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Vibrational relaxation during the retinal isomerization in Bacteriorhodopsin
2 October 1998
Chemical Physics Letters 295 Ž1998. 47–55
Vibrational relaxation during the retinal isomerization in Bacteriorhodopsin Rolf Diller
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Institut fur ¨ Experimentalphysik, Freie UniÕersitat ¨ Berlin, Arnimallee 14, D-14195 Berlin, Germany Received 14 November 1997; revised 3 August 1998
Abstract Femtosecond time-resolved optical pump–infrared probe spectroscopy was employed to characterize the ethylenic ŽC5C.-stretch vibrational dynamics Ž1498–1542 cmy1 . of the retinal chromophore in Bacteriorhodopsin ŽBR. during the initial, all-trans to 13-cis isomerization. The early branching reaction was observed, i.e. formation of the ground-state 13-cis product, K, and partial recovery of the all-trans educt state BR 570 . The BR 570 recovery occurs within 2 ps and thus is faster than K-formation Ž3–4 ps.. The IR transients are described in terms of a kinetic model involving vibrational precursors for K and for the recovering BR 570 , respectively. q 1998 Elsevier Science B.V. All rights reserved.
1. Introduction The light-induced all-trans to 13-cis isomerization of the retinal chromophore in the transmembrane protein Bacteriorhodopsin ŽBR. drives an efficient proton pump process across the cell membrane of Halobacterium salinarium w1x. The retinal cofactor is covalently bound to the lysine-216 residue of the protein by a protonated Schiff’s base. After absorbing a photon, the cofactor runs through a cyclic reaction that is completed on the millisecond timescale and involves the surrounding protein. Thereby protons are moved from the cytoplasmatic to the extracellular side, generating an electrochemical potential that is used by the bacterium to maintain its metabolism under anaerobic conditions. The initiating retinal isomerization in BR has been subject to numerous investigations for several
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Corresponding author. E-mail: [email protected]
reasons. First, it is the starting point for a photosynthetic process based on a retinal chromophore and proton transfer instead of porphyrin chromophores and electron transfer as usually found in plants, algae and bacteria. Second, the isomerization occurs very rapidly, in less than 1 ps w2,3x at a relatively high quantum yield of F f 0.64 w4–7x, obviously competing successfully with other deactivation channels like intra- and intermolecular vibrational relaxation. Third, the specific protein environment allows for a certain control of the isomerization process Žrate, quantum yield and type of isomerization., e.g. by means of steric constraints and localized charges. This was shown by femtosecond time-resolved optical transient absorption experiments employing BR mutants with chemically distinct retinal binding pockets w8x. The high variety of reaction channels in a protein environment involving ultrafast molecular dynamics poses many unanswered questions. The conventional model of the initial photoisomerization of BR is depicted in Scheme 1, as derived
0009-2614r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 8 . 0 0 9 3 2 - 4
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Scheme 1.
from transient optical spectroscopy w2,3x. The reaction starts from the light-adapted state BR 570 Žthe subscript denotes the wavelength of the absorption maximum in nm. with the cofactor in its all-trans configuration. The excited ŽFranck–Condon. electronic state S 1 ŽBRFC . relaxes within 0.2 ps to a twisted retinal configuration ŽBR) .. Then, electronic ground states ŽS 0 . are reached in a branching reaction. Thereby, the J-state is formed within 0.5 ps with a quantum yield F f 0.64, i.e. about 36% of the excited state population is fed back to the all-trans ground-state BR 570 . J, regarded as a torsionally unrelaxed and possibly vibrationally hot state w9–11x, decays within 3–4 ps to the 13-cis K 590 state, which has a much longer lifetime. Ultrafast reaction dynamics in the condensed phase as in this case have been studied intensely during the past few decades. A crucial step in any photoinduced chemical reaction is the transition from the electronically excited state back to the electronic ground-state S 0 . Excess energy, delivered by the absorbed photon will finally become distributed among all vibrational degrees of freedom, including those of the reactants’ environment. However, at early times after photoexcitation the reacting molecule will still exhibit a non-equilibrated energy distribution. With the steady progress in time resolution and spectral accessibility of lasers, especially in the midinfrared region, a more detailed, microscopic picture of the reaction dynamics as well as information about the parameters governing the reaction rates and efficiency can be obtained. Vibrational spectra usually represent a molecular fingerprint concerning structure and dynamics that cannot be easily accessed by electronic absorption spectroscopy. In or-
der to more clearly understand the ultrafast reaction dynamics and mechanisms underlying the complex BR photoisomerization, infrared ŽIR. spectroscopic methods with high time resolution have been developed during the past several years and applied to BR. In a first step w12x, picosecond IR laser technology was employed to obtain the first BR–K infrared difference spectra at ambient temperature. In Ref. w13x, the same method was extended to the femtosecond regime and the isomerization could be followed by monitoring the vibrational dynamics of the ŽC5NH.-stretch vibration of the chromophore. In this Letter, we present femtosecond transient IR experiments on BR with special emphasis on the spectral region of the chromophore ethylenic ŽC5C.-stretch vibration between 1498 and 1542 cmy1 . The frequency of this IR-active mode is sensitive to the degree of p-electron delocalization along the chain of conjugated double bonds. Since the isomerization alters the interaction between the polar protein environment and the protonated Schiff’s base and hence the p-electron density, the ethylenic ŽC5C.-stretch vibration is specifically suited to monitor the isomerization dynamics. We follow the vibrational dynamics which occur as the chromophore excited state population is fed forward to the isomerized K-state with a quantum yield of about 0.64 or back to the all-trans electronic ground state. Further, we relate the results to earlier investigations of the chromophoric ŽC5NH.-stretch vibration w13x, time-resolved resonance Raman ŽRR. studies on BR w9–11x and studies on retinal isomerization in solution w14x. In order to interpret the fast IR kinetics taken within a few picoseconds after excitation, it is important to consider the various phenomena which can
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contribute to the observed absorbance transients: Ž1. chemically distinct molecular species; Ž2. ‘hot bands’, i.e. vibrational transitions originating in vibrationally excited states which contribute to the observed signal at spectral positions according to the mode anharmonicity; Ž3. anharmonic coupling of the specific mode to other modes of the molecule that causes shifts and deformation of the absorption bands at elevated temperature; Ž4. stimulated IR emission if the molecule, on its way to thermalization, runs through a state of an inverted vibrational population distribution.
2. Materials and methods 2.1. Transient IR spectroscopy Õia femtosecond optical gating of a continuous-waÕe IR probe Optical pump, IR probe experiments were performed employing a chirped pulse amplification titanium:sapphire ŽTi:Sa. laser system ŽClarkrMXR. and a home-built liquid nitrogen-cooled carbon monoxide ŽCO. laser. To time-resolve the pump-induced IR absorbance changes, the continuous-wave Žcw. IR probe beam is optically gated by a weak femtosecond pulse in a non-linear crystal after sample transmission. The short pulses of the Ti:Sa laser system Žduration - 180 fs FWHM, energy f 0.6 mJ, rep. rate s 1 kHz. at 790 nm are split into a strong pulse and a weak gate pulse. The strong pulse is focused into a water flow cell, generating a spectral continuum from which the pump pulse at 540 nm is extracted by an interference filter. The pump beam is focused Žca. 270 mm. into the sample non-coaxially Žca. 78. with the IR probe beam Žca. 70 mm.. The transmitted IR is collimated and focused into an AgGaS 2 crystal Ž0.5 mm length.. There, pulses at the sum frequency of the IR and the gate pulse are generated. They are separated from the fundamental frequencies spectrally and via their rotated polarization and fed to a photomultiplier tube. Chopping the pump pulses at half the system repetition rate and using lock-in detection provides the pump induced absorbance change. The IR-probe wavelength is tuned by rotating the grating that serves as the output coupler of the CO laser. The
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time interval between pump and probe event is varied by an optical delay line. As described in the preliminary work w15x, the CO laser was built in order to extend the region of probe wavelengths below ca. 1560 cmy1 , the typical limit of the tuning range of commercially available CO lasers. By cooling the plasma tube with liquid nitrogen, output power increases and the lower tuning range limit shifts down. Thus, vibrational modes like ŽC5C.- and ŽC–C.-stretch vibrations become accessible. The laser runs in a single line mode and the spacing between two spectrally adjacent lines is 2–5 cmy1 . Strong lines reach a power of several hundred milliWatts and the transverse mode is of fairly good TEM00 quality. In order to exclude heating of the sample by the high cw IR power, the IR beam is chopped by an extracavity acousto-optical modulator ŽIntra Action., yielding pulses of 1.5 ms duration at the laser system repetition rate and effectively reducing the duty cycle. The instrument response function after optical excitation and the experimental time zero is established from the IR absorption response of a thin silicon wafer, replacing the sample. When measured as a function of the delay time, the IR transmission forms a sigmoid-shaped curve. From this, the width of the instrument response function is determined to 0.62 ps ŽFWHM.. The IR absorbance is usually proportional to the population density of the various chemical species that absorb at the chosen probe frequency. When the time resolution approaches the dephasing times ŽT2 . of the probed vibrational states, dephasing processes begin to alter the time course of the detected signal. Two types of phenomena are expected in this kind of experiment, i.e. when time resolving the transient IR absorbance by optically gating a narrow-band cw probe beam. First, a bleach signal occurs simultaneously with the pump pulse if the vibrational absorber is spectrally removed far enough from the probe frequency by the optical excitation, e.g. a shift in the electronically excited state or by an ultrafast chemical reaction. Second, a vibrational absorber that is shifted instantaneously from far out into resonance with the IR probe frequency, causes the detected IR absorbance signal to rise with the time constant T2 . In intermediate cases, the detected transient IR sig-
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nals will be modulated by oscillations Žbeat frequencies.. Their observation in the presented data is prevented by the signal-to-noise ratio. If several vibrational absorbers are involved, the corresponding signals will appear superimposed according to the population kinetics. The homogenous widths of the vibrational bands investigated in this study typically amount to 15–20 cmy1 corresponding to T2 of 0.7–0.5 ps. Since this is approximately the experimental system response time, the T2 time-related phenomena are treated qualitatively and not incorporated quantitatively w16,17x into the model. 2.2. Sample preparation Patches of purple membrane ŽPM. of Halobacterium salinarium strain ET1001 were dispersed with water on a calcium fluoride window Ž1.5 in. diameter.. In order to reduce the background absorption in the mid-IR due to bulk water, but at the same time maintain the photocycle, most of the water was evaporated under a 97% humidity atmosphere. This yields a homogenous film of PM. However, even under these conditions, the background IR absorption in the sample Ž1600–1500 cmy1 . due to the vibrational amide I and amide II protein bands is two to three orders of magnitude higher Ž1 OD unit. than the pump-induced absorbance changes ŽmOD scale.. The latter are small since the IR absorption crosssections of the probed vibrational transitions are typically two orders of magnitude smaller than optical absorption cross-sections of allowed electronic transitions. The sample was sealed by a second window and mounted into a holder, rotating and moving up and down during the experiment in order to provide for a fully relaxed but light-adapted sample volume at each laser shot, i.e. each millisecond. The experiments were performed at ambient temperatures.
3. Results IR kinetics were taken in the region between 1498 and 1542 cmy1 , characteristic for the ethylenic ŽC5C.-stretch vibration. They are shown Fig. 1a,b. Displayed is the absorbance difference of the optically excited sample and the unperturbed sample, i.e.
negative signals Žbleach. are due to vibrational transitions belonging to the parent state BR 570 which are spectrally removed under photoexcitation. Positive signals correspond to IR active transitions created by the photoexcitation at the respective probe wavenumber. Kinetics taken at 1498 and 1542 cmy1 Ždata not shown. gave flat curves with zero amplitude within the experimental error. The transients are normalized with respect to a standard. Thus, difference spectra can be derived. Wavenumbers are given with an accuracy of "1 cmy1 . The overall features of the kinetics can be described as follows. At early times a strong bleach signal at about 1529 cmy1 is formed and a weaker positive absorption band appears at about 1511 cmy1 . The bleach recovers partially with the delay time and begins to turn into a positive signal on the low energy side between 1526 and 1519 cmy1 . The kinetics at wavenumbers below ca. 1515 cmy1 appear as positive signals and decay partially. The kinetics are modeled according to Scheme 2. This is similar to Scheme 1 proposed in Ref. w2,3x, but includes two intermediate states, BR educt ) and BR product ) , preceding the reconstituted, fully relaxed BR 570 ground state and K, respectively. The various electronic states of Scheme 2, connected by first-order rate constants, are associated with vibrational states, i.e. ethylenic ŽC5C.-stretch vibrations of the corresponding molecular species. They are the spectroscopic observables in this work. Each of them is characterized by a Lorentzian lineshape with spectral position, spectral width and normalized extinction coefficient. The ethylenic ŽC5C.-stretch vibrational band of the cofactor in BR 570 is found at 1529 cmy1 w18x. BRFC and BR) are considered as kinetically significant species but not as IR–spectroscopic observables Žsee Section 4.. In the isomerized K-state the ethylenic ŽC5C.-stretch vibrational band splits into two adjacent bands at 1519 and 1530 cmy1 , according to RR experiments w19x and model calculations w20x. A precursor of the K-state ŽC5C.-stretch ŽBR product ) . was assigned in Ref. w21x to a RR band with its main contributions at 1518 cmy1 . In the same work, a shoulder of this band at 1510 cmy1 was tentatively assigned to another early intermediate state. The introduction of the state BR educt ) is required as the minimal model extension necessary to account for a kinetic precur-
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Fig. 1. Ža. Transient absorption signals of Bacteriorhodopsin in the infrared at 1502, 1505, 1511 and 1515 cmy1 , respectively. Photoexcitation at 540 nm. Data were taken at room temperature. The solid lines display the model described in the text. Žb. Transient absorption signals of Bacteriorhodopsin in the infrared at 1519, 1526, 1529, 1533 and 1536 cmy1 , respectively. Photoexcitation at 540 nm. Data were taken at room temperature. The solid lines display the model described in the text.
sor of the reformed BR 570 . It will turn out as a result of this work that it can be attributed to a vibrationally not fully relaxed ethylenic stretch vibration of the reconstituted BR 570 with absorbance strength at about 1512 cmy1 .
Within Scheme 2 the IR transients are interpreted as follows Žcp., Fig. 2.. The initial bleach at around 1529 cmy1 is caused by the optical excitation process whereupon the IR absorbance of the ŽC5C.stretch vibration due to the all-trans ground state is
Scheme 2.
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Fig. 2. IR difference spectra of Bacteriorhodopsin at various delay times as derived from the model described in the text.
probably shifted outside w22x the investigated spectral range. Corresponding to the quantum yield of F f 0.64, 64% of the excited BR population ŽBR) . undergoes rapid isomerization to a 13-cis configuration, with the emergence of new absorption strength
ŽBR product ) . in its electronic ground state at energies between 1500 and 1520 cmy1 that undergoes changes on a longer time scale, forming the K-state. Another fraction Ž36%. of the electronically excited molecules is fed back to an unrelaxed educt Žall-trans. configuration ŽBR educt ) . of the electronic ground state and finally to the BR 570 lowest ground state. Correspondingly, the initial bleach at 1529 cmy1 recovers partially. After about 12 ps signal levels are reached that are ‘steady state’ on the timescale of our experiment. In order to model and obtain best agreement with the experimental curves, the whole set of parameters of Scheme 2 Žrate constants, spectral positions and widths, extinction coefficients and quantum efficiency. as listed in Table 1 were divided into two groups. The first includes those parameters that are well accepted in the literature ŽScheme 1. or cannot be well deduced from our data due to insufficient time resolution or signal-to-noise ratio. The second includes those parameters that were deduced from the simulation procedure. The simulation comprised the convolution with the 0.62 ps system response. Since they cannot be fit from the IR kinetics, the time constant for the BR)-formation was fixed at 0.2 ps and that for K-formation was fixed at 3 ps w2,3,13x. The isomerization quantum yield was fixed at F s 0.64. The simulation of the experimental kinetics employing this set of parameters ŽTable 1. is in-
Table 1 ŽC5C.-stretch vibrational band parameters ŽLorentzian lineshape. and kinetic parameters used for the simulation of the IR transients as depicted in Fig. 1a,b Žsolid lines.
BR 570 BR product ) a BR educt ) Ka Kb
Center wavenumber
FWHM
Normalized ext. coefficient Žau.
1529 cmy1 1514 (2) cm y 1 1512 (3) cm y 1 1519 cmy1 1530 cmy1
17 cmy1 18 (2) cm y 1 19 (2) cm y 1 17 (2) cm y 1 17 (2) cm y 1
1 0.8 (2) 0.7 (2) 0.45 (15) 0.55 (15)
Regular typeface: parameters taken from the literature Žcp. text.; italics: parameters deduced from the simulation procedure. The relative contributions of BR educt ) and BR product ) cannot be deduced from the data Žcp. Section 4.. Estimated errors for which the simulations gave acceptable results are given in parentheses. Quantum efficiency for K-formation: 0.64. Formation of BR) : Ž0.2 ps.y1 . Decay of BR) : Ž0.8 ps.y1 , taking into account vibrational dephasing for the appearance of the absorption bands. Formation of K: Ž3 ps.y1 . Decay of BR educt ) : (2(1) ps)y 1 . a
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cluded in Fig. 1a,b as solid lines. From the simulation results difference spectra at various delay times were derived as shown in Fig. 2. Except for the kinetics at 1519 and 1511 cmy1 , respectively, the simulations are in quantitative agreement with the data.
4. Discussion The IR difference spectrum at 12 ps ŽFig. 2. is in accordance with low-temperature FTIR difference spectra, e.g. w23x, where the K-state is trapped cryogenically. At this time all initially excited BR 570 molecules are either fed via K towards the succeeding reaction steps of the photocycle or back to BR 570 . The early part of the decay at 1515 cmy1 is interpreted as the decay of a ŽC5C.-stretch vibrational state, generated from BR) . From its spectral position, it could be due to either the K precursor, BR product ) , or the BR 570 precursor, BR educt ) , e.g. a transiently populated vibrationally hot state of the 1529 cmy1 band of BR 570 or an otherwise not fully relaxed BR 570 ground state. In fact, the overtone of the ethylenic ŽC5C.-stretch Žat 1527 cmy1 . of BR 570 was tentatively assigned w24x to a RR band at 3036 cmy1 , rendering the Õ s 1 to Õ s 2 transition at 1509 cmy1 . The simulation showed that the relative contributions of BR educt ) and BR product ) cannot be distinguished but contribute both to the transient absorption around 1515 cmy1 . However, both states are kinetically necessary in the model in order to account for the observed different formation time constants of the recovering BR 570 Ž2 ps. and K Ž3–4 ps., respectively. A crucial free parameter in our model is the extinction coefficient of the K b component at 1530 cmy1 ŽTable 1.. Its relative intensity as compared to that of the K a component is only known for RR spectra w19x and thus cannot be used here. The numeric value of the K b extinction coefficient was chosen as given in Table 1 in order to account quantitatively for the recovery at 1529 cmy1 according to a quantum yield of 0.64. If there were no absorbing species in this spectral region partially canceling the bleach signal at later times, the apparent recovery could not be accounted for quantita-
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tively by the quantum yield of 0.64. Instead, a quantum yield of about 0.2 would have to be concluded. Such a low value is unreasonable for the BR photoisomerization. In order to rule out experimental or sample artifacts we performed a control experiment at 1640 cmy1 Ždata not shown.. The kinetics, taken at this wavenumber, monitor the bleach and partial recovery of the cofactor ŽC5NH.-stretch vibrational band. Here, the corresponding 13-cis product band is probably shifted to about 1607 cmy1 and hence the partial absorbance recovery reflects quantitatively the quantum yield as was shown in Ref. w13x. We reproduced the earlier observed absorbance recovery of 40% of the initial bleach amplitude within 10%. Another explanation for the initial strong bleach signal, still in accordance with a quantum yield of 0.64, could be a stimulated emission signal, i.e an inverted Õ s 1 to Õ s 0 transition of the ŽC5C.stretch vibration at 1529 cmy1 . However, in view of the high number of vibrational degrees of freedom of the retinal chromophore such a distribution, even at early times, is considered unlikely. In our model, thermally induced broadening and shifts of vibrational bands due to anharmonic coupling among the cofactor vibrational modes are not implemented. No attempts were made to determine a vibrational temperature of the chromophore. This would require a detailed knowledge of the anharmonicity constants of the chromophore vibrational modes and is beyond the scope of this work. Hence, temperature effects are discussed only qualitatively. Thermally induced shifts would, in general, occur towards the low energy side of the specific mode. If the band shift of 5 cmy1 , observed from 1514 cmy1 ŽBR product ) . to 1519 cmy1 Ž K a follows the linear relationship between the frequency of the ethylenic stretch vibration and the lmax of the chromophore w25,26x. between 1 and 12 ps is interpreted as a temperature related shift of a 13-cis ŽC5C.-stretch mode, then, following the lines as described in Ref. w14x, the temperature cannot be high Žca. 400 K.. In that study, the isomerization of Schiff’s base retinal in solution was investigated by means of femtosecond time-resolved IR spectroscopy and from the transient IR band shapes an initial vibrational temperature of 550 K was suggested. As demonstrated here, the overall features of the observed IR kinetics can be explained straightfor-
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wardly by a scheme similar to the conventional model of the retinal all-trans to 13-cis isomerization in BR, well established in the literature. However, this model has been questioned recently by Refs. w27x and w28x. There, a model was suggested, consisting of three electronic potential energy surfaces. The essential feature of this model is that under electronic excitation the system is kept for a few hundred femtoseconds near the Franck–Condon region and then passes rapidly through the reactive region, i.e. the twisted configuration. The scenarios described in the two models provide different starting conditions for the energy distribution of the cofactor vibrational modes immediately after the S1 to S 0 transition, i.e. the energy redistribution among all cofactor modes and the energy relaxation Že.g. thermalization. within each mode. This issue was partly addressed in Ref. w11x. There, a model was found consistent with the BR picosecond-transient anti-Stokes RR data available w10x that assumed the photon excess energy distributed exclusively among the Franck–Condon active modes and not among all the cofactor vibrational modes for a few picoseconds after excitation, leading to a relatively high vibrational temperature, possibly up to 1470 K. However, our data together with those described in Ref. w13x suggest, based on the relatively small observed band shifts, that the ŽC5C.- and ŽC5NH.-stretch modes of the retinal chromophore in BR have a temperature not much higher than 400 K at about 1 ps after photoexcitation. Although this is a contradiction in terms of temperature, the authors of Ref. w10x give the same time constant of about 3 ps for the cooling process. In addition, a molecular dynamics simulation study of the early picosecond events in the BR photocycle w29x suggests that the rise time Ž2.8 ps. of the K-intermediate is determined by cooling of the vibrationally hot Ž350 K. chromophore. It should be pointed out that a time constant of 2 ps was found for the recovery of the all-trans ŽC5C.-stretch vibration at 1529 cmy1 . This is in accordance with the corresponding time constant for the recovery of the relaxed all-trans ŽC5NH.-stretch vibration at 1640 cmy1 as found in Ref. w13x. Thus, relaxation back to BR 570 is faster than K-formation which takes 3–4 ps w2,3x. It seems unlikely that vibrational relaxation in the all-trans or 13-cis species, respectively, takes different times. It is rather
plausible that the reaction towards the fully isomerized 13-cis state takes longer than the reconstitution of the all-trans state because the former involves more structural changes of the surrounding protein than the latter. This suggests that the kinetic precursor of K is not simply a hot K but that the BR product ) –K transition involves other processes, possibly torsional relaxation of the polyene chain as suggested in Refs. w9,10x and protein relaxation.
5. Conclusions Although overlapping vibrational bands complicate the interpretation of the transient IR spectra, the BR 570 ground-state recovery that occurs due to the non-unity photoisomerization quantum yield can be separated spectrally and kinetically from the 13-cis product reaction branch. K-formation and BR 570 recovery occur with different time constants, i.e. 2 and 3–4 ps, respectively. Thus, corresponding precursor states, BR product ) and BR educt ) , respectively, have to be introduced, suggesting that what has been subsumed under the ‘J’-state from optical transient spectroscopy is constituted by contributions of a relaxing 13-cis product state and a recombining Žrecovering. and relaxing all-trans educt state. In contrast to the dynamics of the ŽC5NH.-stretch vibration w13x at 1640 cmy1 , the apparent amplitude of the partial recovery of the BR 570 ŽC5C.-stretch vibration at 1529 cmy1 does not correspond to the isomerization quantum yield of 0.64. This gives evidence for a ŽC5C.-stretch vibration doublet of K with band positions at 1519 and 1530 cmy1 , respectively. Most consistent with our data, including earlier results from Ref. w13x and taking into account the results in Ref. w14x, is a retinal chromophore which reaches a relatively low vibrational temperature within about 1 ps after photoisomerization. A higher temperature has been suggested for trans–cis isomerization of retinal w14x in solution. We have shown that by means of ultrafast IR absorption spectroscopy it is possible to disentangle the pathways of the electronic ground-state reaction dynamics of the all-trans to 13-cis photoisomerization in BR with sub-picosecond time resolution.
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Acknowledgements The author would like to thank Prof. D. Stehlik for encouragement and support and Prof. M. Heyn and I. Wallat for generously providing the BR samples. Extensive contributions to the entire experimental set-up and measurements, as well as the computer program used for fitting the experimental data are due to Dr. Reiner Dziewior in partial fulfillment for his Ph.D. thesis ŽFU Berlin 1998.. This research was supported by the Deutsche Forschungsgemeinschaft ŽDi 405r3-1,-4..
References w1x D. Oesterhelt, W. Stoeckenius, Proc. Natl. Acad. Sci. USA 70 Ž1973. 2853. w2x J. Dobler, W. Zinth, W. Kaiser, D. Oesterhelt, Chem. Phys. Lett. 144 Ž1988. 215. w3x R.A. Mathies, C.H. Brito Cruz, W.T. Pollard, C.V. Shank, Science 240 Ž1988. 777. w4x G. Schneider, R. Diller, M. Stockburger, Chem. Phys. 131 Ž1989. 17. w5x R. Govindjee, S.P. Balashov, T.G. Ebrey, Biophys. J. 58 Ž1990. 597. w6x J. Tittor, D. Oesterhelt, FEBS Lett. 263 Ž1990. 269. w7x S.L. Logunov, M.A. El-Sayed, L. Song, J. Phys. Chem. 100 Ž1996. 2391. w8x L. Song, M.A. El-Sayed, J.K. Lanyi, Science 261 Ž1993. 891. w9x T.L. Brack, G.H. Atkinson, J. Phys. Chem. 95 Ž1991. 2351.
55
w10x S.J. Doig, P.J. Reid, R.A. Mathies, J. Phys. Chem. 95 Ž1991. 6372. w11x A.P. Shreve, R.A. Mathies, J. Phys. Chem. 99 Ž1995. 7285. w12x R. Diller, M. Iannone, B.R. Cowen, S. Maiti, R.A. Bogomolni, R.M. Hochstrasser, Biochemistry 31 Ž1992. 5567. w13x R. Diller, S. Maiti, G.C. Walker, B.R. Cowen, R. Pippenger, R.A. Bogomolni, R.M. Hochstrasser, Chem. Phys. Lett. 241 Ž1995. 109. w14x P. Hamm, M. Zurek, T. Roschinger, H. Patzelt, D. Oester¨ helt, W. Zinth, Chem. Phys. Lett. 268 Ž1997. 180. w15x R. Dziewior, R. Diller, Ber. Bunsenges. Phys. Chem. 100 Ž1996. 2103. w16x B. Locke, R. Diller, R.M. Hochstrasser, Adv. Spectrosc. 21 Ž1993. 1. w17x K. Wynne, R.M. Hochstrasser, Chem. Phys. 193 Ž1995. 211. w18x S.O. Smith, J. Lugtenburg, R.A. Mathies, J. Membr. Biol. 85 Ž1985. 95. w19x T. Althaus, W. Eisfeld, R. Lohrmann, M. Stockburger, Isr. J. Chem. 35 Ž1995. 227. w20x M. Grossjean, P. Tavan, K. Schulten, J. Phys. Chem. 94 Ž1990. 8059. w21x R.v.d. Berg, D.J. Jang, H.C. Bitting, M.A. El-Sayed, Biophys. J. 58 Ž1990. 135. w22x V.F. Kamalov, T.M. Masciangioli, M.A. El-Sayed, J. Phys. Chem. 100 Ž1996. 2762. w23x F. Siebert, W. Mantele, Eur. J. Biochem. 130 Ž1983. 565. ¨ w24x A.B. Myers, R.A. Harris, R.A. Mathies, J. Chem. Phys. 79 Ž1983. 603. w25x L. Rimai, M.E. Heyde, D. Gill, J. Am. Chem. Soc. 95 Ž1973. 4493. w26x B. Aton, A.G. Doukas, R.H. Callendar, B. Becher, T.G. Ebrey, Biochemistry 16 Ž1977. 2995. w27x G. Haran, K. Wynne, A. Xie, Q. He, M. Chance, R.M. Hochstrasser, Chem. Phys. Lett. 261 Ž1996. 389. w28x K.C. Hasson, F. Gai, P.A. Anfinrud, Proc. Natl. Acad. Sci. USA 93 Ž1996. 15124. w29x D. Xu, C. Martin, K. Schulten, Biophys. J. 70 Ž1996. 453.
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Vibrational relaxation during the retinal isomerization in Bacteriorhodopsin Rolf Diller
)
Institut fur ¨ Experimentalphysik, Freie UniÕersitat ¨ Berlin, Arnimallee 14, D-14195 Berlin, Germany Received 14 November 1997; revised 3 August 1998
Abstract Femtosecond time-resolved optical pump–infrared probe spectroscopy was employed to characterize the ethylenic ŽC5C.-stretch vibrational dynamics Ž1498–1542 cmy1 . of the retinal chromophore in Bacteriorhodopsin ŽBR. during the initial, all-trans to 13-cis isomerization. The early branching reaction was observed, i.e. formation of the ground-state 13-cis product, K, and partial recovery of the all-trans educt state BR 570 . The BR 570 recovery occurs within 2 ps and thus is faster than K-formation Ž3–4 ps.. The IR transients are described in terms of a kinetic model involving vibrational precursors for K and for the recovering BR 570 , respectively. q 1998 Elsevier Science B.V. All rights reserved.
1. Introduction The light-induced all-trans to 13-cis isomerization of the retinal chromophore in the transmembrane protein Bacteriorhodopsin ŽBR. drives an efficient proton pump process across the cell membrane of Halobacterium salinarium w1x. The retinal cofactor is covalently bound to the lysine-216 residue of the protein by a protonated Schiff’s base. After absorbing a photon, the cofactor runs through a cyclic reaction that is completed on the millisecond timescale and involves the surrounding protein. Thereby protons are moved from the cytoplasmatic to the extracellular side, generating an electrochemical potential that is used by the bacterium to maintain its metabolism under anaerobic conditions. The initiating retinal isomerization in BR has been subject to numerous investigations for several
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Corresponding author. E-mail: [email protected]
reasons. First, it is the starting point for a photosynthetic process based on a retinal chromophore and proton transfer instead of porphyrin chromophores and electron transfer as usually found in plants, algae and bacteria. Second, the isomerization occurs very rapidly, in less than 1 ps w2,3x at a relatively high quantum yield of F f 0.64 w4–7x, obviously competing successfully with other deactivation channels like intra- and intermolecular vibrational relaxation. Third, the specific protein environment allows for a certain control of the isomerization process Žrate, quantum yield and type of isomerization., e.g. by means of steric constraints and localized charges. This was shown by femtosecond time-resolved optical transient absorption experiments employing BR mutants with chemically distinct retinal binding pockets w8x. The high variety of reaction channels in a protein environment involving ultrafast molecular dynamics poses many unanswered questions. The conventional model of the initial photoisomerization of BR is depicted in Scheme 1, as derived
0009-2614r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 8 . 0 0 9 3 2 - 4
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Scheme 1.
from transient optical spectroscopy w2,3x. The reaction starts from the light-adapted state BR 570 Žthe subscript denotes the wavelength of the absorption maximum in nm. with the cofactor in its all-trans configuration. The excited ŽFranck–Condon. electronic state S 1 ŽBRFC . relaxes within 0.2 ps to a twisted retinal configuration ŽBR) .. Then, electronic ground states ŽS 0 . are reached in a branching reaction. Thereby, the J-state is formed within 0.5 ps with a quantum yield F f 0.64, i.e. about 36% of the excited state population is fed back to the all-trans ground-state BR 570 . J, regarded as a torsionally unrelaxed and possibly vibrationally hot state w9–11x, decays within 3–4 ps to the 13-cis K 590 state, which has a much longer lifetime. Ultrafast reaction dynamics in the condensed phase as in this case have been studied intensely during the past few decades. A crucial step in any photoinduced chemical reaction is the transition from the electronically excited state back to the electronic ground-state S 0 . Excess energy, delivered by the absorbed photon will finally become distributed among all vibrational degrees of freedom, including those of the reactants’ environment. However, at early times after photoexcitation the reacting molecule will still exhibit a non-equilibrated energy distribution. With the steady progress in time resolution and spectral accessibility of lasers, especially in the midinfrared region, a more detailed, microscopic picture of the reaction dynamics as well as information about the parameters governing the reaction rates and efficiency can be obtained. Vibrational spectra usually represent a molecular fingerprint concerning structure and dynamics that cannot be easily accessed by electronic absorption spectroscopy. In or-
der to more clearly understand the ultrafast reaction dynamics and mechanisms underlying the complex BR photoisomerization, infrared ŽIR. spectroscopic methods with high time resolution have been developed during the past several years and applied to BR. In a first step w12x, picosecond IR laser technology was employed to obtain the first BR–K infrared difference spectra at ambient temperature. In Ref. w13x, the same method was extended to the femtosecond regime and the isomerization could be followed by monitoring the vibrational dynamics of the ŽC5NH.-stretch vibration of the chromophore. In this Letter, we present femtosecond transient IR experiments on BR with special emphasis on the spectral region of the chromophore ethylenic ŽC5C.-stretch vibration between 1498 and 1542 cmy1 . The frequency of this IR-active mode is sensitive to the degree of p-electron delocalization along the chain of conjugated double bonds. Since the isomerization alters the interaction between the polar protein environment and the protonated Schiff’s base and hence the p-electron density, the ethylenic ŽC5C.-stretch vibration is specifically suited to monitor the isomerization dynamics. We follow the vibrational dynamics which occur as the chromophore excited state population is fed forward to the isomerized K-state with a quantum yield of about 0.64 or back to the all-trans electronic ground state. Further, we relate the results to earlier investigations of the chromophoric ŽC5NH.-stretch vibration w13x, time-resolved resonance Raman ŽRR. studies on BR w9–11x and studies on retinal isomerization in solution w14x. In order to interpret the fast IR kinetics taken within a few picoseconds after excitation, it is important to consider the various phenomena which can
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contribute to the observed absorbance transients: Ž1. chemically distinct molecular species; Ž2. ‘hot bands’, i.e. vibrational transitions originating in vibrationally excited states which contribute to the observed signal at spectral positions according to the mode anharmonicity; Ž3. anharmonic coupling of the specific mode to other modes of the molecule that causes shifts and deformation of the absorption bands at elevated temperature; Ž4. stimulated IR emission if the molecule, on its way to thermalization, runs through a state of an inverted vibrational population distribution.
2. Materials and methods 2.1. Transient IR spectroscopy Õia femtosecond optical gating of a continuous-waÕe IR probe Optical pump, IR probe experiments were performed employing a chirped pulse amplification titanium:sapphire ŽTi:Sa. laser system ŽClarkrMXR. and a home-built liquid nitrogen-cooled carbon monoxide ŽCO. laser. To time-resolve the pump-induced IR absorbance changes, the continuous-wave Žcw. IR probe beam is optically gated by a weak femtosecond pulse in a non-linear crystal after sample transmission. The short pulses of the Ti:Sa laser system Žduration - 180 fs FWHM, energy f 0.6 mJ, rep. rate s 1 kHz. at 790 nm are split into a strong pulse and a weak gate pulse. The strong pulse is focused into a water flow cell, generating a spectral continuum from which the pump pulse at 540 nm is extracted by an interference filter. The pump beam is focused Žca. 270 mm. into the sample non-coaxially Žca. 78. with the IR probe beam Žca. 70 mm.. The transmitted IR is collimated and focused into an AgGaS 2 crystal Ž0.5 mm length.. There, pulses at the sum frequency of the IR and the gate pulse are generated. They are separated from the fundamental frequencies spectrally and via their rotated polarization and fed to a photomultiplier tube. Chopping the pump pulses at half the system repetition rate and using lock-in detection provides the pump induced absorbance change. The IR-probe wavelength is tuned by rotating the grating that serves as the output coupler of the CO laser. The
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time interval between pump and probe event is varied by an optical delay line. As described in the preliminary work w15x, the CO laser was built in order to extend the region of probe wavelengths below ca. 1560 cmy1 , the typical limit of the tuning range of commercially available CO lasers. By cooling the plasma tube with liquid nitrogen, output power increases and the lower tuning range limit shifts down. Thus, vibrational modes like ŽC5C.- and ŽC–C.-stretch vibrations become accessible. The laser runs in a single line mode and the spacing between two spectrally adjacent lines is 2–5 cmy1 . Strong lines reach a power of several hundred milliWatts and the transverse mode is of fairly good TEM00 quality. In order to exclude heating of the sample by the high cw IR power, the IR beam is chopped by an extracavity acousto-optical modulator ŽIntra Action., yielding pulses of 1.5 ms duration at the laser system repetition rate and effectively reducing the duty cycle. The instrument response function after optical excitation and the experimental time zero is established from the IR absorption response of a thin silicon wafer, replacing the sample. When measured as a function of the delay time, the IR transmission forms a sigmoid-shaped curve. From this, the width of the instrument response function is determined to 0.62 ps ŽFWHM.. The IR absorbance is usually proportional to the population density of the various chemical species that absorb at the chosen probe frequency. When the time resolution approaches the dephasing times ŽT2 . of the probed vibrational states, dephasing processes begin to alter the time course of the detected signal. Two types of phenomena are expected in this kind of experiment, i.e. when time resolving the transient IR absorbance by optically gating a narrow-band cw probe beam. First, a bleach signal occurs simultaneously with the pump pulse if the vibrational absorber is spectrally removed far enough from the probe frequency by the optical excitation, e.g. a shift in the electronically excited state or by an ultrafast chemical reaction. Second, a vibrational absorber that is shifted instantaneously from far out into resonance with the IR probe frequency, causes the detected IR absorbance signal to rise with the time constant T2 . In intermediate cases, the detected transient IR sig-
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nals will be modulated by oscillations Žbeat frequencies.. Their observation in the presented data is prevented by the signal-to-noise ratio. If several vibrational absorbers are involved, the corresponding signals will appear superimposed according to the population kinetics. The homogenous widths of the vibrational bands investigated in this study typically amount to 15–20 cmy1 corresponding to T2 of 0.7–0.5 ps. Since this is approximately the experimental system response time, the T2 time-related phenomena are treated qualitatively and not incorporated quantitatively w16,17x into the model. 2.2. Sample preparation Patches of purple membrane ŽPM. of Halobacterium salinarium strain ET1001 were dispersed with water on a calcium fluoride window Ž1.5 in. diameter.. In order to reduce the background absorption in the mid-IR due to bulk water, but at the same time maintain the photocycle, most of the water was evaporated under a 97% humidity atmosphere. This yields a homogenous film of PM. However, even under these conditions, the background IR absorption in the sample Ž1600–1500 cmy1 . due to the vibrational amide I and amide II protein bands is two to three orders of magnitude higher Ž1 OD unit. than the pump-induced absorbance changes ŽmOD scale.. The latter are small since the IR absorption crosssections of the probed vibrational transitions are typically two orders of magnitude smaller than optical absorption cross-sections of allowed electronic transitions. The sample was sealed by a second window and mounted into a holder, rotating and moving up and down during the experiment in order to provide for a fully relaxed but light-adapted sample volume at each laser shot, i.e. each millisecond. The experiments were performed at ambient temperatures.
3. Results IR kinetics were taken in the region between 1498 and 1542 cmy1 , characteristic for the ethylenic ŽC5C.-stretch vibration. They are shown Fig. 1a,b. Displayed is the absorbance difference of the optically excited sample and the unperturbed sample, i.e.
negative signals Žbleach. are due to vibrational transitions belonging to the parent state BR 570 which are spectrally removed under photoexcitation. Positive signals correspond to IR active transitions created by the photoexcitation at the respective probe wavenumber. Kinetics taken at 1498 and 1542 cmy1 Ždata not shown. gave flat curves with zero amplitude within the experimental error. The transients are normalized with respect to a standard. Thus, difference spectra can be derived. Wavenumbers are given with an accuracy of "1 cmy1 . The overall features of the kinetics can be described as follows. At early times a strong bleach signal at about 1529 cmy1 is formed and a weaker positive absorption band appears at about 1511 cmy1 . The bleach recovers partially with the delay time and begins to turn into a positive signal on the low energy side between 1526 and 1519 cmy1 . The kinetics at wavenumbers below ca. 1515 cmy1 appear as positive signals and decay partially. The kinetics are modeled according to Scheme 2. This is similar to Scheme 1 proposed in Ref. w2,3x, but includes two intermediate states, BR educt ) and BR product ) , preceding the reconstituted, fully relaxed BR 570 ground state and K, respectively. The various electronic states of Scheme 2, connected by first-order rate constants, are associated with vibrational states, i.e. ethylenic ŽC5C.-stretch vibrations of the corresponding molecular species. They are the spectroscopic observables in this work. Each of them is characterized by a Lorentzian lineshape with spectral position, spectral width and normalized extinction coefficient. The ethylenic ŽC5C.-stretch vibrational band of the cofactor in BR 570 is found at 1529 cmy1 w18x. BRFC and BR) are considered as kinetically significant species but not as IR–spectroscopic observables Žsee Section 4.. In the isomerized K-state the ethylenic ŽC5C.-stretch vibrational band splits into two adjacent bands at 1519 and 1530 cmy1 , according to RR experiments w19x and model calculations w20x. A precursor of the K-state ŽC5C.-stretch ŽBR product ) . was assigned in Ref. w21x to a RR band with its main contributions at 1518 cmy1 . In the same work, a shoulder of this band at 1510 cmy1 was tentatively assigned to another early intermediate state. The introduction of the state BR educt ) is required as the minimal model extension necessary to account for a kinetic precur-
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Fig. 1. Ža. Transient absorption signals of Bacteriorhodopsin in the infrared at 1502, 1505, 1511 and 1515 cmy1 , respectively. Photoexcitation at 540 nm. Data were taken at room temperature. The solid lines display the model described in the text. Žb. Transient absorption signals of Bacteriorhodopsin in the infrared at 1519, 1526, 1529, 1533 and 1536 cmy1 , respectively. Photoexcitation at 540 nm. Data were taken at room temperature. The solid lines display the model described in the text.
sor of the reformed BR 570 . It will turn out as a result of this work that it can be attributed to a vibrationally not fully relaxed ethylenic stretch vibration of the reconstituted BR 570 with absorbance strength at about 1512 cmy1 .
Within Scheme 2 the IR transients are interpreted as follows Žcp., Fig. 2.. The initial bleach at around 1529 cmy1 is caused by the optical excitation process whereupon the IR absorbance of the ŽC5C.stretch vibration due to the all-trans ground state is
Scheme 2.
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Fig. 2. IR difference spectra of Bacteriorhodopsin at various delay times as derived from the model described in the text.
probably shifted outside w22x the investigated spectral range. Corresponding to the quantum yield of F f 0.64, 64% of the excited BR population ŽBR) . undergoes rapid isomerization to a 13-cis configuration, with the emergence of new absorption strength
ŽBR product ) . in its electronic ground state at energies between 1500 and 1520 cmy1 that undergoes changes on a longer time scale, forming the K-state. Another fraction Ž36%. of the electronically excited molecules is fed back to an unrelaxed educt Žall-trans. configuration ŽBR educt ) . of the electronic ground state and finally to the BR 570 lowest ground state. Correspondingly, the initial bleach at 1529 cmy1 recovers partially. After about 12 ps signal levels are reached that are ‘steady state’ on the timescale of our experiment. In order to model and obtain best agreement with the experimental curves, the whole set of parameters of Scheme 2 Žrate constants, spectral positions and widths, extinction coefficients and quantum efficiency. as listed in Table 1 were divided into two groups. The first includes those parameters that are well accepted in the literature ŽScheme 1. or cannot be well deduced from our data due to insufficient time resolution or signal-to-noise ratio. The second includes those parameters that were deduced from the simulation procedure. The simulation comprised the convolution with the 0.62 ps system response. Since they cannot be fit from the IR kinetics, the time constant for the BR)-formation was fixed at 0.2 ps and that for K-formation was fixed at 3 ps w2,3,13x. The isomerization quantum yield was fixed at F s 0.64. The simulation of the experimental kinetics employing this set of parameters ŽTable 1. is in-
Table 1 ŽC5C.-stretch vibrational band parameters ŽLorentzian lineshape. and kinetic parameters used for the simulation of the IR transients as depicted in Fig. 1a,b Žsolid lines.
BR 570 BR product ) a BR educt ) Ka Kb
Center wavenumber
FWHM
Normalized ext. coefficient Žau.
1529 cmy1 1514 (2) cm y 1 1512 (3) cm y 1 1519 cmy1 1530 cmy1
17 cmy1 18 (2) cm y 1 19 (2) cm y 1 17 (2) cm y 1 17 (2) cm y 1
1 0.8 (2) 0.7 (2) 0.45 (15) 0.55 (15)
Regular typeface: parameters taken from the literature Žcp. text.; italics: parameters deduced from the simulation procedure. The relative contributions of BR educt ) and BR product ) cannot be deduced from the data Žcp. Section 4.. Estimated errors for which the simulations gave acceptable results are given in parentheses. Quantum efficiency for K-formation: 0.64. Formation of BR) : Ž0.2 ps.y1 . Decay of BR) : Ž0.8 ps.y1 , taking into account vibrational dephasing for the appearance of the absorption bands. Formation of K: Ž3 ps.y1 . Decay of BR educt ) : (2(1) ps)y 1 . a
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cluded in Fig. 1a,b as solid lines. From the simulation results difference spectra at various delay times were derived as shown in Fig. 2. Except for the kinetics at 1519 and 1511 cmy1 , respectively, the simulations are in quantitative agreement with the data.
4. Discussion The IR difference spectrum at 12 ps ŽFig. 2. is in accordance with low-temperature FTIR difference spectra, e.g. w23x, where the K-state is trapped cryogenically. At this time all initially excited BR 570 molecules are either fed via K towards the succeeding reaction steps of the photocycle or back to BR 570 . The early part of the decay at 1515 cmy1 is interpreted as the decay of a ŽC5C.-stretch vibrational state, generated from BR) . From its spectral position, it could be due to either the K precursor, BR product ) , or the BR 570 precursor, BR educt ) , e.g. a transiently populated vibrationally hot state of the 1529 cmy1 band of BR 570 or an otherwise not fully relaxed BR 570 ground state. In fact, the overtone of the ethylenic ŽC5C.-stretch Žat 1527 cmy1 . of BR 570 was tentatively assigned w24x to a RR band at 3036 cmy1 , rendering the Õ s 1 to Õ s 2 transition at 1509 cmy1 . The simulation showed that the relative contributions of BR educt ) and BR product ) cannot be distinguished but contribute both to the transient absorption around 1515 cmy1 . However, both states are kinetically necessary in the model in order to account for the observed different formation time constants of the recovering BR 570 Ž2 ps. and K Ž3–4 ps., respectively. A crucial free parameter in our model is the extinction coefficient of the K b component at 1530 cmy1 ŽTable 1.. Its relative intensity as compared to that of the K a component is only known for RR spectra w19x and thus cannot be used here. The numeric value of the K b extinction coefficient was chosen as given in Table 1 in order to account quantitatively for the recovery at 1529 cmy1 according to a quantum yield of 0.64. If there were no absorbing species in this spectral region partially canceling the bleach signal at later times, the apparent recovery could not be accounted for quantita-
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tively by the quantum yield of 0.64. Instead, a quantum yield of about 0.2 would have to be concluded. Such a low value is unreasonable for the BR photoisomerization. In order to rule out experimental or sample artifacts we performed a control experiment at 1640 cmy1 Ždata not shown.. The kinetics, taken at this wavenumber, monitor the bleach and partial recovery of the cofactor ŽC5NH.-stretch vibrational band. Here, the corresponding 13-cis product band is probably shifted to about 1607 cmy1 and hence the partial absorbance recovery reflects quantitatively the quantum yield as was shown in Ref. w13x. We reproduced the earlier observed absorbance recovery of 40% of the initial bleach amplitude within 10%. Another explanation for the initial strong bleach signal, still in accordance with a quantum yield of 0.64, could be a stimulated emission signal, i.e an inverted Õ s 1 to Õ s 0 transition of the ŽC5C.stretch vibration at 1529 cmy1 . However, in view of the high number of vibrational degrees of freedom of the retinal chromophore such a distribution, even at early times, is considered unlikely. In our model, thermally induced broadening and shifts of vibrational bands due to anharmonic coupling among the cofactor vibrational modes are not implemented. No attempts were made to determine a vibrational temperature of the chromophore. This would require a detailed knowledge of the anharmonicity constants of the chromophore vibrational modes and is beyond the scope of this work. Hence, temperature effects are discussed only qualitatively. Thermally induced shifts would, in general, occur towards the low energy side of the specific mode. If the band shift of 5 cmy1 , observed from 1514 cmy1 ŽBR product ) . to 1519 cmy1 Ž K a follows the linear relationship between the frequency of the ethylenic stretch vibration and the lmax of the chromophore w25,26x. between 1 and 12 ps is interpreted as a temperature related shift of a 13-cis ŽC5C.-stretch mode, then, following the lines as described in Ref. w14x, the temperature cannot be high Žca. 400 K.. In that study, the isomerization of Schiff’s base retinal in solution was investigated by means of femtosecond time-resolved IR spectroscopy and from the transient IR band shapes an initial vibrational temperature of 550 K was suggested. As demonstrated here, the overall features of the observed IR kinetics can be explained straightfor-
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wardly by a scheme similar to the conventional model of the retinal all-trans to 13-cis isomerization in BR, well established in the literature. However, this model has been questioned recently by Refs. w27x and w28x. There, a model was suggested, consisting of three electronic potential energy surfaces. The essential feature of this model is that under electronic excitation the system is kept for a few hundred femtoseconds near the Franck–Condon region and then passes rapidly through the reactive region, i.e. the twisted configuration. The scenarios described in the two models provide different starting conditions for the energy distribution of the cofactor vibrational modes immediately after the S1 to S 0 transition, i.e. the energy redistribution among all cofactor modes and the energy relaxation Že.g. thermalization. within each mode. This issue was partly addressed in Ref. w11x. There, a model was found consistent with the BR picosecond-transient anti-Stokes RR data available w10x that assumed the photon excess energy distributed exclusively among the Franck–Condon active modes and not among all the cofactor vibrational modes for a few picoseconds after excitation, leading to a relatively high vibrational temperature, possibly up to 1470 K. However, our data together with those described in Ref. w13x suggest, based on the relatively small observed band shifts, that the ŽC5C.- and ŽC5NH.-stretch modes of the retinal chromophore in BR have a temperature not much higher than 400 K at about 1 ps after photoexcitation. Although this is a contradiction in terms of temperature, the authors of Ref. w10x give the same time constant of about 3 ps for the cooling process. In addition, a molecular dynamics simulation study of the early picosecond events in the BR photocycle w29x suggests that the rise time Ž2.8 ps. of the K-intermediate is determined by cooling of the vibrationally hot Ž350 K. chromophore. It should be pointed out that a time constant of 2 ps was found for the recovery of the all-trans ŽC5C.-stretch vibration at 1529 cmy1 . This is in accordance with the corresponding time constant for the recovery of the relaxed all-trans ŽC5NH.-stretch vibration at 1640 cmy1 as found in Ref. w13x. Thus, relaxation back to BR 570 is faster than K-formation which takes 3–4 ps w2,3x. It seems unlikely that vibrational relaxation in the all-trans or 13-cis species, respectively, takes different times. It is rather
plausible that the reaction towards the fully isomerized 13-cis state takes longer than the reconstitution of the all-trans state because the former involves more structural changes of the surrounding protein than the latter. This suggests that the kinetic precursor of K is not simply a hot K but that the BR product ) –K transition involves other processes, possibly torsional relaxation of the polyene chain as suggested in Refs. w9,10x and protein relaxation.
5. Conclusions Although overlapping vibrational bands complicate the interpretation of the transient IR spectra, the BR 570 ground-state recovery that occurs due to the non-unity photoisomerization quantum yield can be separated spectrally and kinetically from the 13-cis product reaction branch. K-formation and BR 570 recovery occur with different time constants, i.e. 2 and 3–4 ps, respectively. Thus, corresponding precursor states, BR product ) and BR educt ) , respectively, have to be introduced, suggesting that what has been subsumed under the ‘J’-state from optical transient spectroscopy is constituted by contributions of a relaxing 13-cis product state and a recombining Žrecovering. and relaxing all-trans educt state. In contrast to the dynamics of the ŽC5NH.-stretch vibration w13x at 1640 cmy1 , the apparent amplitude of the partial recovery of the BR 570 ŽC5C.-stretch vibration at 1529 cmy1 does not correspond to the isomerization quantum yield of 0.64. This gives evidence for a ŽC5C.-stretch vibration doublet of K with band positions at 1519 and 1530 cmy1 , respectively. Most consistent with our data, including earlier results from Ref. w13x and taking into account the results in Ref. w14x, is a retinal chromophore which reaches a relatively low vibrational temperature within about 1 ps after photoisomerization. A higher temperature has been suggested for trans–cis isomerization of retinal w14x in solution. We have shown that by means of ultrafast IR absorption spectroscopy it is possible to disentangle the pathways of the electronic ground-state reaction dynamics of the all-trans to 13-cis photoisomerization in BR with sub-picosecond time resolution.
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Acknowledgements The author would like to thank Prof. D. Stehlik for encouragement and support and Prof. M. Heyn and I. Wallat for generously providing the BR samples. Extensive contributions to the entire experimental set-up and measurements, as well as the computer program used for fitting the experimental data are due to Dr. Reiner Dziewior in partial fulfillment for his Ph.D. thesis ŽFU Berlin 1998.. This research was supported by the Deutsche Forschungsgemeinschaft ŽDi 405r3-1,-4..
References w1x D. Oesterhelt, W. Stoeckenius, Proc. Natl. Acad. Sci. USA 70 Ž1973. 2853. w2x J. Dobler, W. Zinth, W. Kaiser, D. Oesterhelt, Chem. Phys. Lett. 144 Ž1988. 215. w3x R.A. Mathies, C.H. Brito Cruz, W.T. Pollard, C.V. Shank, Science 240 Ž1988. 777. w4x G. Schneider, R. Diller, M. Stockburger, Chem. Phys. 131 Ž1989. 17. w5x R. Govindjee, S.P. Balashov, T.G. Ebrey, Biophys. J. 58 Ž1990. 597. w6x J. Tittor, D. Oesterhelt, FEBS Lett. 263 Ž1990. 269. w7x S.L. Logunov, M.A. El-Sayed, L. Song, J. Phys. Chem. 100 Ž1996. 2391. w8x L. Song, M.A. El-Sayed, J.K. Lanyi, Science 261 Ž1993. 891. w9x T.L. Brack, G.H. Atkinson, J. Phys. Chem. 95 Ž1991. 2351.
55
w10x S.J. Doig, P.J. Reid, R.A. Mathies, J. Phys. Chem. 95 Ž1991. 6372. w11x A.P. Shreve, R.A. Mathies, J. Phys. Chem. 99 Ž1995. 7285. w12x R. Diller, M. Iannone, B.R. Cowen, S. Maiti, R.A. Bogomolni, R.M. Hochstrasser, Biochemistry 31 Ž1992. 5567. w13x R. Diller, S. Maiti, G.C. Walker, B.R. Cowen, R. Pippenger, R.A. Bogomolni, R.M. Hochstrasser, Chem. Phys. Lett. 241 Ž1995. 109. w14x P. Hamm, M. Zurek, T. Roschinger, H. Patzelt, D. Oester¨ helt, W. Zinth, Chem. Phys. Lett. 268 Ž1997. 180. w15x R. Dziewior, R. Diller, Ber. Bunsenges. Phys. Chem. 100 Ž1996. 2103. w16x B. Locke, R. Diller, R.M. Hochstrasser, Adv. Spectrosc. 21 Ž1993. 1. w17x K. Wynne, R.M. Hochstrasser, Chem. Phys. 193 Ž1995. 211. w18x S.O. Smith, J. Lugtenburg, R.A. Mathies, J. Membr. Biol. 85 Ž1985. 95. w19x T. Althaus, W. Eisfeld, R. Lohrmann, M. Stockburger, Isr. J. Chem. 35 Ž1995. 227. w20x M. Grossjean, P. Tavan, K. Schulten, J. Phys. Chem. 94 Ž1990. 8059. w21x R.v.d. Berg, D.J. Jang, H.C. Bitting, M.A. El-Sayed, Biophys. J. 58 Ž1990. 135. w22x V.F. Kamalov, T.M. Masciangioli, M.A. El-Sayed, J. Phys. Chem. 100 Ž1996. 2762. w23x F. Siebert, W. Mantele, Eur. J. Biochem. 130 Ž1983. 565. ¨ w24x A.B. Myers, R.A. Harris, R.A. Mathies, J. Chem. Phys. 79 Ž1983. 603. w25x L. Rimai, M.E. Heyde, D. Gill, J. Am. Chem. Soc. 95 Ž1973. 4493. w26x B. Aton, A.G. Doukas, R.H. Callendar, B. Becher, T.G. Ebrey, Biochemistry 16 Ž1977. 2995. w27x G. Haran, K. Wynne, A. Xie, Q. He, M. Chance, R.M. Hochstrasser, Chem. Phys. Lett. 261 Ž1996. 389. w28x K.C. Hasson, F. Gai, P.A. Anfinrud, Proc. Natl. Acad. Sci. USA 93 Ž1996. 15124. w29x D. Xu, C. Martin, K. Schulten, Biophys. J. 70 Ž1996. 453.