Excited state lifetimes and energies of okenone and chlorobactene, exemplary keto and non-keto aryl carotenoids.

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Cite this: Phys. Chem. Chem. Phys., 2015, 17, 13245

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Excited state lifetimes and energies of okenone and chlorobactene, exemplary keto and non-keto aryl carotenoids Dariusz M. Niedzwiedzki*a and Laura Cranstonb Photophysical properties of two typical aryl carotenoids, okenone and chlorobactene, were studied with application of femtosecond and microsecond time-resolved absorption spectroscopies. These carotenoids are structurally similar and differ only by keto-group and character of the aryl ring. The studies have concentrated on aspects of the photochemistry of these carotenoids as possibility of solvent polarity induced formation of intramolecular charge transfer state in okenone, which contains a keto-group directly attached to the carbon–carbon double bond conjugation, estimating the energy of the forbidden first excited singlet electronic state, S1 (21Ag) and testing the photoprotective capabilities of okenone and chlorobactene in real biological systems. The energies of the S1 (21Ag) state obtained for these carotenoids are 12 750 cm1 for okenone and 13 450 cm1 for chlorobactene and are not affected either by temperature or solvent polarity. The effect of cryogenic temperature on the excited states lifetimes

Received 9th February 2015, Accepted 20th April 2015 DOI: 10.1039/c5cp00836k

and energies was also studied at 77 K in 2-methyltetrahydrofuran, which forms a transparent glass upon freezing. The ability to quench bacteriochlorophylls triplets was studied on model bacteriochlorophyll a–carotenoid mixtures with application of flash photolysis. The triplet state lifetime obtained from the anticipated kinetic modelling of the rise and decay of the pool of carotenoid triplets are 2.1 ms for okenone

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and 2.8 ms for chlorobactene.

Introduction Carotenoids are pigments that are naturally synthesized in all phototrophic organisms like bacteria, algae or higher plants, where they play important roles in the process of photosynthesis as accessory light absorbers and photoprotectors of the main photosynthetic pigments – various types of chlorophylls and bacteriochlorophylls.1,2 Carotenoids mostly appear to be yellow, orange or red in colour. Such coloration is due to a stronglyallowed electronic absorption between the electronic ground state, S0 (11Ag) and the singlet excited state, 11Bu+, historically denoted as the S2 state. The lowest-lying excited state, S1 (21Ag) is optically silent due to a lack of change of either symmetry (g 2 u) and pseudoparity (+ 2 ) that are required for onephoton processes as hypothetical S0 (11Ag) 2 S1 (21Ag) transitions.3 The classic three state model quite sufficiently explains typical spectroscopic properties of carotenoids however

a

Photosynthetic Antenna Research Center and Department of Chemistry, Washington University in St Louis, MO 63130, USA. E-mail: [email protected] b Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, Scotland, UK

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many experimental results obtained from time-resolved spectroscopies, broadly introduced in the last past two decades, suggest that other non-optically active electronic states may lie in the vicinity of the S1 (21Ag) and S2 (11Bu+) states, see reviews: ref. 4 and 5. An intramolecular charge transfer state (ICT) is among these so-called dark excited states. The spectroscopic signatures of the ICT state have been recorded for some natural carbonylcontaining carotenoids and synthetic polyene analogues.6–11 It has been shown that introduction of a CQO group into the conjugated p-electron system sometimes may induce a profound effect of solvent polarity on the lifetime of the S1 (21Ag) state as well on the spectral shape of the fully allowed electronic S0 (11Ag) - S2 (11Bu+) absorption. In polar environments, the S1 (21Ag) state lifetime shortens. In addition, transient absorption spectra of these molecules consist of new spectral features including the ICT - S0 (11Ag) stimulated emission. Because the S0 (11Ag) - ICT band is absent in the ground state absorption spectrum, it was postulated that ICT is a transient state that only evolves during energetic relaxation and can participate in an excitation decay pathway.6,7,9,10,12–15 The influence of an ICT state on the temporal properties of the excited states of carbonyl carotenoids is greatly impacted by localization of the CQO in respect to the conjugation backbone and by the number of conjugated carbon–carbon double bonds. The influence

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Fig. 1 (a) Chemical structures of okenone and chlorobactene and (b, c) their steady-state absorption in several organic solvents having various polarity and polarizability taken at room temperature (n-hex – hexane, 2-MTHF – 2-methyl-tetrahydrofuran, MeOH – methanol) and at 77 K (2-MTHF). For details refer to Table 1.

completely fades for carotenoids/polyenes with two symmetrically attached CQO groups or for carotenoids with a number of CQC conjugated double bonds larger than ten.6,16,17

Recently, a considerable effort was made toward better understanding of photophysical properties of specific subgroup of carotenoids called aryl carotenoids. These carotenoids contain one or two aromatic rings that extend the length of the CQC conjugation from the backbone. Interestingly, it was discovered that in some cases, aryl carotenoids that are structurally almost identical such as isorenieratene and renierapurpurin demonstrate very different excited state properties, sometimes even counterintuitive ones.18,19 Further investigations demonstrated that the plane of the terminal ring in these carotenoids can be substantially distorted from the central linear conjugation and the torsion angle of the aryl ring strongly depends on the character of the aryl ring.19 The aryl ring can be either f [C18(18 0 ) methyl group connected with carbon C5(5 0 ) in the ring] or w [C18(18 0 ) methyl group connected with carbon C3(3 0 ) in the ring] (Fig. 1).20 By performing quantum chemical calculations, it was shown that upon relaxation to the S1 (21Ag) state, the w-ring becomes almost planar with the linear backbone, unlike the f-ring that still has significant distortion similar to that of b-ring in non-aryl carotenoids.19 This effect explains the observed shortening of the S1 (21Ag) state lifetime of renierapurpurin (w-ring) in respect to the structurally almost identical isorenieratene (f-ring). In addition, these calculations showed that a CQC double bond in an aryl ring is not equal to a CQC double bond in a b-ring.19 In consequence, even if both types of carotenoids, f-ring aryl and b-ring non-aryl, adapt very similar geometries in both the S2 (11Bu+) and S1 (21Ag) exited states, the effective CQC conjugation in a f-ring aryl carotenoid might be actually shorter, regardless of the fact that the nominal conjugation N is larger by two.

Table 1 Energies of S2 (11Bu+) and S1 (21Ag) states, positions of transient S1 - Sn absorption band and its bandwidths and states lifetimes obtained from global analysis. Fitting uncertainties are o5% and are omitted for table clarity

Solvent Temp. R(n)

P(e)

S0 - S2 (0–0) S1 - Sn IRF lexc S1 - Sn FWHM FWHM tS2 (fs) (nm) (nm) (cm1) S1 (cm1) max (nm) (cm1) (fs) VIS/NIR

tS1 (ps) thS1 (fs) VIS/NIR

tS* (ps) tT (ms) Ref.

Okenone n-hex RT

0.229 0.228 515

515

19 420 12 750

588

870

140/210 160/oIRF

500

3.8/3.7

14.7

2.1

MeOH

0.205 0.913 510

510

19 600 12 650

596

1460

110/240 160/oIRF

400

4.0/4.0

12.0

n.d.

2-MTHF 77 K

0.342 0.922 550

550

18 180 12 650

630

780

210/420 220/oIRF

460

5.4/5.2

10.2

n.d.

clhex Acetone CS2 BAlc CS2

0.256 0.220 0.355 0.313 0.355

RT

RT RT RT RT RT

B590 B590 B650 B640

0.254 0.868 0.353 0.800 0.353

4.2 4.3 4.2 4.6 5.0

Chlorobactene n-hex RT

0.229 0.228 487

487

20 530 13 450

556

1400 210/280 220/oIRF

490

7.1/7.1

n.e.

2.8

MeOH

0.205 0.913 485

485

20 620 13 450

555

1500 150/240 150/oIRF

420

7.3/6.6

n.e.

n.d.

2-MTHF 77 K

0.342 0.922 518

518

19 300 13 450

588

7.4–10.6/8.3 n.e.

n.d.

n-hex RT Benzene RT

0.229 0.228 0.294 0.299

492 509

20 320 19 640

557 579

RT

580 220/420 oIRF/oIRF 540 B1200 B1360

6.7 5.8

This work This work This work 29 29 29 29 24 This work This work This work 18 18

RT – room temperature, n-hex – n-hexane, MeOH – methanol, 2-MTHF – 2-methyl-tetrahydrofuran, clhex – cyclohexane, BAlc – benzyl alcohol, RT – room temperature, IRF – instrument response function, n.d. – not determined, n.e. – not evident, FWHM – full width at half maximum, R(n) – solvent polarizability, P(e) – solvent polarity.

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This study focuses on aspects of the photophysics of two exemplary aryl carotenoids with different ring character that were not the subject of earlier research. These carotenoids are okenone (w-ring) and chlorobactene (f-ring) (Fig. 1). Okenone (Fig. 1a) was first introduced in 1963 as a characteristic carotenoid in bacterium Chromatium okenii21 and is exclusively produced by sulphur purple bacteria from the Chromatiaceae family.22 It is an aromatic carotenoid with terminal w-type aryl ring and methoxy group at C-end. It has also a carbonyl group directly attached to the carbon–carbon double bond conjugation at C4 0 (Fig. 1a). Interestingly, this carotenoid is also an important geochemical biomarker. Its diagenetic product, okenane, can be detected in rock formations that are over a billion years old.23 The purple sulphur bacteria that produce okenone incorporate it into the light harvesting complex 2 (LH2) in which the carotenoid plays the role of a supportive absorber for BChl a.24 Chlorobactene is a major carotenoid present in green sulphur bacteria. It is accumulated in chlorosomes – giant light harvesting complexes consisting of aggregates of tens of thousands of BChl molecules. Chlorobactene plays an important structural role in complex assembly as well as light harvesting supporting pigment and photoprotector.1,25–28 Structurally (Fig. 1a), chlorobactene and okenone share an almost identical CQC conjugation pattern with two differences: the lack of a carbonyl group in chlorobactene that in okenone extends the conjugation by one and the character of the aryl group (f-ring). Both carotenoids had been previously studied by use of transient absorption (TA) spectroscopy. For okenone the studies focused on its role in the LH2 complex24 with particular attention on energetic interactions with BChl a via the S2 (11Bu+) state.29,30 Chlorobactene has been studied as part of broader project in context of influence of conjugated aryl ring on lifetime the S1 (21Ag) state compared to its analogues with other terminal rings – like a b-ring.18 Work presented here concentrates on the photophysical features of both carotenoids that have not been studied before. The first aspect is a possibility of formation of an ICT state in okenone, which contains a keto-group directly attached into the CQC conjugation. As mentioned earlier in the text, an influence of an ICT state fades in carotenoids with number of CQC conjugated double bonds larger than 10. A nominal CQC conjugation of okenone is larger than 10 however, contribution of CQC bonds from the aryl ring to the effective conjugation might be not significant and the effect may still exist. For benchmark purposes, the effect of environment polarity on the non-keto aryl carotenoid chlorobactene was tested. We chose n-hexane as non-polar solvent. As a polar solvent, methanol was used. It is protic solvent and it is known from previous work on other carbonyl-containing carotenoid, peridinin, that ICT character of the S1/ICT will be further enhanced by hydrogen bonding between carbonyl group and the solvent.31 Another aspect is to experimentally define energy of the optically-dark S1 (21Ag) state for both carotenoids and elucidate if it can be perturbed by cryogenic temperature or polarity of solvating medium. These studies have been performed using femtosecond time-resolved transient absorption. The results were also analysed in a broader


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aspect by comparing them with known (and obtained in the same experimental conditions) S1 (21Ag) state energies of various types of carotenoids. The last features that we have elucidated are photoprotective properties of both carotenoids. These carotenoids coexist with bacteriochlorophylls (BChls) in natural light harvesting complexes (LH2s, chlorosomes) and most probably are capable to quench BChl triplets. Because it would be very difficult to perform studies on chlorosomes (due to chlorosome complexity) we have decided to study BChl a–carotenoid mixtures. It was done with application of transient absorption in the microsecond time domain.

Experimental section Sample preparation Okenone was isolated from light harvesting complex 2 (LH2) of the purple bacterium Merichromatium purpuratum (previously Chromatium purpuratum). The bacterial cells were grown and the LH2 complexes were isolated as described previously.32 Chlorobactene was isolated from the cells of green sulphur bacterium Cba. tepidum grown as described previously.33 The pigments were extracted using a mixture of spectroscopic grade acetone/ methanol (1/1, v/v) (Sigma, St. Louis, MO, USA). The extract from each bacterium was first centrifuged using a table top microcentrifuge, the supernatant was collected and dried under a gentle stream of nitrogen gas. The dried pigments were subsequently dissolved in HPLC grade acetonitrile (Sigma, St. Louis, MO, USA) and injected into an Agilent Series 1100 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a reverse-phase Zorbax C-18 column (4.6 250 mm, Agilent Technologies Inc., Santa Clara, CA, USA) operated at 20 1C. The pigments were eluted with a mobile phase consisting initially of 100% acetonitrile (Sigma, St. Louis, MO, USA) with a linear gradient (0–40% within 40 minutes) of tetrahydrofuran (Sigma, St. Louis, MO, USA), pumped at a rate of 1.5 mL min1, collected, dried down under a stream of nitrogen gas and stored in 20 1C until ready to use for spectroscopic experiments. Spectroscopic methods Femtosecond transient absorption experiments at room temperature (RT) and at 77 K were carried out using Helios, a femtosecond transient absorption spectrometer (Ultrafast Systems LCC, Sarasota, FL, USA) coupled to a femtosecond laser system from SpectraPhysics described in detail previously.34 The samples were excited into the (0–0) vibronic band of the S0 (11Ag) - S2 (11Bu+) absorption with pump beam energy of B500 nJ in a spot size of 1 mm diameter corresponding to an intensity of B2 1014 photons per cm2 per pulse (1 kHz repetition of the excitation laser beam). The samples were studied in n-hexane and in methanol (MeOH) at RT and in 2-methyltetrahydrofuran (2-MTHF) at 77 K. The experiments at 77 K were done using a SVP-100 liquid nitrogen cryostat from Janis (Janis, Woburn, MA, USA) and a cryogenic 1 cm, square quartz cuvette from NSG Precision Cells (Farmingdale, NY, USA). Steadystate absorption spectra were recorded using a Shimadzu UV-1800 spectrophotometer with spectral resolution of 1 nm.


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Microsecond time-resolved spectroscopic experiments were performed at RT on the samples in a 1 cm path length quartz cuvette using LP920-K/S flash photolysis spectrometer from Edinburgh Instruments (Livingston, UK) described in detail previously.35 Excitation pulses were delivered from an Opotek Vibrant 355 tuneable laser system equipped with 10 Hz Nd:YAG laser (Quantel), second and third harmonic generators (SHG and THG). Energy of the excitation beam (355 nm, B 1 cm dimension) was set to B10 mJ per pulse consistent to a photon density of B2 1016 photons per cm2. Transient absorption signals (either spectra or kinetic traces) were averaged from 5–10 times depending on samples. For these experiments the carotenoids were mixed with BChl a (as triplet sensitizer) in n-hexane with absorption adjusted to B0.3 for carotenoids and B0.6 for BChl a (Qy) corresponding to concentrations of B2 mM (carotenoids) and B7 mM (BChl a). It was assumed that molar extinction coefficients of both carotenoids are identical of B130 000 cm1 M1 (ref. 36) and for BChl a molar extinction coefficient in pyridine was used.35 The solvent mixture with dissolved pigments was degassed as described in detail previously.35 Triplet measurements were performed under vacuum conditions. In order to prevent spontaneous BChls aggregation and chance of triplet annihilation, a few drops of pyridine were added to the mixtures. Spectroscopic data analysis and fitting Group velocity dispersion in the femtosecond time-resolved TA datasets was corrected using Surface Explorer Pro software (UltrafastSystems LCC, Sarasota, FL, USA) by building a dispersion correction curve from a set of initial times of transient signals obtained from single wavelength fits of representative kinetics. Global analysis of the TA datasets was performed using Carpetview – a data viewing and analysis software for ultrafast spectroscopy measurements from Light Conversion (Light Conversion Ltd, Vilnius, Lithuania). For fitting purposes, the instrument response function (IRF) was assumed to be Gaussian with a variable full width at half maximum (FWHM). Derivative equation modelling the rise and decay of the carotenoid triplet population was solved in Matlab (MathWorks Inc., Natick, MA, USA) and implemented into Origin (OriginLab, Northampton, MA, USA) as a user-defined fitting function.

Results Steady-state absorption Steady-state absorption spectra of okenone and chlorobactene taken at room temperature (RT) in n-hexane and methanol (MeOH) and at 77 K in 2-methyl-tetrahydrofuran (2-MTHF) are shown in Fig. 1b and c. The spectra are associated with the fully-allowed S0 (11Ag) - S2 (11Bu+) transition. For okenone, the influence of the carbonyl group, directly attached to the CQC double bond backbone, on the spectral shape is noticeable. In the polar, protic solvent, MeOH, the absorption spectrum broadens, however this effect is not significant and the vibronic progression of the electronic transition is still partially resolved.


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A limited effect of the carbonyl group on the spectral envelope is related to the long double bond conjugation in this carotenoid. In both solvents, the maximum of the absorption spectrum (0–1 vibronic band) appears at 484 nm. Upon freezing to 77 K, the spectrum shifts to longer wavelengths and demonstrates a substantial increase in vibronic resolution. The bathochromic shift of B35 nm is caused by the increased polarizability of 2-MTHF glass that stabilizes the energy of the S2 (11Bu+) state. The lack of the CQO group in chlorobactene conjugation clearly highlights the influence of the keto group on the electronic absorption of okenone. For chlorobactene, changing the polarity of the solvating medium only minimally affects the shape of the absorption spectrum. A small decrease in the vibronic band resolution visible in this solvent most likely originates from increased de-planarization of the aryl ring (in respect to the rest of the conjugation). The (0–0) and (0–1) vibronic bands appear at 457 and 486 nm, respectively. The vibronic bands in the 77 K absorption spectrum of chlorobactene are better resolved compared to okenone’s counterparts and four vibronic peaks are clearly visible at 427, 453, 482 and 519 nm. The vibronic bands of the S0 (11Ag) - S2 (11Bu+) electronic transition of carotenoids can be modelled as combinations of the progressions of two vibrational modes with frequencies of B1200 cm1, corresponding to totally symmetric C–C stretching vibrations and B1600 cm1, corresponding to totally symmetric CQC stretching vibrations.3 The energetic gap between (0–0) and (0–1) vibronic bands in 77 K absorption spectra can provide some information on which modes mostly contribute. For okenone the energetic spacing is B1240 cm1 suggesting that mostly symmetric C–C stretching vibrations dominate. For chlorobactene this gap is B1480 cm1 and shows that both modes should almost equally contribute – the observed energy gap will be the weighted average of both 1600 cm1 and 1200 cm1 mode. Femtosecond time-resolved absorption at room temperature Transient absorption (TA) results and global analysis of datasets of okenone and chlorobactene taken in n-hexane at RT in the visible (VIS) spectral range are given in Fig. 2. The representative TA spectra taken at various delay times after excitation are shown in Fig. 2a and b. Excitation results in an immediate bleaching of the ground state absorption (GSB) and onset of a TA band located at 588 nm for okenone and at 556 nm for chlorobactene. In general, the transient spectral features follow the trend visible in the steady-state absorption. If the vibronic bands of ground state absorption are well-resolved, the transient absorption band will show up as a narrow peak. Interestingly, this is not the case in these two carotenoids. Regardless of better spectral resolution of the steady-state absorption spectrum of chlorobactene compared to okenone, its transient absorption band is substantially broader in respect to its complement for okenone. This is clearly noticeable if the widths of representative spectra are calculated. For the 2 ps TA spectrum the full width at the half maximum (FWHM) of the transient absorption band is only B870 cm1 for okenone but notably 1400 cm1 for chlorobactene. It is surprising because as mentioned


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Fig. 2 Transient absorption results of okenone and chlorobactene in n-hexane at RT in the VIS spectral range; (a, b) TA spectra taken at various delay times after excitation into (0–0) vibronic band of the S0 (11Ag) - S2 (11Bu+) transition (Table 1); (c, d) results of global analysis according to directed target (okenone) and sequential (chlorobactene) decay models; (e, f) relative components concentrations obtained from the global analysis as the function of time delay. The actual fits of the exemplary kinetic traces extracted from the S1 (21Ag) - Sn band are shown in the insets; SADS – species associated difference spectra, EADS – evolution associated difference spectra. For more details refer to main text and Table 1.

before the appearance of transient bands follows trends observed in the steady-state absorption. Here it seems to be exactly opposite. However, a similar observation was made for another example of an aryl carotenoid with a w-ring: renierapurpurin.19 Broadening of the S1 (11Ag) - Sn TA band is an indication of degree of conformational disorder of the carotenoid in the S1 (11Ag) state. The narrower the band, the smaller the distribution of molecular geometries – in this case, small variations of the aryl ring distortion from the molecular plane. It seems that this spectral attribute is characteristic only for aryl carotenoids with w-ring(s) and could be used for clarification of aryl ring character, if that is ambiguous. A closer look into the TA spectra of okenone reveals that the transient band does not decay equally in the whole TA signal range. From inspecting the TA spectra taken at later delay times, it is apparent that there is an initially hidden band at B550 nm that decays slower than the main transient peak. Numerous TA studies of long conjugated carotenoids show similar spectral feature in TA spectra either dissolved in solvents or bound into


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LH2 complexes.37–45 It had been proposed that this transient band may be associated with a vague excited electronic state that was initially named S*.37 Another hypothesis suggested that S* is simply a vibrationally non-equilibrated ground state.43,45 On the other hand, theoretical modelling of structural geometries of selected carotenoids in the S1 (11Ag) state suggested that S* can contribute to the S1 (11Ag) excited state of a carotenoid having a twisted conformation.39,46 Most recent TA studies performed on b-carotene and rhodopin glucoside using narrowband excitation excluded S* as a vibrationally-excited ground state.47 Another clue about the origin of S* was recently provided by Polı´vka and co-authors who compared bleaching regions of the TA spectrum in time delays that are dominated by transient bands from the S* state.17 These showed that resolution of the vibrational bands of the bleaching part of the TA spectrum is greatly enhanced compared to steady-state absorption, strongly suggesting that there is a pre-existing subset of ground state conformers that upon excitation decay independently from the main group and are solely responsible for the transient features related with S*. Altogether, this points toward an inhomogeneous model of an excitation decay that postulates two molecular sub-populations in the ground state, S0 and S0*, each with distinct S0 - S2 and S0* - S2* transitions and each exhibiting different excited state dynamics and independent decay paths. In general, a TA signal, DA, at any time delay can be described as superposition of n contributions of different components i:48 DAðt; lÞ ¼

n X

Ci ðtÞDAi ðlÞ

(1)

i¼1

Where Ci(t) is concentration and DAi(l) is the species associated difference spectrum (SADS) of component i and the initial concentration is convoluted by the instrument response function (IRF). The individual SADS, DAi(l), are defined as DAi(l) = Ai(l) A0(l) where A0(l) is a ground state absorption and Ai(l) is a transient signal associated with pure spectroscopic species (like excited state, e.g. S1 (11Ag) - Sn). The results, SADS and SADS concentrations (relative to each other) obtained from applying an inhomogeneous model to fit the okenone TA dataset in n-hexane are given in Fig. 2c and e (see embedded schemes), respectively. Based on analysis of the TA spectra it was assumed that the contribution of S* conformers in the ground state absorption might be B20%. In order to reflect it in the fitting model, the amplitudes of the instrument response function (IRF, here taken as Gaussian) convoluting initial concentrations of the S2 and S2* pools were fixed to a 4 : 1 ratio. This fitting gave satisfactory results if five SADS were used. The first two with the same lifetime of 160 fs (due to temporal limitations it is impossible to distinguish between them) correspond to the sum of the initial GSB and stimulated S2 (11Bu+) - S0 (11Ag) emission (between 520 and 650 nm). The 500 fs and 3.8 ps SADS, very characteristic in their spectral lanes, are associated with transient absorption from the vibrationally non-equilibrated (hotS1)49 and fully equilibrated S1 (21Ag) states. Finally, the 14.7 ps SADS should be linked to


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transient features related with the S* state. The rise and decay of SADS concentrations can be traced in Fig. 2e, which also contains the inset with the representative kinetic trace and fit obtained based on applied inhomogeneous model. Inspecting the TA spectra of chlorobactene in n-hexane did not reveal the presence of the long-lived transient band and the dataset was fitted according to a sequential model of the excitation path decay (see Fig. 2d). In general, the results of such fitting of TA data are termed as EADS – evolution associated difference spectra.48 In this particular case, such a sequence very adequately represents the real excitation decay and EADSs are equivalent to SADS. Relative concentrations of each EADS/SADS with inset highlighting the fitted exemplary kinetic traces are given in Fig. 2f. The TA spectra and global analysis results of TA datasets of okenone and chlorobactene taken in n-hexane in the nearinfrared (NIR) spectral range are shown in Fig. 3. The representative TA spectra taken at various delay times after excitation are given in Fig. 3a and b. In the sub-picosecond time regime the spectra are dominated by a strong transition with narrow band having a maximum at B1140 nm for okenone and at 1040 nm for chlorobactene. This band decays within a few hundred fs and other spectral features are formed. These bands are significantly weaker and form a pattern similar to the ground state absorption. Global analysis results (EADS) are shown in Fig. 3c and d. Two spectral components satisfactorily fitted data from both carotenoids. For better comparability the second EADS was multiplied five times. The first EADS has a lifetime shorter than the width of the IRF function. It was previously theoretically and experimentally demonstrated that this transient band is associated with a transition from the S2 (11Bu+) into a higher n1Ag state.50 The characteristic shape and lifetime of the second EADS directs this component toward the S1 (21Ag) - S2 (11Bu+) transient absorption. This transient band was previously recorded for multiple carotenoids.40,51–58 The embedded graphs in Fig. 3c and d show exemplary kinetic traces accompanied by fits obtained from the global analysis of the NIR datasets. The fact that both ground state S0 (11Ag) - S2 (11Bu+) and transient S1 (21Ag) S2 (11Bu+) absorption spectra are recorded allows to calculate the energy of the optically forbidden electronic state S1 (21Ag) using simple arithmetic. First, both profiles require conversion from nonlinear wavelength scale to linear energetic scale, and then should be overlapped. The energetic shift of S1 (21Ag) S2 (11Bu+) spectrum required to obtain best peak-valley coincidence with steady-state complement defines the S1 (21Ag) state energy. The results of spectral overlay are shown in Fig. 3e and d as well calculated energies are also provided. For these calculations the S1 (21Ag) - S2 (11Bu+) profile was taken at B2 ps after excitation. The TA results and global analysis of okenone and chlorobactene taken in MeOH in the VIS spectral range at RT are given in Fig. 4. The large polarity and hydrogen bonding character of the solvent have significant influence on the excited state transition (Fig. 4a). The transient band reveals substantial broadening compared to its complement taken in n-hexane.


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Fig. 3 Transient absorption results of okenone and chlorobactene in n-hexane at RT in the NIR spectral range; (a, b) TA spectra taken at various delay times after excitation into the (0–0) vibronic band of the S0 (11Ag) S2 (11Bu+) transition; (c, d) results of global analysis according to sequential decay model. To enhance comparability, the amplitude of the longer-lived component was multiplied 5 times. The exemplary fits of the kinetic traces extracted from the S1 (21Ag) - S2 (11Bu+) transient band are shown in the insets; (e, f) Estimation of the S1 (21Ag) state energy by overlapping the steady-state absorption (blue) with the transient S1 (21Ag) - S2 (11Bu+) spectrum that prior were converted to wavenumber scale. The shift required to bring both profiles to the best coincidence defines the S1 (21Ag) state energy; EADS – evolution associated difference spectra. For more details refer to main text and Table 1.

The FWHM of the TA peak is 1460 cm1 (870 cm1 in n-hexane), in addition the band shifts to longer wavelengths and is positioned at 596 nm. As might be expected, a polar solvent does not disrupt the excited state properties of chlorobactene (Fig. 4b). Essentially, the spectral shape and position of the S1 (11Ag) Sn do not change. Global analysis results of the TA datasets of both carotenoids are given in Fig. 4c and d. The data were fitted according to the same models as used for data from n-hexane: an inhomogeneous decay path for okenone and a sequential decay for chlorobactene. Despite the fact that for okenone spectral differences are clearly observed between TA data in


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Fig. 4 Transient absorption results of okenone and chlorobactene in MeOH at RT in the VIS spectral range; (a, b) TA spectra taken at various delay times after excitation into (0–0) vibronic band of the S0 (11Ag) - S2 (11Bu+) transition; (c, d) results of global analysis according to directed target (okenone) and sequential (chlorobactene) decay models; (e, f) relative component concentrations obtained from the global analysis as the function of time delay. The actual fits of the exemplary kinetic traces extracted from the S1 (21Ag) - Sn band are shown in the insets; SADS – species associated difference spectra, EADS – evolution associated difference spectra. For more details refer to main text and Table 1.

n-hexane and MeOH temporal properties of the excited states are more or less the same as in n-hexane (Fig. 4c). This is also true for chlorobactene (Fig. 4d). Relative concentrations of the SADS (okenone) and EADS (chlorobactene) in MeOH are given in Fig. 4e and f. These figures contain also embedded graphs with raw kinetic traces taken at the maximum of the S1 (11Ag) - Sn transition (dots) and accompanied fits resulting from global analysis (line). The TA spectra and global analysis results of okenone and chlorobactene taken in MeOH in the NIR spectral range are shown in Fig. 5. Some illustrative TA spectra taken at several delay times after excitation are given in Fig. 5a and b. The narrow, intense transient S2 (11Bu+) - n1Ag band appears to be slightly hypsochromically shifted for both carotenoids (in respect to n-hexane). The band maximum emerges at B1080 nm for okenone and at 1000 nm for chlorobactene. Global analysis results according to a sequential decay path (EADS) are shown in Fig. 5c and d. In both cases, two spectral components satisfactorily fitted the data. For okenone the S1 (21Ag) - S2 (11Bu+) transient absorption band follows the trend observed in steadystate absorption and vibrational structure that was partially


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Fig. 5 Transient absorption results of okenone and chlorobactene in MeOH at RT in the NIR spectral range; (a, b) TA spectra taken at various delay times after excitation into the (0–0) vibronic band of the S0 (11Ag) S2 (11Bu+) transition; (c, d) results of global analysis according to sequential decay model. To enhance comparability, the amplitude of the longer-lived component was multiplied 5 times. The exemplary fits of the kinetic traces extracted from the S1 (21Ag) - S2 (11Bu+) transient band are shown in the insets; (e, f) Estimation of the S1 (21Ag) state energy by overlapping the steady-state absorption (blue) with the transient S1 (21Ag) - S2 (11Bu+) spectrum that were prior converted to wavenumber scale. EADS – evolution associated difference spectra. For more details refer to main text and Table 1.

visible in n-hexane is almost lost in MeOH. For chlorobactene fitting results are very n-hexane-like however, the lifetime of the EADS associated with the S1 (21Ag) state has a slightly smaller value (6.6 ps) than could be anticipated based on remaining results (B7 ps). Fig. 5c and d contain also inserted raw kinetic traces of TA signals. These are presented together with fits obtained from global analysis. The results of spectral overlay of the S1 (21Ag) - S2 (11Bu+) transient band with steady-state absorption profiles are shown in Fig. 5e and f. For chlorobactene the S1 (21Ag) state energy measured in MeOH turns out to be identical as measured in n-hexane but decreases slightly (by 100 cm1) for okenone. The S1 (21Ag) - S2 (11Bu+) profile was taken at B2 ps after excitation. Femtosecond time-resolved absorption at 77 K Fig. 6 shows global analysis results of the VIS and NIR TA datasets of both carotenoids in 2-MTHF at 77 K. Cryogenic temperature substantially affects the spectral shapes of transient bands. The effect is very pronounced for VIS TA of chlorobactene for which the S1 (11Ag) - Sn transition band forms a very


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dataset. The results of spectral overlay of the S1 (21Ag) S2 (11Bu+) transient and ground state absorption spectra are shown in Fig. 6e and f. For these calculations the S1 (21Ag) S2 (11Bu+) profile was taken at B2 ps after excitation. The S1 (21Ag) - S2 (11Bu+) transient spectra are not so complete at 77 K and show only two vibronic bands. This is due to a decreased energetic gap between interacting electronic states. As seen in steady-state absorption, upon freezing to 77 K the S2 (11Bu+) falls to lower energies however but as calculations show, the energy of the S1 (21Ag) state remains unchanged. Sub-microsecond time-resolved absorption

Fig. 6 Transient absorption results of okenone and chlorobactene in 2-MTHF at 77 K in the VIS and NIR spectral ranges: global analysis of the VIS (a, b) and NIR (c, d) TA datasets according to sequential decay model. The exemplary fits of the kinetic traces are shown in the insets; (e, f) Estimation of the S1 (21Ag) state energy by overlapping the steady-state absorption (blue) with the transient S1 (21Ag) - S2 (11Bu+) spectrum that were prior converted to wavenumber scale. The shift required to bring both profiles to the best coincidence defines the S1 (21Ag) state energy; EADS – evolution associated difference spectra. For more details refer to main text and Table 1.

narrow band with maximum at 587 nm and FWHM of only 580 cm1. For simplicity purposes we have not pursued any specific ‘‘target’’ model to fit the data and fitted datasets globally using sequential decay that will allow track evolution of the TA spectra as delay time progresses. Also it needs to be noted that effective (observed) lifetimes of the components are independent from the fitting model. In the VIS spectral range both carotenoids required four kinetic components to obtain satisfactory fits. Cryogenic temperature has a direct impact on the S1 (11Ag) state lifetime that is elongated at 77 K. It is intriguing that for chlorobactene one additional kinetic component with a lifetime of 10.6 ps was required in the VIS TA dataset. However, similar shapes of 7.4 ps and 10.6 ps EADS suggest that upon freezing chlorobactene may accommodate two altered geometries with slightly different effective conjugations affecting their S1 (11Ag) state lifetimes. The third EADS (to be in agreement with the VIS data) was not necessary in global analysis of the NIR TA dataset of chlorobactene (Fig. 6d). However, the lifetime of the second EADS (8.3 ps) matches to the average of 7.4 and 10.6 ps lifetimes, suggesting that probably it was impossible to resolve them due to not so ideal signal-to-noise ratio as it is in the VIS


Transient absorption spectra and kinetic traces of BChl a-carotenoid n-hexane mixtures measured in the microsecond time domain are given in Fig. 7. Fig. 7a shows two representative TA spectra of the BChl a–okenone mix. These spectra are commonly referred to so-called triplet-minus-singlet (T S) spectra. The 100 ns spectral line (black) was slightly adjusted in amplitude in order to match the second spectrum (red). The T S spectrum taken at 100 ns after excitation consists exclusively of the T S profile of BChl a. However, the spectrum recorded at 5 ms reveals also an additional feature with the pronounced sharp band having maximum at 550 nm. This band is associated with a transient T1 - Tn band of okenone. The T S profile of okenone can be simply restored if a 100 ns spectrum (T S spectrum of BChl a only) is subtracted. This is shown as ‘‘difference’’ (blue) in Fig. 7a. A similar reconstruction was done for the T S spectra of the BChl a–chlorobactene mixture and is given in Fig. 7b. The maximum of the T1 - Tn band of chlorobactene appears at 515 nm and demonstrates that relative positioning of the T1 - Tn bands for these two carotenoids follows the same rule as observed for the S1 (21Ag) Sn transient bands. The fact that the triplet state of the carotenoid is not detected at early delay times means that quenching of the BChl a triplet via sensitizing carotenoid molecules is not an instantaneous process. This is also apparent when kinetic traces of the rise and decay of the triplet state of BChl a and the carotenoids are displayed (Fig. 7c and d). BChl a triplets are populated within the IRF (B6 ns) via an intersystem crossing process and the rise of the signal is instrument response limited (see 640 nm ‘‘green’’ line). However, this is not the case for the triplet state of the carotenoids (black line, Fig. 7c and d). A slow rise of the T1 - Tn carotenoid signal is clearly visible on the top of the instantaneously populated T1 - Tn signal of 3BChl a. This is due to the fact the speed of triplet sensitization will vary with the concentrations of interacting compounds. In order to obtain the kinetic trace of the pure T1 - Tn signal of the carotenoids, the contribution from the T1 - Tn signal of BChl a was subtracted. The final BChl a-free trace is shown in Fig. 7c and d as a red line. To prevent introducing more noise into the results, a monoexponential fit of the raw BChl a T1 - Tn signal trace (blue) was used in the subtraction. These final traces (Fig. 7e and f) were further used to estimate the triplet state lifetimes of both carotenoids. Because carotenoid triplets are not instantly populated, fitting the curves according to a sum of exponential decays will


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instantly populated within instrument response time it can be assumed that:


[3BChl] = [3BChl0]exp(kobst)

(3)

Where 3BChl0 is the initial pool of BChl a triplets and kobs = kint + k 0 , where kint is a decay rate of [3BChl] in absence of carotenoid. Then eqn (2) has the following solution: 3

Car

3

¼

BChl0 ðkobs kint Þ 1 kobs kTCar expðkTCar tÞ

(4)

ð1 expððkTCar kobs Þ tÞÞ That can be applied to fit the triplet dynamics of the carotenoids. The fitting results are provided in Fig. 7e and f as black lines. For fitting, one of the parameters, kint was kept fixed at a value of (0.012) (ms1). Previous triplet studies of various (B)Chls showed that the intrinsic decay rate of the triplet of BChl a at this specific concentration should be B1/80 ms1.35 The best agreement of the fit with the raw data was obtained for kobs = (0.028 0.001) (ms1), corresponding to tobs = (35 2) ms. The carotenoid triplet lifetimes resulting from the fitting are (2.1 0.1) ms for okenone and (2.8 0.1) ms for chlorobactene.

Discussion Solvent and temperature effect on lifetimes and energies of the S1 (21Ag) state

Fig. 7 Triplet studies of okenone and chlorobactene. (a, b) Triplet-minussinglet (T S) spectra of BChl a–carotenoid mixture taken at 100 ns (black) and 5 ms (red) after excitation at 355 nm in degassed n-hexane at RT. The blue line represent spectrum corrected for BChl a overlap (c, d) (green) instantaneous population and decay of the BChl a triplets recorded at 640 nm, (black) slow rise and following decay of the carotenoid triplets recorded at maximum of the T1 - Tn band substantially overlapping with signal from BChl a, (red) rise and decay of carotenoid triplet corrected for BChl a fraction; (e, f) fits done according to directed kinetic model from eqn (4). The triplet state lifetimes and uncertainties provided in the figures are obtained from the fitting procedure.

lead to highly overestimated values. In this case, a more sophisticated kinetic modelling has to be applied. Carotenoid triplets (3Car) are products of following second-order reaction that occurs with rate k: 3BChl + 1Car - 3Car. Upon assumption that triplets will later decay with constant kTCar, evolution of a carotenoid triplet population can be described by following derivative equation: d 3 Car ¼ k 1 Car 3 BChl kTCar 3 Car dt

(2)

The T S spectra of carotenoids (Fig. 7a and b, blue lines) show that only B3% of dissolved carotenoid molecules are sensitized to triplet state. Thus it is safe to assume that [1Car] (molecules in the ground state) is a constant number. Then, the triplet population process simplifies to pseudo-first order reaction in which k[1Car] reduces to a new k 0 . Because 3BChl is


Although, an impact of increased solvent polarity even in hydrogen bonding solvent as MeOH on the shape of the ground state absorption of okenone is clearly noticeable, transient absorption results suggest that keto-group directly attached to the molecular backbone of molecule conjugation does not influence the S1 (21Ag) state lifetime thus an ICT state is not formed. The energy of the optically silent S1 (21Ag) electronic state that is determined for okenone to be B12 700 cm1 and chlorobactene to 13 450 cm1 does not fluctuate upon change of molecular environment, neither solvent polarity or temperature. Such large values of the S1 (21Ag) state energies suggest that both carotenoids upon relaxation into the S1 (21Ag) state adapt geometries that have the effective double bonds conjugation (Neff) much shorter than nominal N = 15. The Neff will be B11 for okenone and B10 for chlorobactene. Effect of the terminal rings on the excited state energies Fig. 8 demonstrates the S1 (21Ag) state energy plotted as a function of the S2 (11Bu+) state energy for various types of carotenoids: open chain and with one and two terminal rings. All the energies were obtained in 2-MTHF at 77 K, okenone as an asymmetric keto-carotenoid was not included in the fitting but it is placed in the graph. It is apparent that both states have a mutual relationship that could be very precisely approximated by a linear function. Moreover, it is striking that open chain carotenoids follow a completely different rule that those with terminal ring(s). Such partitioning of the carotenoids into two distinct pools suggests that molecules that seem to be very similar due to presumably identical conjugation (like violaxanthin and neurosporene) are actually


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Conclusions

Fig. 8 The energy of the S1 (21Ag) electronic state plotted as s function of energy of S2 (11Bu+) state (0–0 vibronic band) for various carotenoids obtained at 77 K in 2-MTHF. The carotenoids clearly split into two subgroups: open-chain and ring-ended. The relationship between energies of the electronic states can be very adequately mimicked by liner functions with formulas given in the graph. Data of violaxanthin (Viol), lutein (Lut), zeaxanthin (Zea) and b-carotene (bcar) are taken from ref. 53; neurosporene (Neu), spheroidene (Sph) and spirilloxanthin (Spx) are taken from ref. 40; anhydrorhodovibrin from ref. 52. Data of chlorobactene (Clbn) and okenone (Oke) are from this work.

not really complementary. It may have a direct impact in many comparative studies in which open chain and ring-ended carotenoids are put together and studied simultaneously. Generalized conclusions coming out from these studies could be actually oversimplified. The pragmatic aspect of the observed relationship between electronic states is that an unknown energy of the ‘‘dark’’ S1 (21Ag) state can be simply and accurately estimated on the arithmetic formula even if only the steady-state absorption spectrum is measured. The formulas given in the graph are only adequate for 77 K measurements and for non-keto carotenoids, although it is probable that a similar correlation can be established at RT in selected solvents. Triplet state of chlorobactene and okenone Microsecond TA studies demonstrated that both carotenoids efficiently quench BChl a triplets. The triplet lifetimes of 2.1 ms (okenone) and 2.8 ms (chlorobactene) seem to be quite reasonable. For comparative purposes, triplet state lifetimes of open chain carotenoids with similar effective conjugation lengths can be used. Spirilloxanthin, with conjugation N = 13, bound into light harvesting complex 1 (LH1), has triplet lifetime of 2.9 ms.59 Another open chain carotenoid, anhydrorhodovibrin (N = 12) (also in LH1) shows triplet lifetime of 3.3 ms.59 Spheroidene (N = 10) bound into LH2 has triplet lifetime of 6.2 ms.59 It cannot be ruled out that triplet lifetime values obtained in the solvent dissolved mixtures are slightly underestimated due to effect of concentration self-quenching (even though the carotenoid concentrations in these experiments are low) as was demonstrated by Burke et al.60


We have demonstrated that the ICT state is not formed in okenone in polar, hydrogen bonding solvent. Values of experimentally defined energy of the forbidden S1 (21Ag) state of B12 700 cm1 for okenone and 13 450 cm1 for chlorobactene suggest that in the S1 (21Ag) state both carotenoids adapt geometries with effective conjugations considerably smaller that nominal N. Moreover, comparative analysis of S1 (21Ag) and S2 (11Bu+) state energies of these aryl carotenoids with other carotenoids of various types revealed a mutual relationship between them that can be approximated by linear function. In addition those relations are specific and are different for carotenoids with open chain end closed-ring ends. Both aryl carotenoids quench BChls triplets as we have demonstrated it in the model pigment mixtures. Easiness of triplet state sensitization and relative short triplet lifetime of both aryl carotenoids guarantee their effectiveness of photoprotective capabilities in light harvesting complexes in which they naturally occur.

Acknowledgements The spectroscopic research conducted by DNM was supported by the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC 0001035 to Prof. Robert E. Blankenship. The purple bacteria growth and the LH2 isolation performed by LC were funded by Biotechnology and Biological Sciences Research Council (BBSRC). The authors would like to thank to Prof. Robert E. Blankenship from Washington University in St Louis for helpful discussion and to Greg Orf for help with collecting chlorobactene.

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Potential energy curves and lifetimes of low-lying excited electronic states of CSe studied by configuration interaction method.

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Ultrafast excited-state dynamics of 2,4-dimethylpyrrole.