J. Phys. Chem. B 2008, 112, 1299-1307
1299
Intermolecular Vibrational Coherence in the Bacteriochlorophyll Proteins B777 and B820 from Rhodospirillum rubrum Katherine R. Shelly,† Elizabeth C. Golovich,‡ Kevin L. Dillman, and Warren F. Beck* Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824 ReceiVed: September 4, 2007; In Final Form: October 22, 2007
The low-frequency vibrational coherence in the bacteriochlorophyll (BChl)-containing subunit proteins B777 and B820 from the LH1 light-harvesting complex isolated from Rhodospirillum rubrum G9 exhibits rapidly damped modulation components arising from intermolecular, formally nonbonding interactions between the BChl macrocycle and polar groups in the surrounding detergent or protein. The vibrational coherence observed in the monomeric B777 system resembles that observed previously with BChl in acetone because it contains a pair of broad overlapping line shapes with a mean frequency of 191 cm-1, but the 10:1 intensity ratio of the librational and translational components is distinctive of the motions of the polar head groups in the nonionic detergent micelle that solvates the BChl macrocycle. In contrast, the vibrational coherence observed with the dimeric B820 complex is almost 20 times weaker in intensity and exhibits narrower line shapes and lower average frequencies than observed in B777. The structure of the B820 complex sterically protects the pair of BChl macrocycles from the surrounding solvent, so modulation components assigned to intrinsic interactions between the BChl and the protein and between the pair of BChl’s are revealed. A relatively well-ordered interaction between the BChl macrocycle and a tryptophan residue in each R-helical polypeptide accounts for a 28 cm-1 component with a narrow line shape, but most of the intensity arises from a broader 46 cm-1 component that is assigned to the interaction between the paired BChl macrocycles. The breadth of the line shape for this component is a measure of the disorder in the ensemble of B820 subunits. The results support the hypothesis that the excited-state vibrational dynamics and the optical and/or Marcus charge-transfer reorganization energies of BChl in photosynthetic light-harvesting proteins and reaction centers are strongly controlled by van der Waals modes with neighboring molecules, with dominant contributions to the intermolecular potential arising from the London dispersion and dipole-dipole interactions.
1. Introduction The low-frequency vibrational coherence observed in femtosecond pump-probe experiments with bacteriochlorophyll (BChl)-containing light-harvesting proteins1-5 and reaction centers6-15 from purple bacteria contains especially strong modulation components arising from resonance Raman-active normal modes with frequencies in the 100 cm-1 regime. In reaction centers, these vibrational modes contribute most of the reorganization energy associated with the primary chargeseparation reaction.16-18 Rather than arising from the skeletal motions of the BChl macrocycle, it is likely that these vibrational modes arise from an intermolecular origin. The crucial interactions are likely to be nonbonding, formally van der Waals interactions between the BChl macrocycles and the surrounding protein-derived medium. This hypothesis is supported by the results of two recent studies from this laboratory on the nature of the rapidly damped vibrational coherence from BChl in polar solution.19,20 The modulation components observed in femtosecond pump-probe, dynamic-absorption transients from the dipyridine complex of BChl in neat pyridine exhibit two distinct damping regimes. A * Corresponding author. E-mail:
[email protected]. Phone: 517355-9715 x213. Fax: 517-353-1793. † Current address: Beckman Coulter, Inc., 1000 Lake Hazeltine Blvd., Chaska, Minnesota 55318. ‡ Current address: Pacific Northwest National Laboratory, P.O. Box 999, Mail Stop K5-25, Richland, Washington 99352.
slowly damped set of modulations spans the 100-8000 fs probe delay range; the modulation components exhibit damping times in the >1 ps regime and mode frequencies ranging from 10220 cm-1. The intensity spectrum obtained by Fourier transformation is comparable to the low-frequency region of the conventional resonance Raman spectrum from BChl in solution, films, and the reaction center,21,22 so these slowly damped features are assigned to the low-frequency modes of the BChl macrocycle.19 A much stronger and very rapidly damped set of modulation components is observed over the sub-picosecond time scale. These features were modeled in the time domain using inhomogeneously broadened, asymmetric Gaussian line shapes. The average frequency of the rapidly damped vibrational coherence spans the 100-160 cm-1 range and shows a monotonic increasing dependence on the gas-phase dipole moment of the solvent. The trend is consistent with the natural frequency of a van der Waals intermolecular potential in which the London dispersion and dipole-dipole interactions make the largest contributions. These results suggest that clustered solvent molecules in the first solvation shell make a dominant contribution to the excited-state dynamics of BChl in condensed phases.20 In this contribution, we consider the structural origin of the vibrational coherence from BChl in B777 and B820, which are derived from the purple-bacterial LH1 light-harvesting complex. The recent low-resolution X-ray crystal structure obtained by Isaacs, Cogdell, and co-workers23 shows that LH1 assembles
10.1021/jp077103p CCC: $40.75 © 2008 American Chemical Society Published on Web 01/09/2008
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Figure 1. The Hu and Schulten32 model for the B820 subunit in LH1 from Rhodobacter sphaeroides, showing the pair of BChl macrocycles and the transmembrane R helices in ribbon and surface renderings.
around the photosynthetic reaction center as a ring of 15 identical subunits called B820, each of which contains two transmembrane R helices and a pair of BChl macrocycles. Each R helix binds a single BChl using the side chain of a histidine residue as an axial ligand to the MgII ion.24,25 The LH1 and B820 systems exist in a dynamic equilibrium that is shifted to favor B820 by the addition of a nonionic detergent.26,27 Further addition of detergent causes a reversible dissociation of the B820 subunits to form single-chromophore/single-polypeptide systems called B777.24,26-29 Van Grondelle, Vo¨lker, and their colleagues have shown that the B777 systems consist exclusively of proteinbound BChl.30,31 An atomic-resolution model for an LH1 16-mer complex of B820 was obtained using molecular dynamics techniques by Hu and Schulten.32 As shown in Figure 1, the two BChl’s in the B820 subunit form a face-to-face van der Waals complex in between the two R helices, producing a structure that resembles in several aspects the primary electron donor P in the photosynthetic reaction center.33,34 Van Grondelle and coworkers assign the near-infrared electronic absorption transitions of the B820 subunits to π f π* transitions of BChl pairs in the strong electronic coupling limit.35-42 We discuss here the results of a series of femtosecond pumpprobe, dynamic-absorption experiments performed with preparations of B820 and B777 that examine the rapidly damped vibrational coherence that is driven by resonant excitation of the lowest π f π* transition. The results show that the BChl macrocycles in B777 are exposed to interactions with the surrounding detergent that give rise to modulation features that resemble those observed previously from BChl in acetone solution.20 Because the BChl’s in B820 are sterically protected from first-shell interactions with the detergent, however, the rapidly damped vibrational coherence from B820 is much weaker in intensity and consists of narrower line shapes that are assigned to interactions with the adjacent polypeptide and to the BChl-BChl intrapair mode. These findings strongly support the proposal that the dominant contributions to the optical and/or Marcus charge-transfer reorganization energies of BChl in proteins arise from van der Waals interactions rather than from intrachromophore modes. 2. Experimental Section 2.1. Sample Preparation. Chromatophores from Rhodospirillum rubrum G9 were isolated as described previously34 using
Shelly et al. the procedures described by Loach and co-workers.26,43 After resuspension and ultracentrifugation in 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Research Organics)-NaOH, pH 7.5, the chromatophores were stored as pellets at -20 °C for up to 1 week prior to further use. The B820 subunit was obtained from the chromatophores using the procedure described by Miller et al.26 in the presence of 0.75% (w/v) n-octyl β-D-glucopyranoside (βOG, Aldrich). B777 was prepared using the procedure of Visschers et al.36 using 3.0% (w/v) n-octyl-rac-2,3-dipropylsulfoxide (ODPS, Bachem). In both cases, the detergent was dissolved in the 100 mM HEPES-NaOH buffer solution at pH 7.5. The dissociation of the LH1 light-harvesting complex into the B820 or B777 subunits upon addition of detergent was monitored spectrophotometrically by observing the decay of the 873 nm absorption peak from LH1 (B873) and the formation of the peaks at 820 nm for B820 and at 777 nm for B777. In our preparations, the B873 h B820 h B777 equilibrium strongly favors B777 in the presence of ODPS, so it is possible to obtain a B777 preparation that is essentially free of B820 at a fairly low detergent concentration. B820 is maintained over a fairly wide βOG concentration range; it was not possible to obtain a B777 solution that was judged to be free of B820 except at very high βOG concentrations (>5%) that were considered less suitable for use in the femtosecond pump-probe experiments because of their light-scattering properties and high viscosity. For use in the femtosecond pump-probe experiments, the B820 and B777 preparations were diluted in the βOG or ODPS detergent solution to obtain an absorbance of 0.5-0.7/mm pathlength at the center of the laser spectrum, as detailed below, after passing through a 0.22 µm microfilter. The samples were held in the femtosecond pump-probe spectrometer at room temperature (22 °C) in a fused-silica flow cuvette (1 mm path length). A 10 mL reservoir of sample was pumped through the cuvette at 6 mL/min by a peristaltic pump. The sample’s absorption spectrum was monitored during the experiment for changes arising from photochemistry or permanent photobleaching. 2.2. Absorption and Fluorescence Spectra. Absorption spectra were obtained at 22 °C with a Hitachi U-2000 spectrophotometer (2 nm bandpass). Fluorescence spectra were acquired at 22 °C with a Hitachi F-4500 spectrofluorimeter (5 nm bandpass for the excitation and emission monochromators). As presented as a function of wavenumber, the fluorescence intensities are multiplied by the square of the wavelength in order to compensate for the fixed (in wavelength units) spectral bandpass of the emission spectrometer.44,45 2.3. Femtosecond Spectroscopy. Studies of vibrational coherence were performed using the dynamic-absorption technique46-49 with a pump-probe spectrometer consisting of a self-mode-locked Ti:sapphire oscillator, a prism-pair pulse compressor, and a rapid-scanning, modified Mach-Zehnder interferometer. The present experiments employ ∼60 fs pulses and a fairly narrow transmitted probe bandwidth, which corresponds to the approach used by Champion and co-workers in studies of low-frequency vibrational coherence in heme proteins.50-52 Our previous work on B820 was performed with very short pulses (18 fs) and predominantly detected relatively high-frequency modes from the BChl macrocycle.34 The apparatus employed in these experiments was as described in our recent study of the rapidly damped vibrational coherence in BChl solutions.20 The light source is a Coherent Mira 900F Ti:sapphire oscillator equipped with Coherent’s X-wave broad-tuning-range cavity optics; the oscillator is
Intermolecular Vibrational Coherence in B777 and B820
Figure 2. Qy-band region of the continuous-wave absorption and fluorescence spectra from B777 at room temperature (22 °C), plotted as the dipole strength, A/ν and F/ν3, respectively, and normalized to unit area. The Ti:sapphire oscillator’s output spectrum (dashed curve), as tuned in the experiments with B777, is superimposed with arbitrary scaling. The shaded region marks the 4 nm bandpass of transmitted probe light that was selected by the monochromator and passed to the detector in the femtosecond pump-probe experiments.
pumped by a Coherent Verdi pump laser (5 W version). Extracavity group-delay precompensation employs a doublepassed pair of SF10 Brewster-angled prisms; the separation between the prisms is adjusted to minimize the width of the zero-background autocorrelation function, as measured at the position of the sample. For the experiments reported in this paper, the autocorrelation width was 90 fs; the spectral bandwidth was 11.5 nm. The pump-probe delay is scanned continuously by a galvanometer-driven retroreflector (ClarkMXR, ODL-150). The pump beam’s amplitude is modulated at 100 kHz by a photoelastic modulator (Hinds Instruments, PEM-90), which is set up as a λ/2 retarder at the center of the laser spectrum, followed by a calcite polarizer. At the sample’s position, the incident planes of polarization of the pump and probe beams are oriented 45° apart; after passing through the sample, the probe beam is analyzed by a calcite polarizer oriented at 90° relative to the pump beam’s plane of polarization. An amplified photodiode (Thorlabs PDA55) and a lock-in amplifier (SRS, SR850) referenced to the pump-modulation frequency are used to detect the pump-probe signal from a 2 nm bandpass of the probe beam, which is selected by an ActonResearch SP-150 monochromator. A photomultiplier (1P28, in a Clark-MXR housing) and a lock-in amplifier (Femto, LIAMV-200H) are used to detect the autocorrelation signal. The pump-probe and autocorrelation signals are recorded and averaged simultaneously by a sample-and-hold amplifier and digitizer system, as described previously.53 3. Results 3.1. Vibrational Coherence in B777. Figure 2 shows the Qy-band absorption and fluorescence spectra from preparations of B777 in ODPS at 22 °C. The spectra are plotted as relative dipole strengths44,54,55 as a function of wavenumber (ν), A/ν and F/ν3, respectively. Also shown in Figure 2 is the output spectrum from the Ti:sapphire oscillator, as it was tuned in the femtosecond dynamic-absorption experiments with B777. The shaded region marks the 4 nm section of the transmitted probe spectrum that was passed by the monochromator to the photodiode in the pump-probe spectrometer. These conditions favor detection of ground-state wave packet motion launched by the resonant impulsive stimulated Raman scattering mechanism.53,56,57 The contribution to the pump-probe signal from excited-state wave
J. Phys. Chem. B, Vol. 112, No. 4, 2008 1301
Figure 3. Femtosecond pump-probe, dynamic-absorption transient from B777. The inset shows a magnified view of the oscillatory portion of the signal. The ordinate is normalized with respect to the pumpinduced change in transmission observed at the end of the record.
packet motion was minimized by tuning the detected bandpass to the blue of the onset of the stimulated-emission spectrum. The dynamic-absorption transient obtained from B777 under these experimental conditions is shown in Figure 3. Following an intense spike58-60 near the zero of time, the transient exhibits a short series of damped oscillations marked by three relatively strong recurrences prior to the 600 fs delay point. A much weaker and more slowly damped set of oscillations is observed out to at least the 8 ps delay point. The signal strongly resembles the signals from BChl in pyridine or acetone.20 The rapidly damped, sub-picosecond part of the vibrational coherence from B777 was obtained from the dynamic-absorption transient shown in Figure 3 using the methods we described previously.20 To avoid contributions from nonresonant background signals,60 the signal prior to the 150 fs delay point was truncated. The oscillatory part of the signal was then isolated as the residual from a triple-exponential decay function (∑i aie-t/τi, with a1 ) 0.34, τ1 ) 190 fs, a2 ) 0.22, τ2 ) 1.1 ps, a3 ) 0.48, and τ1 ) 8.1 ps and with the amplitudes (ai) normalized to unity) fitted to the signal from the 150 fs point through to the end of the record at 12 ps. The oscillatory residual was then fit to a multicomponent time-domain model that describes asymmetric, inhomogeneously broadened line shapes in the frequency domain. We previously showed that this model provides a good description in the time domain of the rapidly damped vibrational coherence from BChl in polar solution; the reader is directed to that work for a detailed discussion of the model.20 Each modulation component with phase φi is defined by an integral over a distribution of damped cosinusoids
I(t) )
∑i ∫0 dω Li(ω, Ai, ω0i, ∆ωi, Fi) cos(ωt - φi)e-t/γ ∞
(1)
which was implemented in a nonlinear regression program as a sum of discrete cosinusoids of frequency ω. The models that follow for B877 and B820 set the distributions (Li(ω)) as lognormal (asymmetric Gaussian) line shapes, which are parametrized by the area (Ai), center frequency (ω0i), width (∆ωi), and asymmetry (Fi).61 In the time domain, the damping of each component arises from a convolution in the frequency domain of the Lorentzian line shape corresponding to the intrinsic damping time (γ), here fixed arbitrarily to 1.5 ps for all of the components so as to be in the range observed for the slowly damped features that are assigned to intramolecular (skeletal) modes from the BChl macrocycle,19,20 with the asymmetric Gaussian line shape defined by the width (∆ωi) of the
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Shelly et al.
TABLE 1: Model Parameters for the Rapidly Damped Vibrational Coherence Observed in B777 and B820 component 1
2
3
sum a
parametera
B777b
B820c
ω0 ∆ω F φ A ω0 ∆ω F φ A ω0 ∆ω F φ A ω ∑i A i
147 cm-1 79 cm-1 -1.3 1.51 rad 0.138 184 cm-1 202 cm-1 1.1 1.56 rad 1.37
28 cm-1 19 cm-1 1.4 1.30 rad 0.0197 46 cm-1 74 cm-1 -1.8 0.54 rad 0.0588 102 cm-1 31 cm-1 -1.5 -0.035 rad 0.0055 41 cm-1 0.084
191 cm-1 1.508
See eqs 1 and 2 and the text. b See Figure 4. c See Figure 7.
distribution (Li(ω)). Note that this particular choice of γ has little effect on the widths (∆ωi) returned by the fitting procedure because the observed line shapes are relatively broad; a Raman echo experiment would be required to obtain a discrete measurement of the homogeneous line broadening.62,63 The signs of the tabulated values (see Table 1) for the asymmetries (Fi) indicate whether the broader side of the fitted line shapes is directed to high (Fi > 1) or to low frequency (Fi < 1); because the models employ the minimum number of components that yields a satisfactory description of the signal, the line shapes and asymmetries might arise from a superposition of unresolved components. The tabulated areas (Ai) report the integral from zero frequency, Ai ) ∫∞0 dω Li(ω); they are normalized with respect to the intensity of the ground-state depletion part of the signal (see the ordinate scaling for Figure 3) and to the integrated area of the vibrational coherence observed with BChl in pyridine solvent.20 The mean frequency (〈ω〉) was calculated from the sum of the line shapes, M(ω) ) ∑i Li(ω), using the normalized mean-value relation,
∫0∞dω M(ω)ω 〈ω〉 ) ∞ ∫0 dω M(ω)
(2)
Figure 4 shows that a good description of the rapidly damped vibrational coherence in B777 is obtained with two components (Li(ω)) with a 10:1 area ratio: a stronger component centered at 184 cm-1 with a width of 202 cm-1 and a weaker component centered at 147 cm-1 with a width of 79 cm-1. This model (and the B820 model that follows) is robust with respect to the choice of the starting parameters used in the nonlinear regression procedure; the final fit parameters have uncertainties on the order of 10%, but the frequencies are known to a confidence range of (5 cm-1. 3.2. Vibrational Coherence in B820. Figure 5 shows the Qy-band absorption and fluorescence dipole-strength spectra from preparations of B820 in OG at 22 °C. Figure 5 also shows how the output spectrum from the Ti:sapphire oscillator was tuned in the experiments with B820 to the lower-energy side of the peak of the lower-energy exciton band. The shaded region marks the 4 nm bandpass of the transmitted probe spectrum that was selected by the monochromator in these experiments. These pump and probe spectra favor detection of ground-state vibrational coherence; as with B777, overlap with the stimulated-
Figure 4. Expanded view of the rapidly damped oscillation observed in the dynamic-absorption transient from B777 (see Figure 3) superimposed with a model defined by the sum of two independent lognormal distributions (Li(ω)) of damped cosinusoids, each defined by eq 1. The scaling of the ordinate is relative to the magnitude of the pump-probe ground-state depletion signal, as shown in Figure 3. Bottom: Plots of Li(ω) for the two components observed in B777 and their sum (M(ω)) (thick curve). The model parameters are provided in Table 1.
Figure 5. Qy-band region of the continuous-wave absorption and fluorescence spectra from B820 at room temperature (22 °C), plotted as the dipole strength, A/ν and F/ν3, respectively, and normalized to unit area. The Ti:sapphire oscillator’s output spectrum (dashed curve), as tuned in the experiments with B820, is superimposed with arbitrary scaling. The shaded region marks the 4 nm bandpass of transmitted probe light that was selected by the monochromator and passed to the detector in the femtosecond dynamic-absorption experiments.
emission spectrum is minimized to minimize the contribution of excited-state vibrational coherence to the pump-probe signal, and in addition, we avoid excitation of the upper exciton band to avoid detection of oscillations arising from electronic coherence driven by simultaneous excitation of the two exciton bands.64 The dynamic-absorption transient obtained from B820 under these experimental conditions is shown in Figure 6; it is essentially identical in appearance to the signal observed previously under comparable conditions by Kumble et al.65 Following the spike near the zero of time, the transient exhibits a series of rapidly damped oscillations, with two relatively strong recurrences prior to the 2 ps delay point. The intensity of this part of the vibrational coherence in B820 is about 20 times less intense and lasts about 4 times as long as the comparable section of the signal in B777. As in B777, a slowly damped series of
Intermolecular Vibrational Coherence in B777 and B820
J. Phys. Chem. B, Vol. 112, No. 4, 2008 1303 weaker, higher-frequency oscillations in the signal over the 800-2000 fs region. The three line shapes (Li(ω)) span only the 0-120 cm-1 region of the spectrum, with the two strongest components centered at 28 and 46 cm-1. The weakest component of the three (7% of the area) is centered at 102 cm-1; its line shape is intermediate in breadth (∆ω ) 31 cm-1) between those of the other two components. The 28 cm-1 component exhibits the narrowest line shape (∆ω ) 19 cm-1) and accounts for about 23% of the area; the 46 cm-1 component exhibits a very broad line shape (∆ω ) 74 cm-1) extending to 0 cm-1 and accounts for 70% of the area in the spectrum.
Figure 6. Femtosecond pump-probe, dynamic-absorption transient from B820. The inset shows a magnified view of the oscillatory portion of the signal. The ordinate is normalized with respect to the pumpinduced change in transmission observed at the end of the record.
Figure 7. Expanded view of the rapidly damped oscillation observed in the dynamic-absorption transient from B820 (see Figure 6) superimposed with a model defined by the sum of three independent lognormal distributions (L(ω)) of damped cosinusoids, each defined by eq 1. The scaling of the ordinate is relative to the magnitude of the pump-probe ground-state depletion signal, as shown in Figure 6. Bottom: Plots of L(ω) for the three components observed in B820 and their sum (M(ω)) (thick curve). The model parameters are provided in Table 1.
comparatively weak oscillations lasting at least to the 8 ps time region is observed in the signal from B820; this part of the vibrational coherence is comparably intense in the B820 and B777 signals and is qualitatively comparable (based on Fourier transform spectra, not shown) to that observed previously from BChl in pyridine solution.19 The rapidly damped, sub-picosecond part of the vibrational coherence from B820 was obtained from the dynamic-absorption transient shown in Figure 6 using the same approach as that used for the B777 signal. Again, the signal prior to the 150 fs delay point was truncated. The oscillatory part of the signal was obtained as the residual from a double-exponential decay function (∑i aie-t/τi, with a1 ) 0.8, τ1 ) 100 fs, a2 ) 0.2, and τ2 ) 15 ps and with the amplitudes (ai) normalized to unity) fitted to the signal from the 150 fs point through to the end of the record at 12 ps. Figure 7 shows that the oscillatory part of the signal from B820 is well described by the sum of three inhomogeneously broadened components (see eq 1). The model describes only the rapidly damped part of the signal; it does not describe the slowly damped vibrational coherence, which contributes the
4. Discussion The results show that the low-frequency vibrational coherence observed with resonant excitation of the Qy absorption band of the B777 and B820 light-harvesting proteins contains relatively strong, rapidly damped modulation components that are mostly confined to the
