J. Phys. Chem. 1993,97, 5519-5524
5519
Optical Spectroscopy of a Highly Fluorescent Aggregate of Bacteriochlorophyll c Timothy P. Causgrovqt Peiling Cheng,t Daniel C. Brune, and Robert E. Blankenship' Department of Chemistry and Biochemistry, and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287- 1604 Received: January 1 1 , 1993; In Final Form: March 1 1 , 1993
Bacteriochlorophyll (BChl) c and a similar model compound, Mg-methyl bacteriopheophorbide d, form several types of aggregates in nonpolar solvents. One of these aggregates is highly fluorescent, with a quantum yield higher than that of the monomer. This aggregate is also unusual in that it shows a rise time in its fluorescence emission decay a t certain wavelengths, which is ascribed to a change in conformation of the aggregate. An analysis of fluorescence depolarization data is consistent with either a linear aggregate of four or five monomers or preferably a cyclic arrangement of three dimers.
Introduction
Bacteriochlorophyll (BChl) c (or d or e) is the major lightharvesting pigment in the chlorosome antenna complexes found in green photosynthetic bacteria.' In uiuo, the Qy absorption peak of BChl c at -750 nm is highly red-shifted from that of the extracted pigment in methanol (667 nm). Bystrova et a1.2 were first to show that BChl c aggregates spontaneously in nonpolar solvents to form oligomers with absorption spectra very similar to that of BChl c in chlorosomes. Further experiments have shown that the UV-vis a b ~ o r p t i o nand ~ , ~ fluorescen~e,~ infrared abs~rption,~,~J resonance Raman? circular dichroism,4*10J1 and NMRI2 spectra of the in vitro oligomers are also similar to those of BChl c in chlorosomes,prompting the conclusion that BChl c oligomers actually exist in uiuo. The possible role of proteins in organizing the pigments in chlorosomes has been COORJ controver~ia1,~J although the emphasis of recent discussions Figure 1. Structure of PEF-BChl c and of Mg-MBPd. In BChl c, R I is on how the chlorosomeproteins might interact with the pigment = CH2CH2CH3, R2 = CH3, R3 = ClsH3o (farncsyl). In Mg-MBPd, R I oligomers. CH2CH3, R2 = H, R3 CH3. The aggregation of various homologs of BChl c and related pigments as a function of concentration and solvent has been In this paper we report additional photophysical properties of extensively in~estigated.~-l~ In addition to the 747-nm oligomer, the 710-nm aggregate of BChl c, including a size determination at least two other distinct aggregates with absorption peaks at by rotational diffusion. Because the long hydrocarbon tail of 680 and 710 nm are formed.5,7,'4,'5,18.'9The 710-nm species PEF-BChl c makes modeling of diffusion difficult, we have used exhibits unusual spectroscopic properties, in particular an intense a model compound recently synthesized by Cheng et al.I8 This fluorescence emi~sion,5-~J~J9 compound is similarto BChl d (see Figure 1) but lacksthe famesyl The structure of the 4-propyl-5-ethyl famesyl homolog of BChl tail, thus giving a relatively rigid shape in solution. The model c from Chlorobium limicola is shown in Figure 1. One of the compound has been found to form aggregates similar to the 680-, distinguishingfeatures of BChl c, d, e is the 2 4 1-hydroxyethyl) 710-, and 747-nm aggregates of BChl c, but with absorption substituent; there is evidence2 that this group is responsible for maxima at 672, 693, and -730 nm, the same wavelengths as aggregation with the 2a hydroxyl oxygen acting as a magnesium aggregates formed by BChl d.18 ligand. The PEF-BChl c homolog has been found to form only the 680- and 7 10-nmaggregatesinCHzCl2, CHC13,and cc14.14*Is Experimental Section At higher concentrations, the 710-nm species is formed nearly quantitatively. PEF-BChl c was isolated from Chlorobium limicola f. In contrast to the situation with aggregates of chlorophyll a,Zo thiosulfatophilum as described by Olson and Pedersen;l4 Mgthe presence or absence of small amounts of water in the solutions MBPd was synthesized from Chl a as described by Cheng et a1.18 does not significantly affect the properties of aggregates of Solutions of 7 10-nm aggregate of PEF-BChl c were formed by bacteriochlorophylls c, d, or ea7,21 Presumably, this is because dissolving dried pigment in CCl, and dilutingwith an equal amount the latter pigments contain a nucleophilic group as part of the of hexane. Aggregates of Mg-MBPd were formed by dissolving molecule (the 2a hydroxyl group) and lack the C-10 carboxydried pigment in a minimum amount of CHC13 and diluting with methyl substituent which participates in the bridging by water CC4. Rotational diffusion of aggregates was measured in these in chlorophyll a aggregates.22 However, the presence of very solvents; depolarization of monomers was measured in the same tenaciously bound water in the bacteriochlorophyll c, d, and e solvent as the aggregates but with a small amount of methanol aggregates cannot be ruled out. (0.5%) added to prevent aggregation. Fluorescence lifetimes were measured as described previously6 Current address: CLS-4, M.S.3567, Los Alamos National Laboratory, and analyzed using global methods to obtain decay associated Los Alamos, NM 87545. spectra. In depolarization experiments, the emission polarizer I Current address: Department of Biochemistry, University of Nebraska, Lincoln, NE 68583. was fixed to detect vertical polarization and the excitation +
0022-3654/93/2097-55 19$04.00/0
0 1993 American Chemical Society
Causgrove et al.
5520 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 PEF-BChl c Em. 758 n m
PEF-BChl c 1.21
:
1.0
I
- Exc. spectrum
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em. 755 nm
Em. spectrum exc. 460 nm
t
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.--
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Figure 2. Fluorescence exicitation (solid), emission (dashed), and polarization (dotted) spectra of PEF-BChl c in CC14/hexane. For the polarization spectrum the sample was diluted with mineral oil to slow
s
rotation.
polarization was varied from vertical (parallel) to horizontal (perpendicular)using a broadband polarization rotator. The data were fit according to the time-resolved fluorescence polarization equations Z , , ( t ) = P(t)[l
+ 0.8r(t)]
ZL(t) = P ( t ) [ l - 0.4r(t)] where Zll(t) and Zl(t) are the fluorescence intensity polarized parallel and perpendicular to the excitation polarization, respectively.23 Fitting of parallel and perpendicular polarized decays was done with a global analysis program2* in which the magic angle decay P ( t ) was measured independently and fixed and the parameters in the function r(t) (which is equal to 2.5 times the anisotropy) were allowed to vary. The function r(t) was modeled as either a single exponential with lifetime T , or~ a~biexponential function of the form (1 - a) exp(-t/.rr) u exp(-t/n). Parallel, perpendicular, and magic angle decays were collected in varying chronological orders to check for effects due to sample damage. Molecular sizes of monomers and various aggregates were estimatedfrom molecular modelingusing the QUANTA program package on a Silicon Graphics Iris 4D80GT workstation. Rotational diffusion times were calculated from equations given by Perrin25 for an asymmetric ellipsoid, using numerical integration of the elliptical integrals.
+
Results The absorption, fluorescence excitation and emission spectra of the 710-nm aggregate of BChl c are different from those of other chlorophyll aggregates in several respects. For example, the excitation and a b ~ o r p t i o n ~ spectra J ~ ~ ' ~are ~ ' ~broad and show pronounced structure, including a prominent shoulder at -680 nm (see Figure 2). The presence of this peak is also evident in the fluorescence polarization spectrum, indicating that it has a dipole orientation different from the 710-nm peak. Also, the emission spectrum is not a mirror image of the absorption spectrum, consistent with the assignment of the 680-nm shoulder to a higher exciton level.l5 The 7 10-nm aggregate of BChl c has been previously described as highly fluorescent.536J9 Determination of the fluorescence quantum yield resulted in values of 0.5-0.6 (19, D. C. Brune, R. E. Blankenship, and G. R. Seely, unpublished), compared to a monomer quantum yield of 0.3 in methylene chloride with 0.5% methanol.26 Therefore, the 7 10nm aggregate of BChl c is more fluorescent than the monomer, although at room temperature all other known aggregates of chlorophylls and bacteriochlorophylls are much less than monomer~.2~
-0.5
4
680
700
720
740
760
Emission wavelength (nm)
Figure 4. Decay-associatedemission spectrum of PEF-BChl c in CC14/ hexane with excitation at 700 nm, using four-component exponential analysis.
In a previous paper,6 we ascribed a lifetime of 3.3 ns to the 710-nm aggregate of PEF-BChl c. Similar results have been obtained using BChl c (4-ethyl-5-methyl stearyl ester) isolated from Chloroflexus aurantiacus.5 The excitationdecay associated spectrum (DAS) of a sample containing mostly 710-nm species is shown in Figure 3. The 3.3-11s lifetime is clearly associated with the 7 10-nm species, with other contributions from residual monomer (5.6 ns) and a 1.6-ns lifetime with low amplitude due to what is apparently a breakdown product of BChl c . ~The short component of 180 ps is interesting in that it appears as a negative amplitude, or a rise in fluorescence. This component is most prominent at an excitation wavelength of 700 nm, shifted from the 710-nm absorption peak. This shift was also observed using a different homolog (4-isobutyl-S-methyl/ethyl farnesyl BChl c) in pure CC14 and the model compound Mg-methyl bacteriopheophorbide d (Mg-MBPd; data not shown). The risetime was easily recovered in the kinetic fits to individual decay traces and is clearly not an artifact of the global analysis method. To further investigate the rise in fluorescence, the emission decay-associated spectrum was measured with excitation held constant at 700 nm (Figure 4). The spectrum is again dominated by a long, -3.3-11s component associated with emission from the 710-nm aggregate. The 180-ps rise component is split into two components of 59 and 310 ps, which were probably not resolvable with only negative amplitudes in Figure 3. Both short components shift from positive to negative in amplitude, indicatinga relaxation of the emission to lower energy with time. The temperature dependenceof this relaxation was also measured (using the model
A Highly Fluorescent Aggregate of Bacteriochlorophyll c
The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 5521
Temperature ("C) 30
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loo
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Time ( n s )
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Figure 5. Temperature dependence of the rise component seen in decayassociated spectra of aggregates of Mg-MBPd. The fitted line is a linear regression giving an activation energy of 875 cm-I.
Mg-MBPd
2
i
8
10
Time (ns) Figure 6. Parallel and perpendicular polarized fluorescence decays of Mg-MBPd in CCl4 with 0.5% methanol added. The two decays were fit simultaneously with a singleexponential anisotropy decay of 172 ps, with an overall x 2 of 1.38.
compound Mg-MBPd); thedata were fit using a singleexponential for the rise component. Figure 5 shows an Arrhenius plot of the resulting lifetimes, with the fitted line giving an activation energy of 875 cm-I. Using two exponentials for the rise component slightly improved the fit but greatly increased the scatter in the Arrhenius plot. Information on the size of the 710-nm aggregate in solution was gained by measuring polarized fluorescence, with excitation and emission on the red edge of the aggregate spectrum to avoid intrinsic depolarization. The parallel and perpendicular decays of monomeric BChl c (not shown) did not fit well to a singleexponential anisotropy function but were fit to a biexponential with lifetimes of 100 and 300 ps. The long farnesyl tail of BChl c presumably causes the biexponential rotational relaxation, greatly complicating the analysis of rotational diffusion data. Therefore, depolarization of a BChl d analog, Mg-MBPd (see Figure l), was measured in CCL (Figure 6). The time-dependent anisotropy was fit as described above to a single exponentialwith a rotational lifetime of 172 ps. Fitting with a biexponential function forced both lifetimesto similar values and did not decrease the x2.
Figure 7. Parallel and perpendicular polarized fluorescence decays of Mg-MBPd in CCl+ The two decays were fit simultaneously with a biexponential anisotropy decay with a major lifetime of 1.79 ns (93%) and a minor component of 40 ps (7%); the overall x 2was 1.37. The magic angle decay was modeled as a single exponential with a lifetime of 2.7 ns.
The polarized fluorescence decays of the highly fluorescent aggregateof Mg-MBPd areshownin Figure7. The time-resolved anisotropy of the aggregate was fit to a biexponential function with lifetimes of40 ps and 1.79 ns. The amplitude of the 40-ps component was relatively minor, accounting for about 7% of the total anisotropy amplitude. This extra component was determined to be an artifact arising from incomplete rejection of the opposite polarization. The remaining component represents rotational diffusion, and was quite reproducible. The dominant 3.34s lifetime in the decay-associated spectra in Figures 3 and 4 as well as the definite anisotropy lifetime indicate that the aggregate is a well-defined species and does not contain a range of sizes as has been proposed6J4J69'*for the 747-nm oligomer. A more complete analysisof the rotational diffusion data is given in the discussion.
Discussion The 180-ps rise times observed in the decay-associatedspectra are consistent with a shifting of the emission spectrum with time toward lower energy. This observation is very unlikely to result from trace impurities in the samples. The 1.6-11s component probably does represent a trace impurity; however, it behaves independently from the long-wavelength emitting state, whereas the 180-ps component clearly feeds into the lower energy state, which then fluoresces with a lifetime of 3.3 ns. This would not be expected for a trace impurity that is independent of the major species present in solution. We have also consistently observed the same phenomenon in related pigments isolated from several different species of bacteria, as well as the semisynthetic model compound MBPd. If excitationisdirectly into the red sideof the 7 10-nmabsorption band, no rise component is observed in the fluorescence decay (Figure 3). The presence of a defined peak in the excitation spectrum for the rise component, shifted from the absorption maximum, seems to indicate that a separate state exists which relaxes relatively slowly to the lowest excited state. Several processes could cause the observed red shift, including Fiirster energy transfer between weakly coupledstates,relaxation between excitonic states, vibrational relaxation processes, and a conformational changein theexcited state. In the caseof Farster energy transfer, a depolarizationcomponent of 200 ps (corresponding to the rateof energy transfer) shouldoccur with short-wavelength excitation unless the transition dipoles of the two states are aligned parallel to each other. However, the time-resolved anisotropy
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5522 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993
Causgrove et al.
with excitationat 685 nm shows no such short component. Under most conditions, the rate for Fiirster energy transfer between chlorophyllsthat are in van der Waals contact would be expected to be significantly faster than 200 ps. Relaxation between excitonic states would also be expected to be faster than the -200ps time scale seen here; recent measurements2*have assigned times of -7 ps to this process in chlorosomes. The fluorescence polarization spectrum (see Figure 2) and the circular dichroism spectrumIsJ9 show no evidence of a distinct dipole moment at 700 nm, and a strong temperature dependence as found in Figure 5 would not be expected. The time scale of vibrational relaxation in porphyrins is also typically much faster than several hundred picoseconds29 and would not be expected to show a large temperature dependence. A final possibility, which we prefer, is that the aggregate exists as an equilibrium mixture of two conformations, a minor species absorbing at 700 nm and a major one absorbing at 7 10 nm. After excitation at 700 nm, the higher energy conformation can be seen to relax to the other conformation by a shift in the emission. The temperature dependence in Figure 5 then gives the barrier to conformationalchange in the excited state. Such conformational changes may also occur in the ground state but would not be detectable in the current experiments. The excited-state conformational change seems to be the simplest explanation for the observed results. A similar complex time-dependent fluorescence profile has been observed in a derivative of quinacrine dyes.30 These authors also interpret their results in terms of an excitedstate conformational change. The rotational diffusiondata may be compare# with predictions of Debye-Stokes-Einstein theory in order to relate the timedependent anisotropy with molecular size and shape. If the fluorescing species is modeled as an ellipsoid of rotation, then the time-dependent anisotropy is given explicitly by3'
where DIIand D L are diffusion constants for rotation about axes parallel and perpendicular to the symmetry axis, respectively. The coefficients are A(e) = (3 cos2 e - 1 ) ~ / 4
B(e) = 3 cos2 e sin2 e
c(e)= 3 sin4 e/4 and 0 is the angle between the emission dipole and the symmetry axis. The monomer of Mg-MBPd may be approximated as an oblate spheroid with the emission dipole perpendicular to the symmetry axis (0 = goo). The rotation of monomeric Mg-MBPd in solution may be modeled using dimensions of 3.6 A (twice the van der Waals radius of carbon) by 14.6 A (estimated from the averagedistance between two oppositeprimary aliphatic carbons plus twice thevan der Waals radius of carbon). Using theviscosity q of C C 4 as 0.9 CPat room the predicted decay law is r(t) = 0.4[0.25 exp(-t/164 ps) + 0.75 exp(-t/187 ps)]. These two lifetimes are experimentally unresolvable and in good agreement with the observed decay of 172 ps. The observed rotational time for the aggregate (Figure 7) is much longer than that of the monomer (Figure 6), indicating that the aggregate is of considerable size. Unfortunately, there are many possible molecular geometries of the aggregate which could produce a single-exponential rotational decay of 1.79 ns. If the configuration is taken as a linear aggregate such as proposed the monomers would overlap for for most BChl c aggregate~,~-~J3 approximatelyhalf their length with a maximum thickness twice that of the monomer. Therefore, the length of the aggregate is (N 1)/2 times that of the monomer, where N is the number of molecules in the aggregate. A tetramer would then be
+
Figure 8. Possible structure for the 7 IO-nm aggregate of mg-MBPd. (a, top) Schematic depictionshowing bonding pathways. (b, bottom) Energy minimized structure showing all carbons, oxygens as open circles and only the hydroxyl hydrogens.
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approximated by an asymmetric ellipsoid 7.2 A X 14.6 A X 36.2 A. Such a shape predicts a rotational lifetime of 1.38 ns, somewhat shorter than the observed value of 1.79 ns; a pentamer predicts a rotational time of -2.14 ns. A linear model is an open-ended structure in which further aggregation would be expected to occur. The fact that the aggregate is formed nearly quantitatively at relatively low concentrations and does not grow into a larger species at higher concentrations (as judged by the absorption spectrum) indicates that the structure may be a closed loop which permits no further aggregation. Theoretical calculations of radiative and nonradiative decay rates33have also led to the conclusion that the 7 10nm aggregate is not a linear species. In addition, preliminary NMR analysis of the highly fluorescent aggregate have shown that its spectrum is closely related to that of the dimer which absorbs at 680 nm (Cheng, Ma, and Nieman, unpublished). One possible structure is a triangular arrangement of dimers, held together by coordination of Mg by C9 keto oxygens. Such an arrangement is shown schematically in Figure 8a, based on the dimer structure determined by Smith et al." These authors identified two different structures for the dimer used in Figure
A Highly Fluorescent Aggregate of Bacteriochlorophyll c 8a, termed piggyback and face-to-face dimers. Each of these dimers were constructed using molecular modeling software and subsequently used to build the aggregate depicted in Figure 8a. The energy minimized structure of the aggregate based on the piggyback dimer is shown in Figure 8b; oxygens are depicted as open circles, and only the hydroxyl hydrogens are shown. Using the modified force field of Shelnutt et al.32the energy of the aggregate (invacuo) was approximately50 kcal mol-’ lower than that of the separated dimers. This energy is almost certain to be an overestimate,as van der Waals interactions of pigments with thesolvent is not included. The solventinteractions will belessened in the complex comparedto the dimers due to exclusion of solvent from the regions of interaction of the dimers. The face-to-face dimer also produces a hexameric structure with similarinteraction energy (not shown). Structures of dimers that might be the building blocks for the cyclic hexamer have been proposed based on NMR measurements.12J7 The NMR ring current shifts of the cyclic structure would be expected to be generally similar to those of the dimers that make up the building blocks. The angle of the orientation of the three dimers with respect to each other is near to the magic angle at which the interaction vanishes at distances of more than a few angstrom~.~SThe dimer and 710-nm aggregate are in equilibrium with each other in s o l ~ t i o n Under . ~ ~ our experimental conditions the amount of dimer is very small, as indicated by the similarity of the absorption spectrumto the fluorescenceexcitation spectrum for emission at 755 nm (Figure 2) and the absence of the 0.6-11s fluorescence decay component characteristic of the dimer.6 Hydrodynamically, the aggregate shown in Figure 8 can be approximated by an oblate spheroid approximately 14.6 A X 29.2 A X 29.2 A. The rotational diffusion times of such an aggregate in CCld ( q = 0.9 at room temperature) are predicted to be 1.61, 1.66, and 1.86 ns (the coefficients are unknown due to the unknown direction of the emission dipole). These rotational times, however, are in good agreement with the observed time of 1.79 ns for Mg-MBPd aggregates. The structure shown in Figure 8 is only one of several possible cyclic hexamer structures, which differ principally in the geometry of the dimers that form the building blocks of the hexamer. Another possibility is a head-to-tail arrangement in which the two bacteriochlorophyll molecules of the dimer building block are oriented in approximately the same direction, with the C-9 keto of molecule 1 liganded to the Mg of molecule 2, with the C-2a OH of molecule 2 liganded to the Mg of molecule 1. However, this dimer is inconsistent with the NMR results of Smith et al.17 on bacteriochlorophyll d dimers, which almost certainly are very similar in structure to the presumed dimeric building blocks of the 710-nm aggregated species. Geometries of several different dimeric models have been presented by Nozawa et a1.12 The structure shown in Figure 8 predicts that half of the C-9 keto groups are liganded to Mg and half are free and that half the Mg are five coordinate and half are six coordinate. These properties can be probed by vibrational spectroscopy. Uehara et a1.8 presented FTIR data for a very similar aggregate formed by BChl c in water-saturated CC14. They found approximately equal intensities of 1684- and 1651-cm-1 bands, assigned to free and coordinated C-9 keto groups, respectively, in agreement with our proposed structure. Olson and COXIShave concluded from the concentration dependence of absorption spectra of related pigments that the aggregationstate of the 7 10-nm species is a tetramer which forms by association of two dimers. This conclusion relies on deconvolution of absorption spectra of pigment solutions over a range of concentrations,and small errors in the derived spectra of each of the aggregated forms can lead to substantial errors in the conclusions. We have not been able to propose a chemically
The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 5523 reasonable tetramer structure that is not subject to growth to larger oligomers. As discussed above, the properties of the 7 10nm aggregate suggest that it is a closed structure that does not grow. For these reasons, we prefer a cyclic hexamer structure over the tetramer, although this is not definitively established. The most surprising feature of the work reported here is its high fluorescence quantum yield of the 7 10-nm aggregate, measured to be 0.5-0.6 (ref 19 and Brune, Blankenship, and Seely, unpublished), compared to 0.29 for the monomeric pigment in similar solvents.26 This highly fluorescent aggregated species appears to be unique in the chlorophyll literat~re.~’ The precise reasons for this high fluorescence are not clear, although some general indications can be drawn. Fluorescence quantum yield is given by @f = kf/k,, where kfis the radiative rate constant and k,is the total rate constant for deactivation of the excited state. Theserateconstants arethereciprocalsof the natural fluorescence lifetime 70 and the observed lifetime 7 , respectively. Further, k, = kf+ k,,, where k,, is the sum of all nonradiative excited singletstate decay processes such as intersystem crossing and internal conversion. The measured lifetimes and quantum yields of the = 0.29, I monomer, dimer, and 710-nm aggregated species are @ T = 6.5 ns for the monomer, @f = 0 . 0 5 , ~ = 0.64 ns for the dimer, and @f = 0 . 5 , ~= 3.3 ns for the 710-nm aggregate.6J9v26Values of kfand k,, can be calculated from the above relationships and data. For the monomer kf= 4.5 X lo7 s-I, k,, = 1.5 X lo8 s-I, for the dimer kf = 7.7 X lo7s-1, k,, = 1.5 X lo9 s-I, and for the 710 nm aggregate kf = 1.5 X lo8 s-I and k,, = 1.5 X lo8 s-l. The increasing trend of the kfvalues is to be expected from the Strickler-Berg relationship between the oscillator strength and radiative rate constant.36 Because multiple pigments are involved in the aggregated species, the oscillator strength will be increased and the radiative rate constant increased. Point dipole calculations based on molecular modeling coordinates of the cyclic aggregates and dimer building blocks predict oscillator strengths for the lowest exciton component of 3 and 2 times the monomer, respectively, in good agreement with the radiative rates given above. The most interesting trend is with the values of k,,, which increase by a factor 10 upon dimerization and then decrease upon further aggregationto form the 7 10-nm species. Apparently the dimer has a very efficient nonradiative decay channel which is suppressed in the 710-nm aggregate. The fluorescence of pigment dimers is often weak and has been the subject of intense investigation in many systems. In the particular case of chlorophylls, the dry dimer of chlorophyll a is very weakly f l u ~ r e s c e n t ; its ~ ~quantum -~~ yield has been reported to be =0.01 ,39 compared to 0.3 for monomeric pigments.27 Clearly, the nonradiativedeactivation is enhanced in the dimer relative to the monomer. The reasons for this have been discussed at length in the literature, including the possibility of charge-transfer states as efficient decay channels.39 Why the nonradiative decay is so much slower in the 710-nm aggregate is not entirely clear. One possibility is that the rapid nonradiativedecay in the dimer is due to intermolecularvibrational modes or conformationalfluctuations and that the aggregatedspecies is substantially more rigid, thereby suppressing these processes. This increase in fluorescence in more rigid environments due to less efficient nonradiative deexcitation is observed in a wide variety of dye molecules.40 In summary, the 710-nm aggregate of BChl c has been shown here to have very unusual photophysical properties, including a high fluorescence quantum yield, excitonic structure in its absorption spectrum, and a rise component in the fluorescence decay. The rise in fluorescence has been ascribed to a change in conformation of the aggregate in the excited state; the size of the aggregate as determined by fluorescence depolarization is consistent with either a linear aggregate of about four to five monomers, or, more likely, a triangular arrangement of three dimers.
5524 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993
Acknowledgment. This work was supported by Grant DEFG-85ER13388 to R.E.B. from theDivision of Energy Biosciences of the U.S. Department of Energy and Grant DE-FGOS87ER75361from the DOE University Research Instrumentation Program. This is paper no. 139 from the Arizona State University Center for the Study of Early Events in Photosynthesis. The Center is funded by U.S. Department of Energy Grant DE-FG88ER13969 as a part of the USDA/DOE/NSF Plant Science Centers Program. We thank Drs. David Hobbs and John Shelnutt of Sandia National Laboratories for help in molecular modeling of aggregates, Drs. Devens Gust and Thomas Moore for helpful discussions on NMR ring current shifts, and Dr. G. Seely for assistance in the determination of the quantum yield of fluoescence of monomeric and aggregated BChl c.
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. . causgrove et ai. (1 1) Niedermeier, G.; Scheer, H.; Feick. R. G. Eur. J . Biochem. 1992, 204, 6 8 5 6 9 2 . (12) Nozawa, T.; Suzuki, M.; Ohtomo, K.; Morishita, Y.; Konami, H.; Madigan, M. T. Chem. Lett. 1991, 1641-1644. (13) (a) Feick, R. G.; Fuller, R. C. Biochemistry 1984,23, 3693-3700.
(b) Wechsler, T.; Suter, F.; Fuller, R. C.; Zuber, H. FEBS Lett. 1985,181, 173-178. (c) Holnvarth, A. R.;Griebenow,K.;Schaffner,K.Z. Naturforsch. 1990,45~,35-38. (14) Olson, J. M.; Pedersen, J. P. Photosynth. Res. 1990, 25, 25-37. (15) Olson, J. M.; Cox, R. P. Photosynrh. Res. 1991,30,35-43. (16) Olson, J. M. Photosynth. Res. 1991,30,45-48. (17) Smith, K. M.; Bok, F. W.; Goff, D. A.; Abraham, R. J. J . Am. Chem. Soc. 1986,108,11 11-1 120. (18) Cheng, P.; Liddell, P.; Ma, S.X.C.; Blankenship, R. E. Photochem.
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