Femtosecond impulsive excitation of nonstationary vibrational states in bacteriorhodopsin

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CHEMICAL PHYSICS LETTERS

Femtosecond impulsive excitation of nonstationary vibrational states in bacteriorhodopsin S.L. Dexheimer a-b,Q. Wang a-c,L.A. Peteanu ‘, W.T. Pollard ‘, R.A. Mathies ’ and C.V. Shank a.b.c a Materials Sciences Dlvision, Lawrence Berkeley Laboratory, University o/California

at Berkeley, Berkeley, CA 94720, USA

b Department ofPhysics, University o~Cali/ornia at Berkeley, Berkeley, CA 94720, USA c Department ofChemistry, University of California at Berkeley, Berkeley, C-A94720, USA

Received 30 August 1991; in final form 30 September I99 I

Optical pulses of 12 fs duration are used to excite and probe nonstationary states in the photochemlcally active protein bacteriorhodopsin. Time-resolved differential transmittance measurements at 568, 620, and 656 nm reveal oscillations from coherent vibrational motion of the retinal chromophore as well as time-dependent changes reflecting its excited state dynamics and subsequent photochemistry. The oscillatory response is dominated by the nonstationary excitation of the ground state reactant, consistent with a resonant impulsive Raman process. Calculations based on this mechanism show good agreement with the results.

Resonant optical excitation of a molecule on a time scale short compared to a vibrational period generates a coherent superposition of vibrational states on the ground and excited state molecular potential surfaces. The evolution of these nonstationary vibrational states, or wavepackets, can be detected as oscillations and time-dependent spectral shifts in the optical response. Wavepacket dynamics have been observed in dye molecules using femtosecond laser techniques [l-3]. In this paper, we report observations of nonstationary vibrational states in a photochemically active protein, bacteriorhodopsin, in which the retinal chromophore undergoes a light-induced torsional isomerization. The measurements are performed using pulses of 12 fs duration, sufficiently short to impulsively excite all the dominant active modes of the molecule and of sufficient bandwidth to detect the response both within the absorption spectrum of the reactant and at longer wavelengths, encompassing the absorption spectrum of the isomerized photoproduct. The high time resolution of these measurements allows clear observation of both the nonexponential behavior of the excited state reactive dynamics and the oscillatory behavior of the high frequency components of the nonstationary vibrational states. Long-term oscillations observed

throughout the probed spectral region are interpreted in terms of impulsively excited vibrationally coherent states in the ground state reactant form of the molecule, including red-shifted components from the excitation of higher lying ground state vibrational levels that are not populated in thermal equilibrium. The ground state reactant contribution dominates the oscillatory response of this photochemically active system, since rapid photochemical dynamics on the excited state potential surface quickly carry the excited state response outside of the probed optical wavelength region, and the excited state vibrational coherence initially excited in the reactant molecule is not observed in the isomerized photoproduct. The molecule studied in these experiments, bacteriorhodopsin, is a protein found in the purple membrane of Halobacterium halobium that is responsible for the conversion of light energy to electrochemical energy by the generation of a transmembrane proton gradient. The first step in this process is the photoisomerization of the retinal chromophore covalently bound within the protein. The retinal chromophore, initially in the all-trans configuration, undergoes a torsional isomerization around the C, s=C14double bond following excitation to the

0009-2614/92/$ 05.00 Q 1992 Elsevier Science Publishers B.V. All rights reserved.

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S, potential surface, and undergoes a nonradiative transition to form the 13-cis photoproduct. Previous time-resolved optical measurements have shown that the isomerized photoproduct J, characterized by a red-shifted absorption spectrum, appears within 500 fs [4,5]. The quantum yield for photoisomerization has been variously measured to be 0.6 or 0.3 [6], with the remainder of the molecules returning to the initial all-trans configuration. These processes are shown with the schematic potential energy surfaces in fig. 1.

Previous measurements of dynamic hole burning in bacteriorhodopsin with 60 fs pump pulses and broadband 6 fs probe pulses were interpreted in terms of the motion of an excited state wavepacket along the molecular isomerization coordinate [ 4,7,8]. The motion of this wavepacket, corresponding to the evolution of an initially coherent superposition of low frequency vibrational states, was manifested as the

J

Reaction

Ihis

Coordinote

Fig. I. Schematic potential energy surfaces for the photoisomerization of bacteriorhodopsin, along with the structure of the retinal chromophore in its all-tram form.

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appearance of time-dependent spectral shifts in the optical response of the molecule on a time scale of z 100 fs. In the measurements reported here, optical pulses of 12 fs duration are used to excite and probe nonstationary states involving all the active modes of the molecule, including those extending to high frequencies. The time-dependent optical response is probed at a series of wavelengths: 568 nm, at the center of the absorbtion band of the ground state reactant species, 656 nm, far in the red wing of the reactant absorption band, but within the absorption band of the red-shifted photoproduct, and at the intermediate wavelength of 620 nm, at the center of the optical pulse spectrum. The response at all three of these wavelengths reveals high-frequency oscillations resulting from coherent vibrational motion as well as time-dependent changes reflecting the excited state dynamics and subsequent photochemistry. The oscillatory part of the response is compared to calculations based on a formalism developed for multidimensional harmonic potential surfaces [ 91 using parameters derived from resonance Raman studies of bacteriorhodopsin [ lo]. Optical pulses are generated using a femtosecond laser system consisting of a colliding-pulse modelocked dye laser with a copper vapor laser-pumped dye amplifier that produces 50 fs pulses centered at 620 nm with an 8 kHz repetition rate. These pulses are chirped in an optical fiber and compressed using a sequence of diffraction gratings and prisms [ 111. To increase the energy of the compressed pulses, an additional copper vapor pumped multi-pass amplifier similar to that described in ref. [ 121 is introduced prior to compression of the chirped pulse. The gain medium of this amplifier consists of a mixture of rhodamine 590 and DCM dyes to provide a gain response that nearly covers the spectrum of the chirped pulse, resulting in final compressed pulses of 12 fs duration with an energy of x 100 nJ. Time-resolved differential transmittance measurements are performed with a pump-probe technique, using differential detection with a pair of balanced photodiodes followed by lock-in amplification. Measurements at specific wavelengths are made by passing the probe beam through a 10 nm bandwidth interference filter after it passes through the sample. The energy of the pump pulses is 6 nJ, resulting in dif-

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ferential transmittance signals that are linear with respect to pulse energy. Purple membrane fragments are prepared following published procedures [ 131. Membrane fragments suspended in a buffered salt solution to an optical density of 0.5 in the 200 lrn path length are pumped through a fused silica cell sufficiently rapidly to allow complete replacement of the sample between each pair of laser pulses. Light adaptation, which maintains the chromophore in its active conformation, is ensured by continuous background illumination, and the sample temperature is maintained at 4°C. Measurements of the time-resolved differential transmittance at 568,620, and 656 nm are shown in figs. 2a-2c. Oscillations due to wavepacket motion are clearly evident at all three wavelengths. The oscillations persist on a time scale of picoseconds and their complex structure indicates the presence of a number of frequency components, which are analyzed in detail below. The signals underlying the oscillations reflect the excited state dynamics and subsequent photochemistry. As discussed below, these signals clearly show nonexponential behavior at early

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times, consistent with the evolution of a wavepacket on the reactive excited state potential surface [ 7,8]. At 568 nm (fig. 2a), which lies at the center of the reactant absorption band, the signal underlying the oscillations is dominated by the bleach of the reactant absorption band and its subsequent recovery. This signal is relatively flat until x 200 fs, consistent with a delay in the ground state recovery until the excited state wavepacket reaches the region of the S, potential surface that allows a radiationless transition back to the ground state. The ground state then recovers with a characteristic time of z 450 fs, similar to that observed for the formation of the isomerized photoproduct [ 4,5]. The negative feature at short times is consistent with excited state absorption, which rapidly shifts out of this wavelength region as a result of wavepacket motion on the excited state surface. Additional features at negative delay and near-zero delay at this and other wavelengths result from pump-perturbed polarization free decay and coherent coupling [ 14,7], The response in the far red at 656 nm (fig. 2c), which lies far into the wing of the reactant absorption band, but within the absorption band of the

0.3 -

568

Q

nm

0.2 0.1 0

--?

’

-0.1 0.04

-I

b

620 nm

iv1

0.02 -

I

656 nm

c 0.01 -

-0.02

! -0.2

. 0.2

. 0.6

r

, delay

,

.

.

1.8

c

2.2

’

2.6

’

(k)

Fig. 2. Light-induced differential transmittance (AT/T) of bacteriorhodopsin as a function of time delay between the 12 fs pump and probe pulses at (a) 568 nm, (b) 620 nm, and (c) 656 nm.

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photoproduct, shows contributions from a number of processes. Underlying the oscillations are a sharp transient increase in transmission at short times, followed by an induced absorption and then a slow recovery. The positive feature corresponds to stimulated emission from the S, state, which rapidly disappears following evolution of the system away from the Franck-Condon region on the reactive potential surface [ 4,5,7]. The impulsive excitation used in this experiment would be expected to excite high frequency vibrational modes in the excited state that may contribute to the oscillatory modulation of this initial signal. Following the stimulated emission, the appearance of the initial photoproduct is expected to produce a region of induced absorption, and relaxation of this species to the intermediate K is responsible for the slow recovery [ 15 1. The response at the intermediate wavelength 620 nm (fig. 2b) also shows stimulated emission at short times followed by an induced absorption. This wavelength lies within both the reactant and photoproduct absorption bands and would be expected to include contributions from both species. An analysis of the frequency content of the oscillatory part of the detected response at each of the three wavelengths is presented in the Fourier power spectra in the upper half of each of the panels in figs. 3a-3c. Prior to Fourier analysis, a smooth background consisting of a low-order polynomial fit to the data is subtracted to remove the very low frequency background components. The data at all three wavelengths are transformed over the same time range of 70-2600 fs, with the lower limit constrained by the presence of sharp excited state features. The most striking feature of the comparison of the Fourier power spectra is that the same frequency components are present in all three wavelength regions. Components are evident at frequencies of x 1010, 1160, 1200, and 1525 cm-‘, which correspond well to the dominant vibrational modes detected by resonance Raman spectroscopy for the ground state reactant form of bacteriorhodopsin [ 161. The observation of components at the characteristic frequencies of the ground state reactant species indicates that the resonant impulsive Raman process is the origin of the oscillatory response. The observation of an oscillatory response in the far red indicates a contribution from this process involving 64

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568 NM EXPERIMENTAL

n

I

620NM

EXPERIMENTAL

656 NM EXPERIMENTAL

800

1000

frequency

1200

I

1400 (cm

1600 -‘)

Fig. 3. Fourier power spectra of the oscillatory part of the measured response at (a) 568 nm, (b) 620 nm, and (c) 656 nm, and ofthe calculated response at 580 nm, 620 nm, and 650 nm. Components are evident at frequencies of 1010, I160,1200, and 1525 cm-‘. The frequence resolution of the Fourier spectra of the measured response is z 12 cm-‘/point. The choice of wavelengths for the calculated response reflects differences between the experimental pulse spectrum and that of the model used in the calculations.

higher lying vibrational levels in the ground electronic state of the reactant that are not populated in thermal equilibrium and therefore do not contribute to the normal absorption spectrum. This process is depicted in fig. 4, which shows the generation of a displaced ground state wavepacket by two successive interactions with a pump pulse of finite duration. Since, the duration of the pump pulse used in these measurements is roughly half of the relevant vibrational periods, the resulting wavepacket would be expected to be broadened, with nonzero amplitude across the potential well. Such a broadened wavepacket will also contribute a nonoscillatory component to the measured differential transmittance that

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s, SO

Fig. 4. Schematic representation of the generation of wavepacket motion on the ground state potential surface by the resonant impulsive Raman process. Aninitialinteraction of the molecule with the pumppulse brings a wavepacket up to the excited state surface, where it propagates within the duration of the pump pulse. A second interaction with the pumppulse brings the wavepacket back to a displaced position on the ground state potential surface, where it subsequently oscillates.

would be expected to decay with the characteristic vibrational relaxation time, typically on the order of picoseconds. Fourier power spectra of the oscillatory part of the calculated pump-probe response are shown in the lower half of each of the panels in fig. j, The calculations are performed using a third-order perturbation expansion assuming a potential surface consisting of 29 uncoupled harmonic modes [ 91 with frequencies and excited state displacements derived from a resonance Raman intensity analysis for the reactant form of bacteriorhodopsin [ lo]. Only the ground state contribution to the time-resolved response is included in the calculations. The Fourier spectra of calculated pump-probe response show good agreement with the main features of the experimental results, further supporting the conclusion that the resonant impulsive Raman process is the dominant contribution to the detected oscillatory response. Although the detection wavelength of 656 nm lies well within the photoproduct absorption band, oscillations are not observed at the characteristic vibrational frequencies of the isomerized photoprod-

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uct, which are shifted relative to those of the all-trans reactant [ 16,171. The absence of an oscillatory response from the isomerized photoproduct is most likely a result of damping of the initial impulsively excited coherent vibrational motion in the excited state as the system evolves on an anharmonic reactive potential surface prior to formation of the ground state photoproduct. Oscillations might be expected to occur on the excited state surface during the initial part of this evolution; however, in the measurements presented here, a contribution directly from the excited state is observed only for very short times, as the reactive motion on the S, surface quickly and irreversibly carries the system out of the FranckCondon region of the potential surface. As a result of these effects, which originate from the photochemically active nature of the molecule, the dominant contribution to the oscillatory response in this impulsively excited system comes from the dynamics of the ground state reactant surface. This behavior differs dramatically from that of nonreactive systems, as exhibited by the organic dye molecule nile blue, in which the oscillatory response is dominated by stimulated emission originating from nonstationary states oscillating on a bound, largely harmonic excited state potential surface [ 1,18 1. Although the same vibrational frequencies are present at each of the three detected wavelengths, the relative intensities of the Fourier peaks depend on the detected wavelength. The relative intensities in the Fourier spectrum of the 568 nm data correspond well to those observed in the resonance Raman spectrum. A dramatically different intensity profile is seen at 620 nm, which falls on the red side of the reactant absorption band and at the center of the laser pulse spectrum. Here, the intensity profile appears inverted. At 656 nm, the relative line intensities are again more similar to those of the Raman spectrum. The relative amplitudes of the oscillatory components and their dependence on the detection wavelength are consistent with a resonant impulsive Raman process generated and probed by a pulse of finite duration. In the limit of a delta-function pulse, no ground state oscillations can be excited unless the transition moment is dependent on the nuclear coordinate, an effect that is generally not significant for resonant optical excitation into a strongly allowed absorption band. For excitation pulses of finite du65

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ration, evolution of the system on the excited state surface within the duration of the pump pulse results in a displaced ground state wavepacket when the system is coupled back to the S,, state, and the amplitude of the nonstationary state generated by the resonant impulsive Raman mechanism depends an the length of the vibrational period relative to the excitation pulse as well as the relative displacement of the ground and excited state potential minima. The dependence of the oscillatory response on the detection wavelength arises from the manner in which vibrational modes with differing frequencies modulate the absorption spectrum and from the manner in which the probe pulse interacts with the resulting timedependent polarization [ 91. Both the pulse duration and detection wavelength dependence of the resonant impulsive Raman process are represented in the calculated response, which shows a good qualitative agreement with the measurements. In conclusion, the femtosecond pump-probe measurements on bacteriorhodopsin clearly show the impulsive excitation of the dominant active modes of a complex polyatomic molecule. The nonstationary vibrational states persist on a picosecond time scale, sufficiently long for the frequencies to be resolved by Fourier analysis. Because of the rapid excited state reactive motion, the ground state reactant dynamics dominate the oscillatory response, a result that may serve as a guide for the interpretation of fast time-resolved measurements on other photochemically active systems. The measurements presented here clearly show the initial behavior of the excited state reactive dynamics. Femtosecond measurements at other wavelengths [ 19,201 may provide additional insight into the nonstationary state dynamics as the system approaches the transition state region of the excited state reactive surface. This work was supported by the US Department of Energy under Contract No. DE-AC03-76SF00098. RAM acknowledges support from the NSF (CHE 8615093) and the NIH (GM 44801). SLD acknowl-

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edges support through a University of California President’s Postdoctoral Fellowship. LAP was supported by an NIH Postdoctoral Training Grant (EY 07043 ) .

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