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Chemical Physics 323 (2006) 109–116

Primary reaction dynamics of halorhodopsin, observed bysub-picosecond IR – vibrational spectroscopy

Frank Peters a, Johannes Herbst b, Jorg Tittor c, Dieter Oesterhelt c, Rolf Diller d,*

a Schering AG, Research Laboratory, 13342 Berlin, Germanyb APE, Berlin, Germany

c MPI f. Biochemie, Am Klopferspitz, 82152 Martinsried, Germanyd Fachbereich Physik, Technische Universitat Kaiserslautern, 67663 Kaiserslautern, Germany

Received 1 March 2005; accepted 16 August 2005Available online 28 September 2005

Abstract

The primary all-trans to 13-cis chromophore isomerization of the light driven chloride pump halorhodopsin has been studied bymeans of transient absorption spectroscopy in the visible and mid-infrared regime at a time resolution of better than 100 and 220 fs,respectively. The picosecond vibrational dynamics are dominated by two time constants, i.e., 2 and 7.7 ps in accordance with the biphasicdecay of the retinal excited electronic state and electronic ground state formation with 1.5 and 6.6 ps. The transient vibrational spectra ofthe participating electronic states strongly suggest the existence of two distinct S1 populations as a result of an early branching reaction.It is shown that the 13-cis product is formed with the fast time constant, whereas the all-trans educt state is repopulated via both timeconstants. Concomitant protein dynamics are indicated by spectral changes on a similar time scale in the amide region.� 2005 Elsevier B.V. All rights reserved.

1. Introduction

Halorhodopsin (HR) acts as a light driven Cl�-pump inHalobacteria [1–3]. Together with the proton pump bacte-riorhodopsin (BR) and the photoreceptors sensory rhodop-sin I and II, HR belongs to the family of archaebacterialrhodopsins, photoactive 7-a-helical transmembrane pro-teins that utilize a retinal chromophore, bound covalentlyto a lysine residue via a protonated Schiff base. It appearsthat in all these systems the same chromophore undergoesa photoinduced all-trans to 13-cis isomerization on a (sub-)picosecond time scale [4–9], however, the slightly differentprotein environment brings about significantly altered reac-tion properties as isomerization quantum yield and reac-tion rates. Not only from a theoretical point of view it isa challenge to understand this kind of protein-inducedreaction control, i.e., the impact of the chromophore–pro-tein interaction on the shape of the corresponding elec-

0301-0104/$ - see front matter � 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.chemphys.2005.08.036

* Corresponding author. Tel.: +49 631 2052323; fax: +49 631 2053902.E-mail address: diller@physik.uni-kl.de (R. Diller).

tronic potential energy surfaces of the chromophore ineach specific system. Presupposition is an accurate descrip-tion of the reaction dynamics in the various systems.

In HR the chromophore all-trans to 13-cis isomerizationoccurs with a quantum yield of 34% [1]. Via thermally dri-ven subsequent reaction steps the ground state HR578 (kmax

as subscript) is recovered with the chromophore again inall-trans configuration [10–13]. In various studies, the pri-mary reaction dynamics has been studied by means of tran-sient absorption spectroscopy in the visible, addressing thechromophore electronic state dynamics. Earlier reportsfound a time scale of 5 ps [14] and less than 2.3 ps [15]for the decay of the excited electronic state and a kmax ofabout 600 nm [6,14,16] for the electronic ground stateabsorption of the first reaction product HRK. In experi-ments with improved time resolution (0.1 ps) a biexponen-tial excited state decay was found [6] with time constants of1.5 and 8.5 ps. A kinetic reaction scheme was suggestedinvolving a branching in the S1 state, with one branch beingnon-productive and leading back to the all-trans educt stateHR578 and the second branch leading partly to the product

110 F. Peters et al. / Chemical Physics 323 (2006) 109–116

electronic ground state HRK and partly repopulatingHR578 [6]. However, no unequivocal assignment of thetwo apparent time constants to the productive, respec-tively, the non-productive reaction path could be obtaineddue to the strong similarity of the decay associated spectraderived from a global fit analysis.

Despite their high temporal resolution, optical transientabsorption experiments do not provide direct informationabout the structural dynamics, which, in contrast, can beobtained from time resolved resonance Raman (RR) orinfrared (IR) experiments. In the latter, changes of anyIR transition moments or transition frequencies of eitherthe chromophore or the protein that are induced by the vis-ible excitation pulse are recorded. These changes occur,e.g., upon electronic excitation of the chromophore (S1-dynamics), its conformational and configurational evolu-tion (especially, its trans–cis isomerization) and stericallyinduced conformational changes of the surrounding pro-tein. Thus, the temporal evolution of the recorded differen-tial IR absorption spectra provides direct insight into thedynamics of these processes. This has been demonstratedrecently in the case of BR, where the isomerization processwas time-resolved for the first time on a vibrational spec-troscopic basis [9]. For HR, resonance Raman[12,13,17,18] and Fourier transform infrared (FTIR) stud-ies [19–22] have characterized and analyzed only later reac-tion cycle intermediate states and their kinetics, as well asthe vibrational spectra of the HR ground state and of the(frozen) K-state under various chemical conditions. How-ever, to our knowledge the primary isomerization reactionhas not been investigated so far by means of sufficientlytime resolved vibrational spectroscopy. In this work, we re-port on transient IR absorption experiments at a time res-olution of better than 220 fs. Thereby, the vibrationaldynamics during the retinal isomerization can be correlatedwith the dynamics of its electronic states.

2. Materials and methods

2.1. Material

Halorhodopsin from Halobacterium salinarum strain D2was prepared in a membrane bound form as described ear-lier [23]. In this study not the lowest band from a sucrosegradient (25–45%) was used but the one next to it. Thisband containing 80–90% of the total expressed halorho-dopsin, was isolated and washed two times and resus-pended in 1 mM Tris buffer at pH 7 and 15 mM KCl.

2.2. Transient absorption spectroscopy

2.2.1. Sample preparation

For the time resolved absorption experiments in the vis-ible and the mid-IR, films of HR were prepared on CaF2 orBaF2 windows of 1.5 in. diameter and sealed by a second,similar window. The films were hydrated just sufficientlyto ensure full protein stability and function and to avoid

unnecessary absorption losses by water. In all experiments,the sample absorbance was set to about 0.5 OD units atkmax = 578 nm, the absorption maximum of light-adaptedHR in its ground state. This corresponds to an absorbanceof about 1 OD unit in the region of amide absorption. Dur-ing the experiments, the sample was rotated and moved lat-erally to the focused exciting and probing laser beams inorder to provide for exchange of the excited sample volumeat each laser shot. On average, a sampled volume wasre-excited (and probed) again only after about 1–2 s. Tocheck for sample degradation, steady-state absorptionspectra were taken before and after each experiment. Allmeasurements were performed at room temperature.

2.3. Pump–probe spectrometer

Short excitation and probe pulses were generated bymeans of non-linear optical devices, driven by laser pulses(160 fs FWHM, 775 nm, 0.7–1 kHz repetition-rate) of acommercial Titanium–Sapphire laser amplifier system(CPA1, CLARK-MXR Inc., Dexter/MI, USA) seeded bya fiber laser (Femtolite 780, IMRA).

For the experiments in the visible regime (VIS/VIS) twohome built non-collinear optical parametric amplifiers,independently tunable between 450 and 750 nm were em-ployed. After pulse compression in pairs of prisms thewidth of the instrumental response function (IRF) wasdetermined to ca. 70 fs (FWHM) by cross-correlation ofthe pump and probe pulses in a non-linear crystal. Thepump pulse energy was kept low enough to avoid multiplesample excitation. After transmitting the sample the probepulses were dispersed in a monochromator and detected bya photodiode. The signal, i.e., the pump-induced absor-bance difference DA(t,kpr), was measured as a function ofthe delay time t between pump and probe pulse at specificprobe wavelengths kpr by lock-in amplification of the pho-todiode signal and chopping the pump beam at half thesystem repetition rate. The quantitative evaluation of thetime dependent absorbance changes was restricted to delaytimes t > 100 fs (see below).

For the mid-IR experiments (VIS/IR) visible excitationpulses of ca. 100 fs duration were obtained as describedabove. Probe pulses in the mid-IR were generated bynon-linear mixing procedures in several steps. First, a weakspectral continuum was induced in a sapphire window andused as seed pulse for the parametric generation of signal(1.2–1.5 lm) and idler photons (1.7–1.9 lm) in a BBO crys-tal, pumped by the fundamental pulses at 775 nm. In a sec-ond step, the signal pulses served as seed in a furtheramplification process, again in a BBO crystal and pumpedby 775 nm pulses. Thus, signal and idler pulses at energiesof ca. 15 lJ were obtained. They were mixed in a AgGaS2

crystal to finally generate stable mid-IR pulses at their dif-ference frequency (800–2500 cm�1), a spectral width of ca.100 cm�1 (FWHM), temporal width of down to 160 fs(FWHM and pulse energy of up to ca. 1 lJ). For the probeprocess the mid-IR pulses were strongly attenuated. The

–30

–20

–10

0

0

probe

probe

= 570 nm

10

20

30 = 490 nmλ

λ

λ

OD

)A

bso

rban

ce c

han

ge

(mO

D)

Ab

sorb

ance

ch

ang

e (m

OD

)

= 687 nmprobe

F. Peters et al. / Chemical Physics 323 (2006) 109–116 111

volume transmitted by the mid-IR pulses was purged bydry air in order to avoid their distortion by interaction withrotational–vibrational transitions of water molecules. Afterprobing the pump pulse induced absorption changes in thesample the IR pulses were dispersed in a polychromatorand detected by an array of ten MCT-elements (OPTILAS)with a dispersion of 2.5–5 cm�1 per detector element(equivalent to the spectral resolution employed in thiswork), dependent on the center IR wavelength. In orderto cover a broad spectral range (Figs. 2 and 5), severalspectral windows (‘‘width’’ of 10 detector elements) withsufficient overlap (at least two detector elements) had tobe used. The overlap allowed to normalize the signal ampli-tudes of each spectral window to those of the adjacent win-dow. For each spectral window the IRF (typically 220 fs)and experimental time zero was obtained independentlyby pump–probe experiments on a thin silicon sample.The pump-induced absorbance difference DA(t,kpr) wasmeasured as a function of the delay time t between pumpand probe pulse at specific probe wavelengths kpr on thebasis of single shot detection and chopping the pump beamat half the system repetition rate. The quantitative dataevaluation was restricted to delay times t > 150 fs (see be-low) since the signals at earlier delay times (especially atnegative delay times) are partly obscured by contributionsof the free induction decay as induced by the IR pulse andperturbed by the excitation pulse.

In all experiments, data evaluation was performed by fit-ting the pump-induced absorbance difference DA(t,kpr) to asum of exponentials

DAðt; kprÞ ¼ A0ðkprÞ þXN

1

AiðkprÞ � e�t=si ð1Þ

with A0(kpr) being the pump-induced difference absorptionspectrum after long delay times (30–100 ps in our experi-ments) and Ai (kpr) the spectrum (DAS) associated withthe decay time si. Since the focus of this work lies on thepicosecond time scale, a convolution of this function withthe IRF is not necessary. Eq. (1) was applied to absorbancetransients at each individual probe wavelength as well as ina global analysis (GA) to whole sets of absorbancetransients.

0 10 20 30 40

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–5

0

Ab

sorb

ance

ch

ang

e (m

Time (ps)

Fig. 1. Absorbance transients of halorhodopsin in the visible regime afterexcitation at 570 nm. Shown are experimental data points together with abiexponential fit according to Eq. (1) and time constants of 1.5 ± 0.3 and6.6 ± 0.8 ps (average). At kpr = 687 nm, an additional third time constantof 0.3 ps improved the fit significantly.

3. Results and discussion

3.1. VIS/VIS experiments

3.1.1. Dynamics of electronic states

It is known for HR and BR that spectral and dynamicproperties of the respective photocycles depend on the spe-cific sample preparation, e.g., on ion strength, pH andwater content. Since the IR experiments reported here re-quire HR films instead of aqueous suspension it was neces-sary to first characterize the photoinduced electronic statedynamics of the chromophore–protein complex in thissample (film) preparation. Therefore, transient absorption

experiments were performed with excitation at 570 nmand at the specific probe wavelengths 490, 570 and687 nm, i.e., the spectral position of predominant excitedstate absorption, ground state absorption and stimulatedemission, respectively [6,15]. The results are shown inFig. 1. At 490 nm, the nearly instantaneous excited stateabsorption decays on the picosecond time scale and the sig-nal levels to a small negative value, due to the residualground state bleach at this wavelength. At 570 nm the ini-tial ground state bleach signal decays on the same timescale to a residual value, partially cancelled by a positiveabsorption signal of the photoproduct HR600 in its elec-tronic ground state. At 687 nm the initial stimulated emis-sion signal disappears on the same time scale and the signallevels to a small positive value, due to the residual photo-product absorption at this wavelength. Due to the strongstimulated emission in the red and the significant spectraloverlap between the ground state HR578 and photoproductstate HR600, the initial rise (positive signal) of the latter is

1240 1220 1200 1180 1160 1140

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0

1

2

Wavenumber (cm-1)

Ab

sorb

ance

ch

ang

e (m

OD

)

0.19 ps 2.5 ps 7 ps 60 ps

Fig. 2. IR difference spectra in the fingerprint region after excitation at570 nm at various delay times.

112 F. Peters et al. / Chemical Physics 323 (2006) 109–116

not seen at the investigated spectral positions. On the pico-second time scale all absorbance transients are fitted wellby a biexponential form of Eq. (1) with average time con-stants of s1 = 6.6 ± 0.8 and s2 = 1.5 ± 0.3 ps. Only at theprobe wavelength of 687 nm, an additional time constantof 0.3 ps was necessary to improve the fit significantlyand indicates a very fast depopulation of the Franck–Con-don state. These results are in accordance with those byArlt et al. [6] (i.e., 1.5 ± 0.7 and 8.5 ± 1.5 ps) who used asuspension (10 mM sodium phosphate buffer, pH 7) ofmembrane bound HR from H. salinarum (strain D2) andshow that the electronic state dynamics during the primarychromophore isomerization are not changed in the filmpreparation as compared to suspension.

3.2. Variation of excitation wavelength

In order to take into account an inhomogeneous elec-tronic ground state distribution of light-adapted HR (fromH. salinarum) – e.g., by a certain amount (ca. 17–25%) ofHR with the chromophore in 13-cis,15-syn-configurationas discussed in [18,20] – analogous experiments were per-formed with excitation at 540, 570 and 620 nm, therebysampling the HR absorption band at its maximum andits wings, and probing at the wavelengths as describedabove (data not shown). The analysis showed that withinthe experimental error neither the observed time constantsnor the ratio A1(kpr)/A2(kpr) of their amplitudes changeswith excitation wavelength. In particular, the averaged ra-tio A1(kpr)/A2(kpr) at kpr = 490 nm (excited electronic stateabsorption) amounts to 0.7 ± 0.2 and to 0.6 ± 0.1 at kpr =570 nm. Within this experimental error the results give noindication for a kinetic component that could specificallybe associated with a photoreaction of the 13-cis,15-syn

chromophore. The observed time constants are thereforestrongly suggested to represent the electronic reactiondynamics of the protein population initially hostingexclusively the all-trans chromophore. Further evidencefor this conclusion was obtained by VIS/IR-experiments(cp. 13-cis,15-syn-species).

3.3. VIS/IR experiments

Transient IR absorption experiments in the region be-tween 1580 and 1100 cm�1 were performed with excitationat 570 nm. The recorded differential absorption transientsdisplay the vibrational dynamics of the chromophore–pro-tein complex during its photoreaction. For an overviewFigs. 2 and 5 present transient absorption spectra of HRfilms in the mid-IR after excitation at 570 nm at delay timesof 0.19, 2.5, 7 and 60 ps. The difference spectra show neg-ative contributions due to the vibrational modes of thebleached electronic ground state HR578 and positive contri-butions due to the vibrational modes of the states createdby the optical excitation at the specific delay time. Thus,the transient signals monitor the decay of the excited elec-tronic state vibrational bands, the partial recovery of the

HR578 vibrational bands and the formation of the reactionproduct. The 60 ps spectrum depicts the difference betweenHR600 and HR578.

3.4. Structural chromophore dynamics

The structural dynamics of the chromophore duringits all-trans to 13-cis isomerization are monitored by var-ious chromophore vibrational bands. Their assignment tovibrational modes is substantially based on the carefulvibrational analysis of BR by means of isotopically la-beled chromophores and normal mode calculations [24–28]. Both systems host the same all-trans-retinal Schiffbase chromophore in similar protein binding pocketsand the resonance Raman spectra of BR570 and HR578

[12,13] as well as their FTIR difference spectra (e.g.low temperature HR578/HRK difference spectra [19,21])are highly correlated. However, dissimilarities indicatethe different chromophore–protein interaction in HRwith respect to BR, which consequently causes a substan-tially different isomerization dynamics, especially, the bi-phasic and slower S1 decay in contrast to the faster(0.5 ps) excited state decay in BR [4,5]. Important markerbands in this respect are first the fingerprint C–C stretchvibrations coupled to CH bending modes between 1100and 1250 cm�1, indicating chromophore configuration(13-cis vs. all-trans), and second the ethylenic C@Cstretch vibrations around 1530 cm�1, reflecting the p-elec-tron delocalization in the polyene part of thechromophore.

Fig. 2 presents IR absorption difference spectra in thefingerprint region at delay times between 0.19 and 60 ps.Bleaching bands are observed at 1203 and 1167 cm�1, asexpected for the all-trans chromophore electronic groundstate [12,19,21,22], and a product band rises at around1195 cm�1 as observed in low temperature [19] and nano-second time resolved HR578/HRK FTIR difference spectra[21,22]. In analogy to BR, this difference pattern indicatesthe configurational change from all-trans to 13-cis. Thetransient spectra already suggest on a vibrational spectro-scopic basis that the isomerization process is complete after

F. Peters et al. / Chemical Physics 323 (2006) 109–116 113

7 ps. The global fit analysis of this spectral region yieldstwo time constants, i.e., 1.8 ± 0.1 and 6.9 ± 0.5 ps similarto the time constants of the excited electronic state decayand of the electronic ground state formation as observedin the VIS/VIS experiments (see above). The correspondingdecay associated spectra (DAS) as shown in Fig. 3 aremarkedly different. The 6.9 ps DAS exhibits a large ampli-tude at the position of the educt (all-trans state) bandaround 1203 and 1167 cm�1 but an almost zero amplitudearound 1195 cm�1, the position of the 13-cis product band.The 1.8 ps DAS as well contributes at 1203 and 1167 cm�1

but, in contrast to the 6.9 ps DAS, shows a strong ampli-tude at 1195 cm�1. The spectrum A0 in this analysis isessentially the same as the experimental difference spectrumobtained at long delay times (e.g., 60 ps in Fig. 2) as ex-pected. These observations strongly suggest that the educt(all-trans) ground state recovers biexponentially with theobserved time constants, whereas the product (13-cis) for-mation is determined by the faster (1.8 ps) process. InFig. 4, IR absorbance transients at 1203 cm�1 (educt bandposition) and at 1195 cm�1 (product band position) aregiven together with a biexponential fit as the result of the

1240 1220 1200 1180 1160 1140

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Wavenumber (cm-1)

A0-spectrum

6.9 ± 0.5 ps 1.8 ± 0.1 ps

Fig. 3. Decay associated IR absorption difference spectra (DAS) as theresult of the global analysis of the fingerprint region (excitation at570 nm).

0 10 20 30 40–5

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Time (ps)

1195 cm-1

1203 cm-1

Fig. 4. IR absorbance transients in the fingerprint region at 1203 cm�1

(educt band position) and at 1192 cm�1 (product band position) afterexcitation at 570 nm together with a biexponential fit as the result of theglobal analysis with time constants of 1.8 ± 0.1 and 6.9 ± 0.5 ps.

global analysis in this region. A monoexponential analysisof each transient (data not shown) yields time constants of3.5 ps (at 1203 cm�1) and 2.0 ps (at 1195 cm�1), with aclearly non-zero fit residuum for the transient at1203 cm�1 and an acceptable fit residuum for the transientat 1195 cm�1. This again corroborates the view that theisomerization process is governed by the faster time con-stant whereas the educt ground state recovery has to bedescribed biexponentially.

The strong bleach signal at 1525 cm�1 (Fig. 5) is as-signed to an in-phase C@C stretching vibration (ethylenicstretch) of the chromophore [12]. Its position reflects thep-electron delocalization in the polyene part of the chro-mophore; as for BR, a smaller wave number correspondsin a linear dependency [29–31] to a red-shifted opticalabsorption maximum (kmax) of the respective intermediatestate of the HR photocycle. In particular, since the kmax ofca. 600 nm for the HRK chromophore is slightly red shiftedwith respect to HR578, one expects a corresponding down-shift of the ethylenic stretch frequency. In fact, the smallabsorbance differences after 60 ps (Fig. 5) with a negativepeak at 1525 cm�1 and a positive signal around1511 cm�1 are consistent with the difference betweenstrongly overlapping ethylenic stretching bands of theHRK and the HR578 chromophore. The global fit in thisspectral region (Fig. 6) yields time constants of 2.2 ± 0.1and 8.5 ± 0.7 ps similar to those in the fingerprint region(see above) with different DAS describing the correspond-ing dynamics. At 1526 cm�1 (Fig. 7), the center of the all-trans ethylenic stretch, both DAS exhibit a large amplitudewhereas at around 1512 cm�1 the 2.2 ps DAS again exhib-its a large amplitude but the 8.5 ps DAS is about zero.These observations indicate the biphasic repopulation ofthe all-trans ground state and the formation of an elec-tronic ground state with a red shifted absorption maximumwith only one time constant, i.e., 2.2 ps and are thus fullyconsistent with the observations made in the fingerprintregion.

So far in this discussion, only vibrational ground stateshave been considered. This is a reasonable assumption for

1600 1560 1520 1480

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Wavenumber (cm-1

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Fig. 5. IR difference spectra in the ethylenic stretch region after excitationat 570 nm at various delay times.

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2

Time (ps)

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1526 cm-1

1512 cm-1

Fig. 7. IR absorbance transients in the ethylenic stretch region at1526 cm�1 (educt band position) and at 1512 cm�1 (product bandposition) together with a biexponential fit as the result of the globalanalysis with time constants of 2.2 ± 0.1 and 8.5 ± 0.7 ps (excitation at570 nm).

1600 1560 1520 1480

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2

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Wavenumber (cm-1

)

A0-spectrum

8.5 ± 0.7 ps 2.2 ± 0.1 ps

Fig. 6. Decay associated IR absorption difference spectra (DAS) as theresult of the global analysis of the ethylenic stretch region (excitation at570 nm).

114 F. Peters et al. / Chemical Physics 323 (2006) 109–116

equilibrated electronic states and vibrational frequenciesabove 1000 cm�1 at ambient temperature. However,concomitant to the ultrafast excited electronic state decay,vibrationally excited states on the S0 surface are expectedto become transiently populated. Accordant observationshave been made for BR by RR experiments [32,33] in termsof anti-Stokes vibrational bands and for BR [34] as well asfor protonated schiff base of all-trans retinal in solution [35]by IR experiments in terms of vibrational band shifts. Thepresented IR data on HR lack obvious evidence for ‘‘hot’’vibrational states of the educt chromophore (occurrence oftransient positive absorbance on the low-energy side of thecorresponding educt absorption bands), possibly becausethey are obscured by other vibrational bands of the HRK

chromophore–protein complex or simply due to kineticreasons: If the cooling rate is fast against the feeding rateor comparable with it (e.g., 1–3 ps as suggested in [9,34]against the S1 decay times of ca. 2 and 7 ps reported here),the transient concentration of excited vibrational states re-mains small.

3.5. Vibrational dynamics of the excited electronic state

The early transient difference spectra in Figs. 2 and 5 ex-hibit a number of positive bands that appear almost instan-taneously and are assigned to vibrational bands of theexcited electronic state S1 of the retinal chromophore.Prominent absorbance signals of this kind are observedat 1143, 1222 and 1560 cm�1. At present, their assignmentto specific chromophore vibrational bands can only bemade based on group frequency and intensity argumentsin analogy to electronic ground state vibrations. Thus,the bands at 1143 and 1222 cm�1 are tentatively assignedto C–C single bond stretch vibrations, the band at1560 cm�1 is assigned to the ethylenic stretch vibration ofthe S1-state in accordance to a blue-shifted S1 absorptionmaximum between 470 and 520 nm [6,15]. On the timescale of our experiment these three bands appear equallyfast, at least within 300 fs, do not exhibit a spectral evolu-tion and, as shown by the DAS in Figs. 3 and 6 follow verydistinct kinetics, in fact each of them decays with either theshort or the long decay time, very much alike those foundfor the S1 decay. In other words, the DAS are very differentwith respect to the vibrational bands of the excited elec-tronic state. This finding does not exclude the possibilityof S1 vibrational bands with biphasic decay in other partsof the spectrum but exemplifies the existence of two S1 pop-ulations of the chromophore that are formed quickly afterthe Franck–Condon state has been occupied and that exhi-bit different molecular structures, which, at present, cannotbe deduced from the S1 vibrational spectrum. Thus, theseobservations suggest an early branching reaction on theS1 potential energy surface leading to two HR states,Sloss

1 and Siso1 . Adding the results of the preceding para-

graph it can be concluded that Sloss1 decays exclusively back

to the educt state HR578 with a time constant of 6.6 ps andSiso

1 decays to both the isomerized product state HRK andto HR578 with a time constant of 1.5 ps. Note the pairwisematch between the time constants obtained by the VIS/VISand the VIS/IR experiments that show the correlation be-tween the decay of the S1 states and the change of therespective vibrational spectra. The proposed reactionscheme is summarized in Fig. 10. Provided that the isomer-ization is complete on the time scale of the experiment (seeabove) the isomerization quantum yield is roughly given bythe averaged amount (70%) of the partial decay of theeduct bleach bands in the fingerprint region, i.e., the bandsat 1203 and 1167 cm�1, assuming that they are spectrallyisolated sufficiently to reflect the recovered all-trans popu-lation. This number, i.e., 100% � 70% = 30%, compareswell with the reported HR quantum yield of 34% [1]. Thebranching ratios of the scheme in Fig. 10 are then obtainedas follows. Assuming equal absorption cross-sections forthe excited electronic states Sloss

1 and Siso1 , the first branch-

ing ratio (FC) is given by the ratio A1(kpr)/A2(kpr) � 0.66at kpr = 490 nm, equivalent to populations ofSloss

1 and Siso1 of 40% and 60%, respectively. The second

branching ratio ðSiso1 Þ then amounts to �1.

0 10 20 30 40 50 60

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Time (ps)

1535 cm-1

Fig. 9. IR absorbance transient at 1535 cm�1 after excitation at 570 nmtogether with a biexponential fit (2.2 ± 0.1 and 8.5 ± 0.7 ps) as the resultof the global fit in the ethylenic stretch region.

F. Peters et al. / Chemical Physics 323 (2006) 109–116 115

3.6. Vibrational dynamics of the protein

The spectral region between 1575 and 1480 cm�1 (Fig. 5)not only includes the strong chromophore ethylenic stretchvibrations (S0 and S1) as discussed above but as well thebroad and inhomogeneous amide II band (C–N-stretch/NH-bend) of the peptide backbone. Possible amide II con-tributions are observed at 1538 and 1552 cm�1. At latetimes, i.e., in the 60 ps difference spectrum and the A0-spec-trum, they appear as positive, respectively, negative band.Similar bands were observed in low temperature and nano-second time resolved FTIR spectra of the HR578–HRK

transition [19,21,22]. RR experiments do not indicate thesebands as chromophore vibrations of HR578. The temporalevolution at these positions is complicated due to superpo-sition with chromophore bands.

At 1554 cm�1 (Figs. 5 and 6) a bleach signal appearswith 2.3 ps and decays partially with 15 ps. This is the re-sult of a single wavelength fit shown in Fig. 8 (due to thestatistical weight of the 2 and 7.7 ps components, the timeconstant of 15 ps is suppressed in the global fit DAS). Twopossible kinetic schemes are discussed. First, an almostinstantaneous bleach becomes apparent due to the decayof the overlapping S1 chromophore vibrational band (cp.above). Second – and more likely, assuming a protein bandat this position – the isomerization, taking place with ca.2 ps, perturbs a specific part of the protein backbone andchanges the corresponding amide II frequency or oscillatorstrength. Similar observations were made for bacteriorho-dopsin [36] with a rise time of 0.5–1 ps instead of 2 psand a decay time of again 15 ps. Note that the decay timesare the same for both systems whereas the rise times followthe time scale of the respective isomerization. This findingsuggests an intrinsic time scale for protein relaxation afterchromophore isomerization.

At 1535 cm�1, an initial bleach signal becomes slightlypositive with a dominant amplitude of the 2.2 ps compo-nent (Figs. 5 and 9, a monoexponential fit at this wavenum-ber yields a time constant of 3.0 ps) whereas the 8.5 ps DAS

0 20 40 60 80 100

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Time (ps)

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ang

e (m

OD

)

1550 cm-1

- 1558 cm-1

Fig. 8. IR absorbance transient at 1554 cm�1 (signal average between1550 and 1558 cm�1) after excitation at 570 nm together with a biexpo-nential fit in only this spectral region (2.3 ± 0.6 rise time and 15 ± 7 psdecay time).

in Fig. 6 exhibits a minimum of its absolute value close tothis position. This observation suggests that here an amideII vibrational state is generated concomitantly to the isom-erization, possibly the counterpart to the vibrational statepopulation that disappears at 1554 cm�1.

3.7. 13-cis,15-syn-Species

In order to account for the small fraction of HR with thechromophore in 13-cis,15-syn configuration (cp. above:VIS/VIS experiments), analogous IR experiments wereperformed with excitation at 520, 570, 620 nm. Theyneither yield changes in the time constants of the globalfit nor changes in the difference spectra except for the rela-tive contribution of the negative band (shoulder) at1538 cm�1 at earliest delay times (Fig. 5, spectrum at0.19 ps). This band can be assigned to the ethylenic stretchvibration of the 13-cis,15-syn-chromophore [18]. As ex-pected considering the blue-shifted kmax (ca. 560 nm [18])of the dark-adapted species, its amplitude becomes weakerwith increasing excitation wavelength. This dependency onthe excitation wavelength is quantitatively not observed forthe respective DAS. Thus, it is concluded that the contribu-tion of the 13-cis,15-syn-species to the observed isomeriza-tion dynamics are sufficiently small to be neglected in thecontext of this work.

h

1.5 ps

6.6 ps

0.3 ps578HR FC

1lossS

1isoS

KHR

ν

1.5 ps

6.6 ps

0.3 ps

Fig. 10. Reaction scheme for the halorhodopsin isomerization reaction.FC: Franck–Condon state; Sloss

1 , Siso1 : different states of the S1 potential

energy surface.

116 F. Peters et al. / Chemical Physics 323 (2006) 109–116

4. Conclusions

Sub-picosecond time resolved IR vibrational spectros-copy was employed to study the all-trans to 13-cis retinalisomerization in halorhodopsin from H. salinarum. Thevibrational spectra of the various participating electronicstates that are highly structured as compared to the corre-sponding electronic absorption bands, serve to unravel thedynamics of the excited electronic state, the isomerizedproduct state and the partially recovering educt state.The results suggest the reaction scheme in Fig. 10. The13-cis product is formed via the fast reaction path with atime constant of 1.5–2 ps and a quantum yield of about30%, whereas the all-trans educt state is repopulated viaboth the fast and the slow (6.6–7.7 ps) reaction path. Noevidence was found for an alternative reaction scheme, thatcould explain the biphasic S1 decay and the formation ofthe 13-cis photoproduct by the existence of an inhomoge-neous HR ground state distribution and related parallelphotoreactions.

Besides the structural dynamics of the chromophore,protein contributions are observed in the amide II regionbetween 1530 and 1560 cm�1. They suggest an almostimmediate response of at least parts of the peptide back-bone (of BR and HR) to the steric perturbation as inducedby the chromophore isomerization and a second dynamiccomponent of ca. 15 ps, which is independent of the specificsystem (BR, respectively, HR) and thus an intrinsic prop-erty of the protein moiety.

Acknowledgment

This work was supported by the Deutsche Forschungs-gemeinschaft Sfb 450.

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