Regular paper Vol. 59, No 1/2012

Vibrational dynamics of the excited state in the light-harvesting complex (LH1) have been investigated by femtosecond stimulated Raman spectroscopy (FSRS). The native and reconstituted LH1 complexes have same dynamics. The ν(1) (C=C stretching) vibrational mode of spirilloxanthin in LH1 shows ultrafast high-frequency shift in the S(1) excited state with a time constant of 0.3 ps. It is assigned to the vibrational relaxation of the S(1) state following the internal conversion from the photoexcited S(2) state.


INTRODUCTION
Carotenoids (Cars) play important roles in photosynthesis.In the antenna complexes, they act as lightharvesting and photo-protecting pigments (Frank & Cogdell, 1996;Polívka & Sundström, 2004).Light energy absorbed by carotenoids is transferred to bacteriochlorophyll (BChl) with high efficiency.In the photo-protective function, carotenoids efficiently quench the BChl triplet state to prevent sensitized generation of reactive singlet oxygen.The S 0 ground state of alltrans-carotenoids has A g -symmetry assuming that their linear polyene backbone has C 2h point group symmetry.The lowest singlet excited state, S 1 (the 2A g -state), is optically forbidden.The S 2 (1B u + ) state is the lowest optically allowed state.The energy transfer to BChl has been reported to occur from both the S 2 and S 1 states.Recently, the S* state has been reported to have considerable importance in the light-harvesting function (Cong et al., 2008;Nakamura et al., 2011).It is the precursor on the reaction pathway toward triplet formation and plays a critical role in efficient excitation energy deactivation in the LH1 complex.
The vibrational dynamics of the S 1 state in carotenoids have been attracted much interest.The tunable photoexcitation of the S 2 state has revealed that the excess vibrational energy induced in the S 2 state remains even after the internal conversion to the S 1 state and affects the relaxation kinetics (Kosumi et al., 2005).The efficient energy transfer from the vibrational hot level of the S 1 state to BChl has been reported in the light-harvesting complexes (Wehling & Walla, 2005).Therefore, further investigations of the vibrational dynamics of the S 1 state are needed.
Femtosecond stimulated Raman spectroscopy (FSRS) has been recognized as a powerful method for studying vibrational dynamics in ultrafast phenomena.Combination of narrowband Raman pump pulse and ultrashort probe pulse has enabled femtosecond temporal resolution and a few tens wavenumber of spectral resolution (Yoshizawa & Kurosawa, 2000;Yoshizawa et al., 2001).The progresses in this decade have achieved the tunable Raman pump pulse which is useful for selective measurement of the transient state (Shim & Mathies, 2008).
In this work, we used native LH1 complex from Rhodospirillum rubrum S1 and reconstituted LH1 with purified spirilloxanthin that is hereafter called LH1(Spx).LH1(Spx) is used to remove the effect of other kinds of carotenoids involved in the native LH1.The vibrational dynamics of the S 1 state of carotenoid in the LH1 complexes have been investigated by the FSRS.

MATERIALS AND METHODS
Sample preparation.The native LH1 complex from wild-type Rhodospirillum rubrum S1 contains spirilloxanthin as a major carotenoid (Evans et al., 1988).In this study, the reconstituted LH1(Spx) was prepared as described elsewhere (Nakagawa et al., 2008).The solution of LH1(Spx) was dispersed in a poly-vinyl alcohol film (PVA, Kuraray Co., Ltd., PVA-217) on a glass plate (Nakamura et al., 2011).The solution of the native LH1 was measured using a 1 mm flow cell.During the measurements, the film sample was translated linearly and the solution was circulated to avoid degradation and the accumulation of any potential photoproducts.
Laser spectroscopy.The femtosecond dispersed time-resolved absorption spectroscopy setup shown in Fig. 1 is based on an amplified mode-locked Ti:Sapphire laser system operating at 1 kHz (Kosumi et al., 2010).A fraction of the amplified pulses was used to drive two independent optical parametric amplifiers (OPA).The excitation pulse resonant to the S 0 →S 2 transition of spirilloxanthin (500 nm, 100 fs) and the Raman pump pulse resonant to the S 1 →S m transition (620 nm, 20 cm -1 ) were obtained.The supercontinuum generated by a sapphire plate was used as a broadband probe pulse.The polarizations of all the beams were set to be parallel to one another.A probe pulse after the sample was dispersed onto a linear image sensor with a spectrometer.The excitation and Raman pump beams were modulated at 250 and 500 Hz, respectively, by a mechanical chopper, which was frequency locked to the laser pulse train.

Stationary absorption and Raman spectra
The stationary absorption spectrum of the LH1(Spx) film is shown in Fig. 2. The characteristic vibronic profile of the S 0 →S 2 transition of carotenoid (spirilloxanthin) appears in the region of 470-550 nm.The Q x and Q y bands of BChl are observed at 590 nm and 880 nm, respectively.The wavelengths of the absorption peaks are identical in the native LH1 solution.
Figure 3 shows the stationary spontaneous Raman spectra of the native LH1 solution and the LH1(Spx) film.The major observed signals are assigned to the Car S 0 ground state, because the Raman pump pulse (532 nm) is resonant to the Car S 0 →S 2 transition.The signals in the native LH1 solution and the LH1(Spx) film have almost the same structures.This suggests that spirilloxanthin is in the same environment both in the native LH1 and the reconstituted LH1.The three major Raman peaks at 1559 cm −1 (ν 1 ), 1149 cm −1 (ν 2 ), and 1001 cm −1 (ν 3 ) are assigned to the C=C symmetric stretching, the C-C symmetric stretching, and the methyl-in-plane rocking mode, respectively (Saito & Tasumi, 1983).

Transient absorption spectra
The transient absorbance change following the Car S 2 excitation in the LH1(Spx) film is shown in Fig. 4. The native LH1 solution has almost same transient spectra.
The observed signals are explained using a previously proposed model (Nakamura et al., 2011).The broad transient absorption due to the Car S 2 state is observed at 0.1 ps in the infrared region.The relaxation to the S 1 state and the energy transfer to the BChl Q x state occur simultaneously with a time constant of 70 fs (Kosumi et al., 2011).The well-known Car S 1 →S m absorption (620 nm) and the bleaching of the BChl Q y absorption (890 nm) are clearly observed at 1.0 ps.The relaxation from the hot S 1 state to the S 1 state is observed as temporal spectral change of the S 1 →S m absorption.Then, the S 1 state relaxes to the S 0 ground state with a lifetime of 1.6 ps.At 10 ps, the signal due to the Car S 1 state decreases, but the peak at 580 nm remains.It is assigned to the Car S* state that has a lifetime of 5.7 ps.

Femtosecond stimulated Raman spectra
The time-resolved stimulated Raman signals on the Stokes side are shown in Fig. 5.The Raman pump pulse was tuned at 620 nm to be resonant to the Car S 1 →S n transition.The signals observed in the native LH1 solution and the LH1(Spx) film show essentially the same spectral pattern.The signals without the 500 nm excitation pulse (top curves) are assigned to the S 0 ground state.The ν 1 , ν 2 , and ν 3 modes are observed consistently with the stationary Raman measurement.After the excitation, the ν 1 and ν 2 modes of the S 0 state decrease instantaneously and broad new signals appear around 1750 and 1250 cm -1 .They are assigned to the ν 1 and ν 2 modes in the S 1 state, respectively, as well as the signals observed in b-carotene (Yoshizawa et al., 2001).The high-frequency shift of the S 1 excited state is explained in terms of the vibronic coupling through the vibrational mode with A g symmetry, because they have same A g symmetry (Hashimoto & Koyama, 1989;Noguchi et al., 1989).On the other hand, the ν 3 mode does not show clear change.Since the ν 3 mode is the methylin-plane rocking mode, it has smaller coupling with the S 1 state than the ν 1 and ν 2 modes.The signals assigned to the S 1 state decrease with the 1.6 ps lifetime of the S 1 state.The broad foot observed at the ν 1 mode of the S 0 state decays with a lifetime of 5.4 ps.It is assigned to the hot S 0 state generated by the internal conversion from the S 1 state.
The remarkable feature observed in the FSRS signal is transient peak shift of the ν 1 mode of the S 1 state.The ν 1 signal just after the excitation has a peak at 1740 cm -1 , then it shifts to 1767 cm -1 with a time constant of 0.3 ps.The transient absorption shown in Fig. 4 shows temporal spectral change with the same time constant.The S 1 transient absorption has broad spectrum at 0.1 ps, then the peak shifts to shorter wavelength and the spectrum becomes narrower.These changes are explained in terms of the vibrational relaxation in the S 1 state (Kosumi et al., 2005).The hot S 1 state is generated by the internal conversion from the initially excited S 2 state, then it relaxes to the vibrational ground level of the S 1 state.However, the ν 2 mode of the S 1 state does not show the temporal spectral change.This means that the hot S 1 state of the ν 1 mode is generated because it is the coupled mode of the internal conversion from the S 2 state to the S 1 state similar to that from the S 1 state to the S 0 state.
The vibrational features of spirilloxanthin in the native LH1 and the reconstituted LH1(Spx) have been investigated by the stationary and time-resolved Raman spectroscopies.The vibrational modes of the S 0 ground state observed by the ordinary spontaneous Raman spectroscopy have the same frequencies.The environment of the reconstituted spirilloxanthin is identical to that of the native LH1 complex as previously revealed by the Stark spectroscopy (Nakagawa et al., 2008).The dynamics following the excitation of the Car S 2 state are also the same in the native LH1 and LH1(Spx).The S 1 state in the LH1 complex is in the vibrational excited level just after the formation from the S 2 state.The relatively slow vibrational relaxation of the ν 1 mode is expected to be a universal feature of carotenoids even when they are bound to the LH complexes.Our findings provide the importance of the vibrational dynamics in the energy transfer mechanisms of the LH complexes.