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J. Phys. Chem. B 2001, 105, 1072-1080
Near-Infrared Time-Resolved Study of the S1 State Dynamics of the Carotenoid Spheroidene Toma´ sˇ Polı´vka,† Donatas Zigmantas,† Harry A. Frank,‡ James A. Bautista,‡ Jennifer L. Herek,† Yasushi Koyama,§ Ritsuko Fujii,§ and Villy Sundstro1 m*,† Department of Chemical Physics, Lund UniVersity, Box 124, S-221 00 Lund, Sweden, Department of Chemistry, UniVersity of Connecticut, U-60, 55 North EagleVille Road, Storrs, Connecticut 06269-3060, and Department of Chemistry, Faculty of Science, Kwansei Gakuin UniVersity, Uegahara, Nishinomiya 662-8501, Japan ReceiVed: June 16, 2000; In Final Form: October 3, 2000
Using a novel experimental approach based on near-infrared femtosecond absorption spectroscopy, we have determined the energy of the S1 state of the carotenoid spheroidene. The energy of this state is 13 400 ( 90 cm-1 at both 293 and 186 K, showing that there is no temperature-induced shift of the S1 level. A discrepancy of about 800 cm-1 between the S1 energy determined here and that obtained from previous fluorescence and resonance Raman measurements is explained in terms of the different conformational species coexisting in the S1 excited state. Measurements of kinetics in the near-infrared region revealed that at least three relaxation processes take place after excitation of spheroidene into its S2 state. Ultrafast S2fS1 internal conversion occurs within the first 300 fs, followed by vibrational cooling in the S1 state, which occurs on a time scale of ∼700 fs. The S1 lifetime is 8 ps at 293 K, in good agreement with previous measurements of the S1 f SN transition. A somewhat longer S1 lifetime of 9.5 ps is observed at 186 K.
Introduction Carotenoids are a class of natural pigments that play important roles in many biological systems. In photosynthetic organisms, they assist (bacterio)chlorophylls in the light-harvesting function, efficiently covering the spectral region between 420 and 550 nm.1 In addition, they play an integral role in regulating energy flow in plants2 and are antioxidants that prevent damage of various physiological systems by quenching of triplet states and scavenging singlet oxygen.3 The light-harvesting function of carotenoids relies on their ability to transfer energy to (bacterio)chlorophylls from low-lying excited states, two of which are denoted S1 (2Ag- in the idealized C2h group) and S2 (1Bu+).4 Because the conjugated chain of the carotenoid molecule has C2h symmetry, a transition between the ground-state S0 (1Ag-) and the S2 state is optically allowed, resulting in a strong absorption of carotenoids in the spectral region 420-550 nm. In contrast, the S0 f S1 transition is optically forbidden, because the S1 state has the same symmetry as the ground state.5-16 Therefore, to fully understand the mechanisms and pathways of energy transfer between carotenoids and (bacterio)chlorophylls involving the carotenoid S1 state, the precise location of the S1 energy of carotenoids is necessary. The S1 energies of carotenoids with conjugation lengths less than nine CdC bonds have been determined because weak fluorescence from the S1 level can be observed.17 As the conjugation length of carotenoid increases, however, Kasha’s rule is violated and fluorescence from the S2 state dominates.17-19 Thus, it is very difficult to determine the position of the S1 level in physiologically important carotenoids, as most of these have a conjugation length greater than nine CdC bonds.20 * Corresponding author. E-mail:
[email protected]. Fax: +46-46-2224119. † Lund University. ‡ University of Connecticut. § Kwansei Gakuin University.
Attempts to determine the S1 energy of various carotenoids have used the dynamics of the excited state. Measurements of the decay of S1 f SN excited-state absorption appearing in the visible part of spectrum revealed that the S1 lifetimes of carotenoids with 9 to 11 conjugated double bonds are in the range 3-30 ps.21 Knowledge of the S1 lifetimes, together with the S1 energies and dynamics of shorter carotenoids, enabled the extrapolation of the S1 energy of nonfluorescent carotenoids using the energy gap law.21,22 This approach has been employed to determine energies of the S1 state of spheroidene analogues with conjugation lengths ranging from 7 to 13 double bonds,19 as well as for other physiologically important carotenoids.23,24 In the cases of highly substituted carotenoids, e.g., peridinin, a significant deviation from the energy gap law has been reported.25 Also, for violaxanthin and zeaxanthin, two important carotenoids involved in photoprotection in higher plants, values of the S1 energies determined directly deviate from those calculated by means of the energy gap law.26,27 Fluorescence spectroscopy has enabled detection of the weak S1 f S0 fluorescence in carotenoids with 10 and 11 CdC bonds.17,27-29 Resonance Raman spectroscopy has proven a useful alternative to detection of the weak S1 f S0 fluorescence,30 but the necessity to use crystalline samples at low temperature makes a direct comparison with results obtained in solution or even in protein environment difficult. A new experimental approach based on femtosecond spectroscopy was recently introduced to determine the S1 energies of carotenoids by scanning the S1 f S2 excited-state absorption in the near-infrared region (5500-12 000 cm-1) shortly after S2 excitation.26 In contrast to the S0 f S1 transition, the S1 f S2 transition is allowed, providing the possibility to overcome problems with weak signals originating from the S1 f S0 fluorescence. This approach has been used to determine the S1 energies of zeaxanthin and violaxanthin.26 The location of the S1 levels of these carotenoids determined by this method is about
10.1021/jp002206s CCC: $20.00 © 2001 American Chemical Society Published on Web 01/12/2001
S1 State Dynamics of the Carotenoid Spheroidene
Figure 1. Molecular structure of spheroidene.
450 cm-1 lower in energy than those obtained from fluorescence spectroscopy.27 Spheroidene (Figure 1) is a carotenoid with 10 conjugated double bonds that occurs in light-harvesting systems and reaction centers of the purple bacterium Rhodobacter (Rb.) sphaeroides. A 15,15′-cis-spheroidene is present in the reaction center of this extensively studied photosynthetic organism, whereas the alltrans form of spheroidene is an important light-harvesting pigment in both LH1 and LH2 antenna complexes.31,32 Because energy transfer pathways in these complexes may involve both the S2 and S1 states of the carotenoid,28,33-35 knowledge of the location of the S1 state is essential for understanding the mechanism of carotenoid to bacteriochlorophyl (BChl) energy transfer. Recent results obtained from fluorescence spectroscopy36 and low-temperature resonance Raman spectroscopy30 put the S1 level of spheroidene at 14 200 cm-1, in very good agreement with the value calculated by the energy gap law.19 A different experimental technique based on two-photon absorption has been used to determine the S1 level of spheroidene in its natural protein environment in the LH2 antenna complex of Rb. sphaeroides.37 Because the S0fS1 transition is allowed for two-photon absorption, the two-photon excitation profile was measured by detection of LH2 BChl-a fluorescence, revealing the S1 level to be at 13 900 cm-1. However, to obtain this value, the intensity of the 0-0 transition was supposed to be about 40 times weaker than that corresponding to the 0-1 transition, making an unambiguous assignment difficult. Here we report the S1 f S2 transient absorption spectrum of spheroidene in n-hexane at both room and low temperatures. The S1 energy obtained from these data differs significantly from that obtained with other techniques.30,36 This discrepancy and its implications for further understanding of the photochemistry and photophysics of the excited states of carotenoids are discussed. Experimental Section Spheroidene was extracted from whole cells of Rb. sphaeroides 2.4.1 grown anaerobically in modified Hutner’s medium.38 Approximately 5 g of wet packed cells was mixed with 150 mL of methanol and 20 mL of acetone, and the mixture was shaken until a dark green solution was obtained. The pigments were extracted using 100 mL of petroleum ether and dried using a rotary evaporator. The carotenoids were separated from the rest of the pigments by loading them onto a DEAE sephacel (Sigma I 6505) column using acetone as the eluting solvent. The carotenoids were dried using a gentle stream of nitrogen, redissolved in 1 mL of petroleum ether and loaded onto an alumina column preequilibrated with petroleum ether. Solutions of petroleum ether containing from 0.5 to 4.0% ethyl acetate were used to elute the spheroidene. The fraction containing spheroidene was identified by absorption spectroscopy, dried, redissolved in acetone, and injected into a Millipore Waters E 600 high-performance liquid chromatograph (HPLC) employing a Nova-Pak C18 column with the mobile phase programmed as follows: 0-12 min, isocratic methanol/acetonitrile (95/5 v/v); 12-27 min, linear gradient to methanol/n-hexane (95/5 v/v); 27-45 min linear gradient to methanol/acetonitrile (95/5 v/v). The eluent was monitored using the Model 996 single diode array detector and the peak corresponding to all-trans-spheroi-
J. Phys. Chem. B, Vol. 105, No. 5, 2001 1073 dene was collected and dried using a gentle stream of nitrogen. The purified sample was stored in the dark at -55 °C. Before each experiment, all-trans-spheroidene was dissolved in n-hexane to yield a sample having an optical density of ∼0.2/ mm at the excitation wavelength and kept at room temperature in a 2 mm optical cell. Low-temperature measurements were performed with the same optical cell inserted into an optical cryostat with temperature controller enabling precise adjustment of the sample temperature. The temperature during these measurements was chosen to be 186 K ((0.5 K), about 10 K above the freezing point of n-hexane, to maintain the sample in the liquid phase, thus facilitating a direct comparison with the room temperature (293 K) measurements. The femtosecond spectrometer used in these studies is based on an amplified Ti:sapphire laser system, with tunable pulses obtained from two optical parametric amplifiers. Femtosecond pulses obtained from the Ti:sapphire oscillator operating at a repetition rate of 82 MHz were amplified by a regenerative Ti:sapphire amplifier pumped by a Nd:YLF laser operating at a repetition rate of 5 kHz and producing ∼120 fs pulses with an average output power of ∼1 W and central wavelength of 800 nm. The amplifier output was then divided by a 75/25 beam splitter to pump two independent parametric amplifiers for generation of the pump and probe pulses. The parametric amplifier used for generation of probe pulses was controlled by a computer, enabling direct scanning of the probe pulses over the spectral region 850-1800 nm (12 000-5500 cm-1), where the S1 f S2 transition is expected. In all measurements, the mutual polarization of the pump and probe beams was set to magic angle (54.7°) using a polarization rotator placed in the pump beam path. The instrument response function was measured by frequency mixing the pump and probe pulses in a LiIO3 crystal; the obtained cross-correlation was fitted to a Gaussian function with a fwhm of 160-180 fs, depending slightly on the wavelength of probing pulses. A detection system based on a three-diode arrangement in combination with a single grating monochromator provided spectral resolution better than 40 cm-1. To prevent any photochemical damage of the sample during measurement, the excitation pulses were attenuated using neutral density filters to typical energies of 400 nJ/pulse. No sample degradation was observed during the course of measurement. Furthermore, absorption spectra were measured before and after measurements to ensure that no permanent photochemical changes occurred over the duration of experiment. Results Steady-State Absorption. Absorption spectra of spheroidene at both 293 and 186 K are shown in Figure 2. The absorption maximum of the 0-0 band of the S0-S2 transition at room temperature is located at 20 730 cm-1, while at 186 K this transition is shifted significantly to lower energy, having a maximum at 20 370 cm-1. Pronounced vibrational structure is evident at both temperatures with a characteristic peak-to-peak separation of ∼1350 cm-1 as a result of combinations of the two totally symmetric vibrational modes having frequencies of 1150 cm-1 (CsC stretch) and 1600 cm-1 (CdC stretch).18 However, at 186 K the 0-1 absorption band is clearly asymmetric and the fitting procedure resolves the two vibrational modes. The observed energy of the 0-0 band at room temperature (20 730 cm-1) and a weak absorption at around 28 500 cm-1 (not shown, absorption ratio A28500/A20730 ≈ 0.09), known as a marker of cis-isomer present in sample,39,40 suggest that the all-trans isomer is the dominating species in the studied spheroidene sample.
1074 J. Phys. Chem. B, Vol. 105, No. 5, 2001
Polı´vka et al.
Figure 2. Steady-state absorption spectra of spheroidene in n-hexane at 293 K (dashed line) and 186 K (solid line).
Transient Absorption Spectra. In Figure 3, near-infrared transient absorption spectra of spheroidene are presented. Spheroidene was excited to the lowest vibrational band of the S0 f S2 transition at 20 400 cm-1 at 293 K; at 186 K the excitation was tuned correspondingly with the shift of the absorption spectrum to 20 150 cm-1. These excitation conditions exclude any contribution from relaxation within the vibrational manifold of the S2 state to the transient absorption signal. The transient spectra at both temperatures were recorded 3 ps after excitation. At this time delay, most of the spheroidene molecules are in their S1 state (as the S2 f S1 relaxation is finished within 300 fs33) and the S1 lifetime of spheroidene is known to be approximately 8 ps.19 The room-temperature transient spectrum in Figure 3 exhibits a shape very similar to the vibrationally resolved S0fS2 transition band (Figure 2). Furthermore, the energy gaps between the four peaks in the transient absorption spectrum located at 7310, 8770, 10 050, and 11 270 cm-1 match very well with those observed in the strongly allowed S0 f S2 transition shown in Figure 2. Therefore, we assign the observed transient absorption spectrum to the S1 f S2 transition. At 186 K, only the profile of the lowest-energy band was measured. The temperature-induced spectral shift of the transient absorption spectrum is about the same (∼350 cm-1) as that detected in the S0 f S2 absorption spectrum (Figure 2); the lowest energy transition is at 6980 cm-1. Despite the general similarity of the transient and steadystate absorption spectra shown in Figures 2 and 3, it is important to note that the lowest (0-0) transition of the transient absorption spectrum, shown in detail in Figure 3b, exhibits an apparent asymmetry, which is not observed in the S0 f S2 transition. A Gaussian fitting procedure of the 0-0 band gives the best results with two bands located at 7310 cm-1 (6850 cm-1) and 6860 cm-1 (6550 cm-1) at 293 K (186 K). This asymmetry is likely an intrinsic feature of the S1 state of spheroidene and will be discussed in detail in the Discussion. Kinetics. To be certain that the observed transient absorption spectra shown in Figure 3 indeed correspond to the S1 f S2 transition, kinetics were measured at various energies covering the transient absorption spectrum. Kinetic traces obtained at both temperatures are shown in Figure 4. At the maximum of the 0-0 transition (probing at 7300 cm-1), the decay is singleexponential and characterized by a decay time of 8 ps at 293
Figure 3. Transient absorption spectra of spheroidene in n-hexane at 293 K (open squares) and 186 K (filled squares) in the spectral region 5500-12 000 cm-1 (a) and detailed profile of the 0-0 band in the spectral region 6000-8000 cm-1 (b). Transient spectra were recorded 3 ps after excitation. Solid curves represent Gaussian fits of the transient absorption spectra.
K, in good agreement with the S1 lifetime measured by the decay of S1 f SN excited-state absorption.19 This 8 ps component is also present in kinetics measured at the maximum of the 0-1 transition (8700 cm-1), but an additional fast component with a decay time of 270 fs dominates the decay. A similar behavior is revealed when the shoulder of the 0-0 transition is probed at 6900 cm-1. Here two additional kinetic components are required to obtain a satisfactory fit, with decay times less than 140 fs (47%) and 800 fs (16%). The remaining decay (37%) is again characterized by an 8 ps time constant. The kinetics probed at the maximum of the 0-0 band exhibit a noticeable rise component characterized by a time constant of about 290 fs, though no rise components were observed at other probing wavelengths at 293 K. Generally, the same behavior was observed at 186 K. The primary decay, which is obviously the lifetime of the S1 level, is slightly slower at this temperature, having a time constant of 9.5 ps as observed when probing at
S1 State Dynamics of the Carotenoid Spheroidene
J. Phys. Chem. B, Vol. 105, No. 5, 2001 1075
Figure 5. Transient absorption spectra of the 0-0 band of the S1 f S2 transition of spheroidene in n-hexane at 186 K recorded at different time delays of 1 ps (open circles), 3 ps (filled squares), and 8 ps (open triangles) after excitation. Both transient spectra are normalized to the maximum of the lowest energy transition.
Figure 4. Kinetics of spheroidene in n-hexane at 293 K (left column) and 186 K (right column), recorded at different energies: at the maximum of the 0-1 band of the S1 f S2 transition (a, d), at the maximum of the 0-0 transition (b, e) and at the shoulder of the 0-0 transition (c, f).
TABLE 1: Time Constants Extracted from Multiexponential Fits of Kinetics Measured at Various Probing Wavelengths at Both 293 and 186 K T (K) E (cm-1) τrise (ps) τ1 (ps) A1 (%) τ2 (ps) A2 (%) τ3 (ps) A3 (%) 293 293 293 293 293 186 186 186
11100 10000 8700 7300 6900 8130 6950 6530
0.28 0.25 0.27
98 96 87
0.14 0.25
47 94
0.29 0.14 0.65 0.15
8.1 8.5 7.8 8.0 8.1 9.7 9.5 9.9
2 4 13 100 37 6 100 44
0.8
16
0.75
56
the maximum of the 0-0 transition at 186 K (6950 cm-1). The rise component observed here is ∼650 fs, thus significantly slower than at 293 K. Moreover, at 186 K it is clear that the additional fast components observed at 8130 cm-1 (maximum of the 0-1 transition at 186 K) and at 6530 cm-1 (the shoulder of the 0-0 transition) originate from two different processes: at 8130 cm-1 this component has a time constant of 250 fs and is considerably faster than that observed at the shoulder of the 0-0 transition (750 fs). Furthermore, there is an observable rise time of about 150 fs at the shoulder of the 0-0 transition at 186 K, which is not seen at 293 K. All decay times measured at various energies and at both temperatures are summarized in Table 1. To establish the origin of the two subpicosecond components observed in the decays, we measured the spectral profile of the 0-0 band at 186 K at three different time delays, as shown in Figure 5. The transient spectra were normalized to the maximum
of the lowest energy band. At 1 ps, when there is a substantial signal coming from the subpicosecond (750 fs) process observed at the very red edge of the transient absorption spectrum (see Table 1), the 0-0 band is considerably broader than at later decay times. This observation suggests that the 750 fs process can be ascribed to vibrational cooling in the S1 state and that 3 ps after excitation spheroidene is indeed in its thermally relaxed S1 state. This conclusion is supported by the fact that there is almost no change in the shape of the 0-0 transition when measured at 3 ps and at 8 ps (Figure 5). The ultrafast ( (0-1) > (0-2), are explained, but the absolute spacings are not. Method b. We also tried to determine directly the displacements of the potential minima in the S2 and S1 states by the use of the near-infrared absorption spectrum shown in Figure 3 (at 293 K). The displacements along the ν1 and ν2 coordinates were determined to be ∆1′ ) 1.1 and ∆2′ ) 0.8. The relative positions of those potential minima are depicted in Figure 6b, and the result of spectral simulation is shown in Figure 7b. If one assumes that the shifts of the S2 potential minima with respect to the S0 potential minima, i.e., ∆1 ) 1.3 and ∆2 ) 0.9, are the same as in Method a, it is impossible to obtain a satisfactory simulation of the fluorescence spectrum published previously (data not shown).36 Discussion From the S0 f S2 and S1 f S2 spectra can one calculate the energy of the S1 level by subtraction E(S0 f S2) - E(S1 f S2). On the basis of the assignment of the lowest energy peak in the transient absorption spectrum to the 0-0 transition, the energy of the S1 level is calculated to be 13 420 ( 80 cm-1 at 293 K and 13 390 ( 80 cm-1 at 186 K, leading to the conclusion that there is no temperature-induced shift of the S1 energy. The most striking result, however, is the fact that the energy of the
Figure 7. Near-infrared absorption spectrum (squares) and the results of simulations (solid lines) using the differences between the S1 and S2 potential minima: (a) ∆1′ ) 0.1 and ∆2′ ) 1.0 and (b) ∆1′ ) 1.1 and ∆2′ ) 0.8 (see text for details). A Gaussian function with fwhm of 800 cm-1 was used in the simulations of vibrational bands. All spectra are shifted to have their 0-0 transitions at zero energy.
S1 level measured by the near-infrared femtosecond technique is about 800 cm-1 lower than the energy of 14 200 cm-1 determined by both fluorescence and resonance Raman spectroscopies. The same value of 14 200 cm-1 is also the predicted S1 energy based on extrapolation from shorter spheroidene analogues.19 Previous direct measurements of the spheroidene S1 energy have relied on spectral information only.30,36 In the current work, we have added information about the dynamics of the S1 level, which helps to assign the initial and final states of the transient absorption spectrum. The kinetics measured here show that the lifetime of the S1 state is slightly shorter than that measured by Frank et al. (8.7 ( 0.1 ps); our analysis yields the lifetime 7.88.5 ps, varying slightly with probe wavelength. According to the energy gap law, a shorter lifetime should correspond to a lower S1 level, but this small difference cannot account for the 800 cm-1 energy discrepancy.21 The observation of a slightly shorter S1 lifetime in the present study may suggest the presence of a cis-spheroidene isomer in our sample. The isomer could be, in principle, responsible for the asymmetry of the lowest energy transition in the transient absorption spectra, since it could have a different S1 energy than the all-trans species. Experiments on locked 15,15′-cis-
S1 State Dynamics of the Carotenoid Spheroidene spheroidene revealed an S1 lifetime of 7.1 ( 0.1 ps.39 The shortening of the S1 lifetime observed in our experiments could be therefore ascribed to the presence of the isomer. Then, the calculated energy of 13 420 cm-1 would be the S1 energy of the isomer, while the S1 energy of all-trans-spheroidene would correspond to the shoulder, giving an S1 energy of 13 900 cm-1. This result is much closer to the 14 200 cm-1 obtained from fluorescence and resonance Raman measurements, and taking into account an experimental error of about 90 cm-1, it is actually within the accuracy given by kT at 293 K. However, the absorption spectra do not show any features such as cispeaks39,40 attributable to the presence of the isomer, significant enough to make the S1 f S2 transition of the isomer stronger than that of the all-trans species. Moreover, similar and even more pronounced shoulders in the transient absorption spectra were observed in our previous study of violaxanthin and zeaxanthin, where the observed S1 lifetimes matched perfectly those measured by the S1 f SN transition and no sign of an isomer was seen in the absorption spectra.26 Thus, we conclude that the features observed in our spectra are not due to the presence of the spheroidene isomer. The essential factor enabling a reliable assessment of the S1 energy is proper assignment of the peaks observed in the S1S2 transient absorption spectrum. On the basis of the energy gap between the peaks observed in the transient spectrum shown in Figure 3, the absorption bands reflect the vibrational levels of the S2 state. However, it is possible that the shoulder on the red edge of the peak with lowest energy could reflect a splitting of the two prevailing vibrational modes (CsC and CdC stretches), which are separated by about 450 cm-1, approximately the same value as the energy gap between the shoulder and the main band. Such a splitting is very clear in carotenoids with shorter conjugation lengths.18 This explanation would lead directly to the conclusion that the lowest energy peak observed in the transient spectrum must be due to the 0-1 transition, since such a splitting cannot occur in the 0-0 transition. A calculation based on this assignment locates the S1 energy at 14 800 cm-1, which would be considerably too high in comparison with 14 200 cm-1 obtained by different methods. Two effects, Stokes shift and vibrational modes of false origin may account for this difference. When the S1 energies from absorption and fluorescence methods are compared, the effects of a Stokes shift should be considered. Unfortunately, there is no direct information on the magnitude of the Stokes shift of the S1 level of carotenoids. The only experimental evidence comes from recent resonance Raman experiments on spheroidene, which gives exactly the same value of the S1 energy (14 200 cm-1) as measured via fluorescence.30 Because resonance Raman has the same selection rules as one-photon absorption, these results suggest that there is no Stokes shift in the S1 level of spheroidene. In contrast, a Stokes shift of ∼300 cm-1 was revealed in the polyene undecapentaene in EPA glass at 77 K.42 Measurements of fluorescence excitation spectra of shorter polyenes revealed that there is a difference of about 100 cm-1 between the spectral origins of the 0-0 band obtained by one-photon and two-photon excitation. This shift is caused by the fact that in the case of optically forbidden one-photon excitation, the 0-0 bands are built on nontotally symmetric (bu) vibrational modes of false origin that mix the S1 and S2 states.15-18,43 This effect may also reflect the difference between the spectral origin of the 0-0 bands obtained by fluorescence spectroscopy (forbidden S1 f S0 transition) and our near-infared femtosecond spectroscopy (allowed S1 f S2 transition). However, keeping in mind that
J. Phys. Chem. B, Vol. 105, No. 5, 2001 1077 these vibrational modes of false origin are in-plane bending modes with frequencies of about 100 cm-1 in polyenes, we expect these modes to contribute by approximately this amount in shifting the spectral origin. Hence, we conclude that the combined effect of the Stokes shift and vibrational modes of false origin cannot justify the assignment of the lowest energy peak in the transient absorption spectrum (Figure 3) to the 0-1 transition and does not explain the 600 cm-1 difference as obtained with various methods. Furthermore, if the lowest energy peak in Figure 3 is indeed the 0-1 transition, then the 0-0 transition must be completely suppressed as there are no bands observed at lower energy. Such a dramatic change of Franck-Condon factors corresponding to the 0-1 and 0-0 transitions is not expected. Simulations of the S1 f S2 absorption spectrum presented in Figure 7 show that the vibrational progression in the calculated S1 f S2 spectrum is in good agreement with the experimentally measured transient absorption spectrum presented in Figure 3, again suggesting that the lowest-energy transition in the transient absorption spectrum is indeed the 0-0 transition. Nevertheless, when the parameters resulting from the simulated S1 f S2 transient absorption spectra are compared in Figure 7a,b, it is obvious that the different simulations give different values of the displacements of the S1 and S2 potential surfaces, in particular for the ν1 mode. Moreover, the simulations based on the S1 f S2 transient absorption spectrum cannot explain the fluorescence spectrum observed in ref 36. This result may be explained in terms of either the presence of different conformations existing in the S1 state or the effect of solvent reorganization. However, since we do not observe any dynamic feature in the shape of the 0-0 band of the transient absorption spectrum between 3 and 8 ps (Figure 5), the solvent effect is not likely a candidate to explain this discrepancy. As final support of the assignments of the peaks, in Figure 8 both the steady state (S0 f S2) absorption spectrum and the S1 f S2 transient spectrum of spheroidene are shown, shifted in energy such that their lowest energy bands are superimposed. The spacing of vibrational peaks observed in the steady-state absorption spectrum is very well conserved in the transient absorption spectrum. Looking at the spectral widths of the vibrational bands, it is apparent from Figure 8 that the lowest energy transition of the S1 f S2 spectrum at 293 K (7310 cm-1) is too narrow to be assigned as the 0-1 transition. However, its width matches very well with that of the 0-0 transition of the steady-state absorption spectrum suggesting that the 7310 cm-1 peak in the S1 f S2 spectrum should be ascribed to the 0-0 transition. The same conclusion is valid for the S1 f S2 spectrum measured at 186 K, where only the profile of the lowest energy peak was measured. All these facts lead to the conclusion that the 7310 cm-1 (6980 cm-1) band at 293 K (186 K) is due to the 0-0 transition, locating the S0 f S1 0-0 transition of spheroidene in n-hexane solution at 13 400 ( 90 cm-1 at both 293 and 186 K. Then, two important questions remain: (1) What is the reason for the 800 cm-1 mismatch between the S1 energy determined here and that measured by other methods and (2) what is the origin of the asymmetry displayed in the 0-0 band of the transient absorption spectrum? It is important to emphasize that the crucial difference between the near-infrared femtosecond absorption experiment and those performed earlier is the fact that both fluorescence and resonance Raman measurements deal with the forbidden S1 T S0 transition, while here the allowed S1 f S2 transition is measured. All the results presented here suggest that the
1078 J. Phys. Chem. B, Vol. 105, No. 5, 2001
Figure 8. Steady-state absorption spectrum (solid curves) superimposed with the S1 f S2 transient absorption spectrum (squares) of spheroidene in n-hexane at 186 K (a) and 293 K (b). All spectra are shifted to have their 0-0 transitions at zero energy.
asymmetry of the 0-0 transition in the transient absorption spectrum is due to an intrinsic property of the S1 state of spheroidene, indicating that the S1 potential surface has more complex structure than was previously thought, creating the asymmetry of the 0-0 band. Recent ab initio calculations performed on short polyenes show that if nontotally symmetric modes are taken into account, the S1 potential surface will be indeed more complex.44 Very recently, time-resolved CARS experiments revealed a double-well character of the S1 state of diphenylhexatriene, based on observation of two different vibrational frequencies belonging to the same totally symmetric CdC stretching mode.45 The S1 potential surface is explained in terms of symmetry breaking and perturbation due to nontotally symmetric bu modes. There is also experimental evidence suggesting that the S1 potential surface of carotenoids has a more complex structure than the S2 surface. Some carotenoids have dual emissions, making it possible to measure fluorescence from both their S2 and S1 states.17-19 In such carotenoids, including spheroidene, there is a striking difference in the shape of the fluorescence spectra originating from the S1 and S2 states. Despite the fact that both fluorescence spectra reflect the vibrational structure of the S0 ground state, the resolution of the vibrational bands is much better in the case of the S2 f S0 fluorescence.27,36 On the other hand, the S1 f S0 fluorescence is rather broad, with vibrational peaks only partially resolved.
Polı´vka et al. It is also worth noting that in the S1 f S0 fluorescence spectra of polyenes, which have well resolved vibrational structure, there is a clear shoulder at the low-energy side of the 0-0 band of the fluorescence spectrum.12 These observations support the consideration of a more complicated structure of the S1 potential surface of carotenoids; both the difference between the S2 f S0 and S1 f S0 fluorescence spectra and the shoulder at the low-energy side of the 0-0 transition in some polyenes can be explained as due to more than one fluorescing state of the S1 potential surface. Such a scenario may also account for the present results obtained by near-infrared femtosecond spectroscopy. If the S1 energy is calculated from the shoulder of the 0-0 band of the room-temperature transient absorption spectrum shown in Figure 3, a satisfactory agreement with the S1 energy measured previously from the forbidden S1 f S0 transition is obtained. The same conclusion is reached for two xanthophylls, violaxanthin and zeaxanthin, which have been measured very recently by both femtosecond transient absorption26 and fluorescence spectroscopy.27 Taking into account the shoulder, which is clearly visible in the transient absorption spectra shown in ref 26, and calculating the S1 energies of both carotenoids from this shoulder, one obtains, in fact, perfect agreement with the S1 energies measured by fluorescence. The experimental results described above can be fully explained by a model in which the S1 potential surface of spheroidene has a complicated structure corresponding to the presence of different conformers. These conformers exist only in the S1 state as a result of excitation of torsional modes by the S2 f S1 internal conversion process. Then, after vibrational relaxation in the S1 state, a statistical distribution of these conformers will exist in the S1 state. Some of the conformations, however, deviate from the idealized C2h symmetry of all-trans spheroidene, making the forbidden S1 f S0 transition more allowed than that of the all-trans species. In such a case, the S1 f S0 transition belonging to the conformers that deviate from the idealized C2h symmetry will dominate in spectroscopic methods relying on the forbidden transition, as it is more allowed than the S1 f S0 transition of the all-trans species. However, this is not the case for the near-infrared femtosecond approach presented here, because we explore the allowed S1 f S2 transition and thus there are no restrictions due to the forbidden nature of the observed transition. It is therefore natural to ascribe the S1 energy of 13 400 cm-1 to the all-trans spheroidene, while the value of 14 200 cm-1 extracted from fluorescence experiments is the S1 energy of the twisted species deviating from C2h symmetry. The low-energy shoulder in the 0-0 band of our transient spectra, giving an S1 energy of 13 900 cm-1, is then due to the S1 f S2 transition originating from these twisted species. This value corresponds within experimental error to 14 200 cm-1 obtained by fluorescence spectroscopy, taking into account the accuracy given by a thermal population at room temperature. At 186 K, the difference is too big to be within kT, but there is a rather big error given by the fitting procedure, as the 186 K transient absorption spectrum is noisier than that at 293 K. Despite this progress toward reconciling different results for the carotenoid S1 energies, there are still discrepancies between various techniques that remain to be explained in future experiments. The results obtained by resonance Raman spectroscopy, which give the value of 14 200 cm-1, can hardly be explained by the model described above, because those results were obtained for crystalline spheroidene at 100 K. Moreover, the model presented here requires excitation to the S2 state to
S1 State Dynamics of the Carotenoid Spheroidene create the twisted conformers in the S1 state, which is certainly not the case of the resonance Raman approach. However, the crystalline spheroidene at low temperature might also consist of a mixture of different conformational states, making the situation comparable to that observed in the liquid phase. To understand these discrepancies, it is highly desirable to use both theoretical approaches and other experimental techniques, among which two-photon absorption spectroscopy seems to be the most promising. The results presented here have important implications for understanding energy transfer mechanisms from carotenoids to bacteriochlorophylls in photosynthesis. It is known that carotenoids can transfer energy from both their S1 and S2 excited states. In the case of Rb. sphaeroides, the acceptor states of bacteriochlorophylls are associated with the Qx and Qy transitions of the LH2 antenna complex with energies of 16 950 cm-1 (Qx), 12 500 cm-1 (Qy of B800), and 11 800 cm-1 (Qy of B850). The S1 lifetime of spheroidene bound to the LH2 complex is as short as 1.7 ps, exhibiting a significant shortening due to energy transfer from the S1 level,28 suggesting that the S1 pathway has a significant role in Rb. sphaeroides. As pointed out by Zhang et al.,28 in the case of spheroidene in Rb. sphaeroides, the efficiency of the S1-Qy energy transfer pathway is about 80%. However, despite successful calculations of the S2-Qx transfer rates in different kinds of purple bacteria as Rhodopseudomonas (Rps.) acidophila and Rhodospirillum (Rs.) molischianum, attempts to calculate energy transfer rates of the S1-Qy energy transfer were not able to give satisfying agreement between calculated and observed rates.46,47 To understand the mechanisms of this energy transfer pathway, an improvement of our knowledge about the S1 state is needed. The results presented here show that, at least in solution, the S1 state probably has a more complex character than previously thought and that different conformational states of the carotenoid can change the S1 energy substantially. Therefore, it is probably not straightforward to use the S1 energies obtained from experiments performed in solution directly in calculations of energy transfer rates in the antenna complexes. The known structures of the LH2 complexes from the purple bacteria Rps. acidophila48 and Rs. mollischianum49 show that the carotenoid is twisted by the protein environment. Such a structural modification may affect the S1 energy and change the forbidden nature of the S1 T S0 transition. Thus, a direct measurement of the S1 energies in the protein environment is needed to improve our understanding of the role of the S1 energy in the light-harvesting process in photosynthesis. The experimental method described here holds promise in achieving this objective as there are no obvious restrictions to using this approach for studies of carotenoid dynamics in their natural protein environment. Acknowledgment. We thank to Dr. Jian-Ping Zhang for valuable discussion. The work at Lund University was performed with funding from the Swedish Natural Science Research Council, the Wallenberg Foundation, and the Crafoord Foundation. The work in the laboratory of H.A.F. was supported by grants from the National Science Foundation (MCB-9816759), the National Institutes of Health (GM-30353), and the University of Connecticut Research Foundation. The work at Kwansei Gakuin University was supported by grants from the Science Research Promotion Fund and from the Japan Society for the Promotion of Science. References and Notes (1) Frank, H. A.; Cogdell, R. J. Photochem. Photobiol. 1996, 63, 257. (2) Demmig-Adams, B.; Adams, W. W. Trends Plant Sci. 1996, 1, 21.
J. Phys. Chem. B, Vol. 105, No. 5, 2001 1079 (3) Edge, R.; McGarvey, D. J.; Truscott, T. G. J. Photochem. Photobiol. B 1997, 41, 189. (4) Here we use the standard notation S2 for the 1Bu+ state. However, recent resonance Raman experiments on spheroidene suggested the presence of another dark state 1Bu- lying below the 1Bu+ state: (a) Sashima, T.; Nagae, H.; Kuki, M.; Koyama, Y. Chem. Phys. Lett. 1999, 299, 187. (b) Sashima, T.; Koyama, Y.; Yamada, T.; Hashimoto, H. J. Phys. Chem. B. 2000, 104, 5011. (5) Christensen, R. L. In Photochemistry of Carotenoids; Frank, H. A., Young, A. J., Britton, G., Cogdell, R. J., Eds.; Kluwer Academic Publishers: Dodrecht, The Netherlands, 1999; Chapter 8. (6) Schulten, K.; Karplus, M. Chem. Phys. Lett. 1972, 14, 305. (7) Hudson, B. S.; Kohler, B. E. Chem. Phys. Lett. 1972, 14, 299. (8) Schulten, K.; Ohmine, I.; Karplus, M. J. Chem. Phys. 1976, 64, 4422. (9) Tavan, P.; Schulten, K. J. Chem. Phys. 1986, 85, 6602. (10) Tavan, P.; Schulten, K. Phys. ReV. B 1987, 36, 4337. (11) Orlandi, G.; Zerbetto, F.; Zgierski, M. Z. Chem. ReV. 1991, 91, 867. (12) Cosgrove, S. A.; Guite, M. A.; Burnell, T. B.; Christensen, R. L. J. Phys. Chem. 1990, 94, 8118. (13) Andersson, P.-O.; Gillbro, T. J. Chem. Phys. 1995, 103, 2509. (14) Buma, W. J.; Zerbetto, F. J. Chem. Phys. 1995, 103, 10492. (15) Petek, H.; Bell, A. J.; Choi, Y. S.; Yoshihara, K.; Tounge, B. A.; Christensen, R. L. J. Chem. Phys. 1995, 102, 4726. (16) Kohler, B. E.; Terpougov, V. J. Chem. Phys. 1998, 108, 9586. (17) DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J.; Farhoosh, R.; Frank, H. A. Biochim. Biophys. Acta 1992, 1102, 107. (18) Christensen, R. L.; Goyette, M.; Gallagher, L.; Duncan, J.; DeCoster, B.; Lugtenburg, J.; Jansen, F. J.; van der Hoef, I. J. Phys. Chem. A 1999, 103, 2399. (19) Frank, H. A.; Desamero, R. Z. B.; Chynwat, V.; Gebhard, R.; van der Hoef, I.; Jansen, F. J.; Lugtenburg, J.; Gosztola, D.; Wasielewski, M. R. J. Phys. Chem. A 1997, 101, 149. (20) Frank, H. A., Young, A. J., Britton, G., Cogdell, R. J., Eds. The Photochemistry of Carotenoids: Kluwer Academic Publishers: Dodrecht, The Netherlands, 1999. (21) Chynwat, V.; Frank, H. A. Chem. Phys. 1995, 194, 237. (22) Engelman, R.; Jortner, J. J. Mol. Phys. 1970, 18, 145. (23) Frank, H. A.; Cua, A.; Chynwat, V.; Young, A.; Gosztola, D.; Wasielewski, M. R. Photosynth. Res. 1994, 41, 389. (24) Frank, H. A.; Cua, A.; Chynwat, V.; Young, A.; Gosztola, D.; Wasielewski, M. R. Biochim. Biophys. Acta 1996, 1277, 243. (25) Bautista, J. A.; Hiller, R. G.; Sharples, F. P.; Gosztola, D.; Wasielewski, M. R.; Frank, H. A. J. Phys. Chem. A 1999, 103, 2267. (26) Polı´vka, T.; Herek, J. L.; Zigmantas, D.; Åkerlund, H.-E.; Sundstro¨m, V. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4914. (27) Frank, H. A.; Bautista, J. A.; Josue, J. S.; Young, A. J. Biochemistry 2000, 39, 2831. (28) Zhang, J.-P.; Fujii, R.; Qian, P.; Inaba, T.; Mizoguchi, T.; Koyama, Y.; Onaka, K.; Watanabe, Y.; Nagae, H. J. Phys. Chem. B 2000, 104, 3683. (29) Onaka, K.; Fujii, R.; Nagae, H.; Kuki, M.; Koyama, Y.; Watanabe, Y. Chem. Phys. Lett. 1999, 315, 75. (30) Sashima, T.; Shiba, M.; Hashimoto, H.; Nagae, H.; Koyama, Y. Chem. Phys. Lett. 1998, 290, 36. (31) Sundstro¨m, V.; Pullerits, T.; van Grondelle, R. J. Phys. Chem. B 1999, 103, 2327. (32) Koyama, Y.; Fujii, R. In The Photochemistry of Carotenoids; Frank, H. A., Young, A. J., Britton, G., Cogdell, R. J., Eds.; Kluwer Academic Publishers: Dodrecht, The Netherlands, 1999; pp 161-188. (33) Ricci, M.; Bradforth, S. E.; Jimenez, R.; Fleming, G. R. Chem. Phys. Lett. 1996, 259, 381. (34) Andersson, P. O.; Cogdell, R. J.; Gillbro, T. Chem. Phys. 1996, 210, 195. (35) Desamero, R. Z. B.; Chynwat, V.; van der Hoef, I.; Jansen, F. J.; Lugtenburg, J.; Gosztola, D.; Wasielewski, M.; Cua, A.; Bocian, D. F.; Frank, H. A. J. Phys. Chem. B 1998, 102, 8151. (36) Fujii, R.; Onaka, K.; Kuki, M.; Koyama, Y.; Watanabe, Y. Chem. Phys. Lett. 1998, 288, 847. (37) Krueger, B. P.; Yom, J.; Walla, P. J.; Fleming, G. R. Chem. Phys. Lett. 1999, 310, 57. (38) Cohen-Bazire, G.; Sistrom, W. R.; Stanier, R. Y. J. Cell. Physiol. 1957, 49, 25. (39) Bautista, J. A.; Chynwat, V.; Cua, A.; Jansen, F. J.; Lugtenburg, J.; Gosztola, D.; Wasielewski, M. R.; Frank, H. A. Photosynth. Res. 1998, 55, 49. (40) Jiang, Y.-S.; Kurimoto, Y.; Shimamura, T.; Ko-Chi, N.; Ohashi, N.; Mukai, Y.; Koyama, Y. Biospectroscopy 1996, 2, 47. (41) Fujii, R.; Onaka, K.; Nagae, H.; Koyama, Y.; Watanabe, Y. J. Lumin., in press. (42) Christensen, R.; Kohler, B. Photochem. Photobiol. 1973, 18, 293. (43) Geldof, P. A.; Rettschnick, R. P. H.; Hoytinck, G. Chem. Phys. 1971, 10, 549.
1080 J. Phys. Chem. B, Vol. 105, No. 5, 2001 (44) Garavelli, M.; Celani, P.; Bernardi, F.; Robb, M. A.; Olivucci, M. J. Am. Chem. Soc. 1997, 119, 11487. (45) Werncke, W.; Hogiu, S.; Pfeiffer, M.; Lau, A.; Kummrow, A. J. Phys. Chem A 2000, 104, 4211. (46) Krueger, B. P.; Scholes, G. D.; Jimenez, R.; Fleming, G. R. J. Phys. Chem. B 1998, 102, 2284.
Polı´vka et al. (47) Damjanovic, A.; Ritz, T.; Schulten, K. Phys. ReV. E 1999, 59, 3293. (48) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornwaiteLawless, A.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature 1995, 374, 517. (49) Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. Structure 1996, 4, 581.
