Excited-State Vibrational Coherence and Anisotropy Decay in the

Cynthia K. Larive, Susan M. Lunte, Min Zhong, Melissa D. Perkins, George S. Wilson, Giridharan Gokulrangan, Todd Williams, Farhana Afroz, Christian ...
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J. Phys. Chem. B 1998, 102, 2776-2786

Excited-State Vibrational Coherence and Anisotropy Decay in the Bacteriochlorophyll a Dimer Protein B820 William M. Diffey, Bradley J. Homoelle, Maurice D. Edington,† and Warren F. Beck* Department of Chemistry, Vanderbilt UniVersity, 5134 SteVenson Center, P.O. Box 1822-B, NashVille, Tennessee 37235 ReceiVed: July 15, 1997; In Final Form: February 3, 1998

We have employed dynamic absorption spectroscopy to monitor coherent wave packet dynamics and anisotropy decays following impulsive excitation of the B820 subunit of the LH1 light-harvesting complex, which was isolated from Rhodospirillum rubrum G9. When the lower exciton-state transition of the bacteriochlorophyll a dimer is pumped, the time-resolved pump-probe spectrum exhibits contributions from a fully Stokes shifted stimulated-emission spectrum and a nonstationary vibrational character within 40 fs of excitation. Coherent wave packet motion in both the ground state and the excited state is observed via modulations of singlewavelength transients. The photobleaching portion of the spectrum exhibits strong components only at low frequencies, 20-60 and 180 cm-1, and a weaker component is observed at 400 cm-1. The stimulated-emission portion of the spectrum exhibits weak modulation components at 20-60 and 180 cm-1, but strong components are observed at fairly high frequencies: 360, 400, 470, 600, and 730 cm-1. An anisotropy decay observed in the stimulated-emission region reports a prompt >20° tilt of the photoselected transition-dipole moment. A possible explanation for these results is that an intradimer charge-transfer event occurs on a very short time scale following optical preparation of the lower π f π* exciton state of the bacteriochlorophyll a dimer at room temperature.

1. Introduction The recent publication of the X-ray crystal structure of the LH2 light-harvesting complex from purple non-sulfur bacteria1 has renewed interest in the function in photosynthesis of strongly coupled pairs of chromophores.2,3 LH2 is a membrane-bound structure that contains a “storage ring”4 of eight or nine subunits, each of which contains a pair of bacteriochlorophyll a molecules bound between two trans-membrane R helices in addition to a monomeric bacteriochlorophyll a molecule in the plane of the membrane.1 Electron microscopic studies5 indicate that the related light-harvesting complex known as LH1 consists of a ring of 16 subunits comparable to those in LH2 that contain just a pair of bacteriochlorophylls. The pairs of bacteriochlorophylls in LH2 and probably in LH1 are remarkably similar to the “special pair” primary electron donor P in the purple bacterial reaction center.6,7 The photophysical function of LH1 and LH2 as light-harvesting arrays has been recently reviewed.8,9 It is indeed interesting that nature would have favored use of strongly-coupled bacteriochlorophyll dimers arranged in a near-parallel arrangement (see Figure 1) for use in lightharvesting proteins10,11 and reaction centers.12 Such an arrangement produces two exciton states, which arise from admixture of the two bacteriochlorophyll site (monomer) electronic states, but only one of the exciton states would be expected to be useful in a light-harvesting role. The near-parallel orientation of the two site transition dipoles places nearly all of the oscillator strength in the transition to the lower-energy exciton state. Nevertheless, the upper exciton state of P has been implicated in the coupling of energy transfer via the Fo¨rster mechanism * Corresponding author: 615-343-0348 (voice and fax); warren.f.beck@ vanderbilt.edu. † Current address: Department of Chemistry, Duke University, P.O. Box 90349, Durham, NC 27708.

from the LH1 antenna chromophores; the primary electrontransfer event is supposed to occur following nonradiative transfer of population to the lower exciton state.13 Comparable interexciton-state radiationless decay paths are thought to be important in the trapping function of allophycocyanin in the core of the phycobilisome in cyanobacteria.14,15 Bacteriochlorophyll dimers might be favored in reaction centers because of their charge-transfer properties, which may contribute to the high quantum efficiency and spatial directionality of the initial electron-transfer events. Several investigators have discussed the possibility that the π f π* electronic excited states of P are mixed with charge-transfer states.16-21 This admixture of states is thought to afford the lower exciton state of P an unusually large dipole moment, as detected by Stark spectroscopy by Boxer and co-workers22,23 and Feher and coworkers.24 The coupling of the π f π* excited state to the charge-transfer state is thought to be the origin25-27 of the extremely weak (or even absent) zero-phonon holes that are observed with burn wavelengths across the absorption band in persistent photochemical hole-burning experiments16,17,28-30 at low temperature. However, it is not generally thought that the lower exciton state carries enough charge-transfer character to be thought of as a real intradimer radical pair state.18,19 Timeresolved infrared pump-probe experiments by Hochstrasser and co-workers suggest that the charge-transfer states lie several thousand wavenumbers above the π f π* state.31-33 This interpretation is consistent with the semiempirical molecular orbital calculations by Warshel and Parson.18,19 Calculations by Thompson et al.34,35 have indicated that charge-resonance states contribute to the red shift of the lower exciton state P. It is likely that charge-transfer states contribute to the lowest exciton state in the LH1 and LH2 systems as well. Prior to the determination of the X-ray crystal structure of LH2,1 Boxer and

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Figure 1. Comparison of the structures of two bacteriochlorophyll a dimer systems: (a) the primary electron donor P in the reaction center of Rhodopseudomonas Viridis;6 (b) the B850 system in the LH2 complex of Rhodopseudomonas acidophila.1a Both systems are shown in two views that are related by a right-handed 90° rotation about the horizontal axis. The two sets of structures are shown with the same scaling.

co-workers36 used Stark spectroscopy to show that the bacteriochlorophyll a dimer that gives rise to the B850 band in LH2 has an unusually large excited-state dipole moment; further, they observed a very unusual electric-field-dependent quenching of the fluorescence. Recent Stark spectroscopy studies by van Grondelle and co-workers37 have further indicated that chargetransfer states are mixed with the dimer exciton states in LH2. How the charge-transfer character of the bacteriochlorophyll dimer impacts on the light-harvesting function or excited-state dynamics of the LH1 and LH2 systems has not been directly addressed. We have performed a series of femtosecond spectroscopic studies on the B820 subunit system of LH1 in order to focus on the special properties of the bacteriochlorophyll dimer in the absence of energy transfer or electron transfer to adjacent acceptors. As isolated from Rhodospirillum rubrum G9 (the carotenoidless mutant), B820 contains a heterodimeric system consisting of two bacteriochlorophyll a molecules arranged in a near-parallel orientation38 held between two R helices, as is present in LH2. The system is heterodimeric in that the two R helices are not identical in sequence or length, and accordingly the site energies for the two bacteriochlorophylls may not be quite the same. The present experiments employ a femtosecond dynamic absorption technique39-47 at room temperature in order to monitor the vibrational wave packet motion that arises on the lower exciton-state potential-energy surface. The results discussed here provide the first time-resolved spectra of an isolated bacteriochlorophyll a dimer that directly show the development of a Stokes shifted stimulated-emission spectrum and a nonstationary vibrational character. Single-wavelength transients evidence excited-state coherent wave packet motion that is not the same as that arising on the ground-state surface from resonant impulsive stimulated Raman scattering (RISRS). The anisotropy decay indicates that a >20° tilt of the photoselected transition-dipole moment occurs in synchrony with the dynamic Stokes shift of the stimulated emission. These results suggest, among several possibilities, that an intradimer chargetransfer event occurs promptly following optical preparation of the lower π f π* exciton state, with the wave packet motion observed in the stimulated emission arising from the chargetransfer event itself.

2. Experimental Procedures Sample Preparation. Rs. rubrum G9 cultures were grown in 3-L carboys under low-intensity incandescent light (one 60-W bulb at a distance of 1 ft) in the presence of 5% CO2/95% N2 in modified Hutner’s medium, as described by Cohen-Bazire et al.48 Cells were harvested by centrifugation at 5000g for 5 min in a Sorvall GSA rotor late in the exponential phase of growth; the cells were stored at -10 °C in the dark as pellets in the growth medium. Chromatophores were isolated as described by Loach and co-workers,49,50 except that the benzene extraction step was not required (the G9 mutant does not contain carotenoids). The pelleted chromatophores were diluted in a buffer solution containing 10 mM HEPES-NaOH at pH 8.0 to obtain a final absorbance at 880 nm of 25-35 for a 1-cm path. The procedure described by Visschers et al.51 was used to isolate B820 subunits from the chromatophore preparations. To a suspension of chromatophores, n-octyl-rac-2,3-dipropyl sulfoxide (ODPS, Bachem) was added to establish a ∼1.5% (w/v) solution while monitoring the absorbance at 817 nm; small additions of the ODPS solution were made until no further change in absorbance at 817 nm was detected. The detergenttreated chromatophores were centrifuged at 40 000g for 1 h in a Sorvall SS-34 rotor to pellet any insoluble material. Aliquots (450-µL) from the supernatant were injected onto a FPLC column (Superose 6/HR, Pharmacia) that was equilibrated with 10-mM HEPES-NaOH buffer solution at pH 8.0 containing 0.6% (w/v) ODPS and 400 mM KCl. The column was eluted at a flow rate of 0.3 mL/min. The peak fractions corresponding to the B820 subunit were collected and passed through a 0.22µm filter prior to use in femtosecond spectroscopy. All steps in the chromatographic procedure was conducted in semidarkness at room temperature (22 °C). Femtosecond Spectroscopy. The femtosecond laser spectrometer and detection system has been described in operational detail in another paper.52 The light source is a self-mode-locked titanium-sapphire oscillator that was constructed following the design of Murnane and co-workers53 using some of the components from a Clark Instrumentation NJA-4 kit. For the experiments reported in this paper, the oscillator was adjusted to produce 18-fs pulses (sech2). The output spectrum was centered at 805 nm and exhibited a width of 68 nm (fwhm). As shown

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Diffey et al.

Figure 2. Comparison of the continuous absorption spectrum (dotted curve) obtained at room temperature (22 °C) with B820, as isolated in ODPS from Rhodospirillum rubrum G9, with the output spectrum of the 18-fs (sech2) pulses obtained from the pulse-picked titaniumsapphire laser (solid curve) used in the experiments reported in this paper. The laser spectrum was obtained as a single scan of an intensified silicon-diode-array detector positioned at the focal plane of a 0.125-m spectrograph (used with 0.1-nm spectral band pass); the B820 absorption spectrum was obtained with 2-nm band pass.

in Figure 2, the laser spectrum overlaps well with the absorption spectrum arising from B820’s lower exciton-state transition. The laser spectrum extends well to the red of the 830-nm maximum of the continuous fluorescence-emission spectrum. The oscillator’s repetition rate was reduced to 3 MHz using a singlepass pulse picker constructed from a Harris H101 fused-silica Bragg cell and Camac Systems CD5000 and PB1800 RF electronics. Following group-delay dispersion (pre)compensation by a pair of LaKL21 prisms, the pulse-picked train of pulses was injected into a modified Mach-Zehnder interferometer.52 A rapidscanning galvanometer-driven delay stage (Clark Instrumentation ODL-150) is used in the pump arm of the interferometer. The pump beam is amplitude modulated at 100 kHz by a photoelastic modulator (PEM-90, Hinds Instruments). The linear polarizations of the pump and probe beams are set 45° apart by Glan laser calcite polarizers. A second calcite polarizer is placed in the transmitted probe beam to allow polarization analysis at any angle relative to the polarization of the pump beam. The transmitted probe beam passes through a 0.270-m monochromator (Spex 270M), which was operated with 4-nm spectral band pass, before detection by an amplified silicon photodiode (Thorlabs). Single-wavelength transients were obtained with a rapid-scanning technique; the signal from the photodiode was demodulated at the pump-beam amplitudemodulation frequency by a SRS 850 lock-in amplifier, and the output of the lock-in amplifier was digitized and averaged by a Tektronix digital oscilloscope. Averaged scans were downloaded and accumulated in 256-scan batches by an Apple Macintosh IIsi computer, which controlled the data-acquisition process using LabVIEW (National Instruments) routines. B820 samples were flowed at room temperature (22 °C) at 0.24 mL/min rate through a fused-silica cuvette (1-mm path length) at room temperature. The maximum absorbance of the sample was 0.45 at 817 nm. Fused-silica neutral-density filters were employed to reduce the pump and probe pulse energies at the sample position to 150 and 8 pJ per pulse, respectively. 3. Results Time-Resolved Dynamic Absorption Spectra. Figure 3 shows a series of isotropic dynamic absorption spectra obtained

Figure 3. Time-resolved dynamic absorption spectra obtained at room temperature with isotropic detection with preparations of the B820 subunit. The spectra are shown with the same ordinal scaling but offset arbitrarily; the dotted lines mark the zero level for each spectrum.

with B820 at room temperature. The dynamic-absorption spectra shown in Figure 3 were constructed from a series of singlewavelength, 2-fs/point transients. The transients were acquired at 5-nm intervals across most of the titanium-sapphire laser’s output spectrum. Several transients were acquired at each wavelength over the duration of the data-acquisition period; the transients were not acquired in wavelength order in order to avoid effects from systematic changes in the sample or apparatus, though none were observed. The entire set of transients was used to construct a time-wavelength surface; a grid defined by 5-nm by 4-fs bins was used to accumulate all of the ordered triples (delay, wavelength, pump-induced change in transmission ∆T/T) obtained from the set of single-wavelength transients. The spectra shown in Figure 3 are unsmoothed slices taken across the surface at the indicated delay points. The time-resolved dynamic absorption spectra exhibited by B820 exhibit a time evolution arising from a rapid Stokes shift of the stimulated-emission component and a blue shift of an excited-state absorption feature. The zero-time spectrum is characterized by a photobleaching/stimulated-emission feature centered near 818 nm and flanked on both sides by regions of net absorption near 790 and 845 nm. The 790-nm excited-state absorption region develops over time by rapidly shifting to the blue. The shift of the stimulated-emission band to the red is complete by the 40-fs spectrum. The photobleaching component of the spectrum exhibits a modest broadening, especially over the 40-250-fs delay interval. The broadening causes the peak intensity in the 818-nm region arising from photobleaching to decrease so that the stimulated emission in the 830-nm region eventually is more intense. The appearance of the dynamic absorption spectrum at long delays resembles qualitatively the conventional pump-probe absorption-difference spectrum reported for B820 by van Grondelle and co-workers, as obtained with nanosecond38 or subpicosecond54 pulses. The pump-probe spectrum is deriva-

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Figure 5. Analysis of the vibrational coherence in the dynamic absorption transient observed with B820 at 810 nm. The smooth trace overlaid on the data points is a LPSVD fit to the decaying, oscillatory portion of the parallel-polarized transient in panel (a) of Figure 4. The lower trace shows the residual function (data - fit). A spectral representation of the LPSVD fit is shown in Figure 7. Table 1 shows the parameters that describe the LPSVD fit. Data points in the transient that fell before the 50-fs delay point were excluded from the LPSVD analysis. Figure 4. Single-wavelength dynamic absorption transients observed with B820 at room temperature: (top panel) probe detection at 810 nm; (bottom panel) probe detection at 830 nm. The two transients shown in each panel correspond to detection of the transmitted probe beam with polarization analysis parallel or at the “mystic” angle (63.44°) with respect to the pump beam’s plane of polarization (the latter setting yielding isotropic detection). The transient obtained with parallel pump-probe polarization is the more intense one of the pair in each panel.

tive-shaped, with a maximum photobleaching/stimulated-emission region near 830 nm and a region of excited-state absorption beginning at a zero crossing at ∼810 nm and proceeding to the blue. The general features of this spectrum can be explained by a model involving a strongly coupled pair of bacteriochlorophyll molecules with a mostly parallel organization of the transition-dipole moments; according to the simple theory discussed previously,14 this situation produces a pump-probe spectrum with a relatively sharp excited-state absorption feature lying to the blue of the main photobleaching/stimulated-emission feature. The excited-state absorption arises in this case from transitions from the lower exciton state to the doubly excited exciton state. The dynamic absorption spectrum we observe with B820 at room temperature is broader in the photobleaching/stimulatedemission region than the pump-probe spectrum observed by van Grondelle and co-workers. The dynamic absorption spectrum is broader in the wings than that observed with longer pump pulses because of the impulsive preparation of nonstationary vibrational states in both the ground state and the excited state. This phenomenon has been described theoretically by Pollard et al.42 Single-Wavelength Transients. Figure 4 compares a set of single-wavelength transients obtained at room temperature with B820 with detection at 810 and 830 nm. The two transients report dynamics associated with the photobleaching and stimu-

lated-emission regions of the time-resolved spectrum, respectively. The two transients shown in each panel correspond to detection of the transmitted probe beam with polarization analysis parallel or at the “mystic” angle (63.44°) with respect to the pump beam’s plane of polarization (the latter setting yielding isotropic detection for the case of a 45° angle between the pump and probe planes of polarization55). The 810-nm transients shown in Figure 4a exhibit a rapid decay following the transmission maximum that arises from solvation-induced line broadening and the Stokes shift of the stimulated emission.56 Following the maximum, the transients at both wavelengths exhibit a cosinusoidal modulation extending certainly out to the 1-ps delay region. The modulation exhibited by the 830-nm transients in Figure 4b is of greater amplitude and of higher frequency than observed at 810 nm. A cursory examination of the beats in the 830-nm transients obtains a spacing of ∼5060 fs, while the beats in the 810-nm transients are spaced by ∼180 fs. The decaying, modulated portion of the parallel-polarized transients shown in Figure 4 was fit to a series of damped sinusoids of the form a0 exp(-t/τ) sin (ωt + φ) by a linearprediction, singular-value-decomposition (LPSVD) procedure.57,58 The program we used to perform the LPSVD analyses was a modified version of one that was generously provided to us by Professor Anne Myers (University of Rochester); a discussion of the application of this LPSVD program to an analysis of RISRS transients has been described previously.58 Figures 5 and 6 compare the signals with the fitted curves obtained from the LPSVD analysis. The parameters returned directly from the LPSVD analysis for these transients are shown in Table 1. We estimate that the frequencies are known with a precision of 20 cm-1, the phases to within 20°, and the amplitudes to within 10%. The number of components listed represents the minimum number of components required to describe reasonably the decay and modulation patterns we

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Figure 6. Analysis of the vibrational coherence in the dynamic absorption transient observed with B820 at 830 nm. The smooth trace overlaid on the data points is a LPSVD fit to the decaying, oscillatory portion of the parallel-polarized transient in panel (b) of Figure 4. The lower trace shows the residual function (data - fit). A spectral representation of the LPSVD fit is shown in Figure 7. Table 1 shows the parameters that describe the LPSVD fit. Data points in the transient that fell before the 50-fs delay point were excluded from the LPSVD analysis.

observed. The signal/noise ratio and frequency content of the transients well prior to the pump-probe interaction region was used in comparison to the residual function obtained from a given set of components as a guide in this analysis. The data points falling before the 50-fs delay point were excluded from the analysis. Note that components returned by the LPSVD analysis at frequencies above 900 cm-1 were dropped from the fit because the pump and probe pulses lack significant impulsive frequency content at frequencies above 900 cm-1 (vide infra). Figure 7 shows a spectral representation of the LPSVD results, with the amplitudes for the modulation components normalized by the intensity of the most intense feature observed (the 600-cm-1 component in the 830-nm transient) and by the Fourier transform of the pump-probe instrument-response function, which is depicted as the dashed curve in Figure 7b. The latter normalization accounts for the frequency windowing59 that arises from the linear convolution of the pump-probe instrument-response function with the sample response. A comparable procedure has been used by Lotshaw and McMorrow in their analyses of the modulation patterns that are observed in nonresonant optical Kerr effect transients in liquids.59-63 The frequency components in the spectra shown in Figure 7, in principle, exhibit the relative intensity scaling that would be directly observed in an experiment employing δ-function excitation. Note, however, that it is possible that higher frequency components lying outside the impulsive spectral content of the pulses used in the experiment would contribute if shorter pulses were available. The transients shown in Figure 4 were also subjected to a frequency analysis using discrete Fourier transformation. The decaying portion of the transients was removed by subtracting a fitted double-exponential function from the data points, and the residual function was fed to the Fourier transform subroutine. The resulting frequency spectra (not shown) are quite comparable to the results obtained from the LPSVD analyses. Table

Diffey et al. 1 includes the frequencies obtained directly from the Fourier amplitude spectrum that correspond to those obtained from the LPSVD analysis. Additional low-intensity components in the Fourier amplitude spectrum that were not easily discernible from the noise background are not tabulated. The LPSVD analysis indicates that the 810- and 830-nm transients observed with B820 preparations are both modulated by very low frequency components in the 20-60 cm-1 region and by a 180-cm-1 component. The 180-cm-1 component is the source of the main structure observed in the 810-nm transient. The highest frequency component that was detected in the 810-nm transient was a fairly weak component at 400 cm-1. The 830-nm transient differs from the 810-nm transient in being fairly strongly modulated by higher frequency components in the 360-730-cm-1 region. The 600- and 730-cm-1 components contribute to the modulation with a 50-60-fs period that is especially obvious in the early portion of the transient. Removal of either of these components from the LPSVD fit produces a residual function (not shown) that clearly exhibits a damped sinusoid, well above the noise level. The LPSVD analysis of the 810-nm transient suggests that modulation is absent at these frequencies or at most 20% of that observed at 830 nm (see Figures 4 and 7). This confidence interval was obtained by careful analysis of the residual function for the 810nm parallel-polarized transient as synthetic 600- and 730-cm-1 components were added to the fit. The 20-fs portion of the data; the fit is described by r(t) ) 0.04 exp (-t/100 fs) + 0.28. The largest anisotropy value, 0.42, was observed at delay t ) 0 fs. Data points prior to that point are not shown.

system from Rs. rubrum G9. The strongest mode in this region was a 727-cm-1 feature.70 Anisotropy Decays. Figure 8 shows an anisotropy decay we obtained with B820 preparations at room temperature with detection of the probe beam at 835 nm, well into the stimulatedemission region of the dynamic absorption spectrum. The data points in the anisotropy trace were computed directly from transients recorded with polarization analysis parallel (A|) and perpendicular (A⊥) with respect to the pump beam’s plane of polarization as r(t) ) (A| - A⊥)/(A| + 2A⊥). The largest anisotropy value, 0.42 ( 0.02, was observed at delay t ) 0 fs. The undulations in the anisotropy trace arise from noise in the parallel and perpendicular traces (not shown). Anisotropy decays were measured across the entire probe range covered by the laser spectrum, but the 835-nm anisotropy transient is to be favored for use in subsequent discussion. This probe wavelength is removed as far as our current sensitivity permits from the region dominated by photobleaching and excited-state absorption in the blue region of the dynamic absorption spectrum. The anisotropy decay shown in Figure 8 exhibits an abrupt, nonexponential decrease to the ∼0.32 ( 0.03 level within 40 fs. Note that the initial decay is terminated in the 20-40-fs delay region, which coincides with the initial motion of the stimulated-emission part of the dynamic absorption spectrum (see Figure 3). As the probe wavelength is tuned to the blue, the magnitude of the initial anisotropy decay observed at the 40-fs delay point decreases owing to an increased contribution from ground-state depletion, which contributes an anisotropy near the 0.40 level. Following the initial abrupt anisotropy decay, a slower decay is observed to the 0.28 ( 0.03 level. This decay can be approximately described by an exponential trend with a 100-fs time constant: r(t) ) 0.04 exp(-t/100 fs) + 0.28. Control anisotropy experiments (not shown) were conducted under the same measurement conditions with the thiacarbocyanine dye 3,3′-diethylthiatricarbocyanine iodide (DTTCI) dissolved in ethylene glycol. The anisotropy reached at probe delays past time zero was 0.39 ( 0.02. With this sample, strong modulations are observed in isotropic and polarized transients, and the anisotropy exhibits a weak modulation as well. No net

Diffey et al. decay of the anisotropy was observed over the first picosecond of delay. These control results indicate that the prompt anisotropy decay observed with B820 arises from a real process and not a measurement artifact. In previous work, Hochstrasser and co-workers64 employed 35-fs pulses to measure an integrated anisotropy decay in B820 centered around 800 nm. Owing to the narrower bandwidth of the 35-fs pulses, the signal they obtained is most sensitive to the photobleaching and excited-state absorption region. The initial anisotropy they observed (∼0.6) was significantly larger than the value we observe in the stimulated-emission section of the spectrum; they observed a fast decay, with a 30-fs time constant, to an anisotropy of ∼0.22, followed by a partial recovery (300 fs) and subsequent decay (600 fs) to the same anisotropy level. This anisotropy decay was interpreted in terms of a heavily damped oscillation with a ∼500-cm-1 frequency. Our survey of the anisotropy decay in B820 using a dispersedprobe detection scheme failed to detect initial anisotropies that were larger than 0.42, and no dichroic oscillations were observed; further, the magnitude of the anisotropy decay increases and levels off as the probe detection is tuned from the photobleaching/excited-state absorption region in the blue into the stimulated-emission region in the red part of the dynamic absorption spectrum. Aside from the use of a wavelengthselective technique, our results may also differ from those of Hochstrasser and co-workers owing to the use of very low pump-pulse energies (180 pJ/pulse), which have been shown to be preferred in the measurement of anisotropies by Jonas et al.71 Further, the bandwidth of the excitation pulses we used in our experiments does not overlap extensively with the region of the ground-state absorption spectrum that pumps the transition to the upper exciton state, so we avoid the anisotropy oscillations that would arise from electronic quantum beats associated with a coherent excitation of both exciton transitions. Beats of this type have been observed by Struve and co-workers in Fenna-Matthews-Olson trimers isolated from Chlorobium tepidum.72 4. Discussion The production of ground-state wave packet motion following impulsive excitation by a femtosecond pulse on resonance with an electronic transition has been discussed by Pollard et al.40-42 and by Johnson and Myers58 in terms of a RISRS mechanism. Jonas et al.66 have presented an alternative picture in terms of classical and quantum-mechanical models for the propagation of a ground-state hole wave packet, but the expectations of this theory within its limits of applicability are equivalent to those of the RISRS theory. As in the conventional resonance Raman effect,73 ground-state wave packet motion arises from the displacement of the excited-state potential energy surface along one or more vibrational coordinates with respect to the minimum of the ground-state potential energy surface. In the RISRS picture, the ground-state wave packet arises from a coherence established by the resonant pump pulse: a femtosecond pump pulse launches a wave packet on the excited-state surface, where it evolves for a short time until a second interaction with the pump pulse deposits a portion of the excited-state wave packet at a displaced position on the ground-state surface. Motion of this ground-state wave packet gives rise to the modulation of the pump-probe signal that is commonly observed in the wavelength region overlapping with the ground-state absorption spectrum. For a bound electronic-state surface with the same shape as the ground-state surface, the same modulation components and

Vibrational Coherence and Anisotropy in B820 frequencies would be expected to arise from the wave packet that is still moving on the excited-state potential energy surface. Thus, the structural displacement of the excited-state surface from the ground-state surface along specific system modes is the origin of both the excited-state and ground-state vibrational coherences in pump-probe experiments.40-42 Of course, if the shape of the excited-state surface differs somewhat from that of the ground-state surface or if the surfaces are sufficiently anharmonic, the modulation frequencies may be shifted from those arising from the ground-state wave packet’s motion (depending on the vibrational level prepared on the excitedstate surface by the Franck-Condon transition). In addition, a large change in the electronic structure in the excited state might lead to new vibrational frequencies arising from Duschinsky mixing.73,74 This type of frequency shift apparently accounts for an excited-state modulation component observed by Scherer and co-workers74 in one-color pump-probe experiments with the blue copper protein plastocyanin when the ligand-field transition near 800 nm was pumped. One of the main results of this paper is that the stimulatedemission region of the dynamic absorption spectrum exhibited at room temperature by B820 is strongly modulated by vibrational frequencies that apparently do not strongly modulate the photobleaching region. This probe-wavelength dependence allows us to tentatively conclude that the 360-, 470-, 600-, and 730-cm-1 components detected in the 830-nm transient (see Figures 4, 5, and 6) arise from excited-state wave packet motion. The 20-40- and 180-cm-1 components, most strongly observed in the 810-nm transient and weakly observed in the 830-nm transient, are assigned for now to RISRS components. Both the 810- and 830-nm transients exhibit components in the 390400-cm-1 region, so an assignment of these components to wave packet motion on a particular surface cannot be made at this point. A conclusive assignment of the oscillations to excited-state or ground-state wave packet motions will require a detailed analysis of the dependence of the phase of the components with respect to the detection wavelength. Wave packet motion on a given potential energy surface is expected to display a 180° phase shift for the modulation arising from a given mode at the displacement corresponding to the minimum energy point of the surface,46,58,65,66 which would lead to a 180° phase shift for ground-state wave packets at the wavelength matching the absorption maximum and for excited-state wave packets near the wavelength matching the stimulated-emission maximum. At this point we can report that a 180° phase shift occurs near 830nm for the 730-cm-1 mode and near 818 nm for the 180-cm-1 mode; this supports the tentative assignments made above because the ground-state absorption maximum is near 818 nm and the fluorescence-emission maximum is at 830 nm. The apparent absence of the four high-frequency components from the ground-state wave packet motion despite the use of ample impulsive bandwidth (see Figure 7b) might be explained by known phenomena involving the interaction of the probe spectrum with the nonstationary vibrational character or from the electronic structure of the bacteriochlorophyll dimer in the lower π f π* exciton state. 1. The work by Pollard et al.42 shows that use of very short pump pulses can cause the amplitude of ground-state wave packets to be attenuated by limiting the distance travelled by the excited-state wave packet away from the Franck-Condon region prior to the second, scattering interaction with the pump field; however, this attenuation would be expected to be most

J. Phys. Chem. B, Vol. 102, No. 15, 1998 2783 severe for low frequencies, the opposite of the situation being considered here. 2. The relatiVe amplitude of detected RISRS components is known to depend on the probe wavelength; in the work on bacteriorhodopsin by Dexheimer et al.,42,75 the modulation components were observed to vary significantly in intensity as the probe wavelength was tuned over the region coinciding with ground-state depletion, but all of the components made a distinct contribution at each probe wavelength. The present situation in B820, however, involves large changes in modulation amplitudes as the probe wavelength is tuned from regions that are dominated by ground-state depletion to regions that are dominated by stimulated emission. 3. Harmonics of fundamental frequencies might make a contribution to the signal. Fundamental frequencies are expected to be strongest near the turning points for the wave packet’s motion; second-harmonic components are observed near the center of the surface as the wave packet passes twice per period.57,58,66,67 The relatively intense modes detected in the stimulated-emission region at 830 nm are probably fundamentals rather than harmonics of the less intense modes observed to lower frequency. None of the components in table 1 can be assigned to second harmonics, however, because they do not appear at twice the frequency of a more intense lower frequency fundamental component. 4. The present results do not exclude the possibility of new frequencies arising from the Duschinsky effect, but consider that at least the 730-cm-1 mode that we observe in the stimulated emission corresponds to a frequency that has been detected in the Qy resonance Raman spectra of the monomeric bacteriochlorophylls in the photosynthetic reaction center. Note that Boxer, Mathies, and co-workers observed that the special pair P in the reaction center exhibited more Raman intensity in the lower frequency modes (below 200 cm-1) than did the monomeric bacteriochlorophylls BL and BM, which exhibited more intensity in the middle frequency modes (200-1000 cm-1).76 In this respect the excited-state vibrational coherence in B820 might be said to have a more monomeric character than does the ground-state vibrational coherence, but it should be kept in mind that resonance Raman intensities are not expected to parallel directly the amplitudes of components observed in vibrational coherence, given the findings of Johnson and Myers.58 Owing to the lack of a convincing explanation for the excitedstate vibrational coherence in B820 from the alternatives discussed above, we suggest that at least part of the modulation pattern observed in the stimulated emission in B820 arises from a mechanism involving excited-state chemistry. If the wave packets that modulate the stimulation emission are riding on a bound surface, the theory by Pollard et al.42 does not provide a ready explanation for an observation of oscillatory components in the stimulated emission or fluorescence that lack groundstate counterparts. If the excited-state wave packet moves away from the Franck-Condon region and crosses to a new surface (or section of the surface, if the strongly coupled limit is applicable26,27) associated with a chemically distinct product structure, however, the stimulated emission might exhibit an altered wave packet modulation. Whether new modulation components are actually observed, however, depends on several factors: the mode-specific displacement from the ground-state geometry for the product surface, the electronic coupling strength between the initial and product surfaces, the vibrational relaxation parameters,77,78 and the time it takes for the wave packet to move between the surfaces. One or more FranckCondon active system modes serve in this case as promoting

2784 J. Phys. Chem. B, Vol. 102, No. 15, 1998 modes. A comparable suggestion was made by Champion and co-workers79-81 in the discussion of coherent wave packet motion on the ground electronic state of deoxymyoglobin following photoinduced dissociation of NO from myoglobin. The breaking of the Fe2+-NO bond is thought to take the system ballistically into a region of the ground-state potential energy surface that is displaced along Fe2+-histidine and hemedoming coordinates.80 There are a couple of possible assignments for a product state in B820 that would be formed promptly following preparation of the lower π f π* exciton state. 1. One suggestion is that a dynamic distortion, such as a Jahn-Teller breaking of symmetry, causes a structural deformation of one or both of the bacteriochlorophyll a molecules in the pair. 2. As suggested by the discussion of charge-transfer states in the Introduction, the product might be an intradimer chargetransfer state. The 360-730-cm-1 range of frequencies observed in the stimulated-emission region can be assigned, in comparison to previous bacteriochlorophyll a Raman spectra (as reviewed by Cherepy et al.76 in a discussion of the Raman spectrum arising from bacteriochlorophylls in the reaction center), to in-plane deformation modes of the porphyrin macrocycle. The relative resonance Raman cross sections for these modes would not be necessarily expected to be strongly sensitive to the oxidation state of the bacteriochlorophyll molecule. But QCFF/PI calculations by Warshel82 on a bacteriochlorophyll dimer suggest a significant increase in the Huang-Rhys displacement factor for a 750-cm-1 mode when a bacteriochlorophyll dimer is oxidized, so it is possible that a transfer of charge between the bacteriochlorophylls would cause enhanced vibrational activity in the excited state involving at least the 730-cm-1 mode. The anisotropy decay observed in the stimulated-emission region of the dynamic absorption spectrum (probe detection at 835 nm, Figure 8) can be used to assess the two possibilities. Although the structure of the bacteriochlorophyll a dimer in the B820 subunit is not known from X-ray crystallography at this point, the projection map for LH1 obtained from electron microscopy5 suggests that a given subunit of the LH1 system (i.e., the B820 subunit) is isomorphous with one of the subunits of the LH2 system, for which the structure is known.1 So, it is reasonable to use the pair of bacteriochlorophyll a chromophores in the LH2 structure as a starting point for the analysis of the anisotropy decay in B820. A recent discussion by van Grondelle and co-workers on this point suggests that the Mg-Mg distance in B820 may be somewhat larger than that indicated by the LH2 structure.37 The two bacteriochlorophyll a molecules in B820 are strongly coupled,38,51,54,83-85 so the transition-dipole moment that is photoselected in the anisotropy experiment is that of the lower exciton state, |+〉. This state is formed by the symmetric linear combination of the two site bacteriochlorophyll a transitiondipole moments, which are pointed 19.7° apart in the LH2 structure1 (see Figure 9, where the pair structure that is considered is that involving overlap of rings III and V. As shown in Table 2, the initial anisotropy observed in the B820 system, 0.42, implies that the two Qy moments are actually 20° apart. Given that the finite width of the instrument-response function will cause a decrease in the largest anisotropy observed,86-88 this estimate represents the lower limit for the angle between the two Qy moments in B820. The ultrafast anisotropy decrease observed with B820 (Figure 8) indicates that some mechanism causes a prompt rotation of

Diffey et al.

Figure 9. Representation of Qy transition moments for the bacteriochlorophyll a monomers in the B820 subunit. Also shown is the MgMg vector. The structures were rendered from the paired bacteriochlorophyll a molecules in the LH2 subunit from Rhodopseudomonas acidophila, as determined in the crystal structure by Cogdell and coworkers.1a

the photoselected transition-dipole moment away from the direction that is initially photoselected by the pump pulse. The orientation of the transition-dipole moment of the |+〉 state can be taken as the vector sum of the two Qy moments. The direction of this moment is not dependent on whether the Qy moment is pointed along the vector between the nitrogen atoms in the bacteriochlorophyll a macrocycle, as has been taken previously in calculations based on the LH2 structure,96 or whether the moment is rotated 5° further, as suggested by the work by Boxer and co-workers with Zn-chlorophyllidesubstituted hemoglobin systems.97 Table 2 also summarizes the anisotropy decay expected for mechanisms that would cause the photoselected transition-dipole moment to tilt away from the direction defined for the transition-dipole moment of the |+〉 state: 1. If the anisotropy decay arises from energy transfer between the two bacteriochlorophyll a molecules in a given B820 subunit, the theory discussed by Wynne and Hochstrasser93-95 and by Knox and Gu¨len2,3,92 anticipates that the anisotropy should decrease to a terminal anisotropy r(∞) of 0.37. This final anisotropy reflects an equilibration of site population over the two bacteriochlorophyll a molecules. Prior to reaching its final value, the anisotropy should be modulated at a frequency corresponding to the interaction energy between the transition dipoles of the bacteriochlorophyll a molecules.98,99 As pointed out above, however, the isotropic and parallel-polarized singlewavelength transients we obtained exhibit the same modulation patterns, which arise from coherent wave packet motions rather than from energy transfer. 2. If the anisotropy decay arises from a dynamic Jahn-Teller distortion, as suggested above, one would need to know the nature of the motion that leads to symmetry lowering in order to calculate the expected anisotropy decay. Distortions of the bacteriochlorophyll a rings owing to in-plane deformation modes such as the ones observed in the stimulated-emission region would not be expected to rotate the Qy moments very far; the orientation of the transition-dipole moment of the |(〉 state would not be expected to rotate at all owing to the vector-sum cancellation of coherent motions of the two bacteriochlorophyll a molecules. Similarly, owing to the rigidity of the bacteriochlorophyll a macrocycle, one would not necessarily expect a significant out-of-plane motion of either of the Qy moments in the bacteriochlorophyll a dimer. 3. Finally, if the anisotropy decay arises from transfer of an electron between the two bacteriochlorophyll a molecules, the photoselected transition-dipole moment would be expected to turn to a direction along the motion of charge, which, to a first approximation, should be oriented along the Mg-Mg vector. The LH2 structure indicates that this vector is oriented 17.3°

Vibrational Coherence and Anisotropy in B820

J. Phys. Chem. B, Vol. 102, No. 15, 1998 2785

TABLE 2: Transition Moment Angles and Anisotropies for the Bacteriochlorophyll a Dimer in B820 angle (deg)a

anisotropy

angle between the two bacteriochlorophyll Qy transition-dipole moments angle between the transition-dipole moment of the lower exciton state and the Mg-Mg vector between the two bacteriochlorophyll a molecules anisotropy decay owing to incoherent energy transfer between the two bacteriochlorophyll a molecules (equilibration)

19.7 17.3

r(0) ) 0.42b r(∞) ) 0.35c

19.7

r(∞) ) 0.37d

angle determined from observed initial anisotropy transition in B820 (at 40 fs) angle determined from observed final anisotropy (at 250 fs)

21.4 26.6

r(∞) ) 0.32c r(∞) ) 0.28c

a Measured angles were determined using the geometry of the paired bacteriochlorophyll a molecules in the LH2 crystal structure.1a The orientation for the Qy transition-dipole moments was taken as pointing between the two nitrogen atoms in the bacteriochlorophyll a macrocycle. b Initial anisotropy calculated using r(0) ) 0.3 ((1 - cos2 φ)/(1 + cos2 φ)), as determined for equivalent paired chromophores by Matro and Cina.89 c Terminal anisotropy calculated using r(∞) ) (3 cos2 φ - 1)/5, as required by a pure tilt of the photoselected transition-dipole moment.90-92 d Terminal anisotropy calculated using r(∞) ) 0.1(1 + 3 cos2 φ), as defined by theory determined for equivalent paired chromophores by Wynne and Hochstrasser93-95 and by Knox and Gu¨len.3,92

away from the direction of the transition-dipole moment of the |(〉 state, so the terminal anisotropy r(∞) should be 0.35. Figure 8 shows that the prompt anisotropy decay observed with B820 is to a value of ∼0.32, with a subsequent decay to a value of 0.28 according to a 100-fs time constant. We suggest that the initial anisotropy decay is more consistent with intradimer charge transfer because the observed decay is larger than that expected for any of the other mechanisms. Following the prompt charge-transfer event, the two oppositely charged bacteriochlorophyll a macrocycles would be expected to move with respect to each other. As the two molecules slide against each other in a stacking motion in an effort to lower the Coulomb energy, the Mg-Mg vector would increase in angle with respect to the direction of the originally photoselected |(〉 transition-dipole moment. This structural relaxation should be strongly dependent on the temperature and composition of the surrounding medium. Perhaps this sort of motion is responsible for the slower, 100-fs phase of anisotropy decay following the initial abrupt decrease. The suggestion that an intradimer charge-transfer state is populated directly from the |(〉 exciton state with retention of vibrational coherence conflicts with the simplest interpretation of the recent measurements of the Stark effect on the groundstate absorption spectrum of B820 by van Grondelle and coworkers, who observed that B820 exhibits an even smaller ∆µA than does the polypeptide-bound bacteriochlorophyll a monomeric system called B777 that results upon detergent-induced dissociation of B820. In contrast, a large Stark effect was observed for the intact LH1 system, so a charge-transfer character is present when the bacteriochlorophyll a molecules in adjacent B820 subunits interact with each other.37 At present, it is not easy to understand how the B820 and LH1 results can be reconciled with each other without invoking some kind of detergent-induced change in the structure of the B820 system under the conditions of the Stark experiments. However, the same group recently reported100 that there is a strong temperature dependence to the fluorescence and triplet-state quantum yield in the B820 system; above 170 K, a nonradiative decay mechanism becomes activated. Van Grondelle and co-workers conclude that intradimer charge transfer is not the correct choice for the origin of the nonradiative decay channel.100 Note that this conclusion is based on the previously reported Stark spectroscopy work,37 which was conducted at 77 K, under conditions that suppress the nonradiative decay channel. Whatever the case, it would be interesting to characterize the Stark effect on the spontaneous fluorescence from B820; this would allow one to examine the extent of intradimer charge separation following thermalization and other dynamics in the excited state.21

Acknowledgment. This work is supported by the Molecular Biophysics program of the National Science Foundation. W.M.D. and B.J.H. were supported by National Institutes of Health graduate traineeships in Molecular Biophysics. We are grateful to Professor Anne Myers (University of Rochester) for providing us access to the LPSVD analysis program and to Professor John Jean (Washington University, St. Louis) for a discussion on vibrational coherence associated with potentialenergy surface crossings. We also were aided by discussions with Professor Julio de Paula (Haverford College) and Professor David Bocian (University of California, Riverside). We would also like to thank Professor Richard Cogdell (University of Glasgow) for providing us access to the X-ray crystal structure of LH2 from Rhodopseudomonas acidophila prior to its general release by the Brookhaven Protein Data Bank. References and Notes (1) (a) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaccs, N. W. Nature 1995, 374, 517-521. (b) Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. Structure 1996, 4, 581-597. (2) Rahman, T. S.; Knox, R. S.; Kenkre, V. M. Chem. Phys. 1979, 44, 197-211. (3) Knox, R. S.; Gu¨len, D. Photochem. Photobiol. 1993, 57, 40-43. (4) Ku¨hlbrandt, W. Nature 1995, 374, 497-498. (5) Karrasch, S.; Bullough, P. A.; Ghosh, R. EMBO J. 1995, 14, 631638. (6) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. J. Mol. Biol. 1984, 180, 385-398. (7) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730-5734. (8) Pullerits, T.; Sundstro¨m, V. Acc. Chem. Res. 1996, 29, 381-389. (9) Monshouwer, R.; van Grondelle, R. Biochim. Biophys. Acta 1996, 1275, 70-75. (10) van Grondelle, R. Biochim. Biophys. Acta 1985, 811, 147-195. (11) van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstro¨m, V. Biochim. Biophys. Acta 1994, 1187, 1-65. (12) Parson, W. W. In Photosynthesis; Amesz, J., Ed.; Elsevier: 1987; pp 43-61. (13) Jean, J. M.; Chan, C.-K.; Fleming, G. R. Isr. J. Chem. 1988, 28, 169-175. (14) Edington, M. D.; Riter, R. E.; Beck, W. F. J. Phys. Chem. 1996, 100, 14206-14217. (15) Edington, M. D.; Riter, R. E.; Beck, W. F. J. Phys. Chem. B 1997, 101, 4473-4477. (16) Meech, S. R.; Hoff, A. J.; Wiersma, D. A. Chem. Phys. Lett. 1985, 121, 287-292. (17) Boxer, S. G.; Lockhart, D. J.; Middendorf, T. R. Chem. Phys. Lett. 1986, 123, 476-482. (18) Warshel, A.; Parson, W. W. J. Am. Chem. Soc. 1987, 109, 61436152. (19) Warshel, A.; Parson, W. W. J. Am. Chem. Soc. 1987, 109, 61526163. (20) Friesner, R. A.; Won, Y. Biochim. Biophys. Acta 1989, 977, 99122. (21) Boxer, S. G.; Goldstein, R. A.; Lockhart, D. J.; Middendorf, T. R.; Takiff, L. J. Phys. Chem. 1989, 93, 8280-8294. (22) Lockhart, D. J.; Boxer, S. G. Biochemistry 1987, 26, 664-668.

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