645%
J . Phys. Chem. 1992, 96, 64584464
6896 and 6870 Components of the Rhodobacter sphaerotdes Antenna: A Hole Burning Studyt N. R. S. Reddy,t R. Picorel,# and G. J. SmaU**t Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 5001 1 , and E. E. Aula Dei, CSIC,Apdo. 202, 50080-Zaragoza, Spain (Received: January 6, 1992; In Final Form: March 26, 1992)
Novel nonphotochemical hole burning action spectra are persented that yield the low-temperature absorption profiles of B896 and B870 and their underlying structures (linear electron-phonon coupling and site inhomogeneous broadening). The results establish that B896 and B870 are associated with the far more intense B875 and B850 bacteriochlorophyll a absorption bands, respectively, of the light harvesting I and I1 complexes. The homogeneous widths of the B896 and B870 zero-phonon holes are the same within experimental uncertainty, 3.2 cm-' at 4.2 K, which corresponds to a total optical dephasing time of 6.6 ps. A number of interpretations for B870 and B896 are considered. Favored is one in which they are due to the lowest energy levels of the B850* and B875* exciton bands (asterisk denoting the S,(Q,) state). Based on studies of the dephasing of excitons in organic crystals, the 6.6-p dephasing of B896* is attributed to exciton scattering with energetic inequivalent neighboring unit cells. Such scattering and B870 to B875 energy transfer are suggested to be contributors to the dephasing of B870*. The effect of glasslike structural heterogeneity on the optical selection rules for unit cells of cyclic symmetry is also considered.
I. Introduction Excitons and their role in optical excitation transfer (ET) in photosynthetic antenna protein-chlorophyll complexes have long been subjects of intere~t.l-~ However, our understanding of excitonic effects in such complexes, in contrast with aromatic molecular crystals, is quite poor. To put our level of understanding in perspective, we note that the following have been subjects of thorough experimental and theoretical study in organic crystals: unit cell exciton level (Davydov) and exciton band structure,6-I3 inter-exciton level relaxation via scattering of and coherent and incoherent exciton (wave packet) transport.I8 These works illustrate that one cannot adequately understand the latter two without detailed information on unit cell and exciton band structure, the latter being dependent on interactions between molecules belonging to different unit cells. Whether or not excitons are important in an antenna system or protein-pigment complex contained therein depends on which aspect of excitonic behavior is being addressed: for example, manifestations of excitonic coupling in circular dichroism and linearly polarized spectra, the kinetics of downward energy cascading, the mechanism of transport between structurally distinct complexes, and coherent exciton transport. For consideration of such, exciton level structure and bandwidth, pure dephasing frequencies of exciton levels, electron-phonon coupling, and diagonal energy disorder stemming from structural heterogeneity are important factors. Thus, the temperature of the system need often be specified when the above question is posed. For example, the unit cell excitonic interactions of crystals such as naphthalene, phenanthrene, and anthracene are sufficiently strong to result in the linear polarizations of the SIstate Davydov components of the exciton band at room temperature being dictated by the unit cell factor group (symmetry). However, coherent transport, by which we mean that the coherence length (mean free path) of the exciton wave packet is greater than the unit cell length,I9 is observed only at very low temperatures and for ultrapure 'strain-free" crystals.18 Research in photosynthesis on excitons has been largely limited to understanding the relationship between protein-pigment structure and elementary exciton level structure. As will be further illustrated20-21 here, persistent nonphotochemical hole burning 'Presented, in part, at the Optical Society of America sponsored topical meeting on "Persistent Spectral Hole Burning: Science and Application" (Sept 26-28, 1991, Monterey, CA). *Iowa State University. E. E. Aula Dei, CSIC.
0022-365419212096-6458$03.00/0
(NPHB) is an important frequency domain technique for probing exciton level structure and exciton relaxation/scattering processes. The first application of NPHB to the problem of excitonic behavior in an antenna protein complex was for the base-plate BChl a (bacteriochlorophyll a ) complex of Prosthecochloris aestuarii which is devoid of the reaction center. The results showed that20szzthe structured Q,, (SI)absorption spectrum should be interpreted in terms of delocalized exciton levels of a C3trimer of subunits, each of which contains seven symmetry-inequivalent BChl a m o l e c ~ l e s . The ~ ~ .unstructured ~~ lowest energy absorption band at 824 nm was shown to be due to two states separated by -40 cm-l. It was concluded that they are trimer states polarized parallel and perpendicular to the C3symmetry axis. Fluorescence studies later confirmed these polarization^.^^ Furthermore, the zero-phonon hole profiles (4.2 K)obtained with a wide range of burn wavelengths (A,) led to -200-fs total dephasing times for the higher energy exciton levels which were20interpreted in terms of =lOO-fs downward inter-exciton level relaxation via phonon emission. Importantly, the ZPH widths for the lowest energy exciton level a t 827 nm yielded a much longer dephasing time at 4.2 K of 40 ps, which in view of the results presented here for the antenna complex of Rhodobacter sphaeroides (Rb. sphaeroides) and our understanding of exciton-defect scattering processes in organic crystals can be given a physical interpretation. The above NPHB studies showed, for the first time, that inter-exciton level scattering via phonon emission (which we will refer to as the Davydov mechanism9) can lead to ultrafast downward energy cascading in a protein-Chl complex. Following the studies of the above complex of P. aestuarii, NPHB was applied to the antenna of the purple bacterium Rb. sphaeroides. The antenna of Rb. sphaeroides is comprised of two main complexes, B8WB850 (LH 11) and B875 (LH I), with the former peripheral to the latter, which surrounds the RC. Various structural models have been proposed for B80&B850.2635 The models have in common that the cornerstone of the structure is an cu,B polypeptide pairz8capable of binding one BChl and two BChl at the cytoplasmic and periplasmic sides of the membrane, respectively. The Q,and Q, dipoles of the latter two are believed to lie approximately in and perpendicular to the membrane plane, respectively, while the Q,and Q, dipoles of the single BChl at the cytoplasmic side are both approximately parallel to the planesz6 The center-to-center distance between the two BChl at the eriplasmic side has been estimated to be no greater than 15 This distance can lead to an excitonic matrix element as large as about 100 cm-ISz9The distance between the center of either of these and the other BChl has been estimated to be no greater than
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0 1992 American Chemical Society
Rhodobacter sphaeroides Antenna
The Journal of Physical Chemistry, Vol. 96, No. 15, 1992 6459
21 A. However, it is the organization of the a,j3 pairs into a cyclic arrangement and the two-dimensional lattice commensurate with this arrangement that determine the optical absorption and CD proper tie^.^^ There appears to be general agreement that an aggregate of the two BChl at the periplasmic side is associated with B850 and that its structure may be similar to that of B875. In the original Zuber mode131.32the B8WB850 unit cell was taken to be a hexamer of a,@ pairs, (a,& with c6 symmetry. In a revised model,%which becomes apparent when a two-dimensional lattice of these hexamers is constructed, the unit cell is a trimer of a$? pairs with the two members of the pair belonging to adjacent hexamers of the original Zuber model. This means that the B850 unit cell is a trimer of BChl tetramers (12 BChl molecules per unit cell). Scherz and c o - ~ o r k e r s ~have ~ - ~also ~ proposed trimer of tetramer models which are distinctly different from the revised Zuber model. The reader is referred to the article by P e a r l ~ t e i n ~ ~ for a detailed discussion. It seems unlikely that one will be able to determine which of the trimer of tetramer models for B850 is correct (if any) or to confidently discount the original Zuber model as well as others until a crystal structure is available. There are several reasons for this including (i) the difficulty in determining the relative contributions from excitonic and protein-BChl interactions to the red shifts of B800, B850, and B875 relative to the energy of the Qy state of "isolated" (monomer) BChl a at -780 nm and (ii) that the CD spectrum is sensitive to weak excitonic interaction^.^^ We note that the models of Scherz and co-workers are based on the assumption that, in LH2, a strong pairwise exciton interaction between the BChl of the a,(?pair is responsible for the red shift of the Qy transition to 850 nm. However, recent site directed mutagenesis experiments have36 suggested that protein-BChl a interactions do contribute significantly to the red shift of the BChl Q,, transition. Energy (singlet) transfer in the light harvesting (LH) antenna complex has been extensively studied by picosecond techniques. The B850 to B875 transfer time is 35 ps at room temperat~re,~' whereas the B875 to RC (open) transfer time at T = 77 K is about 60 p3* for chromatophores. The B800 to B850 transfer has been reported to be much faster, 2 f 1 ps at 77 K.37 More recently, experiments with femtosecond resolution have led to a transfer time of 0.7 ps at room temperature for the isolated B800-B850 complex.39 Nonphotochemical hole burning of B800 established that the ) B800* is 4.8 ps at pumped helium total dephasing time ( T ~ of temperatures,@which was later proven to be due to downward energy transfer to B85OS4l Contrary to the findings of van der Laan et ala,@NPHB of B850 is as facile as for B800.21941Furthermore, our hole burned spectra at 4.2 K yielded a B800 ZPH width of 4.2 f 0.5 cm-I, which leads to a lifetime of 2.4 f 0.2 ps for B800*. Within experimental uncertainty this value for the hole width agrees with that determined by van der Laan et ale@ Thus, B800 to B850 transfer is weakly temperature dependent even though the 170-cm-l bandwidth of B800 is predominantly due to site inhomogeneous broadening (r,)and the ABdependent hole spectra show that the excitonic interaction between B800 and B850 BChl a is weak. However, the hole profile of B850 obtained with A B located in B850 (fwhm 280 cm-I) is dominated by a broad hole possessing a width of 210 cm-I at 4.2 K which is invariant to AB. This proved that the homogeneous broadening (rH) is -210 cm-', which is comparable to kT at room temperature. The weak temperature dependence could, as a consequence, be understood.41 A dominant 750-cm-I vibronic hole, which builds on the broad B850 origin hole, was observed with a Franck-Condon factor of 0.05 and a width of only 60 cm-l. This novel vibronic hole narrowing phenomenon is due to FranckCondon factor narrowing of the exciton b a n d ~ i d t h . ~ In ' . ~a~ straightforward manner it was determined that a minimum value for the B850* exciton bandwidth is -210 cm-I. Furthermore, the 750-cm-I vibration was assigned as the dominant mode for B800 to B850 Farster transfer. The results for B875 were similar to those for B850 and led to a minimum exciton bandwidth of -210 cm-l for B875. For both B850 and B875 it was argued that r H is a consequence of exciton level structure and ultrafast
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Figure 1. Qyregion absorption spectra (4.2 K) of NF57 mutant (solid line) and wild type (dashed line) chromatophores of Rhodobacter sphaeroides. The labeis B800, B850, and B875 are based on the positions of the absorption maxima at room temperature. At 4.2 K these bands are at 799.36 nm (12 510 cm-I), 852.52 nm (1 1 730 cm-I), and 888.57 nm (1 1 254 cm-I).
inter-exciton level scattering.4l The site inhomogeneous broadenings (I'J for B850 and B875 were found to be 60 and 80 cm-I, The values for rrfor B850 and B875 bands have error margins of f 5 and f 1 0 cm-I, respectively. In this paper we report the results of NPHB studies on B870 and B896, due to two additional states of the Rb. sphaeroides antenna which time domain and other have implicated as possible "shuttle* states for transfer from B850 to B875 and from B875 to P870 (primary electron donor of the reaction center), respectively. Although B870 and B896 are due to BChl a molecules,the nature of the B870* and B896* states is not understood. The B870 and B896 components have not been isolated by any biochemical technique. Novel NPHB action spectra are presented that allow for the resolution of the absorption profiles of the weakly absorbing B896 and B870 components and the determination of their underlying structure. The results presented together with those for the base-plate antenna complex of P. aestuarii indicate that B870* and B896* states are the lowest energy exciton level of the B850* and B875* exciton bands, respectively. For B896* (and B870*) the ZPH widths yield a total dephasing time of 6.6 ps at 4.2 K. Interpretations for this dephasing are considered including one in which it is the result of exciton scattering in a lattice rendered imperfect by structural heterogeneity. This interpretation is suggested by data for excitons in doped organic crystals.
II. Experimental Section The Rb.sphaeroides NF57 strain (a RC-LH I depleted mutant) and M2192 strain (a RC-LH I1 depleted mutant) were grown aerobically in the dark following the procedures of Picorel et al.4' and Hunter et al.," respectively. Chromatophore membranes were isolated from these mutants as reported earlier47and resuspended in 50 mM tris-HC1 (pH 8) buffer diluted with 50% glycerol. Good glass samples were obtained by quick cooling of glycero1:water mixture (60:40 ratio) from 277 to 4.2 K in a Janis liquid helium optical cryostat. No detergent was added to the sample. Absorption and hole burned spectra (sample optical density at B850 K0.8) were obtained using a Bruker 120 HR Fourier transform spectrometer which has a resolving power of l e . High-resolution spectra were recorded a t a resolution of 0.5 cm-l. A Coherent CR899 Tisapphire laser (line width = 0.06 cm-*), pumped by an argon ion laser (Coherent Innova 200), was used for hole burning. 111. Results
Figure 1 shows the 4.2 K absorption spectra of wild type chromatophores and the NF57 mutant which is devoid of B815 and reaction center. It was reported earlier2's4l that the hole profiles of B850 and B875 are dominated by a broad (-200 cm-l)
6460 The Journal of Physical Chemistry, Vol. 96, No. 15, 1992 750
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Reddy et al. 895
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13000 12000 1 1 000 WAVENUMBERS ( c m - 1 )
Figure 2. Hole burned spectrum (4.2 K) of wild type chromatophores. Bum conditions are 300 mW/cm2 for 20 d n , AB = 11 187 cm-l, and read resolution = 4 ad.Absorbance change at the ZPH is 0.025. The labels
B800, B850, and B875 mark positions of the absorption maxima. Inset shows a 28-cm-l high-resolution (read resolution = 0.5 cm-l) scan of a hole at 11 148 c d . Hole width = 3.2 cm-l. feature which is invariant to the location of AB within the respective alporption bands. Further studies have shown that relatively sharp ZPH can be burned when AB is located on the low-energy sides of B850 and B875 in the above systems as well as others.49 In Figure 2 the hole burned spectrum for wild type chromatophores, obtained with AB = 893.9 nm (red edge of B875), reveals a ZPH a t AB plus the aforementioned broad B875 hole. The sharp spike just to the right of the ZPH at AB is due to the interference between the red-shifted antihole of the ZPH at AB and its real-phonon sideband hole (PSBH) with the pseudo-PSBH. Such an interference is not uncommon and has been thoroughly studied for cresyl violet in poly(viny1 alcohol) films.% The high-energy satellite hole near the B800 arrow is the 1210-cm-' vibronic hole that builds on the broad B875 hole. (See Reddy et a1.21for a detailed analysis and discussion.) The broad high-energy satellite hole labeled as B850 is the aforementioned B850 hole. Its appearance in the spectrum is interesting since our deconvolution indicates that the absorption of B850 a t 893.9 nm should be negligible. Possible reasons for its appearance are considered in section IV. The left inset of Figure 2 shows a ZPH obtained with a read resolution of 0.5 cm-' and AB = 897.02 nm. Its fwhm is 3.2 f 0.2 cm-'for a fractional OD change of 0.03. Shallower holes did not lead to a reduction in the width which was found to be independent of AB in the 890-903-nm range studied. Figure 3 shows a novel NPHB action spectrum obtained with a constant burn fluence of 180 J cm-2 for the B896 ZPH. For the sake of clarity nonresonant holes accompanying each of the ZPH are not shown; the ZPH are suitably offset to have a common baseline. Assuming that the NPHB efficiency is independent of AB, the action spectrum represents the absorption spectrum of the BChl a state being bumed. The profile peaks at 895.3 nm (1 1 170 cm-I) and has a fwhm of -70 cm-I. The ZPH could not be observed for AB < 886 nm. The uncertainty in the estimation of zero absorbance change for the ZPH leads to an error of about f 2 0 cm-' in the B896 profile maximum. For AB tuned to the low-energy side of the B850 band, relatively narrow ZPH could be burned. ZPH for AB < 858 nm in B850 were not observed. A NPHB spectrum obtained with AB = 864.01 nm is shown in Figure 4 for the NF57 mutant. The ZPH a t AB plus its real- and pseudo-PSBH a t W B f 20 cm-' are interfered with by the broad B850 hole (solid arrow) which was reported on earlier4'ss1 and discussed in the Introduction. The antihole indicated by the dashed arrow (labeled 1) is that of the B850 hole. The hole structure to higher energy of the antihole at 845 nm is due to real-vibronic holes which build on the B850 hole. (See refs 21 and 42 for a detailed discussion.) For example, the 750-cm-I hole is due to the BChl a 750-cm-l excited-state fundamental vibration whose Franck-Condon factor is 0.05; cf. Introduction. The broader hole to the left of the B800 maximum is due to three closely spaced excited-state fundamentals (920-980 ~ m - l ) The .~~
11250
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WAVENUMBERS (cm- 1) Figure 3. Constant-fluence (1 80 J/cm2) hole burned spectra (4.2 K) in
the B896 band. ZPHs are offset to have a common baseline, and nonresonant holes accompanying the ZPH are not shown. The maximum absorbance change shown is 0.025.
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Figure 4. Hole burned spectrum (4.2 K) of mutant NF57 chromatophores. Burn conditions are as in Figure 2, with AB = 11 574 cm-". The
numbers 750 and 920 label the real vibronic holes that build on the ZPH at B850 (labeled 2). Feature labeled 3 refers to the nonresonant hole in the residual B875 band, and feature 1 is the antihole that builds on the B850 hole. Inset a shows a 33-cm-l high-resolution (read resolution = 0.5 cm-I) scan of a hole at 11 520 cm-I. Hole width = 3.2 cm-]. Inset b is an expanded view of the ZPH and the associated phonon sideband holes (marked by dashed arrows). upper inset (a) of Figure 4 shows a high-resolution scan of a typical ZPH associated with burning on the low-energy side of B850. Its width is 3.2 f 0.2 cm-', which is, within experimental uncertainty, the same as the widths of the ZPH burned on the low-energy side of B875. The width of the B85O ZPH was found to be invariant to AB in the 864-875-nm range studied. The B850 ZPH action spectrum of the type shown in Figure 3 for B875 was found to peak at 87 1.08 nm (1 1 480 cm-I) and to possess a fwhm of -60 cm-I. The estimate for the B870 peak has the same margin of error (f20 cm-I) as for B896. Thus, the MITOW ZPH hole burning characteristics of B850 and B875 are very similar. (Narrow B850 ZPH with a width of 3.2 cm-'were also observed for the wild type chromatophore, not shown.) The similar burning characteristics are interesting in view of the fact that structural models indicate that the spatial arrangements for B850 and B875 molecules are similar. The lower inset (b) of Figure 4 shows an expanded view of a B850 ZPH and its associated real- and pseudo-PSBH. The mean phonon frequency is w, 20 cm-' and S 0.3 based on the relative integrated intensities of the ZPH and real-PSBH. Thus, as has been observed for all antenna protein-chlorophyll
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The Journal of Physical Chemistry, Vol. 96, No. 15, 1992 6461
complexes studied thus far,51,53 the coupling is weak.
IV. Discussion A. Nature of B870 and B896. There are several possible interpretationsfor the low-energy BChl a ZPH (fwhm = 3.2 cn-') observed for B850 and B875 which serve to identify the B870 and B896 components including (i) unnatural BChl a sites, (ii) special (natural) BChl a sites which are not associated with the B850 and B875 exciton bands, (iii) a self-trapped exciton, as defined by Toyozawa,EOassociated with the B850 and B875 exciton bands, and (iv) the lowest energy exciton level of the B850 and B875 bands. The first is highly improbable since the ZPH widths and action spectra are the same for B870 and B896 in wild type chromatophoresand the NF57 and M2192 mutants. Furthermore, a ZPH width of 3.2 cm-I, which corresponds to a total dephasing time T~ of 6.6 ps, is about 2 orders of magnitude greater than that expected from pure dephasing and spectral diffusion for an isolated chromophore at 4.2 K. For example, Boxer et al.55have studied the pure dephasing/spectral diffusion of the ZPH of zinc pyrochlorophyllide in apomyoglobin and observed a ZPH width (at 4.2 K) of only 0.027 cm-', which is similar to those observed for l m * states of a wide variety of molecules imbedded in glassy hosts.56 For consideration of the last three interpretatioins,it is necessary to review the findings of time domain studies in energy-transfer processes within the antenna and from the antenna to the RC as well as the inferences of structural models for the exciton bandwidths of B800, B850, and B875. The overall excitation transfer time from the antenna to open and closed RCs is ~ 6 and0 -200 ~ ~ ps37957at 77 K. Under our CW burning conditions, 1300 mW cm-2, the RCs are certainly closed since bum intensities of -40 mW cm-2 were found earlier to produce about -20% bleaching of P870 at 4.2 K due to formation of P+Q- (Q quinone).58 As discussed in the Introduction, the B800 to B850 energy transfer time is 0.7 and 2.3 ps at room temperature and 4 K, respectively. There is some variation in the time constants reported for B800-B850 to B875 energy transfer. From picosecond absorption recovery experiments it was concluded that45excitation transfer time between B8WB850 and B875 is 35 ps at T = 296 K whereas time-resolved fluorescence experiments have indicated59a much shorter time (
