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Comparing photoinduced vibrational coherences in bacteriorhodopsin and in native and locked retinal protonated Schiff bases
Chemical Physics Letters 381 (2003) 549–555 www.elsevier.com/locate/cplett
Comparing photoinduced vibrational coherences in bacteriorhodopsin and in native and locked retinal protonated Schiff bases Bixue Hou
a,1
, Noga Friedman b, Michael Ottolenghi a, Mordechai Sheves b, Sanford Ruhman a,*
a
b
Department of Physical Chemistry, and The Farkas Center for Light Induced Processes, The Hebrew University, Givat Ram, Jerusalem 91904, Israel Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel Received 3 August 2003; in final form 9 October 2003 Published online: 30 October 2003
Abstract Low frequency excited state vibrational coherences induced by impulsive photoexcitation in bacteriorhodopsin are detected via femtosecond pump–probe spectroscopy, and compared with similar data in retinal protonated Schiff bases of native and locked retinals. At delays above 100 fs a single vibration below 200 fs dominates the detected spectral modulations. Its frequency of 120 in retinal protonated Schiff base is virtually unchanged by locking the C13 @C14 bond in the trans or cis configurations, but is increased to 170 cm 1 within the protein environment. The implications of this result on the part played by the protein in directing the reactivity of the retinal within bacteriorhodopsin is discussed. Ó 2003 Published by Elsevier B.V.
1. Introduction The storage and subsequent utilization of photon energy in retinal proteins is a long standing focus of scientific interest. To this end, Bacterio-
*
Corresponding author. fax: +97225618033. E-mail address: [email protected] (S. Ruhman). 1 Present address: Center for Ultrafast Optical Science, University of Michigan, 2200 Bonisteel Boulevard, Ann Arbor, MI 48109-2099, USA. 0009-2614/$ - see front matter Ó 2003 Published by Elsevier B.V. doi:10.1016/j.cplett.2003.10.038
rhodopsin (BR), the light energized proton pump embedded in the purple membrane of Halobacterium salinarum [1–3] has been investigated using femtosecond pump–probe spectroscopy to elucidate the initial stages of translating photon energy into biological activity [4–8]. Dramatic spectral changes take place in BR on the sub-picosecond timescale following light absorption, the most prominent being the buildup of strong absorption and emission bands peaking at 460 and 950 nm, respectively. Both are assigned to the fluorescent excited state ÔI460 Õ, 460 being the absorption peak
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B. Hou et al. / Chemical Physics Letters 381 (2003) 549–555
of the intermediate in nanometers. Both decay concertedly on a 0.5 ps timescale, giving way to the first reactive ground state intermediate ÔJ625 Õ. In a series of recent experiments, contrary to earlier assertions, the formation of the fluorescent state has been demonstrated to require negligible torsional flexibility around the C13 @C14 bond. This was achieved by comparing the photoinduced response of native BR to that obtained in an artificial pigment where the prosthetic chromophore was replaced with synthetically locked retinals [9,10]. These experiments further show that the stage of retinal isomerization, crucial to the biological activity, must coincide with the 0.5 ps decay of the fluorescent state. Finally, in an effort to probe the dynamics of the ongoing isomerization, the fluorescent state was probed by stimulated emission pumping at various times during its lifetime, curiously exhibiting no evolution in the fluorescing population as it decays [11]. The only dynamics uncovered in that experiment were spectral modulations indicative of wave packet motions along bound vibrational coordinates in impulsively excited ÔI460 Õ. A central objective in the research of retinal proteins is understanding how the surrounding protein controls the retinalÕs early response to photoabsorption. Accordingly the photoinduced dynamics of the free protonated Schiff base (PSB) of retinal has also been studied using ultrafast methods, including a recent extension to PSBs of locked retinal where isomerization around the C13 @C14 bond is blocked [12]. The emerging picture from these studies is that the protein hastens the internal conversion of the retinal and allows it to take place only via isomerization of the specific double bond. Another important result was the observation of spectral modulations associated with excited state vibrational coherences in free PSB transient transmission scans as well. These coherences have recently been addressed theoretically, and assigned to skeletal motions involving much of the polyene [13]. In this report, we present analysis of the modulations appearing in pump–probe signals of BR and of retinal PSB and locked PSB in solution. The objective of this analysis is to investigate which molecular deformation is responsible for
these coherences, and how the binding to the protein or the locking structures influence the frequency of the observed modulations. The main result of this analysis is that while the locking of the retinal C13 @C14 double bond does little to alter the frequency of the observed skeletal reverberations unleashed by light absorption, inclusion of the retinal within the protein increases this frequency almost twofold, possibly reflecting the forces exerted by the protein which direct the photochemistry of the prosthetic group in BR.
2. Experimental The locked retinal analogs were prepared according to previously described methods [9,10,12] H. salinarum was grown from the S9 strain, and purple membranes containing bacteriorhodopsin were isolated as previously described [14]. Potassium phosphate buffer was used to adjust the pH to 7. The laser system consisted of a home made multipass amplified Ti:sapphire arrangement producing a 1 kHz train of 30 fs pulses, centered at 790 nm [15]. Excitation pulses at 395 nm used for exciting the PSBs were derived by doubling a small portion of the amplifier output in a 0.1 mm BBO crystal. Pulses for exciting BR were generated in a NOPA (Clark MXR) producing 20 fs pulses centered at 670 nm. Probe pulses were generated by interference filtering from a white continuum produced in a 3 mm sapphire plate. The origin of pump/probe delay was determined by conducting Optical Kerr Effect scans in the sample cell. Dispersion in either arm was precompensated by prism pairs to provide the shortest pump/probe cross-correlation in the sample cell. Amplified photodiodes and lock-in detection were used to measure transmission changes as a function of probe delay, using a pump fluance which was reduced to ensure linear power dependence of the data. The samples were peristaltically circulated, under a nitrogen atmosphere in the case of the PSBs, through a 100 lm path length cell constructed of stainless steel, and equipped with 150 lm thick quartz windows. Flow rates ensured sample replenishment between laser pulses.
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3. Results and discussion The transient spectral changes following photoexcitation of BR and of the retinal PSBs in ethanol solution have been reported earlier and will not be presented here. In the native and locked PSBs the ground state absorbs near 440 nm in ethanol producing an excited state emitting above 580 nm, and exhibiting an absorption peaking near 500 nm, which partially overlaps the emission. Due to the opsin shift in BR, the energies of ground state and excited state absorption and also emission of the later are shifted down with respect to the corresponding bands in the PSBs [16]. The ground state absorption is shifted from 440 to 570 nm. The stimulated emission band in BR, peaking at 950 nm, has been shown to be red shifted with respect to spontaneous fluorescence indicating overlap with an excited state absorption which is almost completely erased [17,18]. In BR and the PSBs the most obvious spectral modulations were obtained when probing at wavelengths to the blue of the observed stimulated emission peak, where transient transmission changes were minimal. This may be due to cancellation of excited state absorption and emission, contributing to accentuating the modulations with respect to the background signal. Accordingly the modulations were extracted with high signal to noise only when probing at 790 nm. The fact that this cancellation does not simultaneously erase the modulations as well may in the future teach us about the topologies of the three potential surfaces involved in transient spectroscopy near 800 nm. Here we concentrate on the fact that regardless of the leading transition, the modulations may surely be assigned to vibrational coherences in the reactive excited state. The transient signal obtained following excitation at 570 nm is presented in Fig. 1 along with the modulations extracted in the lower panel. The signal is one of weak excess transmission at all delay times measured. The modulations were obtained by subtracting a multiexponential fit of the signal from the data. We have intentionally discarded the initial 150–200 fs of the residual for its form is very sensitive to the model used for subtraction of the background signal. We will return to this point when interpretation of these results is discussed.
Fig. 1. Transient transmission at 800 nm following 570 nm excitation of BR. Lower panel shows residual obtained by subtracting biexponential fit from the data. See text for details.
Similar results for retinal PSBs are portrayed in Fig. 2. The trans-locked PSB has the C13 @C14 bond constrained by a 2 carbon aliphatic bridge connecting C12 to C14 . 13-cis-Locked retinal maintains the same bond in the cis configuration via a 3 carbon bridge connected to its ends [12]. Unlike the BR data, in 13-cis-locked and native all trans retinal PSB a rapid transition from excess absorption to net emission shows up at early delay times. In the trans-locked case an initial excess absorption decays rapidly almost to zero but no change in signal sign is observed. The residuals shown are those collected at the probing wavelength providing the best signal to noise ratio, and are similarly extracted by subtracting a slowly varying background which is not directed by any kinetic model, and serves only to separate out the rapid oscillatory component of the signals. One of these fits is displayed for the 13 cis-locked PSB in the upper panel of Fig. 2. In all three molecules, readily observable periodic undulations which damp out within 2 ps are seen. The FFTs of the modulations depicted in Figs. 1 and 2, along with those obtained from similar scans probing at other frequencies, are shown in Fig. 3. As observed, at all wavelengths,
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Fig. 2. Transient transmission signals for retinal PSBs in ethanol solution, exciting at 400 nm, and probing at 600 nm. As in Fig. 1, lower panel shows oscillating residuals extracted by subtracting fit from the data. One such multiexponential fit is shown for the 13 cis locked PSB in the upper panel.
the long lived modulations recovered can be rationalized in terms of the first and second harmonics of a single damped oscillation in the 100–200 cm 1 range. It is not surprising that frequencies significantly above this are not observed since the time resolution of our experiment excludes this. Clearly discernable from Fig. 3 is the fact that while the locking of the retinals does little to change the frequency of the recorded modulations (only a slight reduction seems to accompany trans locking), in the protein the extracted frequency increases by more than 50% – a change which lies well outside the estimated error of 20 cm 1 . The relative component of first and second harmonic modulations is dictated by the frequency of the probe pulses. This is most clearly demonstrated by comparing the residuals at various frequencies for native all-trans retinal PSB as shown in Fig. 4. The probe frequency dependence of the harmonics detected in the modulations can be explained qualitatively by realizing that a coherent wave packet will be detected twice in every period
Fig. 3. Normalized FFTs of the residuals obtained from pump– probe data in BR and the PSBs, probing at wavelengths designated in the figure.
when observed at a frequency corresponding to the transition energy at the center of the motion, but only once when observed by a pulse which probes at frequencies corresponding to end points of the motion etc [15]. This effect can be used to obtain the contours of the effected band, or alternatively to extinguish the fundamental modulation components in order to allow unhindered detection of higher harmonics in impulsive vibrational coherence experiments [19]. A gradual doubling of the modulation period can be observed in Fig. 4 upon shifting the detection wavelength from 550 to 750 nm, reflecting the approach and transition through the emission band maximum, and causing an observable change in the relative intensities of the fundamental and first harmonic peaks in the FFT. Another indication that this wavelength dependence of the residuals is correct is the change of sign in the modulation structure when moving beyond the emission bands center.
B. Hou et al. / Chemical Physics Letters 381 (2003) 549–555
Fig. 4. Residuals extracted from all trans retinal PSB transient transmission scans at different probing wavelengths, designated in the figure.
Assuming that a single low frequency mode dominates the persistent spectral modulations in all the tested molecules including the BR, the remaining discussion deals with the significance of the observed vibrational mode, and what the comparison teaches us about excited state dynamics. The activity of this mode must by definition make it important in the dynamics since it reflects a substantial geometrical change induced by promotion to the excited state. Theoretical efforts to simulate impulsive excited state dynamics predict photoexcitation will induce activity along a number of low frequency modes. The fact that FFT analysis can be based on a single mode may result from rapid dephasing of all but one of the modes, or alternatively reflect the combined action of a number of closely spaced modes. In the spirit of choosing the simplest model which is consistent with the data, we tentatively assume an identical mode is being probed in all 4 systems. One aspect which is common to all of the active low frequency modes arising in the retinal PSB
553
simulations is that they involve bends and/or torsions which are distributed over a large portion of the molecular backbone, and, therefore, should be sensitive to steric restrictions imposed by the protein. Comparative studies of retinal PSB and BR photochemistry, show that the protein surroundings increase the rate of internal conversion by a factor of at least 4, and induce specificity of isomerization around the C13 @C14 double bond. It stands to reason that this must be accomplished by constraining the conformational motions of the retinal backbone. Such structural restriction would naturally lead, on average, to increased restoring forces for displacement of the skeletal carbons and torsions around the bonds. One consequence of such restrictions would be an increase in the frequency of low lying modes which involving these degrees of freedom. This is exactly what we observe in the present study – a mode which must involve deformation of a large portion of the retinal chain, increases its frequency from 120 to 170 cm 1 upon covalent binding to the protein. Frequency modulation of stretching mode coherence with this period were recently observed in 5 fsec pulsed experiments in BR, see Ref. [26]. Another noteworthy trend in our results concerns the lack of significant changes in the frequency of PSB modulations upon locking of the C13 @C14 bond in either the cis or trans configurations. This finding is compatible with an expanding number of studies, including previous publications from our own lab, which maintain that isomerization around the active double bond, or for that mater around any of the double bonds in the retinal chain, is not the initial light driven response in BR or in the retinal PSB [9,10,18,20– 24]. Had this not been true, double bond torsions would be a major ingredient in low frequency impulsive responses, and the locking would be expected to result in significant alterations to the vibrational period. It is interesting to compare our results with those of the extensive study reported by Lin et al. [25] on the low frequency Raman active modes in the related rhodopsin and 11 cis retinal PSB. Remarkably, the lowest frequency strongly active mode is in the range of 120–130 cm 1 both in the PSB and in rhodopsin, and vibrational analysis of
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the PSB identifies this mode as being made up mainly of torsions around various CAC single and double bonds along the retinal backbone. In their experiments the ground state frequency of this mode is not found to be sensitive to incorporation of the Schiff base into the protein, and changes in band positions appear to be limited to higher frequency and more localized modes of vibration. Keeping in mind the basic differences both in the spectroscopies employed, and in the dynamics of these two related retinal proteins, the apparent discrepancy in the proteinÕs effect on low frequency retinal vibrations is worthy of continued investigation. An improvement in the time resolution of our pump–probe setup would allow an observation of many more active modes in the excited state, and is a major focus of our future experiments. None the less, the limited time resolution of the current study may be a blessing in disguise, since it allows us to concentrate attention on the very low frequency end of the vibrational spectrum which may be obscured by higher frequency modulations. As stated above, since deformations involving large portions of the retinal are involved, the effect of the protein should be most easily observed for these modes of vibration.
4. Conclusions Low frequency vibrational coherences generated in the excited state by impulsive excitation of bacteriorhodopsin and retinal protonated Schiff bases of native and locked retinals in solution are detected via periodical modulations that they induce in transient transmission signals. The pattern of these modulations which supercedes the first 100 fs of delay can be interpreted in terms of a single damped harmonic oscillation. In native all trans retinal PSB, as well as analogues with the C13 @C14 locked in the cis or trans configuration the fundamental frequency of the modulations is in the range 115–125 cm 1 whereas in bacteriorhodopsin the frequency is shifted up to 170 cm 1 . The change in frequency is suggested to result from forces imposed by the protein, which are instrumental in facilitating the bond specificity of isomerization and the enhanced rate of BR inter-
nal conversion relative to the relevant PSB. The different protein effect on the retinal vibration observed in BR and rhodopsin might reflect different chromophore-protein interactions in the two systems. In this respect we note that C13 @C14 isomerization in BR involves alterations in the Schiff base vicinity whereas such alterations were not detected in rhodopsin isomerization.
Acknowledgements We thank M. Olivucci, M. Garavelli, and S. Haacke for fruitful discussions and sharing data before publication. This work was supported by the Human Frontier Science Program (HFSP), and the Israel Science Foundation (ISF). The latter is administered by the Israel Academy of Sciences and the Humanities. The Farkas Center is supported by the Minerva Gesellschaft, GmbH, Munich, Germany.
References [1] M. Ottolenghi, M. Sheves (Eds.), Isr. J. Chem. 35 (3/4) (1995). [2] J. Lanyi (Ed.), Biochim. Biophys. Acta Bioenerg. 1460 (2000). [3] U. Haupt, J. Tittor, D. Oesterhelt, Ann. Rev. Biophys. Biomol. Struct. 21 (1999) 367. [4] J. Hurley, T.G. Ebrey, B. Honig, M. Ottolenghi, Nature 270 (1977) 540. [5] R.A. Mathies, C.A. Brito Cruz, T.W. Pollard, C.V. Shank, Science 240 (1988) 777. [6] J. Dobler, W. Zinth, K. Kaiser, D. Oesterhelt, Chem. Phys. 144 (1988) 215. [7] T. Kobayashi, M. Terauchi, T. Kouyama, M. Yoshizawa, M. Taiji, SPIE 1403 (1990) 407. [8] A.V. Sharkov, A.V. Pakulev, S.V. Chekalin, U.V.A. Matveets, Biochim. Biophys. Acta 808 (1985) 94. [9] Q. Zhong, S. Ruhman, M. Ottolenghi, M. Sheves, N. Friedman, G.H. Atkinson, J.K. Delaney:, J. Am. Chem. Soc. 118 (1996) 12828. [10] T. Ye, N. Friedman, Y. Gat, G. Atkinson, M. Sheves, M. Ottolenghi, S. Ruhman, J. Phys. Chem. B 103 (1999) 5122. [11] S. Ruhman, B. Hou, N. Friedman, M. Ottolenghi, M. Sheves, J. Am. Chem. Soc 124 (2002) 8854. [12] B. Hou, N. Friedman, S. Ruhman, M. Sheves, M. Ottolenghi, J. Phys. Chem. 105 (2001) 7042. [13] A. Cembran, F. Bernardi, M. Olivucci, M. Garavelli, J. Am. Chem. Soc. 125 (2003) 12509.
B. Hou et al. / Chemical Physics Letters 381 (2003) 549–555 [14] D. Oesterhelt, W. Stoeckenius, Meth. Enzymol. 31 (1974) 667. [15] E. Gershgoren, J. Vala, R. Kosloff, S. Ruhman, J. Phys. Chem. A 105 (2001) 5081. [16] K. Nakanishi, V. Balogh-Nair, M. Arnaboldi, K. Tsujimoto, B. Honig, J. Am. Chem. Soc. 102 (1980) 7945. [17] K.C. Hasson, F. Gai, P.A. Anfinrud, Proc. Natl. Acad. Sci. 93 (1996) 15124. [18] G. Haran, K. Wynne, A. Xie, Q. He, M. Chance, R.M. Hochstrasser, Chem. Phys. Lett. 261 (1996) 389. [19] F. Rosca, D. Ionascu, A.T.N. Kumar, A.A. Demidov, P.M. Champion, Chem. Phys. Lett. 337 (2001) 107.
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[20] M. Garavellei, F. Negri, M. Olivucci, J. Am. Chem. Soc. 121 (1999) 1023. [21] R. Akiyama, A. Yoshimori, T. Kakitani, Y. Imamoto, Y. Schichida, Y. Hatano, J. Phys. Chem. A 101 (1997) 417. [22] L. Song, M.A. El-Sayed, J. Am. Chem. Soc. 120 (1998) 8889. [23] J. Herbst, K. Heyne, R. Diller, Science 297 (2002) 822. [24] S. Haacke, S. Schenkl, S. Vinzani, M. Chergui, Biopolymers 67 (2002) 306. [25] S.W. Lin, M. Groesbeek, I. vanderHoef, P. Verdegem, J. Lugtenburg, R.A. Mathies, J. Phys. Chem. B 102 (1998) 2787. [26] T. Kobayashi, T. Saito, H. Ohtani, Nature 414 (2001) 531.
Comparing photoinduced vibrational coherences in bacteriorhodopsin and in native and locked retinal protonated Schiff bases Bixue Hou
a,1
, Noga Friedman b, Michael Ottolenghi a, Mordechai Sheves b, Sanford Ruhman a,*
a
b
Department of Physical Chemistry, and The Farkas Center for Light Induced Processes, The Hebrew University, Givat Ram, Jerusalem 91904, Israel Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel Received 3 August 2003; in final form 9 October 2003 Published online: 30 October 2003
Abstract Low frequency excited state vibrational coherences induced by impulsive photoexcitation in bacteriorhodopsin are detected via femtosecond pump–probe spectroscopy, and compared with similar data in retinal protonated Schiff bases of native and locked retinals. At delays above 100 fs a single vibration below 200 fs dominates the detected spectral modulations. Its frequency of 120 in retinal protonated Schiff base is virtually unchanged by locking the C13 @C14 bond in the trans or cis configurations, but is increased to 170 cm 1 within the protein environment. The implications of this result on the part played by the protein in directing the reactivity of the retinal within bacteriorhodopsin is discussed. Ó 2003 Published by Elsevier B.V.
1. Introduction The storage and subsequent utilization of photon energy in retinal proteins is a long standing focus of scientific interest. To this end, Bacterio-
*
Corresponding author. fax: +97225618033. E-mail address: [email protected] (S. Ruhman). 1 Present address: Center for Ultrafast Optical Science, University of Michigan, 2200 Bonisteel Boulevard, Ann Arbor, MI 48109-2099, USA. 0009-2614/$ - see front matter Ó 2003 Published by Elsevier B.V. doi:10.1016/j.cplett.2003.10.038
rhodopsin (BR), the light energized proton pump embedded in the purple membrane of Halobacterium salinarum [1–3] has been investigated using femtosecond pump–probe spectroscopy to elucidate the initial stages of translating photon energy into biological activity [4–8]. Dramatic spectral changes take place in BR on the sub-picosecond timescale following light absorption, the most prominent being the buildup of strong absorption and emission bands peaking at 460 and 950 nm, respectively. Both are assigned to the fluorescent excited state ÔI460 Õ, 460 being the absorption peak
550
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of the intermediate in nanometers. Both decay concertedly on a 0.5 ps timescale, giving way to the first reactive ground state intermediate ÔJ625 Õ. In a series of recent experiments, contrary to earlier assertions, the formation of the fluorescent state has been demonstrated to require negligible torsional flexibility around the C13 @C14 bond. This was achieved by comparing the photoinduced response of native BR to that obtained in an artificial pigment where the prosthetic chromophore was replaced with synthetically locked retinals [9,10]. These experiments further show that the stage of retinal isomerization, crucial to the biological activity, must coincide with the 0.5 ps decay of the fluorescent state. Finally, in an effort to probe the dynamics of the ongoing isomerization, the fluorescent state was probed by stimulated emission pumping at various times during its lifetime, curiously exhibiting no evolution in the fluorescing population as it decays [11]. The only dynamics uncovered in that experiment were spectral modulations indicative of wave packet motions along bound vibrational coordinates in impulsively excited ÔI460 Õ. A central objective in the research of retinal proteins is understanding how the surrounding protein controls the retinalÕs early response to photoabsorption. Accordingly the photoinduced dynamics of the free protonated Schiff base (PSB) of retinal has also been studied using ultrafast methods, including a recent extension to PSBs of locked retinal where isomerization around the C13 @C14 bond is blocked [12]. The emerging picture from these studies is that the protein hastens the internal conversion of the retinal and allows it to take place only via isomerization of the specific double bond. Another important result was the observation of spectral modulations associated with excited state vibrational coherences in free PSB transient transmission scans as well. These coherences have recently been addressed theoretically, and assigned to skeletal motions involving much of the polyene [13]. In this report, we present analysis of the modulations appearing in pump–probe signals of BR and of retinal PSB and locked PSB in solution. The objective of this analysis is to investigate which molecular deformation is responsible for
these coherences, and how the binding to the protein or the locking structures influence the frequency of the observed modulations. The main result of this analysis is that while the locking of the retinal C13 @C14 double bond does little to alter the frequency of the observed skeletal reverberations unleashed by light absorption, inclusion of the retinal within the protein increases this frequency almost twofold, possibly reflecting the forces exerted by the protein which direct the photochemistry of the prosthetic group in BR.
2. Experimental The locked retinal analogs were prepared according to previously described methods [9,10,12] H. salinarum was grown from the S9 strain, and purple membranes containing bacteriorhodopsin were isolated as previously described [14]. Potassium phosphate buffer was used to adjust the pH to 7. The laser system consisted of a home made multipass amplified Ti:sapphire arrangement producing a 1 kHz train of 30 fs pulses, centered at 790 nm [15]. Excitation pulses at 395 nm used for exciting the PSBs were derived by doubling a small portion of the amplifier output in a 0.1 mm BBO crystal. Pulses for exciting BR were generated in a NOPA (Clark MXR) producing 20 fs pulses centered at 670 nm. Probe pulses were generated by interference filtering from a white continuum produced in a 3 mm sapphire plate. The origin of pump/probe delay was determined by conducting Optical Kerr Effect scans in the sample cell. Dispersion in either arm was precompensated by prism pairs to provide the shortest pump/probe cross-correlation in the sample cell. Amplified photodiodes and lock-in detection were used to measure transmission changes as a function of probe delay, using a pump fluance which was reduced to ensure linear power dependence of the data. The samples were peristaltically circulated, under a nitrogen atmosphere in the case of the PSBs, through a 100 lm path length cell constructed of stainless steel, and equipped with 150 lm thick quartz windows. Flow rates ensured sample replenishment between laser pulses.
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3. Results and discussion The transient spectral changes following photoexcitation of BR and of the retinal PSBs in ethanol solution have been reported earlier and will not be presented here. In the native and locked PSBs the ground state absorbs near 440 nm in ethanol producing an excited state emitting above 580 nm, and exhibiting an absorption peaking near 500 nm, which partially overlaps the emission. Due to the opsin shift in BR, the energies of ground state and excited state absorption and also emission of the later are shifted down with respect to the corresponding bands in the PSBs [16]. The ground state absorption is shifted from 440 to 570 nm. The stimulated emission band in BR, peaking at 950 nm, has been shown to be red shifted with respect to spontaneous fluorescence indicating overlap with an excited state absorption which is almost completely erased [17,18]. In BR and the PSBs the most obvious spectral modulations were obtained when probing at wavelengths to the blue of the observed stimulated emission peak, where transient transmission changes were minimal. This may be due to cancellation of excited state absorption and emission, contributing to accentuating the modulations with respect to the background signal. Accordingly the modulations were extracted with high signal to noise only when probing at 790 nm. The fact that this cancellation does not simultaneously erase the modulations as well may in the future teach us about the topologies of the three potential surfaces involved in transient spectroscopy near 800 nm. Here we concentrate on the fact that regardless of the leading transition, the modulations may surely be assigned to vibrational coherences in the reactive excited state. The transient signal obtained following excitation at 570 nm is presented in Fig. 1 along with the modulations extracted in the lower panel. The signal is one of weak excess transmission at all delay times measured. The modulations were obtained by subtracting a multiexponential fit of the signal from the data. We have intentionally discarded the initial 150–200 fs of the residual for its form is very sensitive to the model used for subtraction of the background signal. We will return to this point when interpretation of these results is discussed.
Fig. 1. Transient transmission at 800 nm following 570 nm excitation of BR. Lower panel shows residual obtained by subtracting biexponential fit from the data. See text for details.
Similar results for retinal PSBs are portrayed in Fig. 2. The trans-locked PSB has the C13 @C14 bond constrained by a 2 carbon aliphatic bridge connecting C12 to C14 . 13-cis-Locked retinal maintains the same bond in the cis configuration via a 3 carbon bridge connected to its ends [12]. Unlike the BR data, in 13-cis-locked and native all trans retinal PSB a rapid transition from excess absorption to net emission shows up at early delay times. In the trans-locked case an initial excess absorption decays rapidly almost to zero but no change in signal sign is observed. The residuals shown are those collected at the probing wavelength providing the best signal to noise ratio, and are similarly extracted by subtracting a slowly varying background which is not directed by any kinetic model, and serves only to separate out the rapid oscillatory component of the signals. One of these fits is displayed for the 13 cis-locked PSB in the upper panel of Fig. 2. In all three molecules, readily observable periodic undulations which damp out within 2 ps are seen. The FFTs of the modulations depicted in Figs. 1 and 2, along with those obtained from similar scans probing at other frequencies, are shown in Fig. 3. As observed, at all wavelengths,
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Fig. 2. Transient transmission signals for retinal PSBs in ethanol solution, exciting at 400 nm, and probing at 600 nm. As in Fig. 1, lower panel shows oscillating residuals extracted by subtracting fit from the data. One such multiexponential fit is shown for the 13 cis locked PSB in the upper panel.
the long lived modulations recovered can be rationalized in terms of the first and second harmonics of a single damped oscillation in the 100–200 cm 1 range. It is not surprising that frequencies significantly above this are not observed since the time resolution of our experiment excludes this. Clearly discernable from Fig. 3 is the fact that while the locking of the retinals does little to change the frequency of the recorded modulations (only a slight reduction seems to accompany trans locking), in the protein the extracted frequency increases by more than 50% – a change which lies well outside the estimated error of 20 cm 1 . The relative component of first and second harmonic modulations is dictated by the frequency of the probe pulses. This is most clearly demonstrated by comparing the residuals at various frequencies for native all-trans retinal PSB as shown in Fig. 4. The probe frequency dependence of the harmonics detected in the modulations can be explained qualitatively by realizing that a coherent wave packet will be detected twice in every period
Fig. 3. Normalized FFTs of the residuals obtained from pump– probe data in BR and the PSBs, probing at wavelengths designated in the figure.
when observed at a frequency corresponding to the transition energy at the center of the motion, but only once when observed by a pulse which probes at frequencies corresponding to end points of the motion etc [15]. This effect can be used to obtain the contours of the effected band, or alternatively to extinguish the fundamental modulation components in order to allow unhindered detection of higher harmonics in impulsive vibrational coherence experiments [19]. A gradual doubling of the modulation period can be observed in Fig. 4 upon shifting the detection wavelength from 550 to 750 nm, reflecting the approach and transition through the emission band maximum, and causing an observable change in the relative intensities of the fundamental and first harmonic peaks in the FFT. Another indication that this wavelength dependence of the residuals is correct is the change of sign in the modulation structure when moving beyond the emission bands center.
B. Hou et al. / Chemical Physics Letters 381 (2003) 549–555
Fig. 4. Residuals extracted from all trans retinal PSB transient transmission scans at different probing wavelengths, designated in the figure.
Assuming that a single low frequency mode dominates the persistent spectral modulations in all the tested molecules including the BR, the remaining discussion deals with the significance of the observed vibrational mode, and what the comparison teaches us about excited state dynamics. The activity of this mode must by definition make it important in the dynamics since it reflects a substantial geometrical change induced by promotion to the excited state. Theoretical efforts to simulate impulsive excited state dynamics predict photoexcitation will induce activity along a number of low frequency modes. The fact that FFT analysis can be based on a single mode may result from rapid dephasing of all but one of the modes, or alternatively reflect the combined action of a number of closely spaced modes. In the spirit of choosing the simplest model which is consistent with the data, we tentatively assume an identical mode is being probed in all 4 systems. One aspect which is common to all of the active low frequency modes arising in the retinal PSB
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simulations is that they involve bends and/or torsions which are distributed over a large portion of the molecular backbone, and, therefore, should be sensitive to steric restrictions imposed by the protein. Comparative studies of retinal PSB and BR photochemistry, show that the protein surroundings increase the rate of internal conversion by a factor of at least 4, and induce specificity of isomerization around the C13 @C14 double bond. It stands to reason that this must be accomplished by constraining the conformational motions of the retinal backbone. Such structural restriction would naturally lead, on average, to increased restoring forces for displacement of the skeletal carbons and torsions around the bonds. One consequence of such restrictions would be an increase in the frequency of low lying modes which involving these degrees of freedom. This is exactly what we observe in the present study – a mode which must involve deformation of a large portion of the retinal chain, increases its frequency from 120 to 170 cm 1 upon covalent binding to the protein. Frequency modulation of stretching mode coherence with this period were recently observed in 5 fsec pulsed experiments in BR, see Ref. [26]. Another noteworthy trend in our results concerns the lack of significant changes in the frequency of PSB modulations upon locking of the C13 @C14 bond in either the cis or trans configurations. This finding is compatible with an expanding number of studies, including previous publications from our own lab, which maintain that isomerization around the active double bond, or for that mater around any of the double bonds in the retinal chain, is not the initial light driven response in BR or in the retinal PSB [9,10,18,20– 24]. Had this not been true, double bond torsions would be a major ingredient in low frequency impulsive responses, and the locking would be expected to result in significant alterations to the vibrational period. It is interesting to compare our results with those of the extensive study reported by Lin et al. [25] on the low frequency Raman active modes in the related rhodopsin and 11 cis retinal PSB. Remarkably, the lowest frequency strongly active mode is in the range of 120–130 cm 1 both in the PSB and in rhodopsin, and vibrational analysis of
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the PSB identifies this mode as being made up mainly of torsions around various CAC single and double bonds along the retinal backbone. In their experiments the ground state frequency of this mode is not found to be sensitive to incorporation of the Schiff base into the protein, and changes in band positions appear to be limited to higher frequency and more localized modes of vibration. Keeping in mind the basic differences both in the spectroscopies employed, and in the dynamics of these two related retinal proteins, the apparent discrepancy in the proteinÕs effect on low frequency retinal vibrations is worthy of continued investigation. An improvement in the time resolution of our pump–probe setup would allow an observation of many more active modes in the excited state, and is a major focus of our future experiments. None the less, the limited time resolution of the current study may be a blessing in disguise, since it allows us to concentrate attention on the very low frequency end of the vibrational spectrum which may be obscured by higher frequency modulations. As stated above, since deformations involving large portions of the retinal are involved, the effect of the protein should be most easily observed for these modes of vibration.
4. Conclusions Low frequency vibrational coherences generated in the excited state by impulsive excitation of bacteriorhodopsin and retinal protonated Schiff bases of native and locked retinals in solution are detected via periodical modulations that they induce in transient transmission signals. The pattern of these modulations which supercedes the first 100 fs of delay can be interpreted in terms of a single damped harmonic oscillation. In native all trans retinal PSB, as well as analogues with the C13 @C14 locked in the cis or trans configuration the fundamental frequency of the modulations is in the range 115–125 cm 1 whereas in bacteriorhodopsin the frequency is shifted up to 170 cm 1 . The change in frequency is suggested to result from forces imposed by the protein, which are instrumental in facilitating the bond specificity of isomerization and the enhanced rate of BR inter-
nal conversion relative to the relevant PSB. The different protein effect on the retinal vibration observed in BR and rhodopsin might reflect different chromophore-protein interactions in the two systems. In this respect we note that C13 @C14 isomerization in BR involves alterations in the Schiff base vicinity whereas such alterations were not detected in rhodopsin isomerization.
Acknowledgements We thank M. Olivucci, M. Garavelli, and S. Haacke for fruitful discussions and sharing data before publication. This work was supported by the Human Frontier Science Program (HFSP), and the Israel Science Foundation (ISF). The latter is administered by the Israel Academy of Sciences and the Humanities. The Farkas Center is supported by the Minerva Gesellschaft, GmbH, Munich, Germany.
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