Article pubs.acs.org/JPCB
Spectroscopic Studies of Cryptophyte Light Harvesting Proteins: Vibrations and Coherent Oscillations Paul C. Arpin,† Daniel B. Turner,†,¶ Scott D. McClure,† Chanelle C. Jumper,† Tihana Mirkovic,† J. Reddy Challa,‡ Joohyun Lee,‡ Chang Ying Teng,§ Beverley R. Green,§ Krystyna E. Wilk,∥ Paul M. G. Curmi,∥ Kerstin Hoef-Emden,⊥ David W. McCamant,*,‡ and Gregory D. Scholes*,†,# †
Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada Department of Chemistry, University of Rochester, Rochester, New York 14627, United States § Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ∥ School of Physics, The University of New South Wales, Sydney, New South Wales 2052, Australia ⊥ Botanical Institute, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany # Department of Chemistry, Princeton University, Washington Road, Princeton, New Jersey 08544, United States ‡
ABSTRACT: The first step of photosynthesis is the absorption of light by antenna complexes. Recent studies of light-harvesting complexes using two-dimensional electronic spectroscopy have revealed interesting coherent oscillations. Some contributions to those coherences are assigned to electronic coherence and therefore have implications for theories of energy transfer. To assign these femtosecond data and to gain insight into the interplay among electronic and vibrational resonances, we need detailed information on vibrations and coherences in the excited electronic state compared to the ground electronic state. Here, we used broad-band transient absorption and femtosecond stimulated Raman spectroscopies to record ground- and excited-state coherences in four related photosynthetic proteins: PC577 from Hemiselmis pacif ica CCMP706, PC612 from Hemiselmis virescens CCAC 1635 B, PC630 from Chroomonas CCAC 1627 B (marine), and PC645 from Chroomonas mesostigmatica CCMP269. Two of those proteins (PC630 and PC645) have strong electronic coupling while the other two proteins (PC577 and PC612) have weak electronic coupling between the chromophores. We report vibrational spectra for the ground and excited electronic states of these complexes as well as an analysis of coherent oscillations observed in the broad-band transient absorption data.
1. INTRODUCTION Photosynthesis uses proteins called light-harvesting complexes to amplify the spectral and spatial capture of light.1,2 After light is absorbed by an antenna complex, the electronic excitation is transferred through space to a reaction center to drive charge separation. Here, we study spectroscopic properties of lightharvesting complexes known as phycobiliproteins that were isolated from cryptophyte algae.3−5 In these phycobiliproteins, eight chromophores are covalently bound to a protein matrix. The chromophores are straight-chain tetrapyrrole molecules, known as bilins. Transport of excitation (energy transfer and migration) occurs on femtosecond to picosecond time scales within each phycobiliprotein. Femtosecond spectroscopy has contributed extensively to the understanding of energy-transfer dynamics in light-harvesting antenna complexes. In traditional transient absorption spectroscopy, a narrow-band laser pulse excites a limited number of transitions in the sample, and a broad-band probe pulse measures the differential absorption spectrum induced by pumping the sample. Measurements of the transient differential absorption have led to detailed models of energy-transfer pathways and time scales in phycobiliproteins.6 A more sophisticated method known as two-dimensional electronic © 2015 American Chemical Society
spectroscopy (2D ES) maps the dynamics as a function of both excitation and emission frequencies.7,8 Consequently, broadband excitation pulses can be used without sacrificing the spectral resolution of the excitation process. In addition to initiating population dynamics, broad-band laser pulses can excite coherent superpositions of quantum-mechanical states, which appear as oscillations in the measured dynamics.9−13 2D ES measurements are helpful because they contain all of the information available in a four-wave mixing experiment.14 Yet, this power is also a weakness because 2D ES does not readily isolate single physical effects. Sophisticated models are required to understand the spectra, and there is not yet a consensus on interpreting many of the subtle features. For example, 2D ES has identified oscillatory dynamics in photosynthetic complexes.15,16 These experiments have inspired discussion on what role coherence could play in energy transfer.12,17−23 However, there is some debate regarding how to interpret the oscillations:24−31 Are they due to simple intramolecular vibrations? After all, oscillatory dynamics in Received: May 17, 2015 Revised: July 18, 2015 Published: July 18, 2015 10025
DOI: 10.1021/acs.jpcb.5b04704 J. Phys. Chem. B 2015, 119, 10025−10034
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Figure 1. Four species of cryptophyte algae and their primary light-harvesting proteins known as phycobiliproteins. (a) Micrographs show that, compared to other cryptophytes, these four species have very similar physical properties including color and size. Cell lengths: CCMP269: 9.7 ± 0.6 μm, CCAC 1627 B: 10.3 ± 0.9 μm, CCAC 1635 B: 7.5 ± 0.7 μm, CCMP706: 7.0−8.5 μm (the latter according to Lane and Archibald38). (b) Visualizations of the X-ray crystal structures. Each protein contains eight covalently bound chromophores; red, blue, and yellow indicate, respectively, dihydrobiliverdin (DBV), phycocyanobilin (PCB), and mesobiliverdin (MBV). The main difference between the two proteins on the left from the two on the right is due to a ∼73° rotation of the two halves of the tetramer (colored green and black). PC645 and PC630 are “closed”, while PC612 and PC577 are “open”. The result is that the pair of DBVs is separated by about 0.5 nm in PC645 and PC630 while the pair of DBVs in PC612 and PC577 is separated by about 2 nm.
have established that the quaternary structure is a tetrameric α1βα2β association of the subunits.39 One difference among the proteins is the replacement of chromophores to tune the absorption spectrum for the given species; for example, PC645 and PC630 have mesobiliverdins (MBVs) whereas PC577 and PC612 do not.4 Far more importantly for the present study is that the proteins have two distinct conformations:40 the “closed” form of PC645 and PC630, and the “open” form of PC612 and PC577. In the open form, a single aspartic acid inserted near the bilin-binding site of the α-subunit disrupts the hydrogen-bonding network that prevents these proteins from adopting the closed forms. In the closed form, the two central dihydrobiliverdins (DBVs) are about 0.5 nm apart, while in the open form, the DBVs are about 2 nm apart. This enables us to compare spectroscopic data systematically for the cases where there is a central molecular excitonic dimer and for the cases where that electronic coupling is small.
molecular condensed-phase systems have for decades been attributed to the coherent evolution of intramolecular vibrations.32−34 It is challenging for 2D ES to distinguish signatures of coherent molecular vibrations from coherent superpositions of excitonic states.27,29,30,35−37 Moreover, in molecular aggregates, the electronic and vibrational transitions can mix, and it remains an open question of how to interpret and assign the oscillatory dynamics observed in femtosecond spectroscopy experiments.27,30,37 Resolving this issue is crucial for modeling and interpreting the results. Here, we provide a step toward addressing the issues described above. We used two pump−probe spectroscopy experiments with interpretations that are more intuitive than 2D ES: stimulated resonance Raman spectroscopy to isolate oscillatory dynamics in the electronic ground state and broadband transient absorption spectroscopy to isolate oscillatory dynamics in the electronic excited state. We are thus able to measure separately excited-state oscillations and ground-state oscillations (the vibrational spectrum). We assay four related phycobiliproteins. Specifically, we measure phycocyanin 577 (PC577) extracted from Hemiselmis pacif ica CCMP706, phycocyanin 612 (PC612) extracted from Hemiselmis virescens CCAC 1635 B, phycocyanin 630 (PC630) extracted from Chroomonas CCAC 1627 B (marine), and phycocyanin 645 (PC645) extracted from Chroomonas mesostigmatica CCMP269. In Figure 1, we show micrographs of representative algae and the X-ray crystal structures of the phycobiliproteins. Each protein has slight differences in structure and composition that alter the electronic energy levels and vibrational modes of the chromophores, which in turn tunes the energy-transfer dynamics. Phycobiliproteins are tetramers having α1βα2β structure. Crystallography and gene sequencing
2. EXPERIMENTAL METHODS 2.1. Transient Absorption Experiments. We detailed the transient absorption spectrometer in a related manuscript.41 Briefly, a commercial 5 kHz Ti:sapphire laser amplifier pumped a home-built noncollinear optical parametric amplifier (NOPA) to produce broad-band visible pulses.35,42 We tuned the peak of the output spectrum to 580 nm to strongly excite the peak absorption of the DBV chromophores present in all four samples as shown in Figure 2. A weak tail in the laser spectrum extending to about 750 nm probed the sample response beyond the absorption and fluorescence maxima of the PCB chromophores. Grating and prism compressors compensated the second-order and third-order dispersion, leaving a ∼45 fs instrument response function dominated primarily by residual fourth-order dispersion which cannot be compensated with our 10026
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the presented analysis, we assume four parallel, single exponential decay components in the fitting. We have verified that the values of the fastest decay components are sensitive to the number of assumed decay components; however, we focus our conclusions on the relative decay components between proteins which are insensitive to the choice of model. 2.2. Two-Dimensional Electronic Spectroscopy. The reported 2D ES was collected in a four wave mixing apparatus described in detail previously.35,42 Briefly, a commercial Ti:sapphire amplifier pumped a home-built noncolinear optical parametric amplifier (NOPA) to generate broad-band pulses in the visible.44 For the 2D ES measurements, the NOPA was tuned to a central wavelength of 580 nm and a full width at half maximum (fwhm) bandwidth of 70 nm. The pulses were compressed with a combination of a grating compressor and a prism compressor to a duration of 11 fs as measured with transient grating frequency resolved optical gating (TG-FROG) at the sample position.45 The NOPA beam was split into four identical beams arranged in the BOXCARS geometry with a single transmissive diffractive optic. The four beams followed a common path from the diffractive optic to the sample position to maintain passive phase stability. Delays between pulses were controlled with the insertion or removal of glass with wedged prism pairs. The first two beams acted as the pump and excited the sample; the third acted as the probe and initiated the radiation of a signal beam in a background-free direction. The final beam, the local oscillator, was aligned collinear with the signal, and the two were heterodyne detected by the spectrometer. Spectral interferometry enabled extraction of the complex signal electric field.46 The waiting time delay between the excitation and the final pulse, which corresponds to the delay time in the transient absorption measurements, was varied from 0 to 400 fs in 5 fs steps. At each waiting time, the delay between the two excitation pulses was varied from +45 to −45 fs in 0.15 fs steps, and a spectrum was recorded at each step. A Fourier transform of the complex signal extracted from spectral interferometry at each detection wavelength along the excitation pulse delay axis produced a 2D map of the signal at each waiting time as a function of both the excitation wavelength and the emission wavelength. 2.2. FSRS. The femtosecond stimulated Raman spectroscopy (FSRS) setup has been described elsewhere.47−50 Briefly, the 3 ps duration Raman pump and a 100 fs, broad-band probe are concurrently crossed in the sample to produce ground-state stimulated Raman spectra. The Raman pump pulse at 596.5 nm was generated by propagating a narrow-band pulse at 400 nm from a second harmonic bandwidth compressor51 into H2 Raman shifter.52 The probe pulse was generated by focusing the 800 nm fundamental into sapphire. The Raman pump pulse is not a photoinitiating, or actinic, pump. The energy of the Raman pump pulse was chosen53 to avoid severe depletion of ground-state population and was set to 25 or 30 nJ. The pump was chopped at 500 Hz, and the transmitted probe beam was dispersed by a grating spectrograph and was detected via the second-order diffraction collected on a CCD. For higher signalto-noise ratio, the scanning multichannel technique (SMT) method was used.54 The 1 mm sample cuvette was temperature-controlled to 5 °C and was continuously translated at 2 mm/s. For baseline correction, the resulting Raman spectrum was subtracted by a scaled transient absorption background spectrum, obtained by applying a time delay between the Raman pump and the probe. Any residual baseline was
Figure 2. Absorption spectra (black) of PC645, PC630, PC612, and PC577. The laser spectrum used both to excite and to probe the dynamics is shown in gray. The peak of the excitation spectrum is tuned to 580 nm to strongly excite the DBV chromophores present in each of the four phycobiliproteins studied in the present work. The laser spectrum extends to almost 750 nm capturing excited-state absorption signals outside the linear absorption spectrum of the complexes.
current compression scheme and temporal smearing. A transient-grating frequency-resolved optical gating measurement reveals that the pulse was 740 cm−1 with difficulty. The comparison shows consistency in the overlapping spectral region between the two measurements. We do not identify any distinct peaks in the transient absorption measurement that are not present in the FSRS, indicating that the oscillations observed in the nonlinear spectra can be attributed primarily to coherent nuclear dynamics. One of the most visible differences between the TA measurement and the FSRS measurement is the location of the peak near the 500 cm−1 cluster in PC645 and PC630. The peak in the TA spectrum has an apparent red shift relative to the measured FSRS. As we will discuss later, the FSRS exclusively identifies ground-state vibrational frequencies where the transient absorption identifies predominantly electronic excited state vibrational frequencies. This may account for some shift in the peak frequency between the two measurements. However, this particular case is likely an artifact of the measurement. FSRS is an incoherent technique and measures the sum of the intensity of each mode where transient absorption is coherent and consequently sensitive to the relative phase of the overlapping modes which can artificially shift the apparent peak of a cluster of closely spaced oscillation frequencies.57 10029
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Figure 5. Broad-band transient absorption measurements of the four cryptophyte proteins. In the first column, representative raw transient spectra are plotted for waiting time delays from 0 to 1.8 ps. Positive (red) features represent increased signal at a particular wavelength after excitation (ground-state bleach or stimulated emission), and negative (blue) features correspond to additional absorption in the sample after excitation (excited-state absorption). The middle column shows 2-D oscillation maps obtained by subtracting the fitted population dynamics from the transient absorption spectra. In the final column, the wavelength dependent coherent maps are plotted. Bright features indicate high amplitude oscillations at the given frequency in the time trace taken at that probe wavelength. PC577 and PC612 show a minimum in the amplitude of the oscillations at a probe wavelength near 630 nm for each of the dominant oscillation frequencies.
accompanied by abrupt phase changes. The node and phase changes occur at the detection wavelength corresponding to the minimum of the electronic potential well on which the wave packet is oscillating.33,58,65−69 Each amplitude plot contains a node at the wavelength corresponding to the fluorescence maximum of each phycobiliprotein, signifying that the oscillations arise from vibrational wave packets oscillating on the excited electronic state of the low-energy chromophores. Despite complications that might arise when considering multichromophoric systems with multiple nuclear degrees of freedom, the semiclassical model of vibrational wave packets seems to hold in the phycobiliproteins. Several low-amplitude peaks and nodes appear at shorter detection wavelengths. These features may be due to vibrations on the ground electronic state of the PCB chromophores, or they may be due to vibrations on the ground or excited electronic states of other chromophores. In some cases, an additional node is visible closer to 700 nm. This is near the peak of the excited-state absorption on the red side of the spectrum and is consistent with the excitation of vibrational wavepackets oscillating on the excited electronic state to a higher lying electronic state. We cannot assign this excited-state absorption, but its presence provides additional evidence that the dominant contribution to the oscillations is due to oscillations on the excited electronic state.41 Another detail that supports the assignment of excited-state vibrations is that a significant portion of the oscillations appears as excitedstate absorption signals located beyond the red side of the
Table 2. Oscillation Frequencies of the Four Proteins Obtained from Gaussian Fits to the Probe Wavelength Integrated Transient Absorption Oscillation Spectraa PC645
PC630
PC612
PC577
wavenumbers (cm−1)
wavenumbers (cm−1)
wavenumbers (cm−1)
wavenumbers (cm−1)
199 276.0 350 478 657 852 1100
± ± ± ± ± ± ±
3 0.8 4 2 4 5 30
200 272 351 470 600 657 843
± ± ± ± ± ± ±
2 2 3 4 20 4 3
202 261.8 372 465 516 660.4 807
± ± ± ± ± ± ±
2 0.7 3 2 4 0.7 8
208 264 367 468 509 660.5 745 920
± ± ± ± ± ± ± ±
2 1 4 4 7 0.7 7 20
a Values represent the average and standard deviation of fits to five independent data sets. The values are reported in wavenumber units (the oscillation frequency divided by the speed of light).
have studied the detection-wavelength dependence of the oscillations,58−64 as we discussed recently.41 Maps of the oscillation amplitude and phase as a function of emission frequency can further discern ground-state vibrational wave packets from excited-state vibrational wave packets. We therefore inspect amplitude (green) and phase (red) plots of each frequency as a function of detection wavelength; we present the results for three oscillations in Figure 7. In Figure 7, the 270 and 470 cm−1 amplitude and phase plots are representative examples of the character of almost all the modes. The amplitude traces for all four proteins have nodes 10030
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Figure 6. Excited-state and ground-state wavepackets produce specific spectroscopic signals. Broad-band transient absorption spectroscopy (red) identifies the energies of excited-state wave packets, which include vibrational contributions (right red), while stimulated resonance Raman (gray) identifies the energies of ground-state vibrational wave packets that are coupled to the electronic transitions (right, gray). The stimulated resonance Raman spectra show that the chromophores of the four phycobiliproteins have very similar ground-state vibrational modes. These values match those previously published for PC645 and PC577. Broad-band transient absorption spectra, meanwhile, primarily reveal excited-state vibrational modes. The four phycobiliproteins share many excited-state vibrational frequencies.
Figure 7. Plots of the amplitude and phase of specified oscillations from transient absorption spectra as a function of detection wavelength. Excitedstate vibrational wave packets oscillate about the excited-state electronic potential minimum and are therefore characterized by a node in the oscillation amplitude and a phase change at the wavelength of the fluorescence maxima. The vertical dashed lines indicate the fluorescence maxima. In the four proteins, nearly all of the frequencies have nodes and phase shifts consistent with the signatures for excited-state vibrational modes. Previous work on PC645 identified that the 660 cm−1 mode showed unusual characteristics, and therefore, we carefully study it in the bottom panels. The 660 cm−1 amplitude and phase lineouts for PC612 and PC577 indicate an excited-state vibrational wave packet. The 660 cm−1 amplitude and phase lineouts of PC645 and PC630, however, lack the amplitude node. This feature has not been previously observed.
linear absorption spectrum. Excited-state absorption signals cannot contain ground-state oscillations. The 660 cm−1 mode is of particular interest. PC612 and PC577 follow the excited-state vibrational model with an amplitude node and abrupt phase change. However, PC645 and PC630 do not have such a distinct node in the amplitude plot. The difference is statistically significant but does not have a ready explanation. The effect is not due to an interference effect by the MBVs, for example, oscillations centered at slightly different detection wavelengths, because the other modes in PC645 and PC630 do not show the same signature. The most significant challenge to interpretation of the present data is establishing a correlation between excitation frequency and response frequency. As with population dynamics, the
coherence dynamics contain overlapping contributions from oscillations excited in each of the bilins present in the protein. Interestingly, the 660 cm−1 frequency that yields the indistinct node is similar to an electronic frequency gap (of ∼700 cm−1) in the system that has previously been suggested to show coherent electronic oscillations in 2D ES studies of closed structures, particularly PC645, when probed in the highenergy side of the absorption spectrum.11,35,42 We compare a time trace from a transient absorption measurement of PC645 to an analogous measurement obtained from the 2D spectra on PC645 in Figure 8. Two-dimensional electronic spectroscopy resolves both the excitation wavelength and the emission wavelength that contribute to the signal radiated by the sample at a particular time delay. Transient absorption spectroscopy only resolves whether there is a signal at a particular emission 10031
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Figure 9. Comparison of population dynamics in open and closed structure phycobiliproteins. Room-temperature decay associated spectra (DAS). (a) A closed-structure phycobiliprotein (PC645) and (b) an open-structure phycobiliprotein (PC577) after broad-band excitation. Gray shading indicates the uncertainty based on an independent analysis of five separate data sets. (c) Direct comparison of normalized time traces taken at a probe wavelength of 580 nm, overlapping the high-energy DBV absorption peak in the samples. Decay indicates downhill energy flow of the excitation.
according to global fits to data taken with parallel polarization between the pump and probe (data not shown). This suggests that the molecular excitons effectively distribute the excitation, in accord with the theoretical predictions of Huo and Coker.18 Figure 8. Relation between 2D ES and transient absorption (TA) spectroscopy of PC645. (top left) Integrating the 2D ES from previous work over the outlined area produces a value for each waiting time point. The scan range was limited to 400 fs. (top right) Selection from the full TA data set, which ranged from −0.5 to 5 ps. Shading indicates the integrated region to produce the trace below. (bottom) The traces indicate the mean of multiple 2D ES (black) and TA (red) measurements. Error bars for the 2D trace and the shaded region for the TA trace indicate the respective standard deviations. The traces agree quantitatively.
5. CONCLUSIONS We used femtosecond stimulated Raman and broad-band transient absorption spectroscopies to record, respectively, ground- and excited-state coherences in four related photosynthetic proteins: PC577 from Hemiselmis pacif ica CCMP706, PC612 from Hemiselmis virescens CCAC 1635 B, PC630 from Chroomonas CCAC 1627 B (marine), and PC645 from Chroomonas mesostigmatica CCMP269. Two of those proteins (PC630 and PC645) have strong electronic coupling, while the other two proteins (PC577 and PC612) have weak electronic coupling between the chromophores. We identified several oscillatory components in the transient absorption spectra of each of the four proteins that followed a simple prescription for nuclear oscillations on the excited electronic state. The lack of a node as a function of probe frequency for the 660 cm−1 component in two of these proteins prevents us from identifying the mode using the same prescription. This mode appears in all four proteins but shows the unique lack of a node only in the two closed proteins where strong electronic coupling is expected and is at a frequency previously identified in 2D electronic spectroscopy as a candidate for coherent oscillations because of strong electronic coupling. In the present work, we are unable to identify the nature of this oscillation, however, the Raman spectroscopy presented here clearly identifies a ground-state vibrational oscillation at the same frequency. This suggests that a full characterization of this mode will require a model that allows for coupling between the vibration and excitonic gap.27,30,70,71 We anticipate that future experiments can disentangle overlapping contributions to the oscillations by tuning the central frequency of the pump pulse to clearly identify the nature of this oscillation using transient absorption spectroscopy. In the course of this study, we found that energy-transfer dynamics is faster in the closed structure proteins PC645 and PC630 than in the open structures PC577 and PC612.
wavelength at the given waiting time regardless of what excitation frequency initiated the dynamics which contributes to the signal. To reconcile the two measurements, it is necessary to sum the 2D spectrum along the excitation axis to account for all of the possible contributions at that emission wavelength, which can be quantitatively shown with the projection slice theorem. The plotted time traces are taken at a single emission wavelength from the transient absorption measurement and are taken at the same emission wavelength from the 2D spectrum but are summed in the excitation wavelength dimension. The two time traces obtained on different instruments several months apart show quantitative agreement improving confidence in the reproducibility of the conclusions. Oscillations in the transient absorption data are well aligned with those at the notable cross peak in the 2D ES data. Additional insights into the excited-state dynamics can be gained by analyzing the kinetics of the transient absorption data. Here, we introduce our preliminary analysis, highlighting a comparison of the dynamics between one protein having a closed structure (PC645) and one with an open structure (PC577) in Figure 9. We focus our discussion on the fastest decay component which peaks near 580 nm in each protein, the absorption peak of the DBV chromophores. The interesting observation is that excitation energy transfer initiated by excitation on the blue side of the absorption spectra flows to absorption resonances on the red side of the spectra about twice as quickly in the closed structures compared to the open structures (440 ± 50 fs vs 870 ± 80 fs, respectively). Results are consistent across the two closed and the two open structures
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*E-mail: [email protected]. *E-mail: [email protected]. 10032
DOI: 10.1021/acs.jpcb.5b04704 J. Phys. Chem. B 2015, 119, 10025−10034
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Department of Chemistry, New York University, New York, NY 10003.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. P.C.A. and D.B.T. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the Natural Sciences and Engineering Research Council of Canada, DARPA (QuBE), and the United States Air Force Office of Scientific Research (FA9550-13-1-0005). J.R.C., J.L., and D.W.M. were supported by a United States National Science Foundation CAREER award, CHE-0845183, and D.W.M. was supported as an Alfred P. Sloan Research Fellow
(1) Blankenship, R. E. Molecular Mechanisms of Photosynthesis; Blackwell: Oxford, U.K., 2002. (2) Scholes, G. D.; Fleming, G. R.; Olaya-Castro, A.; van Grondelle, R. Lessons from Nature About Solar Light Harvesting. Nat. Chem. 2011, 3, 763−774. (3) Doust, A. B.; Wilk, K. E.; Curmi, P. M. G.; Scholes, G. D. The Photophysics of Cryptophyte Light-Harvesting. J. Photochem. Photobiol., A 2006, 184, 1−17. (4) Wedemayer, G. J.; Kidd, D. G.; Wemmer, D. E.; Glazer, A. N. Phycobilins of Cryptophycean Algae. J. Biol. Chem. 1992, 267, 7315− 7331. (5) Scholes, G. D.; Mirkovic, T.; Turner, D. B.; Fassioli, F.; Buchleitner, A. Solar Light Harvesting by Energy Transfer: From Ecology to Coherence. Energy Environ. Sci. 2012, 5, 9374−9393. (6) Marin, A.; Doust, A. B.; Scholes, G. D.; Wilk, K. E.; Curmi, P. M. G.; van Stokkum, I.; van Grondelle, R. Flow of Excitation Energy in the Cryptophyte Light-Harvesting Antenna Phycocyanin 645. Biophys. J. 2011, 101 (4), 1004−1013. (7) Jonas, D. M. Two-Dimensional Femtosecond Spectroscopy. Annu. Rev. Phys. Chem. 2003, 54, 425−463. (8) Brixner, T.; Stenger, J.; Vaswani, H. M.; Cho, M.; Blankenship, R. E.; Fleming, G. R. Two-Dimensional Spectroscopy of Electronic Couplings in Photosynthesis. Nature 2005, 434, 625−628. (9) Engel, G. S.; Calhoun, T. R.; Read, E. L.; Ahn, T.-K.; Mancal, T.; Cheng, Y.-C.; Blankenship, R. E.; Fleming, G. R. Evidence for Wavelike Energy Transfer through Quantum Coherence in Photosynthetic Systems. Nature 2007, 446 (7137), 782−6. (10) Panitchayangkoon, G.; Hayes, D.; Fransted, K. A.; Caram, J. R.; Harel, E.; Wen, J.; Blankenship, R. E.; Engel, G. S. Long-Lived Quantum Coherence in Photosynthetic Complexes at Physiological Temperature. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 12766−12770. (11) Collini, E.; Wong, C. Y.; Wilk, K. E.; Curmi, P. M. G.; Brumer, P.; Scholes, G. D. Coherently Wired Light-Harvesting in Photosynthetic Marine Algae at Ambient Temperature. Nature 2010, 463 (7281), 644−7. (12) Cheng, Y. C.; Fleming, G. R. Dynamics of Light Harvesting in Photosynthesis. Annu. Rev. Phys. Chem. 2009, 60, 241−262. (13) Ishizaki, A.; Calhoun, T. R.; Schlau-Cohen, G. S.; Fleming, G. R. Quantum Coherence and Its Interplay with Protein Environments in Photosynthetic Electronic Energy Transfer. Phys. Chem. Chem. Phys. 2010, 12, 7319−7337. (14) Mukamel, S. Principles of Nonlinear Optical Spectroscopy; Oxford University Press: New York, 1995. 10033
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Spectroscopy Study of the Alpha-Subunit of Phycoerythrocyanin and Phycocyanin from the Cyanobacterium Mastigocladus Laminosus. J. Raman Spectrosc. 1998, 29, 939−944. (56) Myers, A. B.; Mathies, R. A. Resonance Raman Intensities: A Probe of Excited-State Structure and Dynamics. In Biological Applications of Raman Spectrometry; Spiro, T. G., Ed.; Wiley: New York, 1987; Vol. 2, pp 1−57. (57) Braun, M.; Sobotta, C.; Dürr, R.; Pulvermacher, H.; Malkmus, S. Analysis of Wave Packet Motion in Frequency and Time Domain: Oxazine 1. J. Phys. Chem. A 2006, 110 (32), 9793−9800. (58) Jonas, D. M.; Fleming, G. R. Ultrafast Processes in Ultrafast Processes in Chemistry and Photobiology. In Vibrationally Abrupt Pulses in Pump-Probe Spectroscopy; El-Sayed, M. A., Tanaka, I., Molin, Y.; Oxford, Blackwell Scientific Publications: Oxford, U.K., 1995; p 225. (59) Wand, A.; Kallush, S.; Shoshanim, O.; Bismuth, O.; Kosloff, R.; Ruhman, S. Chirp Effects on Impulsive Vibrational Spectroscopy: A Multimode Perspective. Phys. Chem. Chem. Phys. 2010, 12, 2149− 2163. (60) Vos, M. H.; Rappaport, F.; Lambry, J.-C.; Breton, J.; Martin, J.L. Visulization of Coherent Nuclear Motion in a Membrane Protein by Femtosecond Spectroscopy. Nature 1993, 363, 320−325. (61) Polli, D.; Luer, L.; Cerullo, G. High-Time-Resolution PumpProbe System with Broadband Detection for the Study of TimeDomain Vibrational Dynamics. Rev. Sci. Instrum. 2007, 78 (10), 103108. (62) Horikoshi, K.; Misawa, K.; Lang, R. Rapid Motion Capture of Mode-Specific Quantum Wave Packets Selectively Generated by Phase-Controlled Optical Pulses. J. Chem. Phys. 2007, 127 (5), 054104. (63) Florean, A. C.; Carroll, E. C.; Spears, K. G.; Sension, R. J.; Bucksbaum, P. H. Optical Control of Excited-State Vibrational Coherences of a Molecule in Solution: The Influence of the Excitation Pulse Spectrum and Phase in Ld690†. J. Phys. Chem. B 2006, 110 (40), 20023−20031. (64) Lanzani, G.; Zavelani-Rossi, M.; Cerullo, G.; Comoretto, D.; Dellepiane, G. Real-Time Observation of Coherent Nuclear Motion in Polydiacetylene Isolated Chains. Phys. Rev. B: Condens. Matter Mater. Phys. 2004, 69, 134302. (65) Pollard, W. T.; Fragnito, H. L.; Bigot, J.-Y.; Shank, C. V.; Mathies, R. A. Quantum-Mechanical Theory for 6 Fs Dynamic Absorption Spectroscopy and Its Application to Nile Blue. Chem. Phys. Lett. 1990, 168 (3,4), 239−245. (66) Wang, Q.; Schoenlein, R. W.; Peteanu, L. A.; Mathies, R. A.; Shank, C. V. Vibrationally Coherent Photochemistry in the Femtosecond Primary Event of Vision. Science 1994, 266, 422−424. (67) Kumar, A. T. N.; Rosca, F.; Widom, A.; Champion, P. M. Investigations of Amplitude and Phase Excitation Profiles in Femtosecond Coherence Spectroscopy. J. Chem. Phys. 2001, 114 (2), 701−724. (68) Kumar, A. T. N.; Rosca, F.; Widom, A.; Champion, P. M. Investigations of Ultrafast Nuclear Response Induced by Resonant and Nonresonant Laser Pulses. J. Chem. Phys. 2001, 114 (15), 6795−6815. (69) De Silvestri, S.; Cerullo, G.; Lanzani, G. Coherent Vibrational Dynamics; CRC Press: Boca Raton, FL, 2008. (70) Romero, E.; Augulis, R.; Novoderezhkin, V. I.; Ferretti, M.; Thieme, J.; Zigmatus, D.; van Grondelle, R. Quantum Coherence in Photosynthesis for Efficient Solar-Energy Conversion. Nat. Phys. 2014, 10, 677−683. (71) Kolli, A.; O’Reilly, E. J.; Scholes, G. D.; Olaya-Castro, A. The Fundamental Role of Localised Vibrations in Excitation Dynamics in Photosynthetic Light-Harvesting Systems. J. Chem. Phys. 2012, 137, 174109.
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Spectroscopic Studies of Cryptophyte Light Harvesting Proteins: Vibrations and Coherent Oscillations Paul C. Arpin,† Daniel B. Turner,†,¶ Scott D. McClure,† Chanelle C. Jumper,† Tihana Mirkovic,† J. Reddy Challa,‡ Joohyun Lee,‡ Chang Ying Teng,§ Beverley R. Green,§ Krystyna E. Wilk,∥ Paul M. G. Curmi,∥ Kerstin Hoef-Emden,⊥ David W. McCamant,*,‡ and Gregory D. Scholes*,†,# †
Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada Department of Chemistry, University of Rochester, Rochester, New York 14627, United States § Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ∥ School of Physics, The University of New South Wales, Sydney, New South Wales 2052, Australia ⊥ Botanical Institute, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany # Department of Chemistry, Princeton University, Washington Road, Princeton, New Jersey 08544, United States ‡
ABSTRACT: The first step of photosynthesis is the absorption of light by antenna complexes. Recent studies of light-harvesting complexes using two-dimensional electronic spectroscopy have revealed interesting coherent oscillations. Some contributions to those coherences are assigned to electronic coherence and therefore have implications for theories of energy transfer. To assign these femtosecond data and to gain insight into the interplay among electronic and vibrational resonances, we need detailed information on vibrations and coherences in the excited electronic state compared to the ground electronic state. Here, we used broad-band transient absorption and femtosecond stimulated Raman spectroscopies to record ground- and excited-state coherences in four related photosynthetic proteins: PC577 from Hemiselmis pacif ica CCMP706, PC612 from Hemiselmis virescens CCAC 1635 B, PC630 from Chroomonas CCAC 1627 B (marine), and PC645 from Chroomonas mesostigmatica CCMP269. Two of those proteins (PC630 and PC645) have strong electronic coupling while the other two proteins (PC577 and PC612) have weak electronic coupling between the chromophores. We report vibrational spectra for the ground and excited electronic states of these complexes as well as an analysis of coherent oscillations observed in the broad-band transient absorption data.
1. INTRODUCTION Photosynthesis uses proteins called light-harvesting complexes to amplify the spectral and spatial capture of light.1,2 After light is absorbed by an antenna complex, the electronic excitation is transferred through space to a reaction center to drive charge separation. Here, we study spectroscopic properties of lightharvesting complexes known as phycobiliproteins that were isolated from cryptophyte algae.3−5 In these phycobiliproteins, eight chromophores are covalently bound to a protein matrix. The chromophores are straight-chain tetrapyrrole molecules, known as bilins. Transport of excitation (energy transfer and migration) occurs on femtosecond to picosecond time scales within each phycobiliprotein. Femtosecond spectroscopy has contributed extensively to the understanding of energy-transfer dynamics in light-harvesting antenna complexes. In traditional transient absorption spectroscopy, a narrow-band laser pulse excites a limited number of transitions in the sample, and a broad-band probe pulse measures the differential absorption spectrum induced by pumping the sample. Measurements of the transient differential absorption have led to detailed models of energy-transfer pathways and time scales in phycobiliproteins.6 A more sophisticated method known as two-dimensional electronic © 2015 American Chemical Society
spectroscopy (2D ES) maps the dynamics as a function of both excitation and emission frequencies.7,8 Consequently, broadband excitation pulses can be used without sacrificing the spectral resolution of the excitation process. In addition to initiating population dynamics, broad-band laser pulses can excite coherent superpositions of quantum-mechanical states, which appear as oscillations in the measured dynamics.9−13 2D ES measurements are helpful because they contain all of the information available in a four-wave mixing experiment.14 Yet, this power is also a weakness because 2D ES does not readily isolate single physical effects. Sophisticated models are required to understand the spectra, and there is not yet a consensus on interpreting many of the subtle features. For example, 2D ES has identified oscillatory dynamics in photosynthetic complexes.15,16 These experiments have inspired discussion on what role coherence could play in energy transfer.12,17−23 However, there is some debate regarding how to interpret the oscillations:24−31 Are they due to simple intramolecular vibrations? After all, oscillatory dynamics in Received: May 17, 2015 Revised: July 18, 2015 Published: July 18, 2015 10025
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Figure 1. Four species of cryptophyte algae and their primary light-harvesting proteins known as phycobiliproteins. (a) Micrographs show that, compared to other cryptophytes, these four species have very similar physical properties including color and size. Cell lengths: CCMP269: 9.7 ± 0.6 μm, CCAC 1627 B: 10.3 ± 0.9 μm, CCAC 1635 B: 7.5 ± 0.7 μm, CCMP706: 7.0−8.5 μm (the latter according to Lane and Archibald38). (b) Visualizations of the X-ray crystal structures. Each protein contains eight covalently bound chromophores; red, blue, and yellow indicate, respectively, dihydrobiliverdin (DBV), phycocyanobilin (PCB), and mesobiliverdin (MBV). The main difference between the two proteins on the left from the two on the right is due to a ∼73° rotation of the two halves of the tetramer (colored green and black). PC645 and PC630 are “closed”, while PC612 and PC577 are “open”. The result is that the pair of DBVs is separated by about 0.5 nm in PC645 and PC630 while the pair of DBVs in PC612 and PC577 is separated by about 2 nm.
have established that the quaternary structure is a tetrameric α1βα2β association of the subunits.39 One difference among the proteins is the replacement of chromophores to tune the absorption spectrum for the given species; for example, PC645 and PC630 have mesobiliverdins (MBVs) whereas PC577 and PC612 do not.4 Far more importantly for the present study is that the proteins have two distinct conformations:40 the “closed” form of PC645 and PC630, and the “open” form of PC612 and PC577. In the open form, a single aspartic acid inserted near the bilin-binding site of the α-subunit disrupts the hydrogen-bonding network that prevents these proteins from adopting the closed forms. In the closed form, the two central dihydrobiliverdins (DBVs) are about 0.5 nm apart, while in the open form, the DBVs are about 2 nm apart. This enables us to compare spectroscopic data systematically for the cases where there is a central molecular excitonic dimer and for the cases where that electronic coupling is small.
molecular condensed-phase systems have for decades been attributed to the coherent evolution of intramolecular vibrations.32−34 It is challenging for 2D ES to distinguish signatures of coherent molecular vibrations from coherent superpositions of excitonic states.27,29,30,35−37 Moreover, in molecular aggregates, the electronic and vibrational transitions can mix, and it remains an open question of how to interpret and assign the oscillatory dynamics observed in femtosecond spectroscopy experiments.27,30,37 Resolving this issue is crucial for modeling and interpreting the results. Here, we provide a step toward addressing the issues described above. We used two pump−probe spectroscopy experiments with interpretations that are more intuitive than 2D ES: stimulated resonance Raman spectroscopy to isolate oscillatory dynamics in the electronic ground state and broadband transient absorption spectroscopy to isolate oscillatory dynamics in the electronic excited state. We are thus able to measure separately excited-state oscillations and ground-state oscillations (the vibrational spectrum). We assay four related phycobiliproteins. Specifically, we measure phycocyanin 577 (PC577) extracted from Hemiselmis pacif ica CCMP706, phycocyanin 612 (PC612) extracted from Hemiselmis virescens CCAC 1635 B, phycocyanin 630 (PC630) extracted from Chroomonas CCAC 1627 B (marine), and phycocyanin 645 (PC645) extracted from Chroomonas mesostigmatica CCMP269. In Figure 1, we show micrographs of representative algae and the X-ray crystal structures of the phycobiliproteins. Each protein has slight differences in structure and composition that alter the electronic energy levels and vibrational modes of the chromophores, which in turn tunes the energy-transfer dynamics. Phycobiliproteins are tetramers having α1βα2β structure. Crystallography and gene sequencing
2. EXPERIMENTAL METHODS 2.1. Transient Absorption Experiments. We detailed the transient absorption spectrometer in a related manuscript.41 Briefly, a commercial 5 kHz Ti:sapphire laser amplifier pumped a home-built noncollinear optical parametric amplifier (NOPA) to produce broad-band visible pulses.35,42 We tuned the peak of the output spectrum to 580 nm to strongly excite the peak absorption of the DBV chromophores present in all four samples as shown in Figure 2. A weak tail in the laser spectrum extending to about 750 nm probed the sample response beyond the absorption and fluorescence maxima of the PCB chromophores. Grating and prism compressors compensated the second-order and third-order dispersion, leaving a ∼45 fs instrument response function dominated primarily by residual fourth-order dispersion which cannot be compensated with our 10026
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the presented analysis, we assume four parallel, single exponential decay components in the fitting. We have verified that the values of the fastest decay components are sensitive to the number of assumed decay components; however, we focus our conclusions on the relative decay components between proteins which are insensitive to the choice of model. 2.2. Two-Dimensional Electronic Spectroscopy. The reported 2D ES was collected in a four wave mixing apparatus described in detail previously.35,42 Briefly, a commercial Ti:sapphire amplifier pumped a home-built noncolinear optical parametric amplifier (NOPA) to generate broad-band pulses in the visible.44 For the 2D ES measurements, the NOPA was tuned to a central wavelength of 580 nm and a full width at half maximum (fwhm) bandwidth of 70 nm. The pulses were compressed with a combination of a grating compressor and a prism compressor to a duration of 11 fs as measured with transient grating frequency resolved optical gating (TG-FROG) at the sample position.45 The NOPA beam was split into four identical beams arranged in the BOXCARS geometry with a single transmissive diffractive optic. The four beams followed a common path from the diffractive optic to the sample position to maintain passive phase stability. Delays between pulses were controlled with the insertion or removal of glass with wedged prism pairs. The first two beams acted as the pump and excited the sample; the third acted as the probe and initiated the radiation of a signal beam in a background-free direction. The final beam, the local oscillator, was aligned collinear with the signal, and the two were heterodyne detected by the spectrometer. Spectral interferometry enabled extraction of the complex signal electric field.46 The waiting time delay between the excitation and the final pulse, which corresponds to the delay time in the transient absorption measurements, was varied from 0 to 400 fs in 5 fs steps. At each waiting time, the delay between the two excitation pulses was varied from +45 to −45 fs in 0.15 fs steps, and a spectrum was recorded at each step. A Fourier transform of the complex signal extracted from spectral interferometry at each detection wavelength along the excitation pulse delay axis produced a 2D map of the signal at each waiting time as a function of both the excitation wavelength and the emission wavelength. 2.2. FSRS. The femtosecond stimulated Raman spectroscopy (FSRS) setup has been described elsewhere.47−50 Briefly, the 3 ps duration Raman pump and a 100 fs, broad-band probe are concurrently crossed in the sample to produce ground-state stimulated Raman spectra. The Raman pump pulse at 596.5 nm was generated by propagating a narrow-band pulse at 400 nm from a second harmonic bandwidth compressor51 into H2 Raman shifter.52 The probe pulse was generated by focusing the 800 nm fundamental into sapphire. The Raman pump pulse is not a photoinitiating, or actinic, pump. The energy of the Raman pump pulse was chosen53 to avoid severe depletion of ground-state population and was set to 25 or 30 nJ. The pump was chopped at 500 Hz, and the transmitted probe beam was dispersed by a grating spectrograph and was detected via the second-order diffraction collected on a CCD. For higher signalto-noise ratio, the scanning multichannel technique (SMT) method was used.54 The 1 mm sample cuvette was temperature-controlled to 5 °C and was continuously translated at 2 mm/s. For baseline correction, the resulting Raman spectrum was subtracted by a scaled transient absorption background spectrum, obtained by applying a time delay between the Raman pump and the probe. Any residual baseline was
Figure 2. Absorption spectra (black) of PC645, PC630, PC612, and PC577. The laser spectrum used both to excite and to probe the dynamics is shown in gray. The peak of the excitation spectrum is tuned to 580 nm to strongly excite the DBV chromophores present in each of the four phycobiliproteins studied in the present work. The laser spectrum extends to almost 750 nm capturing excited-state absorption signals outside the linear absorption spectrum of the complexes.
current compression scheme and temporal smearing. A transient-grating frequency-resolved optical gating measurement reveals that the pulse was 740 cm−1 with difficulty. The comparison shows consistency in the overlapping spectral region between the two measurements. We do not identify any distinct peaks in the transient absorption measurement that are not present in the FSRS, indicating that the oscillations observed in the nonlinear spectra can be attributed primarily to coherent nuclear dynamics. One of the most visible differences between the TA measurement and the FSRS measurement is the location of the peak near the 500 cm−1 cluster in PC645 and PC630. The peak in the TA spectrum has an apparent red shift relative to the measured FSRS. As we will discuss later, the FSRS exclusively identifies ground-state vibrational frequencies where the transient absorption identifies predominantly electronic excited state vibrational frequencies. This may account for some shift in the peak frequency between the two measurements. However, this particular case is likely an artifact of the measurement. FSRS is an incoherent technique and measures the sum of the intensity of each mode where transient absorption is coherent and consequently sensitive to the relative phase of the overlapping modes which can artificially shift the apparent peak of a cluster of closely spaced oscillation frequencies.57 10029
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Figure 5. Broad-band transient absorption measurements of the four cryptophyte proteins. In the first column, representative raw transient spectra are plotted for waiting time delays from 0 to 1.8 ps. Positive (red) features represent increased signal at a particular wavelength after excitation (ground-state bleach or stimulated emission), and negative (blue) features correspond to additional absorption in the sample after excitation (excited-state absorption). The middle column shows 2-D oscillation maps obtained by subtracting the fitted population dynamics from the transient absorption spectra. In the final column, the wavelength dependent coherent maps are plotted. Bright features indicate high amplitude oscillations at the given frequency in the time trace taken at that probe wavelength. PC577 and PC612 show a minimum in the amplitude of the oscillations at a probe wavelength near 630 nm for each of the dominant oscillation frequencies.
accompanied by abrupt phase changes. The node and phase changes occur at the detection wavelength corresponding to the minimum of the electronic potential well on which the wave packet is oscillating.33,58,65−69 Each amplitude plot contains a node at the wavelength corresponding to the fluorescence maximum of each phycobiliprotein, signifying that the oscillations arise from vibrational wave packets oscillating on the excited electronic state of the low-energy chromophores. Despite complications that might arise when considering multichromophoric systems with multiple nuclear degrees of freedom, the semiclassical model of vibrational wave packets seems to hold in the phycobiliproteins. Several low-amplitude peaks and nodes appear at shorter detection wavelengths. These features may be due to vibrations on the ground electronic state of the PCB chromophores, or they may be due to vibrations on the ground or excited electronic states of other chromophores. In some cases, an additional node is visible closer to 700 nm. This is near the peak of the excited-state absorption on the red side of the spectrum and is consistent with the excitation of vibrational wavepackets oscillating on the excited electronic state to a higher lying electronic state. We cannot assign this excited-state absorption, but its presence provides additional evidence that the dominant contribution to the oscillations is due to oscillations on the excited electronic state.41 Another detail that supports the assignment of excited-state vibrations is that a significant portion of the oscillations appears as excitedstate absorption signals located beyond the red side of the
Table 2. Oscillation Frequencies of the Four Proteins Obtained from Gaussian Fits to the Probe Wavelength Integrated Transient Absorption Oscillation Spectraa PC645
PC630
PC612
PC577
wavenumbers (cm−1)
wavenumbers (cm−1)
wavenumbers (cm−1)
wavenumbers (cm−1)
199 276.0 350 478 657 852 1100
± ± ± ± ± ± ±
3 0.8 4 2 4 5 30
200 272 351 470 600 657 843
± ± ± ± ± ± ±
2 2 3 4 20 4 3
202 261.8 372 465 516 660.4 807
± ± ± ± ± ± ±
2 0.7 3 2 4 0.7 8
208 264 367 468 509 660.5 745 920
± ± ± ± ± ± ± ±
2 1 4 4 7 0.7 7 20
a Values represent the average and standard deviation of fits to five independent data sets. The values are reported in wavenumber units (the oscillation frequency divided by the speed of light).
have studied the detection-wavelength dependence of the oscillations,58−64 as we discussed recently.41 Maps of the oscillation amplitude and phase as a function of emission frequency can further discern ground-state vibrational wave packets from excited-state vibrational wave packets. We therefore inspect amplitude (green) and phase (red) plots of each frequency as a function of detection wavelength; we present the results for three oscillations in Figure 7. In Figure 7, the 270 and 470 cm−1 amplitude and phase plots are representative examples of the character of almost all the modes. The amplitude traces for all four proteins have nodes 10030
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Figure 6. Excited-state and ground-state wavepackets produce specific spectroscopic signals. Broad-band transient absorption spectroscopy (red) identifies the energies of excited-state wave packets, which include vibrational contributions (right red), while stimulated resonance Raman (gray) identifies the energies of ground-state vibrational wave packets that are coupled to the electronic transitions (right, gray). The stimulated resonance Raman spectra show that the chromophores of the four phycobiliproteins have very similar ground-state vibrational modes. These values match those previously published for PC645 and PC577. Broad-band transient absorption spectra, meanwhile, primarily reveal excited-state vibrational modes. The four phycobiliproteins share many excited-state vibrational frequencies.
Figure 7. Plots of the amplitude and phase of specified oscillations from transient absorption spectra as a function of detection wavelength. Excitedstate vibrational wave packets oscillate about the excited-state electronic potential minimum and are therefore characterized by a node in the oscillation amplitude and a phase change at the wavelength of the fluorescence maxima. The vertical dashed lines indicate the fluorescence maxima. In the four proteins, nearly all of the frequencies have nodes and phase shifts consistent with the signatures for excited-state vibrational modes. Previous work on PC645 identified that the 660 cm−1 mode showed unusual characteristics, and therefore, we carefully study it in the bottom panels. The 660 cm−1 amplitude and phase lineouts for PC612 and PC577 indicate an excited-state vibrational wave packet. The 660 cm−1 amplitude and phase lineouts of PC645 and PC630, however, lack the amplitude node. This feature has not been previously observed.
linear absorption spectrum. Excited-state absorption signals cannot contain ground-state oscillations. The 660 cm−1 mode is of particular interest. PC612 and PC577 follow the excited-state vibrational model with an amplitude node and abrupt phase change. However, PC645 and PC630 do not have such a distinct node in the amplitude plot. The difference is statistically significant but does not have a ready explanation. The effect is not due to an interference effect by the MBVs, for example, oscillations centered at slightly different detection wavelengths, because the other modes in PC645 and PC630 do not show the same signature. The most significant challenge to interpretation of the present data is establishing a correlation between excitation frequency and response frequency. As with population dynamics, the
coherence dynamics contain overlapping contributions from oscillations excited in each of the bilins present in the protein. Interestingly, the 660 cm−1 frequency that yields the indistinct node is similar to an electronic frequency gap (of ∼700 cm−1) in the system that has previously been suggested to show coherent electronic oscillations in 2D ES studies of closed structures, particularly PC645, when probed in the highenergy side of the absorption spectrum.11,35,42 We compare a time trace from a transient absorption measurement of PC645 to an analogous measurement obtained from the 2D spectra on PC645 in Figure 8. Two-dimensional electronic spectroscopy resolves both the excitation wavelength and the emission wavelength that contribute to the signal radiated by the sample at a particular time delay. Transient absorption spectroscopy only resolves whether there is a signal at a particular emission 10031
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Figure 9. Comparison of population dynamics in open and closed structure phycobiliproteins. Room-temperature decay associated spectra (DAS). (a) A closed-structure phycobiliprotein (PC645) and (b) an open-structure phycobiliprotein (PC577) after broad-band excitation. Gray shading indicates the uncertainty based on an independent analysis of five separate data sets. (c) Direct comparison of normalized time traces taken at a probe wavelength of 580 nm, overlapping the high-energy DBV absorption peak in the samples. Decay indicates downhill energy flow of the excitation.
according to global fits to data taken with parallel polarization between the pump and probe (data not shown). This suggests that the molecular excitons effectively distribute the excitation, in accord with the theoretical predictions of Huo and Coker.18 Figure 8. Relation between 2D ES and transient absorption (TA) spectroscopy of PC645. (top left) Integrating the 2D ES from previous work over the outlined area produces a value for each waiting time point. The scan range was limited to 400 fs. (top right) Selection from the full TA data set, which ranged from −0.5 to 5 ps. Shading indicates the integrated region to produce the trace below. (bottom) The traces indicate the mean of multiple 2D ES (black) and TA (red) measurements. Error bars for the 2D trace and the shaded region for the TA trace indicate the respective standard deviations. The traces agree quantitatively.
5. CONCLUSIONS We used femtosecond stimulated Raman and broad-band transient absorption spectroscopies to record, respectively, ground- and excited-state coherences in four related photosynthetic proteins: PC577 from Hemiselmis pacif ica CCMP706, PC612 from Hemiselmis virescens CCAC 1635 B, PC630 from Chroomonas CCAC 1627 B (marine), and PC645 from Chroomonas mesostigmatica CCMP269. Two of those proteins (PC630 and PC645) have strong electronic coupling, while the other two proteins (PC577 and PC612) have weak electronic coupling between the chromophores. We identified several oscillatory components in the transient absorption spectra of each of the four proteins that followed a simple prescription for nuclear oscillations on the excited electronic state. The lack of a node as a function of probe frequency for the 660 cm−1 component in two of these proteins prevents us from identifying the mode using the same prescription. This mode appears in all four proteins but shows the unique lack of a node only in the two closed proteins where strong electronic coupling is expected and is at a frequency previously identified in 2D electronic spectroscopy as a candidate for coherent oscillations because of strong electronic coupling. In the present work, we are unable to identify the nature of this oscillation, however, the Raman spectroscopy presented here clearly identifies a ground-state vibrational oscillation at the same frequency. This suggests that a full characterization of this mode will require a model that allows for coupling between the vibration and excitonic gap.27,30,70,71 We anticipate that future experiments can disentangle overlapping contributions to the oscillations by tuning the central frequency of the pump pulse to clearly identify the nature of this oscillation using transient absorption spectroscopy. In the course of this study, we found that energy-transfer dynamics is faster in the closed structure proteins PC645 and PC630 than in the open structures PC577 and PC612.
wavelength at the given waiting time regardless of what excitation frequency initiated the dynamics which contributes to the signal. To reconcile the two measurements, it is necessary to sum the 2D spectrum along the excitation axis to account for all of the possible contributions at that emission wavelength, which can be quantitatively shown with the projection slice theorem. The plotted time traces are taken at a single emission wavelength from the transient absorption measurement and are taken at the same emission wavelength from the 2D spectrum but are summed in the excitation wavelength dimension. The two time traces obtained on different instruments several months apart show quantitative agreement improving confidence in the reproducibility of the conclusions. Oscillations in the transient absorption data are well aligned with those at the notable cross peak in the 2D ES data. Additional insights into the excited-state dynamics can be gained by analyzing the kinetics of the transient absorption data. Here, we introduce our preliminary analysis, highlighting a comparison of the dynamics between one protein having a closed structure (PC645) and one with an open structure (PC577) in Figure 9. We focus our discussion on the fastest decay component which peaks near 580 nm in each protein, the absorption peak of the DBV chromophores. The interesting observation is that excitation energy transfer initiated by excitation on the blue side of the absorption spectra flows to absorption resonances on the red side of the spectra about twice as quickly in the closed structures compared to the open structures (440 ± 50 fs vs 870 ± 80 fs, respectively). Results are consistent across the two closed and the two open structures
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*E-mail: [email protected]. *E-mail: [email protected]. 10032
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Department of Chemistry, New York University, New York, NY 10003.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. P.C.A. and D.B.T. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the Natural Sciences and Engineering Research Council of Canada, DARPA (QuBE), and the United States Air Force Office of Scientific Research (FA9550-13-1-0005). J.R.C., J.L., and D.W.M. were supported by a United States National Science Foundation CAREER award, CHE-0845183, and D.W.M. was supported as an Alfred P. Sloan Research Fellow
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