data for the FMO complex was viewed as important for consideration of the mechanism for the ultrafast energy-transfer processes of the bacterial RC. A brief account of the initial stages of our work has been published.19 11. Experimental Section
Samples of the water-soluble BChl a antenna-protein complex from Prosthecochloris aestuarii were suspended in a buffered glass forming solution consisting of a mixture of 75% potassium glycerophosphate (in water, K & K Laboratories) and water (2:l). The optical density (OD) of the sample was ~ 0 . at 3 the 814-nm absorption band (4.2 K). Burn irradiation was provided by a Tixapphire laser (Model T-1000, Excel Technology, Inc., Bohemia, NY) pumped by a Nd:YLF laser (Model 1-1000, Excel Technology). The laser was operated at 1 kHz and provided tunable radiation in the area of interest (790-830 nm) with pulse duration of -35 ns. The laser line width for the moderate-resolution spectra presented here was 50 GHz (- 1.7 cm-I). A narrower line width (-500 MHz) was obtained through the use of an intracavity etalon for the highresolution experiments. The absorption and hole-burned spectra were obtained with a Bruker IFS 120 H R Fourier transform spectrometer operating at a read resolution of 2 cm-l for the moderate-resolution scans and at 0.05 cm-l for the high-resolution scans. Samples were mounted and cooled to T = 4.2 K in a Janis Model 8-DT super vari-temp liquid helium cryostat. Samples were plunged into liquid helium to form a glass; however, slower cooling rates yielded no variation in the 4.2 K absorption spectrum. Burn intensities and times are given in the figure captions. Polarized hole spectra were obtained by inserting a Glan/Thompson prism into the spectrometer (normal to the probe beam path) and a (17) Knapp, E. W.; Fischer, S. F.; Zinth, W.; Sander, M.; Kaiser, W.; Deisenhofer, J.; Michel, H. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 8463. (18) Breton, J.; Martin, J.-L.; Fleming, G. R.; Lambry, J.-C. Biochemistry 1988, 27, 8276. (19) Johnson, S. G.; Small, G.J. Chem. Phys. Lett. 1989, 155, 371.
The Journal of Physical Chemistry, Vol. 95, No. 1, 1991 473
Bacteriochlorophyll a Antenna Complex TABLE I: Exciton Components wavelength, component nm (em-') 827.1 ( I 2 090) 824.4 ( 1 2 130) 816.3 (12250) 813.0 (12300) 807.8 (1 2 380) 804.8 ( 1 2 425) 801.3 (12480) 793.6 ( 1 2 600)
I 2 3 4
5 6
7 8
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excited-state decay time' 1 2 0 psb 1 2 0 ps
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Decay timc of cxciton state as measured by burning directly into that state. *Hole width 0.5 em-I for components 1 and 2. cHole width 50 em-' for components 3-8. a
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Figure 3. Hole spectra, AB = 829.0 nm (12063 cm-I). Burn intensity 400 mW/cm2 (1 kHz, -35-ns pulse). Burn times: (A) 35 s, (b) 95 s. Read resolution 2 em-'. T = 4.2 K. Solid arrow indicates As. Dashed arrows indicate satellite holes. Dashed horizontal lines indicate AA = 0. Same vertical scale applies to each spectrum (spectrum A offset for clarity). Solid arrows pointing upward indicate real- and pseudo-FSBH.
.2 -
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Figure 2. Prcburn absorption spectrum (dashed line) and hole-burned absorption spectrum (solid line) for AB = 819.5 nm. These spectra provide the M spectrum for Figure 4C. See Figure 4 caption for details. similar prism in the burn beam, immediately before the cryostat in both cases. The prism in the probe beam path was then rotated (90O) to provide parallel and perpendicular linearly polarized hole spectra. The prism in the burn beam path ensured that the laser polarization was clean. Fluorescence spectra were obtained from a conventional setup in which an excimer laser (Lambda Physik EMG 102) pumped a dye laser (Lambda Physik FL2002) using Rhodamine 800 or Styryl 9 laser dyes. Fluorescence was dispersed by a I-m McPherson 2061 ( R = 160000) monochromator. Signal processing was accomplished with a PMT (RCA C31034) and a Stanford Research SR250 boxcar averager. Fluorescence spectra were normalized for laser jitter. The laser line width for the fluorescence experiments was 0.2 c d . 111. Results The 4.2 K absorption spectrum of the Qy region for the BChl a complex of P. aesruarii (Figure 1) is comparable to a previously published low-T spectrum.1930 Five components at 825, 814, 805, 800, and 794 nm are discernible. The arrows indicate the burn frequencies utilized in this study. For each bum frequency, spectra were obtained for several burn fluences (burn times for a fixed burn intensity). Only a few of the spectra will be given here. The key features of these spectra used, for example, for state assignments were confirmed to be reproducible. The assignments are further supported by spectra not shown. In arriving at the assignments given in Table I, it was important to analyze all spectra in concert. We begin with Figure 2 which illustrates the considerable degree to which hole burning can affect the absorption spectrum. The burn wavelength (AB) employed was 819.5 nm, but similar changes (20) Whitten, W. B.; Olson, J. M.; Pearlstein, R. M. Biochim. Biophys. Acta 1980, 5 9 / , 203.
have been induced with higher and lower values of AB. The A(absorbance) (AA)spectra presented later show that conservation of absorption intensity accompanies hole burning, confirming that the mechanism is nonphotochemical.21q22 The hole spectra are persistent provided the sample is maintained a t the burn temperature, 4.2 K. The two most striking aspects of the spectra in Figure 2 are (i) the significant blue-shifting of the entire absorption spectrum and intensity redistribution produced by hole burning and (ii) that the absorption features to higher energy of AB (as well as lower) are affected. Noting that at 4.2 K only downward energy transfer from the burn frequency is possible, it is immediately apparent that these observations cannot be understood in the absence of connectivity between the different absorption bands (states). The persistent NPHB spectra shown in Figure 3 were obtained with AB = 829.0 nm (12 063 cm-I), which lies on the red edge of the 825-nm absorption band. Burn intensities for both AA spectra were identical, but spectra A and B were obtained with burn times (73 of 35 and 95 s. Both exhibit a zero-phonon hole (ZPH) a t AB with a width of 8.0 cm-I. This width is determined by the burn and read resolutions and saturation b r ~ a d e n i n g . ~The ~ - ~ZPH ~ in spectrum B is saturated and corresponds to a 70%AOD change at AB. Higher resolution scans of much shallower ZPH will be discussed later. According to hole-burning theory:6 the fractional AOD change for the saturated ZPH is given by exp(-S), where S is the Huang-Rhys factor for the protein phonons. This prediction has recently been confirmed by NPHB studies of the laser dye Oxazine 720 imbedded in a glycerol glass and poly(viny1 alcohol) films.26 A AOD change of 70% corresponds to an S value of 0.3. This value is nearly a factor of 3 smaller than observed for Chl a in the light-harvesting complex of photosystem I of spinach' and represents the weakest linear electron-phonon coupling observed to date for antenna complexes. The phonons to which the transition electron couples are responsible for the relatively broad phonon sideband holes (PSBH) which appear at w, ==30 cm-' to higher and lower energy of the ZPH in the spectra of Figure 3 (see also Figure 4A). The former and latter are referred to as the real and pseudo PSBH; they are regular and (21) Jankowiak, R.; Small, G. J. Science 1987, 237, 618. (22) Hayes, J. M.; Small, G. J. Chem. Phys. 1978, 27, 151. (23) Jankowiak, R.; Shu, L.; Kenney, M.; Small, G. J. J. Lumin. 1987, 36, 293. (24) Friedrich, J.; Haarer, D. Angew. Chem., Int. Ed. Engl. 1984, 23, 113. (25) Volker, S. J . Lumin. 1987, 36, 251. (26) Shu, L.; Small, G. J. Chem. Phys. 1990, 141, 447.
The Journal of Physical Chemistry, Vol. 95, No. I , 1991
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Johnson and Small WAVELENGTH (nm)
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Figure 4. Hole spectra, AB = (A) 825.8 nm (12 110 cm-I), (B) 824.0 nm (12 136 cm-I), and (C) 819.5 nm ( 1 2 202 cm-I). Burn intensities: (A) 150 mW/cm2, (B) 230 mW/cm2, and (C) 400 mW/cm2. Burn times: (A) 7 min, 40 s; (B) 8 min, 15 s; and (C) 6 min, 15 s. Arrows, T, read resolution, and horizontal lines same as in Figure 2. Same vertical scale applics to cach spcctrum (spectra A and C offset for clarity). Solid arrows pointing upward indicate real- and pseudo-PSBH.
well-understood features of hole-burned The frequency of the phonons observed (mean frequency of w, =30 cm-I) is typical of that observed for other protein-chlorophyll complexes such as the core and native antenna complexes of PS 12*29 and the primary electron donor and accessory Qypigment states of the PS I 1 R C 3 0 Noteworthy for the BChl a complex of P. aestuarii is that the optical reorganization energy, Sw,, is very small, = l o cm-l. We turn next to the satellite hole structure in Figure 3 which appears to higher energy of AB and which is indicated by the dashed arrows. (The horizontal dashed lines define AA = 0.) The hole and antihole structure approximately satisfy the conservation of absorption intensity expected for NPHB.Z'*2ZThe satellite holes occur at 8 16, 8 13, and 4 0 8 nm, with the latter superimposed on the broad antihole derived from the former two. (NPHB of AT* states is generally characterized by an antihole that is predominantly blue-shifted relative to the hole profikZ) Comparison of Figure 3 with Figure 1 shows that the 816- and 813-nm holes are in the vicinity of the strongest absorption feature at 814 nm and that they correspond to only a 2% AOD change. These two holes are listed as components 3 and 4 in Table I. The widths of the satellite holes, =50 cm-l, do not necessarily reflect uncertainty broadening due to downward energy transfer since, for example, it cannot be assumed that perfect correlation exists between the site excitation distribution function of the state excited at AB and those of the other Qystates. In order'to determine the homogeneous line width of a state, it is necessary to burn directly into it (vide infra). We note that spectra A and B of Figure 2 show that the 81 3/81 6-nm hole intensity ratio increases with increasing burn time. The hole spectra in Figure 4 labeled as A, B, and C correspond to AB = 825.5, 823.5, and 819.5 nm, respectively. The sharp ZPH at AB in A and B correspond to a 70% AOD change. In spectrum A the PSBH at f 3 0 cm-' relative to the ZPH are clearly discernible (solid arrows). Focusing first on the pseudo PSBH at -30 cm-I, we observe that its shape in spectrum B is distorted (fattened). This distortion cannot be explained by linear elec(27) Hayes, J . M.: Gillie, J. K.; Tang, D.; Small, G. J. Biocfiim. Biopfiys. 305. (28) Lee, I.-J.; Hayes, J. M.; Small, G. J. J . Cfiem. Phys. 1989, 91, 3463. (29) Gillie, J. K.; Hayes, J . M.; Small, G. J.; Golbeck, H. M . J . Pfiys. Cfiem. 1987. 91. 5524. (30)Jankowiak, R.:Tang, D.; Small, G. J.: Seibert, M. J . Pfiys. Cfiem. 1989. 93, 1649.
Acta 1988, 932,
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WAVENUMBERS (cm-1) Figure 5. Holc spectra, AB = ( A ) 816.3 nm (12250 cm-I), (B) 815.0 nm (1 2 270 cm-I), and (C) 81 2.5 nm (1 2 308 cm-I). Burn intensities: (A)
700 mW/cm2, (B) 670 mW/cm2, and (C) 567 mW/cm2. Burn times: (A) 4 min, 20 s; (B) 4 min, 50 s; and (C) 4 min, 20 s. Arrows, T, read resolution, and horizontal lines same as in Figure 2. Same vertical scale applies to each spectrum (spectra A and C offset for clarity).
tron-phonon coupling. Rather, it is most likely a manifestation of energy transfer from the isochromat excited at AB to lower energy absorbers of the 825-nm absorption band. This interpretation is supported by the fluorescence data (vide infra). Downward energy transfer is inconsistent with the interpretation that views the 825-nm band as being due to a single state of a seven BChl a containing subunit. Spectrum C of Figure 4 provides additional support for energy transfer. It was obtained with AB = 819.5 nm (12202 cm-I), located in the valley between the 825and 8 14-nm absorption bands. Two holes, indicated by dashed arrows, are observed at 828 and 824 nm. They are denoted as components 1 and 2 in Table I. It is important to note that the centroid of the overall hole profile is close to 828 nm and not 825 nm, which is the maximum of the lowest energy absorption band (Figure 1). All three spectra in Figure 4 exhibit a prominent satellite hole at 814 nm, rather than two at 816/813 nm as in Figure 3. In fact, the 816/813-nm doublet could only be observed for AB located in the low-E side of the 825-nm absorption band. An interpretation for this is provided in section IV. The 8 14-nm hole of Figure 4-8 is probably one and the same as the 8 13-nm hole. Additional satellite holes in Figure 4 appear at 806 and 800 nm (components 5-7 of Table I). Spectra A, B, and C of Figure 5 were obtained with AB = 816.3, 8 15.0, and 8 12.5 nm, respectively (see solid arrows), which range from the low- to high-E sides of the 814-nm absorption band. A weak and relatively sharp ZPH appears at AB in each of the spectra and is superimposed on the broader and most intense hole at 814 nm (third dashed arrow from the left in spectrum C). An interpretation of the weak ZPH is given in section IV. At this point it is sufficient to know that the ZPH are not connected with the 814-nm hole. In spectrum C, four broad satellite holes at 808, 805, 800, and 794 nm are also discernible (dashed arrows). The hole of 808 nm had been observed in Figure 3, while in Figure 4 a relatively broad and shallow feature at =806 nm was observed which, most likely, represents the unresolved 808/805-nm holes. The above four holes are listed as components 5-8 in Table I. Further evidence for the 808/805-nm doublet is provided by the burn time dependent hole spectra shown in Figure 6 (AB = 812.5 nm). These spectra also provide more convincing evidence for a broad and shallow hole a t 794 nm (rightmost dashed arrow). It is instructive to examine the hole structure of Figure 5 near 825 and 81 4 nm on an expanded scale. Figure 7 shows that the centroid of the satellite hole to the red of 825 nm shifts to higher energy as the burn frequency in the 814-nm band increases (see solid vertical line). This type of correlation has been observed
Bacteriochlorophyll a Antenna Complex
The Journal of Physical Chemistry, Vol. 95, No. 1, 1991 475 WAVELENGTH (nm)
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Figure 6. Burn time dependent hole spectra. AB = 812.5 nm (12308 cm-I). Burn time utilized: 5 s, 20 s; 2 min, 20 s; and 4 min, 20 s. Burn intensity same as in Figure 5 .
12000
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WAVENUMBERS (cm-1) Figure 8. Hole spectra, AB = 800.0 nm (12 500 cm-I). Burn intensity of 800 mW/cm2. Burn time of 1 min, 30 s. Arrows, T, read resolution, and horizontal lines same as in Figure 2.
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Figure 7. Samc hole spectra as in Figure 5 with expanded x axis. AB = 8 16.3, 8 15.0, and 8 12.5 nm (A, B, and C). Vertical line is drawn through centroid of hole (-828 nm) for AB = 816.3 nm. See text for details.
Figure 9. Fluorescence line-narrowed spectrum. X, = 796.0 nm. Read resolution 8 cm-I. T = 4.2 K. Power intensity incident on sample IO mW/cmz.
between the lower and upper dimer (Davydov) components of the special pair Qystate of the Rps. viridis and Rb. sphaeroides RC.’-* The hole to the red of 825 nm is also asymmetric due to components I and 2 at 827 and 824 nm; cf. two leftmost dashed arrows in Figures 4C and 5C. This asymmetry is perhaps more apparent in Figure 8, which shows the hole spectrum obtained with AB = 800.0 nm. In this spectrum all hole components except the one at 816 nm are apparent (see arrows). It also shows that the conservation of absorption intensity expected for NPHB is, for all intent and purposes, satisfied. From the hole spectra presented it is apparent that their intensity distribution is determined, in part, by the interplay between the hole and antihole profiles. For example, in Figure 8 the four highest energy holes are obviously superimposed on an antihole derived mainly from the most intense hole at 814 nm. Using the absorption spectrum of Figure I , one can eliminate the interferences from the four highest energy holes to this antihole to arrive at an approximate antihole profile. This profile, which is similar to that observed for Chl a of the PSI-200 antenna complex,* rises quite sharply to the right of the 8 14-nm hole, peaks approximately at 810 nm, and slowly tails out to ~ 7 9 5nm. Therefore, the blue-shifted antihole distribution is spread over a range of about 300 cm-I.
A comparison of this antihole in Figure 8 with that of the hole at 827 nm in the same figure is instructive. If the 827-nm hole was due to only one component (not two as we have suggested), one would have to conclude that its antihole is significantly less sharply rising than the antihole for the 814-nm hole. We are not able to provide a convincing argument to support such a conclusion. It is, however, entirely reasonable to propose that the intensity of the hole at 824 nm (component 2) in Figure 8 is suppressed due to a sharply rising antihole from the 827-nm hole (leftmost arrow). We have explored this possibility by using the shape of the antihole profile from the 814-nm hole to model the antihole of the 827-nm hole and find that the 821- and 824-nm holes have comparable intensities. The 825-nm absorption band in Figure 1 can also be accounted for in terms of two comparable intensity components at 827 and 824 nm (Table I) with widths of 70 cm-l (simulation not shown). In fitting this absorption, the contribution from the low-E tail of the 814-nm absorption band was subtracted out by using a Lorentzian (appropriate since the 814-nm state is homogeneously broadened; vide infra). The simulations also showed that the valley absorption between the 825- and 8 14-nm bands can be accounted for by the tails of these two bands. To provide further support for the two-component proposal for the 825-nm absorption band, fluorescence studies were conducted.
476 The Journal of Physical Chemistry, Vol. 95, No. 1, 1991 TABLE 11: Vibrational Modes for Lowest Enerev State ~~~
~
excited-state freqf cm-'
excited-state freq,b cm-'
199 209 216 273 286 320 350 368 415 456 484 493 510 522
I94
342 379 449
Excited-state (S,) vibrational frequencies; this work. From ref 45. Figure 9 is the spectrum obtained with A,, = 796.0 nm. This wavelength excites both the 800- and 794-nm states (Figure 1 ) but, in addition, can be expected to excite very weak vibronic bands (=500-~m-~ region) which build on the 825-nm band. Thus, one anticipates the observation of sharp zero-phonon lines from vibronic excitati0r-1~~ superimposed on a broad fluorescence band which results from largely uncorrelated downward energy transfer from the 800- and 794-nm states to the relatively long lived emitting state. Such is observed. Four of the ZPL are labeled (in cm-l) with their excited-state BChl a mode frequencies. Table I I lists the mode frequencies observed with this and other values for A,, and compares them with ground-state frequencies determined by resonance Raman. A key observation is that the centroid of the overall fluorescence origin band is at 828.3 nm, which is 40 cm-l lower in energy than the absorption maximum at 825.0 nm. I f it is asumed that the 825-nm band is due to a single state (emitting), then the 40 cm-' represents the Stokes shift (2Sw,) from phonons with w, = 30 cm-l (vide supra). However, we showed earlier that the entire 825-nm absorption band is characterized by an S value of 0.3. Thus, a Stokes shift of 2(0.3)(30) -20 cm-I would be expected. This is significantly different from the observed gap of 40 cm-'. Therefore, we conclude that the observed emission originates from the assigned absorption component state at 827.1 nm. In addition to the wavelengths (frequencies) of the hole components, Table I lists their widths. The widths for components 1 and 2 were obtained from high-resolution scans (cf. section TI) on shallow holes (
