Femtosecond Time-Resolved Transient Absorption Spectroscopy of

22872

J. Phys. Chem. B 2006, 110, 22872-22885

Femtosecond Time-Resolved Transient Absorption Spectroscopy of Xanthophylls Dariusz M. Niedzwiedzki,† James O. Sullivan,† Toma´ sˇ Polı´vka,‡,§ Robert R. Birge,† and Harry A. Frank*,† Department of Chemistry, UniVersity of Connecticut, Storrs, Connecticut 06269-3060, Institute of Physical Biology, UniVersity of South Bohemia, NoVe´ Hrady, Czech Republic, and Biological Centre, Czech Academy of Sciences, Ceske´ 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 S1 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) 486-2844. Fax: (860) 486-6558. E-mail: [email protected]. † 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

10.1021/jp0622738 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/25/2006

Femtosecond Spectroscopy of Xanthophylls

J. Phys. Chem. B, Vol. 110, No. 45, 2006 22873

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 S1 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 S1 state of zeaxanthin is low enough to potentially quench the lowest excited singlet state of Chl, which lies at ∼14 600 cm-1.9 However, the various reported values of the S1 state energy of violaxanthin in solution10-12 and in pigmentprotein complexes13 range from 13 700 ( 300 to 15 580 ( 60

cm-1, indicating that there is still some uncertainty whether violaxanthin also has a sufficiently low energy S1 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, S0, is assigned Ag symmetry in the idealized C2h 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 Ag symmetry and denoted S1 or 21Ag-, 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.17-19 In this notation the

22874 J. Phys. Chem. B, Vol. 110, No. 45, 2006 ground state S0 is 11Ag-. One-photon transitions between S0 (11Ag-) and S1 (21Ag-) are forbidden by group theoretical (g T u) and pseudoparity (+ T -) selection rules. The state into which one-photon absorption from the ground S0 (11Ag-) state is strongly allowed is the 11Bu+ state. The customary practice is to denote this state S2, but both theoretical20,21 and experimental results (see Polivka and Sundstrom22 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 S0 (11Ag-), S1 (21Ag-), and S2 (11Bu+), 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 11Bu- state and have postulated that it provides a route of both deactivation from S2 and energy transfer to Chl. Also, van Grondelle and co-workers24 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 molecule24 and that it provides an alternate path for the depopulation of S2 (11Bu+). However, subsequent studies on spheroidene,25 rhodopin glucoside,26,27 lycopene,28 zeaxanthin,28 and β-carotene28 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 S0 f S2 and S1 f Sn absorption bands and that it decays in several picoseconds. (In this paper, Sn 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.24-26 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 S2 state population using a high-power laser pulse they observed a decrease in the intensity of the S1 f Sn transition but no effect on the S* population. On the basis of this observation and the position and broadness of the S* f Sn transition they argued that, in solution, S* is a vibrationally excited, “hot” ground state populated by a combination of impulsive Raman scattering of the S0 f S2 pump pulse and internal conversion from S1. 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 S1 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

Niedzwiedzki et al. 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 C18 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 0.1-0.3 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 ∼2 × 1014 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



TABLE 1: Dynamics of the S1 (τ1), Vibrationally “Hot” S1 (τ1′), S2 (τ2), and S* (τ3) States of β-Carotene, Zeaxanthin, Lutein, Violaxanthin, and Neoxanthina molecule

f

pump λ (nm)

probe λ (nm)

τ1 (ps)

τ 1′ (fs)

τ2 (fs)

τ3 (ps)

τsb (fs)

fitting method

solvent

reference

β-carotene

497 497 450 450 450 355 481 481 476 504 397 504 397 490 475 475 475 480 480 352 793 794 490 490 490 490 490 490

cont. 594 460 480 550 556 cont. 570 cont. cont. cont. cont. cont. var.g cont. cont. cont. 540 570 n.e. cont. var. 484 546 580 481 544 583

9.5 ( 0.1 9.2 ( 0.3 7.9 ( 0.5 8.1 ( 0.6 10.0 ( 0.5 12.4 ( 0.5 8.9 ( 0.2 9.7 ( 0.4 9.9 8.2 ( 0.2 9.4 ( 0.2 8.4 ( 0.2 9.1 ( 0.2 9.0 9.1 ( 0.5 9.6 ( 0.5 10.7 ( 0.5 9.5 11.0 10 ( 2 8.7 ( 0.9 8.8 ( 0.2 9.0 8.9 9.0 9.0 9.0 9.0

366 ( 10 n.a.d n.d.e n.d. n.d. n.d. 600 ( 100 n.a. n.d. n.d. n.d. n.d. n.d. 500-650 400 ( 100 300 ( 100 400 ( 100 n.d. n.d. n.d. 200 ( 20 600 210 n.d. 650 180 n.d. 500

170 ( 2 n.a. n.d. n.d. n.d. n.d. 180 ( 10 150 ( 50 n.d. 160 ( 40 220 ( 50 140 ( 40 250 ( 50 110-260 140 ( 30 120 ( 30 130 ( 30 250 200 n.d. 163 ( 9 350 ( 50 n.d. 260 140 n.d. 200 110

3.4 ( 0.2 2.9 ( 0.4 n.e.f n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e.

97 n.a. 4000 4000 4000 50 220 ( 20 100 ( 50 250 300 300 300 300 120-160 100 100 100 200 200 6000 80 300 ∼150 ∼150 ∼150 ∼150 ∼150 ∼150

global fit single λ single λ single λ single λ single λ global fit single λ n.d. global fit global fit global fit global fit single λ global fit global fit global fit single λ single λ single λ single λ single λ single λ single λ single λ single λ single λ single λ

pyridine pyridine 3-methylpentane 3-methylpentane 3-methylpentane n-hexane n-hexane n-hexane n-hexane n-hexane n-hexane benzene benzene n-hexane hexane ethanol benzyl alcohol ethanol CS2 var. cyclohexane benzene n-hexane n-hexane n-hexane methanol methanol methanol

this work this work 63 63 63 64 50 50 65 66 66 66 66 52 51 51 51 67 67 68 69 70 52 52 52 52 52 52

zeaxanthin

497 497 266 400 485 490 420 490 490 490 490 490 490 490 485

cont. 596 cont. cont. cont. var. 540 555 484 547 577 481 548 579 560

10.2 ( 0.2 10.3 ( 0.1 9.8 ( 1.0 9.0 ( 0.9 9.2 ( 0.9 8.6 or 8.8 9.0 9.0 9.0 9.6 9.1 9.0 9.1 9.0 9.3

370 ( 5 n.a. 700 ( 70 350 ( 40 350 ( 40 220 or 230 n.d. n.d. 300 n.d. 800 200 n.d. 540 n.d.

146 ( 2 n.a. 180 ( 36 70 ( 14 135 ( 27 nd. n.d. n.d. n.d 270 200 n.d. 280 120 290

2.8 ( 0.2 n.e. 4.9 ( 0.5 2.8 ( 0.3 n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e.

130 n.a. 120-160 120-160 120-160 200 300 ∼150 ∼150 ∼150 ∼150 ∼150 ∼150 ∼150 ∼100

global fit single λ global fit global fit global fit single λ single λ single λ single λ single λ single λ single λ single λ single λ single λ

pyridine pyridine methanol methanol methanol methanol hexane methanol n-hexane n-hexane n-hexane methanol methanol methanol ethanol

this work this work 32 32 32 10 9 71 52 52 52 52 52 52 72

lutein

491 491 490

cont. 571 528

15.6 ( 0.1 15.8 ( 0.1 15

435 ( 5 n.a. n.d.

127 ( 3 n.a. n.d.

2.9 ( 0.1 2.2 ( 0.4 n.e.

121 n.a. ∼150

global fit single λ single λ

pyridine pyridine methanol

this work this work 71

violaxanthin

485 485 420 480

cont. 543 512 var.

26.1 ( 0.1 25.5 ( 0.2 23.9 24.6 or 25.3

582 ( 6 n.a. n.d. 320-380

163 ( 1 n.a. n.d. n.d.

5.0 ( 0.2 2.0 ( 0.6 n.e. n.e.

128 n.a. 300 200

global fit single λ single λ single λ

pyridine pyridine n-hexane methanol

this work this work 9 10

neoxanthin

481 481 467 467

cont. 558 n.a. n.a.

37.6 ( 0.1 37.5 ( 0.8 35 ( 2 35 ( 2

444 ( 5 n.a. n.d. n.d.

110 ( 2 n.a. n.d. n.d.

2.7 ( 0.1 2.7 ( 0.4 n.e. n.e.

120 n.a. 140 140

global fit single λ single λ single λ

pyridine pyridine n-hexane methanol

this work this work 59 59

c

a All data were taken at room temperature. b τs is the instrument response time. c White light continuum. d Not applicable. e Not determined. Not evident. g Various. The fitting of the data was initialized after 1 ps from t0, where component associated with S2 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 1.0.4 (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 (t0) 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

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.36-38 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.39-43 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 -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 S2 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 470-520 nm corresponding to the bleaching of the strongly allowed S0 f S2 absorption band upon excitation and also show a buildup of a strong transient absorption signal in the region of 520-610 nm. This latter peak is associated with the S1 f Sn transition. The fact that the transition is very intense implies that the Sn state has Bu+ symmetry. This is supported by the quantum computations discussed below. The spectrum of this S1 f Sn 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 S1 f Sn 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 S1 f Sn 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 Sn state brought

Femtosecond Spectroscopy of Xanthophylls about by the molecule having one less carbon-carbon double bond in conjugation. The S1 state energy also increases with decreasing conjugation length, but the increase of the S1 (21Ag-) state energy is apparently less than that for the high-energy Bu+ state into which the S1 f Sn 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 S1 f Sn 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 S1 f Sn 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 S0 f S2 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 S1 f Sn 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 S1 f Sn 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 S0 f S2 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 S1 f Sn transition in the region of 520-610 nm and a variable-sized short-wavelength shoulder in the region of 500-550 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 Ai(λ) exp(-t/τi), where Ai(λ) is the preexponential amplitude factor associated with

J. Phys. Chem. B, Vol. 110, No. 45, 2006 22877 decay component i having a time constant τi. This sum of exponentials model represents the dynamic behavior of a number of parallel, noninteracting kinetic components, the amplitude factors, Ai(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., DADSi ) ∑nj cijSADSj where the ith DADS component, DADSi, is identical to the preexponential factor, Ai(λ), in the multiexponential function, S(λ,t), and j is the index for each one of a number, n, of physically real (SADSj) spectra contributing to the DADSi profile. Individual SADSj 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 (χ2) 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 λmax of the S1 f Sn 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 S2 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 S1 f Sn transition, the formation of the S* state, the buildup of its associated S* f Sn 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 S1 f Sn transition and the S* f Sn short-wavelength shoulder. For β-carotene this second amplitude spectrum has a time constant of 366 ( 10 fs, and for the xanthophylls, the


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 S1 state to form a vibrationally equilibrated S1 state.50-52 This is rationalized by the fact that the decay of S2 to S1 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 S1, which populates the upper vibronic levels of the state, is expected to give rise to a broad, red-shifted S1 f Sn spectrum that then decays to a narrower, blue-shifted, S1 f Sn 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 11Bu- excited electronic state,23 theoretically predicted to be in the vicinity of S1 and S2.20,53,54 However, as pointed out by Billsten,32 if this were the case, then such an intense signal implies that the final state should have Ag+ symmetry. The energy of the lowest Ag+ state is known from the location of the S0 f “Ag+” cis-peak. For β-carotene this occurs at ∼29 000 cm-1.55 Subtraction of the approximate energy (∼18 000 cm-1) corresponding to the observed broad transition peaking at ∼ 550 nm would put the 11Bu- state at ∼11 000 cm-1, which is far below the S1 (21Ag-) state energy of β-carotene known to be at ∼14 500 cm-1.56 This would contradict both experimental evidence23 and theoretical predictions53 for the position of the 11Bu- state relative to S1. 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

Niedzwiedzki et al.

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 S2 into the S* state, giving rise to an associated S* f Sn transition. The longest-time DADS component, in all cases, shows the familiar, strongly allowed, vibronically relaxed, positiveamplitude spectrum associated with the S1 f Sn 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 S1 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 S1 decays. For all of the molecules examined here, a third DADS component was observed having a time constant in the range of 2.7-5.0 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 S1 f Sn 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 S1 f Sn band. However, in all cases the negative-amplitude feature tracks precisely the strong positive feature of the final DADS.


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 S1 f Sn transition involving vibrationally hot S1 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 S2 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 S2. The first EADS decays rapidly (110-170 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 S1 state and Sn.51,52 The third EADS in the sequence rises in 366-582 fs and decays with a 2.7-5.0 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, Cij. 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 2.7-5.0 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

J. Phys. Chem. B, Vol. 110, No. 45, 2006 22879 DADS component assigned to the hot S1 state crosses zero. At these wavelengths, the kinetics are free from a contribution of S1 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 S1 and S* states. The values of the S1/S* ratios of the preexponential factors were -4.3 (β-carotene), -15.7 (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 S0 f S2 and S1 f Sn 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 S0 f S2 and S1 f Sn spectra due to conformational disorder. The only exception is neoxanthin where its S1 f Sn 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 S0 f S2 transition for cisisomers of carotenoids are generally blue-shifted compared to their all-trans counterparts57 the S1 f Sn 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 S1 state. A change in S1 lifetime is expected based on variations in the conjugated π-electron chain length that lead to changes in the S1-S0 energy gap. This effect has been well-documented (Table 1). The second kinetic component spans a narrower range (2.7-5.0 ps) and represents the lifetime of the S* state. The wavy, variable-amplitude spectra of the 2.7-5.0 ps DADS components (Figure 6) are suggestive of spectral band shifts. In the wavelength region of the S1 f Sn 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 S1 f Sn transition and the S0 f S2 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.


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 1Ag--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 S1*.

In the longest (fourth) EADS, a shoulder is observed on the short-wavelength side of the S1 f Sn 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 S1 state for these xanthophylls. This is demonstrated by the fact that it persists in the longest DADS and EADS profiles, even for neoxanthin whose S1 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 S1 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 S1 f Sn 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 900-1100 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 S1 to a different higher-energy electronic state than that giving rise to the major S1 f Sn 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.36-38 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 11Bu+ 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 21Ag- 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-



Figure 11. Calculated (MNDO-PSDCI) S1 f Sn and S2 f Sn spectra of β-carotene based on geometries minimized to the specified (cis- or trans-)6-s-conformation. Solid vertical bars indicate transitions to the 1Bu-like states, and gray bars represent transitions to 1Ag-like states. Calculations included full single and double configuration interaction within the π-electron manifold.

amination of Figure 10 indicates that transferring an electron from the filled to the open orbitals creates significant bond order reversal near the center of the polyene chain. Indeed, the bond lengths of the polyene carbon atoms near the center of the polyene chain are within 0.01 Å of one another. Such a high degree of bond order reversal is unexpected in such a longchain polyene. Figure 11 shows the calculated (MNDO-PSDCI) S1 f Sn and S2 f Sn spectra of β-carotene based on geometries minimized to the specified (cis- or trans-) 6-s-conformation. Solid vertical bars indicate transitions to the 1Bu-like states, and gray bars represent transitions to 1Ag-like states. The computations show that β-carotene optimized to a 6-s-cis-conformation displays two separate but neighboring electronic transitions from the S1 (21Ag-) state to different high-energy Bu-like states (top

panel in Figure 11). The higher-energy transition of the two is computed to have approximately half of the oscillator strength of the major transition, which is entirely consistent with the experimental observations of a major S1 f Sn band and a smaller blue-edge shoulder (Figures 5-7). The optimization to the 6-scis-conformation also predicts an S2 f Sn transition in the nearIR region at ∼850 nm (bottom panel in Figure 11). We have observed strong transient absorption signals from xanthophylls in this wavelength region (data not shown). The signals build up and decay within the time duration of the excitation laser pulse and thus are entirely consistent with a transition originating from S2. The details of these observations will be included in a forthcoming paper soon to be submitted for publication. At present it is not possible to distinguish between the two possibilities, but if either of the interpretations is correct, then


Figure 12. Two hypothetical schemes for the origin of S*, the excitedstate model and the hot ground-state model, and associated photoprocesses: a, absorption; ta, transient absorption; ir, impulsive Raman scattering; esa, excited-state absorption. The green dashed arrows represent branching from Sn as suggested by Papagiannakis et al.,27 and the red arrows show branching from S2.

the shoulder observed in the longest-time DADS and EADS line shapes should grow in at the same rate as the major S1 f Sn transition upon vibronic relaxation of the hot S1 state. The second DADS components (dotted lines in Figure 6) show that this is the case. Both the shoulder and the main band feature appear in the same negative (build-up) amplitude DADS for all of the molecules except violaxanthin. For this molecule the amplitude of the DADS spectrum in the region of the shoulder is essentially zero. However, violaxanthin behaves less like a xanthophyll with terminal rings and more analogous to openchain carotenoids where the majority of the S1 f Sn oscillator strength appears to be built into a single, narrow, strong band.50,52,59-61 Thus, the shoulder observed on the shortwavelength side of the S1 f Sn transition at early times (15 ps) is associated solely with S1. Nature of the S* State. Currently, there have been two models proposed to explain the origin and behavior of S* for carotenoids in solution: the excited-state model and the hot ground-state model (Figure 12). In the excited-state model, S* is formed in ∼100 fs after photoexcitation either via a branched decay pathway from S2 which also forms S1 (red dashed lines in Figure 12) or, to account for the nonlinear dependence of the signal amplitudes on pump energy,27 from a higher electronic or vibronic state populated via a second photoexcitation from S2 which then branches to form S* and S1 (green dashed lines in Figure 12). It is important to note, however, that S* is formed with a substantial yield even at very low excitation intensities.24,27 Subsequently, the S* f Sn and S1 f Sn signals decay independently in several picoseconds as S* and S1 are depopulated. In the hot ground-state model, S* is a vibrationally excited ground state populated in part instantaneously by impulsive Raman scattering during the time course of the pump laser excitation.28 In this case, photoexcitation can be thought of as yielding a population of hot ground-state molecules from which absorption still occurs to the S2 state, but the spectral line shape of the S* f S2 transition is expected to be broader and redshifted relative to the normal S0 f S2 transition. The DADS and EADS global fits in conjunction with quantum computational modeling hold the key to understanding

Niedzwiedzki et al. which of these models represents the true behavior of these molecules. Because the DADS are very well approximated by differences in sequential EADS (Figure S4), it is clear that the wavy shape of the 2.7-5.0 ps DADS kinetic components (Figure 6) occurs due to the formation and decay of transient species having distinct spectra in the regions corresponding to both the S1 f Sn and the S0 f S2 transitions. To see this more clearly, Figure 13 shows the spectral profiles of the 2.7-5.0 ps DADS component overlaid with both the S0 f S2 steady-state absorption spectra and the longest-time DADS component, which is dominated by the spectrum of the strongly allowed S1 f Sn transition. Note that the spectra in Figure 13 are all normalized to the amplitude of their largest positive feature. The 2.7-5.0 ps DADS line shapes seen in Figure 13 reveal especially clearly for zeaxanthin, lutein, violaxanthin, and neoxanthin a very broad transition associated with S* and having a significant positive rise starting at 650 nm and gaining intensity as one goes to shorter wavelengths. Built on this broad spectral line shape are features corresponding to the major S1 f Sn transition and the peaks of the ground-state S0 f S2 vibronic transitions. These features appear because neither the DADS (parallel) model nor the EADS (sequential) model perfectly represent the photophysics of decay of the xanthophylls from their excited states. Thus, the third DADS components or equivalently the difference between the third and fourth EADS components (Figure S4) contains a contribution from the S1 f Sn excited-state absorption and ground-state bleaching in addition to the S* f Sn′ transition. Nevertheless, as shown in previous work,28 the S* f Sn′ line shape is broad and featureless in the region of 450-650 nm, and the data presented here indicate that the intensity of this band depends critically on the structure of the xanthophyll. A clear distinction is observed between two groups of molecules: β-Carotene, lutein, and zeaxanthin have a higher yield of S* and a stronger S* f Sn′ transition than violaxanthin and neoxanthin. The major structural difference between these two groups is the extension of conjugation to the terminal ring(s) in the first group (Figure 3), and these data indicate the key role of the conjugated β-ionylidene rings in controlling the dynamics of deactivation from the excited states of the xanthophylls. Figure 11 shows that the calculated (MNDO-PSDCI) S1 f Sn spectrum of β-carotene based on a geometry minimized to a 6-s-cis-conformation predicts two close-lying electronic transitions originating from S1. In contrast, the geometry minimized to a 6-s-trans-conformation shows three relatively strong electronic transitions in the region between 450 and 650 nm, the combined maximum of which is shifted approximately 20 nm to the shorter wavelengths of the 6-s-cis-conformation spectrum. This is the same magnitude of shift observed for the S* f Sn′ transition compared to the S1 f Sn transition (Figure 13). The spectrum of the S* f Sn′ transition may also be broader based on the finding of significant bond order reversal near the center of the polyene chain that could lead to spectral heterogeneity due to conformational disorder.44 The 6-s-transconformation also has a narrower energy gap with the ground state (Figure 9), which, based on the energy gap law for radiationless transitions,62 could account for its faster (3.4 ( 0.2 ps) lifetime compared to the (9.5 ( 0.1 ps) value for the S1 state of β-carotene. Thus, we propose that a branched decay pathway of the photoexcited xanthophylls leads to the population of either a 6-s-cis-conformation (the S1 state) or a 6-s-trans-conformation (the S* state), which then decay independently at different rates back to the 6-s-cis ground state. This is an important point because if the 6-s-trans ground state



Figure 13. Overlay of the third and fourth DADS components with the steady-state spectra of all the molecules: (A) β-carotene, (B) zeaxanthin, (C) lutein, (D) violaxanthin, and (E) neoxanthin. The spectra were all normalized to the amplitude of their largest positive components.

were populated, then the barrier between it and the more stable 6-s-cis ground state would prevent the molecule from reforming the original conformation and would result in a nondecaying bleaching signal. Because this is not observed, all of the excited molecules must ultimately end up back in the original 6-s-cis ground state. The 6-s-trans-conformation accounts for the spectroscopic features and dynamics attributed to the S* state including the transition appearing to the blue of the major S1 f Sn transient absorption band (Figure 5), its broad featureless spectral line shape (Figure 13), and the partial recovery of the ground-state bleaching in 2.7 to 5.0 ps (Figure 7). The remainder of the ground-state bleaching recovers as S1 decays. The presence of carbon-carbon double bonds in the β-ionylidene rings in conjugation with the π-electron polyene systems of β-carotene, zeaxanthin, and lutein facilitate the conformational change and lead to higher yields of S* for those molecules. The fact that the S* state was first detected and described for spirilloxanthin, which has the longest π-electron conjugated system (N ) 13) of any naturally occurring carotenoid, is consistent with this apparent requirement for extended, flexible, terminal π-bonds to produce S* in a significant yield. The model being proposed here is similar to that described by de Weerd et al.51 in which distortion of β-carotene is invoked to account for the time-resolved changes in excited-state

absorption during relaxation. If these models are correct, then the yield of twisted molecules should be influenced by the medium and reduced in high viscosity or frozen solvents. Preliminary studies in our laboratory on zeaxanthin and lutein in 5/5/2 v/v/v diethyl ether/isopentane/ethanol (EPA) glasses at 77 K indicate that this is indeed the case (unpublished results). It should be mentioned that the assignment of the spectroscopic features of S* to an excited state in the first place24 was based in part on the fact that spirilloxanthin showed similar behavior whether it was in solution or bound in the LH1 complex of Rs. rubrum. In particular, the analysis of the S* dynamics revealed it to be a precursor to carotenoid triplet state formation in the LH1 complex, but this photochemical process does not occur for the molecule in solution. It is thus likely that in light-harvesting complexes conformationally perturbed carotenoids may facilitate triplet state formation. The results of this study have detailed the positions and time dependence of the complex, ultrafast spectroscopic features associated with the major xanthophyll pigments in higher plants and have elucidated the origin of the S* state in xanthophylls. The higher yields of S* for β-carotene, zeaxanthin, and lutein compared to those for violaxanthin and neoxanthin and the faster dynamics of S* decay compared to that of S1 suggest S* and its associated twisted conformation may facilitate nonradiative

22884 J. Phys. Chem. B, Vol. 110, No. 45, 2006 relaxation to the ground state as a means of dissipating excess energy in the process of NPQ. In any case, the data are expected to be useful in the ongoing analysis of the ultrafast spectroscopic observables of these same molecules in thylakoid and pigmentprotein complex preparations from higher plants. These studies seek to understand what precise relaxation pathways in proteins pertain to the mechanism of NPQ and control the manner in which plants adapt to high light stress. Acknowledgment. The authors thank George Gibson for expert advice on the operation of the laser spectrometer system. This work is supported in the laboratory of H.A.F. by the National Science Foundation (Grant No. MCB-0314380) and the University of Connecticut Research Foundation and in the laboratory of R.R.B. by the National Institutes of Health (Grant No. GM-34548) and the National Science Foundation (Grant Nos. BES-0412387 and CCF-0432151). Partial support for components of the ultrafast laser spectrometer system was also provided by a grant to H.A.F. from the National Institutes of Health (Grant No. GM-30353). T.P. thanks the Czech Ministry of Education for financial support (Grants Nos. MSM6007665808 and AV0Z50510513). Supporting Information Available: Plot of the ratio of the S1 and S* amplitudes obtained from the third and fourth EADS as a function of pump pulse energy for lutein, comparison of the three- and four-component fits at the λmax of the S1-Sn transition, comparison of the three- and four-component fits at the region of the S*-Sn transition, and overlay of the third DADS components with the difference spectra generated by subtracting the third and fourth EADS components. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhaüser Verlag: Basel, Switzerland, 1995; Vol. 1B. (2) Demmig-Adams, B.; Adams, W. W., III. The xanthophyll cycle. In Carotenoids in Photosynthesis; Young, A. J., Britton, G., Eds.; Chapman and Hall: London, 1993; p 206 (3) Krinsky, N. I. Function. In Carotenoids; Isler, O., Guttman, G., Solms, U., Eds.; Birkhaüser Verlag: Basel, Switzerland, 1971; p 669. (4) Holt, N. E.; Fleming, G. R.; Niyogi, K. K. Biochemistry 2004, 43, 8281. (5) Ruban, A. V.; Phillip, D.; Young, A. J.; Horton, P. Biochemistry 1997, 36, 7855. (6) Horton, P.; Ruban, A. V.; Rees, D.; Pascal, A. A.; Noctor, G.; Young, A. J. FEBS Lett. 1991, 292, 1. (7) Horton, P.; Ruban, A. V.; Walters, R. G. Plant Physiol. 1994, 106, 415. (8) Horton, P.; Ruban, A. V.; Wentworth, M. Philos. Trans. R. Soc. London, Ser. B 2000, 355, 1361. (9) Frank, H. A.; Cua, A.; Chynwat, V.; Young, A.; Gosztola, D.; Wasielewski, M. R. Photosynth. Res. 1994, 41, 389. (10) Polı´vka, T.; Herek, J. L.; Zigmantas, D.; Akerlund, H. E.; Sundstro¨m, V. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4914. (11) Frank, H. A.; Bautista, J. A.; Josue, J. S.; Young, A. J. Biochemistry 2000, 39, 2831. (12) Josue, J. S.; Frank, H. A. J. Phys. Chem. A 2002, 106, 4815. (13) Polı´vka, T.; Zigmantas, D.; Sundstro¨m, V.; Formaggio, E.; Cinque, G.; Bassi, R. Biochemistry 2002, 41, 439. (14) Holt, N. E.; Zigmantas, D.; Valkunas, L.; Li, X. P.; Niyogi, K. K.; Fleming, G. R. Science 2005, 307, 433. (15) Hudson, B.; Kohler, B. Annu. ReV. Phys. Chem. 1974, 25, 437. (16) Hudson, B. S.; Kohler, B. E.; Schulten, K. Linear polyene electronic structure and potential surfaces. In Excited States; Lim, E. D., Ed.; Academic Press: New York, 1982; Vol. 6, p 1. (17) Birge, R. R. Acc. Chem. Res. 1986, 19, 138. (18) Pariser, R. J. Chem. Phys. 1955, 24, 250268. (19) Callis, P. R.; Scott, T. W.; Albrecht, A. C. J. Chem. Phys. 1983, 78, 16. (20) Tavan, P.; Schulten, K. J. Chem. Phys. 1979, 70, 5407. (21) Schulten, K.; Karplus, M. Chem. Phys. Lett. 1972, 14, 305.

Niedzwiedzki et al. (22) Polı´vka, T.; Sundstro¨m, V. Chem. ReV. 2004, 104, 2021. (23) Koyama, Y.; Rondonuwu, F. S.; Fujii, R.; Watanabe, Y. Biopolymers 2004, 74, 2. (24) Gradinaru, C. C.; Kennis, J. T. M.; Papagiannakis, E.; van Stokkum, I. H. M.; Cogdell, R. J.; Fleming, G. R.; Niederman, R. A.; van Grondelle, R. Proc. Natl. Acad. Sci. U.S.A 2001, 98, 2364. (25) Papagiannakis, E.; Kennis, J. T. M.; van Stokkum, I. H. M.; Cogdell, R. J.; van Grondelle, R. Proc. Natl. Acad. Sci. U.S.A 2002, 99, 6017. (26) Wohlleben, W.; Buckup, T.; Herek, J. L.; Cogdell, R. J.; Motzkus, M. Biophys. J. 2003, 85, 442. (27) Papagiannakis, E.; van Stokkum, I. H.; Vengris, M.; Cogdell, R. J.; van Grondelle, R.; Larsen, D. S. J. Phys. Chem. B 2006, 110, 5727. (28) Wohlleben, W.; Buckup, T.; Hashimoto, H.; Cogdell, R. J.; Herek, J. L.; Motzkus, M. J. Phys. Chem. B 2004, 108, 3320. (29) Papagiannakis, E.; Das, S. K.; Gall, A.; Stokkum, I. H. M.; Robert, B.; van Grondelle, R.; Frank, H. A.; Kennis, J. T. M. J. Phys. Chem. B 2003, 107, 5642. (30) Polı´vka, T.; Pullerits, T.; Frank, H. A.; Cogdell, R. J.; Sundstro¨m, V. J. Phys. Chem. B 2004, 108, 15398. (31) Christensen, R. L.; Barney, E. A.; Broene, R. D.; Galinato, M. G. I.; Frank, H. A. Arch. Biochem. Biophys. 2004, 430, 30. (32) Billsten, H. H.; Pan, J.; Sinha, S.; Pascher, T.; Sundstro¨m, V.; Polı´vka, T. J. Phys. Chem. A 2005, 109, 6852. (33) Andersson, P. O.; Gillbro, T. J. Chem. Phys. 1995, 103, 2509. (34) Ilagan, R. P.; Christensen, R. L.; Chapp, T. W.; Gibson, G. N.; Pascher, T.; Polivka, T.; Frank, H. A. J. Phys. Chem. A 2005, 109, 3120. (35) Liu, Z. F.; Yan, H. C.; Wang, K. B.; Kuang, T. Y.; Zhang, J. P.; Gui, L. L.; An, X. M.; Chang, W. R. Nature (London) 2004, 428, 287. (36) Dolan, P. M.; Miller, D.; Cogdell, R. J.; Birge, R. R.; Frank, H. A. J. Phys. Chem. B 2001, 105, 12134. (37) Shima, S.; Ilagan, R. P.; Gillespie, N.; Sommer, B. J.; Hiller, R. G.; Sharples, F. P.; Frank, H. A.; Birge, R. R. J. Phys. Chem. A 2003, 107, 8052. (38) Ilagan, R. P.; Shima, S.; Melkozernov, A.; Lin, S.; Blankenship, R. E.; Sharples, F. P.; Hiller, R. G.; Birge, R. R.; Frank, H. A. Biochemistry 2004, 43, 1478. (39) Ruban, A. V.; Pascal, A. A.; Robert, B.; Horton, P. J. Biol. Chem. 2001, 276, 24862. (40) Das, S. K.; Frank, H. A. Biochemistry 2002, 41, 13087. (41) Peterman, E. J. G.; Gradinaru, C. C.; Calkoen, F.; Borst, J. C.; van Grondelle, R.; van Amerongen, H. Biochemistry 1997, 36, 12208. (42) Peterman, E. J. G.; Dukker, F. M.; van Amerongen, H. Biophys. J. 1995, 69, 2670. (43) Pascal, A.; Gradinaru, C.; Wacker, U.; Peterman, E.; Calkoen, F.; Irrgang, K.-D.; Horton, P.; Renger, G.; Van Grondelle, R.; Robert, B.; Van Amerongen, H. Eur. J. Biochem. 1999, 262, 817. (44) Christensen, R. L.; Kohler, B. E. Photochem. Photobiol. 1973, 18, 293. (45) Kohler, B. E. Electronic structure of Carotenoids. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhaüser Verlag: Basel, Switzerland, 1995; Vol. 1B, p 3. (46) Papagiannakis, E. Shedding light on the dark states of carotenoids. Ph.D. Dissertation, Free University of Amsterdam, Amsterdam, 2004. (47) Holzwarth, A. R. Data analysis of time-resolved measurements. In Biophysical Techniques in Photosyntesis; Amesz, J., Hoff, A. J., Eds.; Advances in Photosynthesis 3; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1996; p 75. (48) van Stokkum, I. H. M.; Larsen, D. S.; van Grondelle, R. Biochim. Biophys. Acta 2004, 1657, 82. (49) Macpherson, A. N.; Arellano, J. B.; Fraser, N. J.; Cogdell, R. J.; Gillbro, T. Biophys. J. 2001, 80, 923. (50) Pendon, Z. D.; Gibson, G. N.; van der Hoef, I.; Lugtenburg, J.; Frank, H. A. J. Phys. Chem. B 2005, 109, 21172. (51) de Weerd, F. L.; van Stokkum, I. H. M.; van Grondelle, R. Chem. Phys. Lett. 2002, 354, 38. (52) Billsten, H. H.; Zigmantas, D.; Sundstro¨m, V.; Polı´vka, T. Chem. Phys. Lett. 2002, 355, 465. (53) Tavan, P.; Schulten, K. J. Chem. Phys. 1986, 85, 6602. (54) Tavan, P.; Schulten, K. Phys. ReV. B 1987, 36, 4337. (55) Tsukida, K. Methods Enzymol. 1992, 213, 291-298. (56) Sashima, T.; Koyama, Y.; Yamada, T.; Hashimoto, H. J. Phys. Chem. B 2000, 104, 5011. (57) Carotenoids; Isler, O., Gutmann, H., Solms, U., Eds.; Birkhaüser Verlag: Basel, Switzerland, 1971. (58) Dreuw, A. J. Phys. Chem. A 2006, 110, 4592. (59) Frank, H. A.; Bautista, J. A.; Josue, J.; Pendon, Z.; Hiller, R. G.; Sharples, F. P.; Gosztola, D.; Wasielewski, M. R. J. Phys. Chem. B 2000, 104, 4569. (60) Polli, D.; Cerullo, G.; Lanzani, G.; De Silvestri, S.; Hashimoto, H.; Cogdell, R. J. Biophys. J. 2006, 90, 2486. (61) Polli, D.; Cerullo, G.; Lanzani, G.; De Silvestri, S.; Hashimoto, H.; Cogdell, R. J. Synth. Met. 2003, 139, 893. (62) Englman, R.; Jortner, J. Mol. Phys. 1970, 18, 145.

Femtosecond Spectroscopy of Xanthophylls (63) Wasielewski, M. R.; Johnson, D. G.; Bradford, E. G.; Kispert, L. D. J. Chem. Phys. 1989, 91, 6691. (64) Hashimoto, H.; Koyama, Y.; Hirata, Y.; Mataga, N. J. Phys. Chem. 1991, 95, 3072. (65) Nagae, H.; Kuki, M.; Zhang, J. P.; Sashima, T.; Mukai, Y.; Koyama, Y. J. Phys. Chem. A 2000, 104, 4155. (66) Kosumi, D.; Yanagi, K.; Nishio, T.; Hashimoto, H.; M., Y. Chem. Phys. Lett. 2005, 408, 89. (67) Shreve, A. P.; Trautman, J. K.; Owens, T. G.; Albrecht, A. C. Chem. Phys. Lett. 1991, 178, 89.

J. Phys. Chem. B, Vol. 110, No. 45, 2006 22885 (68) Bondarev, S. L.; Bachilo, S. M.; Dvornikov, S. S.; Tikhomirov, S. A. J. Photochem. Photobiol., A 1989, 46, 315. (69) McCamant, D. W.; Kukura, P.; Mathies, R. A. J. Phys. Chem. A 2003, 107, 8208. (70) Yoshizawa, M.; Aoki, H.; Hashimoto, H. Phys. ReV. B 2001, 63, 18301. (71) Billsten, H. H.; Bhosale, P.; Yemelyanov, A.; Bernstein, P. S.; Polı´vka, T. Photochem. Photobiol. 2003, 78, 138. (72) Billsten, H. H.; Sundstro¨m, V.; Polı´vka, T. J. Phys. Chem. A 2005, 109.

Femtosecond Time-Resolved Transient Absorption Spectroscopy of

Recommend Documents