Femtosecond Time-Resolved Transient Absorption Spectroscopy of Xanthophylls

Transcription

1 22872 J. Phys. Chem. B 2006, 110, Femtosecond Time-Resolved Transient Absorption Spectroscopy of Xanthophylls Dariusz M. Niedzwiedzki, James O. Sullivan, Tomáš Polívka,, Robert R. Birge, and Harry A. Frank*, Department of Chemistry, UniVersity of Connecticut, Storrs, Connecticut , Institute of Physical Biology, UniVersity of South Bohemia, NoVé Hrady, Czech Republic, and Biological Centre, Czech Academy of Sciences, Ceské BudejoVice, Czech Republic ReceiVed: April 12, 2006; In Final Form: July 20, 2006 Xanthophylls are a major class of photosynthetic pigments that participate in an adaptation mechanism by which higher plants protect themselves from high light stress. In the present work, an ultrafast time-resolved spectroscopic investigation of all the major xanthophyll pigments from spinach has been performed. The molecules are zeaxanthin, lutein, violaxanthin, and neoxanthin. β-carotene was also studied. The experimental data reveal the inherent spectral properties and ultrafast dynamics including the S 1 state lifetimes of each of the pigments. In conjunction with quantum mechanical computations the results address the molecular features of xanthophylls that control the formation and decay of the S* state in solution. The findings provide compelling evidence that S* is an excited state with a conformational geometry twisted relative to the ground state. The data indicate that S* is formed via a branched pathway from higher excited singlet states and that its yield depends critically on the presence of β-ionylidene rings in the polyene system of π-electron conjugated double bonds. The data are expected to be beneficial to researchers employing ultrafast time-resolved spectroscopic methods to investigate the mechanisms of both energy transfer and nonphotochemical quenching in higher plant preparations. Introduction Xanthophylls are the oxygenated derivatives of carotenes and represent a large part of the group of naturally occurring pigments known as carotenoids. Carotenoids are derived from photosynthesis and are responsible for the abundance of yellow, orange, and red colors of many biological organisms. 1 In higher plants, these molecules play particularly important roles in harvesting light, stabilizing protein structures, regulating energy flow, and dissipating excess energy not required by the organism for photosynthetic growth. 2 If this surplus energy is not dissipated, then deleterious reactions may occur between chlorophyll (Chl) and active oxygen species. Especially harmful is the 1 g state of molecular oxygen that is generated by energy transfer from the Chl triplet state formed by intersystem crossing from the photoexcited Chl singlet state. 3 Xanthophylls contribute to both short- and long-term adaptive mechanisms of protection of plants against high light stress. One such mechanism is termed nonphotochemical quenching (NPQ). NPQ has several components that work together to bring about nonradiative dissipation of excess excited singlet states of Chl that limits the photoinduced damage to the photosynthetic apparatus. The most rapid component of NPQ is denoted qe and is sometimes referred to as high-energy or feedbackregulated quenching. 4 For qe to occur, a protein denoted PsbS must be present, the chloroplast thylakoid lumen must be acidified, and the xanthophyll, violaxanthin, must be enzymatically de-epoxidated to zeaxanthin (Figure 1). 4 De-epoxidation of violaxanthin to zeaxanthin has a profound effect on both the structure of the xanthophylls (Figure 1) and * Author to whom correspondence should be addressed. Phone: (860) Fax: (860) harry.frank@uconn.edu. University of Connecticut. University of South Bohemia. Czech Academy of Sciences. Figure 1. Interconversion of violaxanthin and zeaxanthin according to the xanthophyll cycle. Figure 2. Energy level diagram showing possible pathways of energy transfer to and from xanthophylls and chorophylls. their excited-state energy levels (Figure 2). Removing the epoxide groups from the terminal β-ionylidene rings renders them more in the plane of the extended π-electron system of conjugated carbon-carbon double bonds. This is evident in Figure 3, which shows different views of the computationally /jp CCC: $ American Chemical Society Published on Web 10/25/2006

2 Femtosecond Spectroscopy of Xanthophylls J. Phys. Chem. B, Vol. 110, No. 45, Figure 3. Geometry-optimized structures of the all-trans configurations of β-carotene, zeaxanthin, lutein, and violaxanthin. The structure of 9 -cis-neoxanthin was obtained from the coordinates of its crystal structure in the LHCIIb pigment protein complex. 35 optimized xanthophyll structures. De-epoxidation also lengthens the conjugated π-electron system (Figures 1 and 3) and significantly lowers the energies of the excited states of zeaxanthin compared to those of violaxanthin. The structural alterations and the changes in positions of the excited-state energy levels have been implicated separately in different models of how qe functions. The change in xanthophyll structure (shape) upon conversion of violaxanthin to zeaxanthin has been postulated to increase the aggregation state of the major light-harvesting pigment-protein complexes associated with photosystem II (PS II) in higher plants. 5-8 Chl aggregation is thought to generate low-energy exciton traps that in turn may induce qe. This model is termed indirect quenching. 5-8 In an alternative model, a lower-energy excited S 1 state of zeaxanthin compared to violaxanthin may provide a direct route of quenching by energy transfer from the excitedstate Chl (Figure 2). 9 This direct quenching model has been supported by steady-state fluorescence spectroscopic measurements on model polyenes and carotenoids that have shown, either by extrapolation or by direct measurements, that the energy of the S 1 state of zeaxanthin is low enough to potentially quench the lowest excited singlet state of Chl, which lies at cm However, the various reported values of the S 1 state energy of violaxanthin in solution and in pigmentprotein complexes 13 range from ( 300 to ( 60 cm -1, indicating that there is still some uncertainty whether violaxanthin also has a sufficiently low energy S 1 state to enable quenching. Additional evidence from femtosecond time-resolved spectroscopic investigations carried out on thylakoid membranes from spinach and Arabidopsis thaliana reinforce this model of zeaxanthin functioning as a direct quencher of Chl singlet states. 14 However, the mechanism consistent with the ultrafast spectral and kinetic observations is complex and involves excitation transfer from bulk antenna-bound Chl to a special Chl-zeaxanthin heterodimer that undergoes ultrafast (0.1-1 ps) electronic charge separation to form a zeaxanthin cation-chl anion radical pair (Zea + Chl - ) that then recombines nonradiatively in 150 ps. 14 It still remains to identify where in the PS II reaction center such a heterodimer may be situated. An energy level diagram that can be used to describe many of the spectroscopic and photochemical properties associated the xanthophylls is shown in Figure 2. The xanthophyll ground state, S 0, is assigned A g symmetry in the idealized C 2h point group. This is in keeping with the convention derived from an abundance of studies on model polyenes and carotenoids. 15,16 The first excited singlet state is also assigned A g symmetry and denoted S 1 or 2 1 A g -, where the minus sign designates the pseudoparity character of the state derived from orbital pairing relationships when configuration interaction among singly excited configurations is included In this notation the

3 22874 J. Phys. Chem. B, Vol. 110, No. 45, 2006 Niedzwiedzki et al. ground state S 0 is 1 1 A - g. One-photon transitions between S 0 (1 1 A - g ) and S 1 (2 1 A - g ) are forbidden by group theoretical (g T u) and pseudoparity (+ T -) selection rules. The state into which one-photon absorption from the ground S 0 (1 1 A - g ) state is strongly allowed is the 1 1 B + u state. The customary practice is to denote this state S 2, but both theoretical 20,21 and experimental results (see Polivka and Sundstrom 22 for a recent review of this topic) are suggestive of excited states lying near or below this state. (To avoid confusion regarding state ordering, in this work we shall adhere to the customary notation of S 0 (1 1 A - g ), S 1 (2 1 A - g ), and S 2 (1 1 B + u ), and when it is necessary to refer to the positions of the other states, we shall do so using either their symmetry representations or nonnumerical notation.) In particular, Koyama et al. 23 have assigned spectroscopic features to a low-lying 1 1 B - u state and have postulated that it provides a route of both deactivation from S 2 and energy transfer to Chl. Also, van Grondelle and co-workers 24 have invoked a different excited state called S*, to account for the ultrafast dynamics of the carotenoid, spirilloxanthin, in solution and in the LH1 complex from Rhodospirillum rubrum, being different at different probe wavelengths, and in the LH1 complex leading to triplet state formation. Initially, S* was thought to be formed only in the very long (number of conjugated double bonds, N ) 13) spirilloxanthin molecule 24 and that it provides an alternate path for the depopulation of S 2 (1 1 B + u ). However, subsequent studies on spheroidene, 25 rhodopin glucoside, 26,27 lycopene, 28 zeaxanthin, 28 and β-carotene 28 have suggested that S* may occur more commonly. 22,29-31 S* has yet to be fully characterized, and there is considerable debate as to how it is formed. 22,24-26,28,32 The primary spectroscopic characteristics of S* are that it is associated with a transition having a maximum in the wavelength region between the S 0 f S 2 and S 1 f S n absorption bands and that it decays in several picoseconds. (In this paper, S n should be taken to mean a generic high-energy excited singlet state having a symmetry that gives rise to strong allowedness of the transition with which it is associated.) In light-harvesting pigment-protein complexes, S* is proposed to lead to triplet state formation via ultrafast singlet-triplet homofisson In solution, triplet states are apparently not formed from S*. An alternative view of the nature of the S* state has been published by Wohlleben et al. 28 who carried out pump-deplete-probe and transient absorption spectroscopic experiments. Upon selective depletion of the S 2 state population using a high-power laser pulse they observed a decrease in the intensity of the S 1 f S n transition but no effect on the S* population. On the basis of this observation and the position and broadness of the S* f S n transition they argued that, in solution, S* is a vibrationally excited, hot ground state populated by a combination of impulsive Raman scattering of the S 0 f S 2 pump pulse and internal conversion from S 1. They proposed that the lifetime of S* corresponds to vibrational relaxation in the ground state and measured it to be a constant 6.2 ( 0.4 ps for carotenoids having N g 11 and equal to the S 1 lifetime for shorter molecules. A time constant in this range has been previously implicated in vibrational relaxation in the ground state of carotenoids. 33 However, one measure of uncertainty with this assignment is that it implies that S* in solution differs from S* observed in LH2 proteins where it was shown to serve as a donor state in energy transfer from a carotenoid to BChl a. 25 Virtually all of the models proposing to explain how xanthophylls function in the qe component of NPQ have been derived from observations of the spectroscopic and dynamic behavior of the molecules. Given the complexity associated with the spectroscopic properties of xanthophylls and uncertainty in their system of energy levels, to make compelling assignments of their function, it is critical to have a clear understanding of how each of these molecules behaves in the ultrafast time regime. This is particularly important for analyzing the ultrafast spectroscopic observables of xanthophylls present in the multicomponent, spectrally congested, thylakoid and pigmentprotein complex preparations from higher plants. In those samples it is essential to know precisely where the various excited-state transitions occur and how they are contributing to the spectral and temporal line shapes. In this paper we present the results of a systematic, ultrafast, time-resolved spectroscopic investigation of all the major carotenoid pigments in spinach: β-carotene and the xanthophylls, zeaxanthin, lutein, violaxanthin, and neoxanthin. The data reveal the inherent spectral properties and ultrafast dynamics of each of these pigments and address the molecular features of xanthophylls that control S* formation in solution. The results provide compelling evidence for the origin of the S* state and will be of use to researchers employing ultrafast time-resolved spectroscopic methods to investigate the mechanisms of both energy transfer and NPQ in intact thylakoid membranes, isolated pigment-protein complexes, and whole photosynthetic organisms. Materials and Methods All xanthophylls except zeaxanthin were extracted from spinach obtained at a local market. Approximately 10 g of leaves were ground in 50 ml of acetone/methanol (50/50 v/v technical grade), filtered, and dried with a gentle stream of nitrogen gas in the dark at room temperature. The dried pigment extract was redissolved in 87/10/3 v/v/v acetonitrile (Fisher)/methanol (Fisher)/water (Sigma), filtered, and injected into a Millipore Waters 600E high-performance liquid chromatography system (HPLC) employing a 3.9 mm 300 mm Nova-Pak C 18 column. The protocol featured a gradient mobile phase of 100% A to 100% B in 40 min (A, 87/10/3 v/v/v acetonitrile (Fisher)/ methanol (Fisher)/water (Sigma); B, ethyl acetate (Fisher)) with a flow rate of 1 ml/min. Zeaxanthin was obtained from F. Hoffman LaRoche, and β-carotene was purchased from Sigma. Both molecules were purified using the above protocol. The purified pigments from HPLC were dried with a gentle stream of nitrogen gas in the dark at room temperature and stored at -40 C until use. Prior to the transient absorption measurements, the molecules were dissolved in 99.9% grade pyridine (J.T. Baker) to an optical density (OD) of at the excitation wavelength in a 2 mm path length cuvette. Transient absorption spectra were taken using a femtosecond spectrometer system described in detail previously. 34 The xanthophylls and β-carotene were excited into the lowest energy vibronic band (0-0) associated with their absorption spectra in pyridine: 481 nm for neoxanthin, 485 nm for violaxanthin, 491 nm for lutein, and 497 nm for zeaxanthin and β-carotene. The excitation energy was typically 1 µj, but the signals depended slightly on pump energy (see below). The excitation beam was focused into a spot of 1.2 mm in diameter, yielding excitation densities in the range of photons pulse -1 cm -2 for the used excitation wavelengths. The excitation and probe beams were overlapped at the sample, and the relative polarization of the beams was set to the magic angle. Also, a polarizer was placed before the CCD detector to minimize scattered signal from the pump beam. The time resolution (instrument response time) of the spectrometer was obtained as one of the parameters of the global fitting procedure (Table

4 Femtosecond Spectroscopy of Xanthophylls J. Phys. Chem. B, Vol. 110, No. 45, TABLE 1: Dynamics of the S 1 (τ 1 ), Vibrationally Hot S 1 (τ 1 ), S 2 (τ 2 ), and S* (τ 3 ) States of β-carotene, Zeaxanthin, Lutein, Violaxanthin, and Neoxanthin a molecule pump λ (nm) probe λ (nm) τ 1 (ps) τ 1 (fs) τ 2 (fs) τ 3 (ps) τ s b (fs) fitting method solvent reference β-carotene 497 cont. c 9.5 ( ( ( ( global fit pyridine this work ( 0.3 n.a. d n.a. 2.9 ( 0.4 n.a. single λ pyridine this work ( 0.5 n.d. e n.d. n.e. f 4000 single λ 3-methylpentane ( 0.6 n.d. n.d. n.e single λ 3-methylpentane ( 0.5 n.d. n.d. n.e single λ 3-methylpentane ( 0.5 n.d. n.d. n.e. 50 single λ n-hexane cont. 8.9 ( ( ( 10 n.e. 220 ( 20 global fit n-hexane ( 0.4 n.a. 150 ( 50 n.e. 100 ( 50 single λ n-hexane cont. 9.9 n.d. n.d. n.e. 250 n.d. n-hexane cont. 8.2 ( 0.2 n.d. 160 ( 40 n.e. 300 global fit n-hexane cont. 9.4 ( 0.2 n.d. 220 ( 50 n.e. 300 global fit n-hexane cont. 8.4 ( 0.2 n.d. 140 ( 40 n.e. 300 global fit benzene cont. 9.1 ( 0.2 n.d. 250 ( 50 n.e. 300 global fit benzene var. g n.e single λ n-hexane cont. 9.1 ( ( ( 30 n.e. 100 global fit hexane cont. 9.6 ( ( ( 30 n.e. 100 global fit ethanol cont ( ( ( 30 n.e. 100 global fit benzyl alcohol n.d. 250 n.e. 200 single λ ethanol n.d. 200 n.e. 200 single λ CS n.e. 10 ( 2 n.d. n.d. n.e single λ var cont. 8.7 ( ( ( 9 n.e. 80 single λ cyclohexane var. 8.8 ( ( 50 n.e. 300 single λ benzene n.d. n.e. 150 single λ n-hexane n.d. 260 n.e. 150 single λ n-hexane n.e. 150 single λ n-hexane n.d. n.e. 150 single λ methanol n.d. 200 n.e. 150 single λ methanol n.e. 150 single λ methanol 52 zeaxanthin 497 cont ( ( ( ( global fit pyridine this work ( 0.1 n.a. n.a. n.e. n.a. single λ pyridine this work 266 cont. 9.8 ( ( ( ( global fit methanol cont. 9.0 ( ( ( ( global fit methanol cont. 9.2 ( ( ( 27 n.e global fit methanol var. 8.6 or or 230 nd. n.e. 200 single λ methanol n.d. n.d. n.e. 300 single λ hexane n.d. n.d. n.e. 150 single λ methanol n.d n.e. 150 single λ n-hexane n.d. 270 n.e. 150 single λ n-hexane n.e. 150 single λ n-hexane n.d. n.e. 150 single λ methanol n.d. 280 n.e. 150 single λ methanol n.e. 150 single λ methanol n.d. 290 n.e. 100 single λ ethanol 72 lutein 491 cont ( ( ( ( global fit pyridine this work ( 0.1 n.a. n.a. 2.2 ( 0.4 n.a. single λ pyridine this work n.d. n.d. n.e. 150 single λ methanol 71 violaxanthin 485 cont ( ( ( ( global fit pyridine this work ( 0.2 n.a. n.a. 2.0 ( 0.6 n.a. single λ pyridine this work n.d. n.d. n.e. 300 single λ n-hexane var or n.d. n.e. 200 single λ methanol 10 neoxanthin 481 cont ( ( ( ( global fit pyridine this work ( 0.8 n.a. n.a. 2.7 ( 0.4 n.a. single λ pyridine this work 467 n.a. 35 ( 2 n.d. n.d. n.e. 140 single λ n-hexane n.a. 35 ( 2 n.d. n.d. n.e. 140 single λ methanol 59 a All data were taken at room temperature. b τ s is the instrument response time. c White light continuum. d Not applicable. e Not determined. f Not evident. g Various. The fitting of the data was initialized after 1 ps from t 0, where component associated with S 2 state is greatly diminished. 1). The samples were stirred using a magnetic microstirrer to protect them from photodegradation. To confirm the sample integrity, absorption spectra were taken before and after the transient absorption experiments at room temperature. No changes in the absorption spectra were evident. Surface Xplorer Pro (Ultrafast Systems, LLC) software was used to correct for dispersion in the transient absorption spectra using a correction curve based on a set of initial times (t 0 ) of the signal produced from fitting the kinetics at several different wavelengths. ASUfit 3.0 software provided by Dr. Evaldas Katilius from Arizona State University was used for global fitting calculations and for separation of artifacts in the transient absorption spectra associated with the solvent response within 100 fs of excitation. Carotenoid structures (except 9 -cis-neoxanthin) shown in Figure 3 were constructed using ChemDraw Ultra 5.0 software (CambridgeSoft Corp.) and geometrically optimized using HyperChem 5.1 (Hypercube, Inc.) software that employs an AM1 semiempirical method with a Polak-Ribiere algorithm in a vacuum environment. The structure of 9 -cis-neoxanthin

5 22876 J. Phys. Chem. B, Vol. 110, No. 45, 2006 Niedzwiedzki et al. Figure 4. Steady-state absorption spectra of β-carotene (β-car), zeaxanthin (zea), lutein (lut), violaxanthin (viol), and neoxanthin (neo) taken in pyridine solvent at room temperature. The spectra were all normalized at the maximum of their (0-1) vibronic bands and arbitrarily vertically offset for clarity. was obtained from its coordinates in the crystal structure in the major light-harvesting complex (LHCIIb) from spinach. 35 For the quantum mechanical computations carried out on β-carotene, the structure was optimized using density functional methods for the ground state (B3LYP/6-31G(d)) and ab initio methods (CIS(D) and SAC-CI with a D95 basis set) for the low-lying excited singlet states. The excited singlet state calculations were limited to the 32 highest-energy filled orbitals and the 32 lowestenergy unfilled orbitals, and the SAC-CI calculations were carried out by selecting integrals using the level one approximation. The spectroscopic properties were then analyzed using MNDO-PSDCI molecular orbital theory using methods and procedures described previously Results Room-temperature absorption spectra of β-carotene and the xanthophylls in pyridine are shown in Figure 4. Pyridine, which has a refractive index of n 1.51 at 20 C and a polarizability of 0.3, was chosen because the absorption spectra of the molecules in this solvent are well-resolved and occur at wavelengths very close to those observed when they are bound in lipid membranes or in membrane proteins β-carotene and zeaxanthin, both with 11 conjugated carbon-carbon double bonds (N ) 11, Figure 3), display almost identical broad absorption spectral line shapes having Franck-Condon maxima at 468 nm. A profound similarity is expected because the hydroxyl groups attached to the terminal β-ionylidene rings of zeaxanthin (Figure 3) do not perturb the π-electron conjugated system that controls the light-absorption characteristics of these molecules. The absorption spectrum of lutein (N ) 10) shown in Figure 4 is better resolved than those of β-carotene and zeaxanthin, and its Franck-Condon maximum is blue-shifted by 7 nm to 461 nm. The blue shift is due to the decrease in N, and the improved vibronic resolution can be traced to a reduction in conformational disorder, which can cause broadening of the spectral profiles. 31 It is well-known that the presence of the terminal β-rings broadens the distribution of conformations along the π-electron conjugated chain, and that together with disorder owing to variations in the solvent environment leads to spectral broadening. 44,45 The higher vibronic resolution of lutein compared those of to β-carotene and zeaxanthin derives from the fact that one of the rings in lutein, the ɛ-ring on the right-hand side of the structure shown in Figure 3, has a double bond removed from the extended π-electron polyene chain by two carbon-carbon single bonds. Hence, lutein, with one less ring in conjugation than β-carotene and zeaxanthin, has a lesser amount of ring-induced conformational disorder. The maxima in the steady-state absorption spectra of violaxanthin (N ) 9) Figure 5. Transient absorption spectra taken at different time delays after excitation into the (0-0) vibrational level of the S 2 state: (A) β-carotene, (B) zeaxanthin, (C) lutein, (D) violaxanthin, and (E) neoxanthin. The spectra were taken at room temperature from the molecules dissolved in pyridine. and neoxanthin (N ) 9), which appear at 456 and 451 nm, respectively, are blue-shifted relative to the other molecules owing to the presence of epoxide groups in the case of violaxanthin and an epoxide and allene group in the case of neoxanthin (Figure 3). These molecules contain one less conjugated double bond than lutein and show even more improved vibrational resolution because in both instances the terminal β-rings do not contain carbon-carbon double bonds in conjugation with the extended polyene chain. Transient absorption spectra of β-carotene and the xanthophylls in pyridine at room temperature were taken at various delay times after the excitation pulse. The spectral traces are shown in Figures 5A-E. Analogous to their steady-state absorption spectra shown in Figure 4, the transient absorption spectra of β-carotene and zeaxanthin (Figures 5A and 5B) are very similar. They both display broad negative signals in the range of nm corresponding to the bleaching of the strongly allowed S 0 f S 2 absorption band upon excitation and also show a buildup of a strong transient absorption signal in the region of nm. This latter peak is associated with the S 1 f S n transition. The fact that the transition is very intense implies that the S n state has B u + symmetry. This is supported by the quantum computations discussed below. The spectrum of this S 1 f S n transition is broad (56 ( 1 nm, full width at half-maximum (fwhm) measured at a 2 ps delay time) for both β-carotene and zeaxanthin and shows only a slight difference in their maximum positions: 579 nm for β-carotene and 576 nm for zeaxanthin. For lutein, as is observed in its steady-state absorption spectrum (Figure 4) and attributed to reduced conformational disorder, the transient absorption spectrum of this xanthophyll (Figure 5C) in the S 1 f S n transition region is sharper (46 ( 1 nm fwhm at 2 ps) than those of β-carotene and zeaxanthin (Figures 5A and 5B). Also, the main S 1 f S n band has a maximum at 558 nm, which is shifted to a shorter wavelength compared to those of β-carotene and zeaxanthin. This is consistent with an increase in the energy of the S n state brought

6 Femtosecond Spectroscopy of Xanthophylls J. Phys. Chem. B, Vol. 110, No. 45, about by the molecule having one less carbon-carbon double bond in conjugation. The S 1 state energy also increases with decreasing conjugation length, but the increase of the S 1 (2 1 A g - ) state energy is apparently less than that for the high-energy B u + state into which the S 1 f S n transition occurs. The spectrum of lutein also displays a clearly formed shoulder near 525 nm on the short-wavelength side of the main band. For the carotenoids, spirilloxanthin and spheroidene, in LH complexes, this shoulder has been assigned to the S* state. 24,25,27,29,46 A less well-resolved shoulder is seen in this region of the spectra from β-carotene and zeaxanthin (Figures 5A and 5B). Upon close inspection, it is observed that the short-wavelength (shoulder) feature has a different time dependence than the main band and, as recently reported for the carotenoid rhodopin glucoside in the LH2 complex from Rhodopseudomonas acidophila, 27 also a slightly different dependence on pump laser power. The data reveal a slight increase in relative signal intensity in the shoulder region as the pump energy is increased from 600 nj to 2 µj. (See Figure S1 in the Supporting Information and discussion below.) The transient absorption spectrum of violaxanthin is shown in Figure 5D. Due to its shorter π-electron conjugated chain and the absence of terminal β-rings in conjugation compared to β-carotene, zeaxanthin, and lutein, the main S 1 f S n transient absorption peak is shifted even farther to the blue, appearing at 533 nm as a fairly sharp peak with a fwhm of 24 ( 1 nm. This spectrum does show a short-wavelength shoulder at 510 nm, but it is much less intense compared to those of lutein and the other molecules. The transient absorption spectrum of neoxanthin is shown in Figure 5E. Its main S 1 f S n absorption peak has a maximum at 543 nm and a fwhm of 41 ( 1 nm, both values of which are midrange between those for violaxanthin and the other molecules. The position of the maximum suggests that in the excited state neoxanthin has a longer effective π-electron conjugation than violaxanthin but shorter than those of the other molecules. The value of the bandwidth suggests that the extent of conformational disorder for neoxanthin is greater than that in in violaxanthin but less than those in the other molecules. Neither of these two factors follow the same trend seen for neoxanthin in the ground state where its S 0 f S 2 transition is the most blue-shifted of all the molecules and its extent of vibronic resolution is comparable to that of violaxanthin. One other significant spectral feature seen for all of the molecules is a broad, gradually sloping, positive signal that appears on the long-wavelength side of the major S 1 f S n peak. This is observed in all of the transient profiles taken at a 500 fs delay of the pulse beam. (See the dashed lines in Figures 5A-E.) In all cases, this feature builds up and decays before the main S 1 f S n peak (solid line in Figures 5A-E) reaches its full intensity. The transient data can be summarized as follows: In the time range between 0 and 10 ps, upon photoexcitation all of the molecules display an immediate onset of bleaching of the S 0 f S 2 absorption transition in the wavelength range between 450 and 525 nm, the subsequent build up and decay within 1 ps of a broad, sloping, long-wavelength feature, followed by the rise and partial decay of both a strong S 1 f S n transition in the region of nm and a variable-sized short-wavelength shoulder in the region of nm. To gain more insight into the photophysical behavior of these molecules, the entire spectral and temporal datasets were fit simultaneously using a global analysis procedure employing a multiexponential function, S(λ,t) ) i A i (λ) exp(-t/τ i ), where A i (λ) is the preexponential amplitude factor associated with decay component i having a time constant τ i. This sum of exponentials model represents the dynamic behavior of a number of parallel, noninteracting kinetic components, the amplitude factors, A i (λ), which are termed decay associated spectra (DAS), or more appropriately in the present context, decay associated difference spectra (DADS) because difference absorption spectra are recorded. 47,48 As is thoroughly described in the literature, DADS amplitudes do not represent real, physical, spectroscopic profiles of the transient species. Real, physical spectra are termed species associated difference spectra (SADS), but DADS can be expressed as linear combinations of SADS; i.e., DADS i ) j n c ij SADS j where the ith DADS component, DADS i, is identical to the preexponential factor, A i (λ), in the multiexponential function, S(λ,t), and j is the index for each one of a number, n, of physically real (SADS j ) spectra contributing to the DADS i profile. Individual SADS j are often difficult to obtain due to overlapping spectral profiles and comparable kinetic behavior among the transient species. An alternative method is to fit the datasets using a nonbranching, sequential, irreversible scheme A f B, B f C, C f D,... The arrows represent increasingly slower monoexponential processes, and the time constants of these processes correspond to lifetimes of the transient species A, B, C, D,... The spectral profiles of these species are termed evolution-associated difference spectra (EADS). Although EADS in complicated systems do not necessarily correspond to SADS of particular excited states, they provide information about the time evolution of the whole system. 48 Thus, while DADS provide information about spectral profiles of the preexponential factors, EADS give first approximations to the real concentration profiles of the transient species. A detailed analysis of the global fitting methods and their application to various biological systems can be found in ref 48. For all the molecules examined in this work, four (n ) 4) DADS and EADS components were necessary to obtain satisfactory fits based on a chi square (χ 2 ) test and the smallness of their equivalent residual matrices. Single-wavelength fits displayed in Figures S2 and S3 show that the molecules exhibit different time dependences at different probe wavelengths. Although three kinetic components lead to a satisfactory fit at the λ max of the S 1 f S n transition (Figure S2), four components are needed when one probes the wavelength region on the shortwavelength side of this band. This is most clearly illustrated in Figure S3 for β-carotene, zeaxanthin, and lutein where a threecomponent fit is shown to be inadequate, but a four-component fit works nicely. The DADS amplitudes resulting from fitting the transient absorption data to a sum of exponentials kinetic model are displayed in Figure 6. For β-carotene (Figure 6A), a very broad, negative-amplitude DADS component builds up in 170 ( 2 fs. For the xanthophylls, the time for this fast, negative component ranged from 110 to 170 fs (Figures 6B-E). A time constant in this range is associated with the lifetime of the S 2 state of carotenoids. 49 The various assorted negative features appearing in this first DADS component may be attributed to the buildup of a vibrationally hot S 1 f S n transition, the formation of the S* state, the buildup of its associated S* f S n transition, stimulated fluorescence, and stimulated Raman bands arising from the solvent. The second DADS component in all cases has a complex line shape featuring a broad positive (decay) profile at long wavelengths, a zero crossing, and a broad negative (build-up) band that spans the region encompassing the S 1 f S n transition and the S* f S n short-wavelength shoulder. For β-carotene this second amplitude spectrum has a time constant of 366 ( 10 fs, and for the xanthophylls, the

7 22878 J. Phys. Chem. B, Vol. 110, No. 45, 2006 Niedzwiedzki et al. Figure 6. Decay associated difference spectra (DADS) obtained from a global fitting analysis using four kinetic components: (A) β-carotene, (B) zeaxanthin, (C) lutein, (D) violaxanthin, and (E) neoxanthin. values range from 370 to 582 fs. The positive, long-wavelength part of this component has been attributed to the decay of a vibrationally hot S 1 state to form a vibrationally equilibrated S 1 state This is rationalized by the fact that the decay of S 2 to S 1 is so rapid that it brings with it a significant amount of vibrational energy that can only be dissipated after a few hundred femtoseconds. The presence of this excess vibrational energy in S 1, which populates the upper vibronic levels of the state, is expected to give rise to a broad, red-shifted S 1 f S n spectrum that then decays to a narrower, blue-shifted, S 1 f S n transition associated with the vibrationally equilibrated system. The zero crossing and a strong, negative, shorter-wavelength feature observed in the second DADS component are compelling evidence that this is the case for all of the molecules examined here. Yet, it also has been suggested that this kinetic component and the broad, long-wavelength feature correspond to an excitedstate transition originating from the 1 1 B u - excited electronic state, 23 theoretically predicted to be in the vicinity of S 1 and S 2. 20,53,54 However, as pointed out by Billsten, 32 if this were the case, then such an intense signal implies that the final state should have A g + symmetry. The energy of the lowest A g + state is known from the location of the S 0 f A g + cis-peak. For β-carotene this occurs at cm Subtraction of the approximate energy ( cm -1 ) corresponding to the observed broad transition peaking at 550 nm would put the 1 1 B u - state at cm -1, which is far below the S 1 (2 1 A g - ) state energy of β-carotene known to be at cm This would contradict both experimental evidence 23 and theoretical predictions 53 for the position of the 1 1 B u - state relative to S 1. The second DADS component also shows a negative-amplitude shoulder on the short-wavelength side of the strong negative feature. This is most evident at 530 nm in the amplitude spectrum of lutein (dotted line in Figure 6C), to some extent noticeable, but not well-resolved, in the amplitude spectra of β-carotene, zeaxanthin, and neoxanthin, and absent in the Figure 7. Evolution-associated difference spectra (EADS) obtained from a global fitting analysis using four kinetic components: (A) β-carotene, (B) zeaxanthin, (C) lutein, (D) violaxanthin, and (E) neoxanthin. amplitude spectrum of violaxanthin (dotted line in Figure 6D). This negative short-wavelength shoulder may be attributed to the arrival of population from S 2 into the S* state, giving rise to an associated S* f S n transition. The longest-time DADS component, in all cases, shows the familiar, strongly allowed, vibronically relaxed, positiveamplitude spectrum associated with the S 1 f S n transition. For β-carotene the time constant of this component is 9.5 ( 0.1 ps, and for the xanthophylls, zeaxanthin, lutein, violaxanthin, and neoxanthin, the values are 10.2 ( 0.2, 15.6 ( 0.1, 26.1 ( 0.1, and 37.6 ( 0.1 ps, respectively. These correspond well to the values of the S 1 lifetime for these molecules reported in the literature that tend to increase with decreasing π-electron conjugation length (Table 1). This longest-time DADS component also has an associated strong negative signal at a shorter wavelength that represents the recovery of the ground-state bleaching as S 1 decays. For all of the molecules examined here, a third DADS component was observed having a time constant in the range of ps, i.e., between the second and longest-time components. This DADS component has a significant amplitude for β-carotene, zeaxanthin, and lutein but is small and hardly noticeable for violaxanthin and neoxanthin. The component shows a wavy line shape with at least two positive and two negative peaks spanning the entire probe wavelength region. For β-carotene, zeaxanthin, and lutein, it has a negative amplitude on the red side and a positive amplitude on the shortwavelength side of the S 1 f S n transition profile. For violaxanthin and neoxanthin, although the signals are very small (Figures 6D and 6E), this appears to be reversed, with positive amplitude on the long-wavelength side and negative amplitude on the short-wavelength side of the S 1 f S n band. However, in all cases the negative-amplitude feature tracks precisely the strong positive feature of the final DADS.

8 Femtosecond Spectroscopy of Xanthophylls J. Phys. Chem. B, Vol. 110, No. 45, Figure 8. Transient absorption kinetic traces of (9) β-carotene, (0) zeaxanthin, (b) lutein, (O) violaxanthin, and ([) neoxanthin probed at the crossover wavelengths where the contribution from the S 1 f S n transition involving vibrationally hot S 1 is negligible. The amplitudes were normalized to unity, and only every third data point is shown for clarity. The solid lines represent the fits obtained from a sum of exponentials expression as described in the text. The EADS components resulting from a global fitting analysis using a sequential kinetic model are displayed in Figure 7. In all cases, the initial EADS corresponds to the spectrum of the excited S 2 state. It is characterized by a large negative, groundstate bleaching signal between 475 and 525 nm accompanied by a broad, sloping, negative feature at a longer wavelength due primarily to stimulated emission from S 2. The first EADS decays rapidly ( fs) to form the second EADS component that for all the molecules displays a very broad, positive line shape extending significantly to long wavelengths. As mentioned above this feature is assigned to a transition between a vibrationally hot S 1 state and S n. 51,52 The third EADS in the sequence rises in fs and decays with a ps time constant and shows a very strong, broad, positive band, which narrows as the system evolves into a fourth and final EADS. The line narrowing is most evident for β-carotene, zeaxanthin, and lutein. This step is also accompanied by a slight wavelength shift of the major positive feature either to the blue (β-carotene, zeaxanthin, lutein) or to the red (violaxanthin, neoxanthin). Also, from the third to the fourth EADS, the ground-state bleaching signal recovers slightly indicating a portion of the population has relaxed from an excited state to the ground state. Because the DADS components can be expressed as linear combinations of various SADS approximated by the EADS (see above), the interpretation of the shape of the DADS profiles in Figure 6 is straightforward. The individual DADS in Figure 6 can be generated by taking an arithmetic difference between two sequential EADS with only a slight adjustment in coefficient, C ij. The larger the difference in the time constants of the EADS components, the more precise the agreement. For example, subtracting any fourth EADS component from any third EADS component from the same molecule yields almost perfect agreement with its third DADS component given in Figure 6. This is clearly illustrated in an overlay of the EADS difference spectra with the DADS components shown in Figure S4 in the Supporting Information. Thus, the reason for the wavy nature of the ps (third) DADS components of β-carotene, zeaxanthin, and lutein (light solid lines in Figures 6A-C) becomes clear. It is due to shifts in the wavelength positions of the peaks in the fourth EADS component compared to those in the third (Figures 7A-C). The time-resolved data were also analyzed using singlewavelength fits (Figure 8) taken at positions where the second DADS component assigned to the hot S 1 state crosses zero. At these wavelengths, the kinetics are free from a contribution of S 1 vibrational relaxation. The crossover wavelengths are 594 nm for β-carotene, 596 nm for zeaxanthin, 571 nm for lutein, 543 nm for violaxanthin, and 558 nm for neoxanthin (Figures 6A-E). The fits to these specific single-wavelength response profiles are shown in Figure 8 and required two exponential decay components to satisfactorily reproduce the experimental data in all cases except for zeaxanthin where two were not needed because of the vanishingly small amplitude of the second decay component at 596 nm. These two decay components emerging from the single-wavelength fits are associated with the lifetimes of the S 1 and S* states. The values of the S 1 /S* ratios of the preexponential factors were -4.3 (β-carotene), (lutein), 16.5 (violaxanthin), and 6.8 (neoxanthin). The change in sign of the ratio for the latter two molecules is due to the inversion of the amplitude spectrum of the kinetic component associated with S* (Figure 6). The kinetics obtained from the global fitting and singlewavelength analyses have been collected with results from previous experiments on the same molecules in various solvents and are presented in Table 1. Discussion Spectral Features and Kinetic Components. The steadystate and transient absorption spectra of the molecules examined here follow the trends previously observed in both position and broadness of the S 0 f S 2 and S 1 f S n transitions. The longer conjugated carotenoids absorb farther to the red than the shorter molecules, and the systems with terminal β-ionylidene rings having a double bond in conjugation with the extended polyene chain show broader S 0 f S 2 and S 1 f S n spectra due to conformational disorder. The only exception is neoxanthin where its S 1 f S n transition appears broader and more red-shifted than expected. This is the case because neoxanthin adopts a 9 -cisconfiguration as its most stable geometric isomer. Previous work has demonstrated that although the S 0 f S 2 transition for cisisomers of carotenoids are generally blue-shifted compared to their all-trans counterparts 57 the S 1 f S n transitions of cisisomers are typically red-shifted. Recent work in our laboratory comparing the transient absorption spectra of cis- and transisomers of β-carotene and spheroidene have confirmed this to be the case. 50 Thus, neoxanthin would not be expected to follow the trends in position and width set by a series of trans-isomers, and indeed the lack of agreement for this molecule among the others in the series is understandable on this basis. The global fits reveal two kinetic components with lifetimes longer than 1 ps. The first of these ranges from 9.5 to 37.6 ps in going from β-carotene to neoxanthin and is clearly associated with the lifetime of the S 1 state. A change in S 1 lifetime is expected based on variations in the conjugated π-electron chain length that lead to changes in the S 1 -S 0 energy gap. This effect has been well-documented (Table 1). The second kinetic component spans a narrower range ( ps) and represents the lifetime of the S* state. The wavy, variable-amplitude spectra of the ps DADS components (Figure 6) are suggestive of spectral band shifts. In the wavelength region of the S 1 f S n transition, the shift appears to be to longer wavelengths for β-carotene, zeaxanthin, and lutein (see the third DADS component in Figures 6A-C), because a positive feature appears between the S 1 f S n transition and the S 0 f S 2 transition and a negative feature appears at longer wavelengths. The effect is reversed for violaxanthin and neoxanthin (see the third DADS component in Figures 6D and 6E), but to understand the data more thoroughly, the EADS components should be considered.

9 22880 J. Phys. Chem. B, Vol. 110, No. 45, 2006 Niedzwiedzki et al. Figure 10. Key orbitals that make up the configurational description of the first excited singlet state of β-carotene. This state is an optically forbidden 1 A g- -like state characterized by both MNDO-PSDCI and SAC-CI theory as having a high degree of doubly excited character ( 55%). This character produces significant bond order reversal and preferential stabilization of the 6-s-trans geometry, thus providing a more highly correlated singlet state. Figure 9. Approximate adiabatic surfaces for ring torsion in the ground and first two excited singlet states of β-carotene. The ground-state surface minimum has a distorted s-cis geometry, but the lowest excited singlet state has a near-planar s-trans geometry whose conformation is labeled S 1*. In the longest (fourth) EADS, a shoulder is observed on the short-wavelength side of the S 1 f S n transition for β-carotene, zeaxanthin, lutein, and neoxanthin. This shoulder is reminiscent of that assigned to S* state in spirilloxanthin, 24,27,29,46 but the data presented here show unequivocally that the shoulder observed at the longer times must be associated with the S 1 state for these xanthophylls. This is demonstrated by the fact that it persists in the longest DADS and EADS profiles, even for neoxanthin whose S 1 lifetime is 37.6 ps, i.e., more than an order of magnitude longer than the 2.7 ps lifetime assigned to S* state. This indicates that the shoulder seen in the longesttime DADS and EADS components must be associated with the S 1 state, because if it were associated with the S* state, then it would have already decayed away. There are two possibilities for how the shoulder may arise. The first is that it may be associated with a (0-1) vibronic transition accompanying the major (0-0) spectral origin of the S 1 f S n transition. The intensity ratio and energy separation between the (0-0) major feature and the (0-1) shoulder are not inconsistent with this assignment. The energy separation is observed to be in the range of cm -1, which is in agreement with that expected for the difference between the (0-0) spectral origin and a (0-1) vibronic band for these molecules. See, for example, the 25 nm separation of the (0-0) and (0-1) vibronic peaks in the steady-state absorption spectra of β-carotene and zeaxanthin (Figures 4A and 4B), which corresponds to an energy separation of 1100 cm -1. The second possibility is that the shoulder represents a transition from S 1 to a different higher-energy electronic state than that giving rise to the major S 1 f S n absorption band. To examine this option more thoroughly, quantum computations were carried out. Quantum Chemical Computations. The geometry of β-carotene was optimized using density functional methods for the ground state (B3LYP/6-31G(d)) and ab initio methods (CIS(D) and SAC-CI with a D95 basis set) for the low-lying excited singlet states. The spectroscopic properties of the molecule were then analyzed using MNDO-PSDCI molecular orbital theory Experimental and theoretical studies are in agreement that the β-ionylidene ring in both the short-chain retinal polyenes and the longer-chain carotenoids selects a 6-s-cis-conformation in the ground state. 37,58 This observation also applies to the lowlying strongly allowed 1 1 B + u state. In contrast, the approximate adiabatic surfaces for ring torsion in the ground and first two excited singlet states of β-carotene given in Figure 9 show that lowest-lying 2 1 A - g state selects preferentially a 6-s-transconformation. The ground-state surface minimum has a distorted s-cis geometry, but the lowest excited singlet state has a planar s-trans geometry. The origin of this conformational selection is examined in Figure 10 where the key molecular orbitals that participate in the configurational description are shown. Ex-