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Carotenoid fluorescence
Volume 158, number
CHEMICAL
3,4
CAROTENOID
PHYSICS LETTERS
9 June 1989
FLUORESCENCE
Tomas GILLBRO and Richard J. COGDELL Deparrmentof PhysicalChemistry,Umed University, S-901 87 Urned,Sweden and DepartmentofBotany, Universrry of Glasgow,GlasgowGl2 8QQ.Sc~otland, IJK Received
10 January
19S9; in final form 18 April 1989
Fluorescence excitation and emission spectra arc reported from the carotenoids P-carotene, rhodopin and spheroidenone in carbon disulfide. From the small Stokes shift and the high emission anisotropy it is concluded that the fluorescence is emitted durmg the 16,-r lA, transition. From the small quantum yields, of about 3 X IO-’ for spheroidenone and 6 X IO-’ for p-carotene, and the 15ps ground-state recovery lifetime for spheroidenone combined with a natural radiative lifetime of I-10 ns, it is concluded that a fast (30-300 fs) non-radiative relaxation occurs. This probably represents the transition from the IB, to a low-lying 2A, state. Anomalities in the excitation spectra also indicate that this process competes efficiently with vibrational relaxation within the lB, state.
1. Introduction
2. Experimental
Carotenoids have two important functions in the photosynthetic apparatus of bacteria, algae and green plants. One role is as an accessory light-harvesting pigment transferring its excitation energy to chlorophyll or bacteriochlorophyll [ 11. The second function is to protect the photosynthetic organism from harmful photooxidation reactions [ 2 1. In order to increase our understanding of the function of carotenoids in photosynthesis it is of prime interest to study the basic photophysics of these naturally occurring polyenes. This has so far been hampered by the low fluorescence quantum yield of the carotenoids investigated [ 3,4]. In this work we report for the first time fluorescence emission and excitation spectra of three carotenoids, namely p-carotene, spheroidenone and rhodopin in the solvent carbon disulfide. We also report quantum yields, fluorescence anisotropy and picosecond absorption-recovery measurements of some of the carotenoids. The data show that the allowed lB, state is responsible for the weak fluorescence and that there is a fast femtosecond reraxation to a low energy state, probably, the 2A, state, which decays to the ground state on a 10 ps timescale.
The bacterial carotenoids were isolated and purified from species or strains in which they were the major carotenoid component; rhodopin from Chromatium vinosum strain D and spheroidenone from Rhodobacter sphaeroides strain 2.4.1 which was grown photosynthetically and then vigorously aercated (this converts spheroidene to spheroidenone), The carotenoids were extracted from the cells into acetone. The extract was rotary evaporated to dryness, taken up in a little 40-60” b-p. petroleum ether and passed through an activated alumina column. The carotenoids were eluted with diethyl ether/ petroleum ether mixtures and their purity was confirmed spectrophotometrically and by TLC on- silica gel plates [ 51. Throughout this procedure care was taken to keep the extracts, as far as possible, in the dark. p-carotene (all-trans) was obtained from Sigma. Two qualities were used, but they both gave the same result. Freshly prepared solutions in CS, (Fischer Spectral grade) were used in all measurements and precautions were taken in some experiments not to expose the samples to light and oxygen. However, the presence of oxygen did not influence the fluorescence observed in this work. The intensity of the
312
Volume 158, number 3,4
CHEMICAL
PHYSICS LETTERS
fluorescence remained constant even in samples used for several hours. The fluorescence and fluorescence excitation spectra were obtained with a Spex Fluorolog 112 equipped with a cooled Hamamatsu P 928 photomultiplier. The absorbance of the sample was about 0.15 at the excitation wavelength used. The slit widths of the excitation and emission monochromators were set at 7 or 14 nm. Variation in the excitation light intensity was corrected for by means of a rhodamine 10 1 quantum counter. The fluorescence was collected at right angles to the excitation beam. No correction of the fluorescence spectra for photomultiplier sensitivity was made in this work. The fluorescence anisotropy, r, was calculated according to
All measurements ture (23-25°C).
9 June 1989
were made at room tempera-
3. Results In figs. lA-1C fluorescence emission, excitation, and absorption spectra of the carotenoids p-carotene, rhodopin and spheroidenone in carbon disulphide are shown. There is good agreement between the position of the peak maxima in the absorption spectrum and the corresponding peaks in the excitation spectrum. There is also a considerable overlap between the corresponding fluorescence and excitation spectra. We calculated the fluorescence quan-
F\,- F,
r=F,, +2FI
’
where F,, and F, denote fluorescence polarized parallel and perpendicular to the excitation polarization, respectively. Correction to F,, and F, was made for the apparatus sensitivity [ 61. The excitation anisotropy was calculated in an equivalent way. Fluorescence yield measurements were made with rhodamine 6G in methanol with a quantum yield of 0.95 as a standard [ 71. In the quantum yield measurements as well as in the fluorescence and excitation spectra presented in fig. 1 we have subtracted the background signal from a pure solution of carbon disulfide. In the anisotropy measurements this correction was not made. The main reason for choosing carbon disulfide as a solvent was that other solvents such as petroleum ether, acetone, toluene and chloroform all gave interfering Raman lines z 3000 cm- ’ from the excitation wavelength. The picosecond absorption-recovery measurements were performed with the pump-probe technique [ 81. A train of 8 ps long pulses from a cavity-dumped dye laser (laser dye rhodamine 110) were divided by a beam splitter. The most intense part (90%) of the pulse was used to excite the sample, while the weak reflected part (10%) was used to monitor the absorbance change in the sample. The time resolution was obtained by changing the optical pathlength of the analyzing light relative to that of the excitation light. Absorption spectra of the purified carotenoids were recorded on a Beckman DW 70 spectrophotometer.
Fig. I. Absorption (---), fluorescence excitation (-) and emission (-) spectra of [A) p-carotene, (B) rhodopin and (C) spheroidenone in carbon disulfide. The excitation and emission wavelengths are indicated in the figure.
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Volume 158,number 3,4
9 June 1989
CHEMICALPHYSICSLETTERS
turn yields for p-carotene and spheroidenone in carbon disulfide to be about 6 x 10m5 and 3x IO-‘, respectively. In figs. 2 and 3 the fluorescence excitation and emission anisotropies of p-carotene and spheroidenone are shown. For both compounds the anisotropy was found to be high (r=0.35-0.36) over the main absorption and emission bands, while it drops at shorter excitation wavelengths, e.g. at 400 nm r= 0.16 for p-carotene. This indicates that there are two states with different polarization in the main absorption band of p-carotene. For the sake of completeness we also show the absorption recovery of spheroidcnonc in CS, at 555 nm in fig. 4. The recovery follows a single exponential with a lifetime of 15 ps. Similar measurements on the other carotenoids at the available wavelengths above 535 nm normally gave a very
I 0
I
I
25
50
75
I
I
100
125
TlME,p*
Fig. 4. Picosecond absorption recovery kinetics for spheroidenone in carbon disulfide at 555 nm.
weak signal, probably due to excited state absorption.
4. Discussion
1
001 400
435
470
Fig. 2. Fluorescence excitation spectrum and amsotropy otene in carbon &sulfide. Emission at 570 nm.
00
1 550
ofa-car-
I
635
Fig. 3. Fluorescence emission spectrum and anisotropy oidenone in carbon disultide. Excitation at 480 nm.
314
640
505
720
of spher-
From the spectra in fig. 1 there is no doubt that we have observed the fluorescence of several carotenoids. The possibility that we have observed the fluorescence of an impurity instead is excluded for two reasons. Firstly, as can be seen in fig. 1 the excitation spectra show great similarity with the corresponding absorption spectra. The occurrence of impurities with absorption spectra shifting in exactly the same manner as for the carotenoids is highly unlikely. Secondly, the carotenoids were prepared from different starting materials and the purity checked by chromatography. The occurrence of similar impurities in different preparations also seems highly unlikely. The origin of the carotenoid emission is believed to be from the first cxcitcd singlet IB, state, which is populated directly by light absorption by the allowed transition 1B,+- l$. The main reason for this interpretation is the small Stokes shift between the highest energy vibronic transition of the ground state and the lowest energy vibronic transition of the excited state. This shift is of the order of a few hundred cm-’ as can be seen clearly in figs. lb and lc. This is less than the observed linewidths in the absorption spectra.
CHEMICAL PHYSICS LETTERS
Volume 158, number 3,4
If the origin of the emission had been from the forbidden singlet 2A, state, situated below the allowed 1B, state, we would have expected a large Stokes shift similar to that which has been observed in several shorter polyenes [ 9 1. The quantum yield of the fluorescence in CS, is low, i.e. 6x 10P5 for p-carotene and 3 x 10P5 for spheroidenone:
this indicates
that non-radiative
pro-
cesses from
lB, are very fast and compete efficiently with the radiative process. The latter has been calculated to have a lifetime of l-10 ns from the p-carotene absorption spectrum [ 3,4]. Since the extinction coefficient at the absorption maximum as well as the vibrational structure of the absorption spectrum is similar for the bacterial carotenoids used in the present study, i.e. e(max) x 1.5x 10’ M-’ cm-’ [lo], we can safely assume that their natural radiative lifetime, ro, is in the range 1O-8-1O-9 s. Forexamplewithzo=10-9sand@P,=3x10-5we obtain a non-radiative lifetime of 3 X lo-l4 s for the spheroidenone 1B, state, while the measured ground state recovery lifetime, 7g, is 1.5 x 1O- ’ ’ s in the same solvent. Clearly we have to consider a third state in our relaxation scheme (fig. 5 ) in order to explain this large difference. It is now generally accepted that polyenes with more than two conjugated double bonds exhibit a forbidden excited state 2A,, whose energy is lower that the allowed lB, state 191. It
’ B”
w Tnr
2 *g
Fig. 5. Schematic energy level diagram for carotenoids. Here 1A, is the ground state and IB, the allowed excited state. 2A, is a forbidden excited electronic state populated by non-radiative relaxation from the 1B. state. In the figure different radiative and non-radiative processes are indicated.
9 June 198Y
therefore seems likely that the low fluorescence quantum yield is caused by a fast non-radiative relaxation 1B,-,2A,, whose lifetime, r,,, is of the order of 30-300 fs. The lifetime of the 2A, state then determines the rate of ground state recovery, which is about 10 ps for the carotenoids studied thus far [ 4 1. The high anisotropy (r=0.35-0.36) for both excitation and emission of the main transition shows that the emitting state has a polarization close to that of the allowed lA,ulB, transition. It is unlikely that the deviation of the anisotropy from the theoretical optimum value of 0.40 is due to rotation of the carotenoid in CS2 since this is expected to be about two order of magnitude slower than the calculated lifetime of the excited 1B, state [ 111. A possible explanation is that the direction of the excited state transition is changed due to interaction with solventinduced dipoles. From the anisotropy measurements we cannot exclude the possibility that 1A,contributes to the emission, The reason for this is that this transition is forbidden and is induced by intensity borrowing from the strongly allowed lB,-tlA, transition and thus the 2A,-+ lA, transition should have the same polarization as the allowed transition [ 91. As can be clearly seen in fig. 2 there is a decrease of the P-carotene excitation anisotropy at shorter wavelengths. At about 420 nm there is a plateau with an anisotropy of about 0.16. This indicates the presence of a higher excited state with a polarization differing from the 1A,-1 lB, transition. A similar drop in the linear dichroism has been observed for p-carotene in lipid bilaycrs [ 121. There are in our opinion two main reasons why carotenoid fluorescence has escaped detection by conventional spectrofluorometry. These are both due to the choice of solvent. Firstly, we have noted that solvents containing C-H bonds, for instance toluene, acetone or ethanol, all give rise to Raman scattering of the same order of magnitude as the fluorescence. These peaks will occur at N 3000 cm-’ from the excitation wavelength. No such interfering Raman scattering was observed for CSZ over the wavelength interval studied in this work. Secondly, we have observed (to be published) that there is a redshift of the carotenoid absorption maxima with increasing solvent polarizability. This is in accordance with earlier findings for other polyenes [ 13,141. Since 315
Volume 158. number
3,4
CHEMICAL
PHYSICS LETTERS
according to the current theory [ 151 the energy shift of the allowed transition should be larger than that of the forbidden lA,-+2A, transition, one might expect that the coupling between the lB,, and 2A, levels should change (see fig. 5 ) . This would lead to a different rate for the non-radiative relaxation from lB,. We believe that this mechanism might be the reason why B-carotene has a quantum yield of < 10s5 in, e.g., toluene and ethanol [ 3,4] while it is 6x 10m5 in CS,. In the carotenoids studied by us (see fig. 1) the long wavelength (lowest) vibronic transition exhibits higher intensity in the fluorescence excitation spectrum than in the absorption spectrum. A possible explanation for this effect is that the rate of transfer from 1B, to 2A, is of the same order of magnitude as the vibrational relaxation within the lB, state (see fig. 5 ). This seems to be a reasonable assumption since we have calculated the transfer rate from the quantum yield of fluorescence to be 30-300 fs for spheroidenone and 60-600 fs for B-carotene. This should indeed be fast enough to compete with vibrational relaxation, which has been found to be about 80 fs for some dye molecules in liquid solution [ 161. It should also be mentioned that the laser excitation profile of B-carotene fluorescence was shown recently to exhibit similar deviations from the absorption spectrum as found in this work [ 171, We believe that the same explanation holds here as well. Finally, our results provide further support for previous suggestions [ 18-2 1 ] that the energy transfer from excited carotenoids to chlorophylls actually proceeds through a forbidden state below the lB, state. The very short lifetime of the lB, state seems to exclude the Fijrster mechanism [22] as a candidate for efficient energy transfer from carotenoids in some light-harvesting antennae. The most likely mechanism seems to be a short range electron exchange interaction as suggested by Dexter [ 231.
Acknowledgement We are grateful to the Swedish Natural Science Research Council (TG) and the Science and Engi-
316
9 June 1989
neering Research Council (RJC) for financial support. RJC also thanks the British Council for a travel grant which allowed him to visit Sweden. Ms. Linda Ferguson provided expert technical assistance.
References [ 1 ] D. Siefermans-Harms, Biochim. Biophys. Acta 8 I I ( 1985) 325. [2] NJ. Krinsky, Pure Appl. Chcm. 51 (1979) 649. [3] P.S. Song and T.A. Moore, Photochcm. Photobiol. 19 (1974) 435. [4] M.R. Wasielewski and L.D. I&pert, Chem. Phys. Letters 128 (1986) 238. [5] R.J. Cogdell, R.J. Land and T.G. Truscott, Photochem. Photobiol. 38 ( 1983) 723. J.R. Lakowtcz, Principles of fluorescence spectroscopy (Plenum Press, New York, 1983). R.F. Kubin and A.N. Fletcher, J. Luminescence 27 (I 982) 455.
1E. Akesson, V. Sundstrom
and T. Gillbro, Chem. Phys. Letters 121 (1985) 513. B.S. Hudson, B.E. Kohler and K. Schulten, in: Excited states, Vol. 6, ed. E.C. Lim (Academic Press, New York, 1982). [ 10 1B.H. Davies, in: Chemistry and biochemistry of plant pigments, ed. T. W. Goodwin (Academic Press, New York, 1965). [ II] D.H. Waldeck, W.T. Lotshaw, D.B. McDonald and G.R. Fleming, Chem. Phys. Letters 88 (1982) 297. [I* 8. Norden, G. Lindhlom and I. Jonas, J. Phys. Chem. 81 (1977) 2086. 113 L.A. Sklar, B. Hudson, M. Peterson and J. Diamond, Biochemistry 16 (1977) 813. ]l4 K.L. D’Amico, C. Manos and R.L. Christensen, J. Am. Chem. Sot. 102 (1980) 1777. ]l5 S. Basu, Advan. Quantum Chem. 7 ( 1964) 289. [l6 C.H. Brito Cruz, R.L. Fork, W.H. Knox and C.V. Shank, Chem. Phys. Letters 132 (1986) 341. ]l7 M. van Riel, J. Kleinen Hammahs, M. van de Vcn, W. Verwer and Y.K. Levine, Biochem. Brophys. Rev. Commun. II3 (1983) 102. [I* K. Razi Naqvi, Photochem. Photobiol. 3 I (1980) 523. ]J9 M.R. Wasrelewski, P.A. Liddell, D. Barrett, ?‘.A. Moore and D. Gust, Nature 322 (1986) 570. [ 20 1M.R. Wasielewski, D.M. Tiede and H.A. Frank, in: Ultrafast phenomena, Vol. 5, eds. G.R. Fleming and A.E. Siegman (Springer, Berlin, 1986) p. 388. [ 2 I 1T. Gillbro, R.J. Cogdell and V. Sundstrdm, FEBS Letters 235 (1988) 169. [ 221 T. Forster, Discussions Faraday Sot. 27 ( 1965) 7. [23] D.L. Dexter, J. Chem. Phys. 21 (1953) 836.
CHEMICAL
3,4
CAROTENOID
PHYSICS LETTERS
9 June 1989
FLUORESCENCE
Tomas GILLBRO and Richard J. COGDELL Deparrmentof PhysicalChemistry,Umed University, S-901 87 Urned,Sweden and DepartmentofBotany, Universrry of Glasgow,GlasgowGl2 8QQ.Sc~otland, IJK Received
10 January
19S9; in final form 18 April 1989
Fluorescence excitation and emission spectra arc reported from the carotenoids P-carotene, rhodopin and spheroidenone in carbon disulfide. From the small Stokes shift and the high emission anisotropy it is concluded that the fluorescence is emitted durmg the 16,-r lA, transition. From the small quantum yields, of about 3 X IO-’ for spheroidenone and 6 X IO-’ for p-carotene, and the 15ps ground-state recovery lifetime for spheroidenone combined with a natural radiative lifetime of I-10 ns, it is concluded that a fast (30-300 fs) non-radiative relaxation occurs. This probably represents the transition from the IB, to a low-lying 2A, state. Anomalities in the excitation spectra also indicate that this process competes efficiently with vibrational relaxation within the lB, state.
1. Introduction
2. Experimental
Carotenoids have two important functions in the photosynthetic apparatus of bacteria, algae and green plants. One role is as an accessory light-harvesting pigment transferring its excitation energy to chlorophyll or bacteriochlorophyll [ 11. The second function is to protect the photosynthetic organism from harmful photooxidation reactions [ 2 1. In order to increase our understanding of the function of carotenoids in photosynthesis it is of prime interest to study the basic photophysics of these naturally occurring polyenes. This has so far been hampered by the low fluorescence quantum yield of the carotenoids investigated [ 3,4]. In this work we report for the first time fluorescence emission and excitation spectra of three carotenoids, namely p-carotene, spheroidenone and rhodopin in the solvent carbon disulfide. We also report quantum yields, fluorescence anisotropy and picosecond absorption-recovery measurements of some of the carotenoids. The data show that the allowed lB, state is responsible for the weak fluorescence and that there is a fast femtosecond reraxation to a low energy state, probably, the 2A, state, which decays to the ground state on a 10 ps timescale.
The bacterial carotenoids were isolated and purified from species or strains in which they were the major carotenoid component; rhodopin from Chromatium vinosum strain D and spheroidenone from Rhodobacter sphaeroides strain 2.4.1 which was grown photosynthetically and then vigorously aercated (this converts spheroidene to spheroidenone), The carotenoids were extracted from the cells into acetone. The extract was rotary evaporated to dryness, taken up in a little 40-60” b-p. petroleum ether and passed through an activated alumina column. The carotenoids were eluted with diethyl ether/ petroleum ether mixtures and their purity was confirmed spectrophotometrically and by TLC on- silica gel plates [ 51. Throughout this procedure care was taken to keep the extracts, as far as possible, in the dark. p-carotene (all-trans) was obtained from Sigma. Two qualities were used, but they both gave the same result. Freshly prepared solutions in CS, (Fischer Spectral grade) were used in all measurements and precautions were taken in some experiments not to expose the samples to light and oxygen. However, the presence of oxygen did not influence the fluorescence observed in this work. The intensity of the
312
Volume 158, number 3,4
CHEMICAL
PHYSICS LETTERS
fluorescence remained constant even in samples used for several hours. The fluorescence and fluorescence excitation spectra were obtained with a Spex Fluorolog 112 equipped with a cooled Hamamatsu P 928 photomultiplier. The absorbance of the sample was about 0.15 at the excitation wavelength used. The slit widths of the excitation and emission monochromators were set at 7 or 14 nm. Variation in the excitation light intensity was corrected for by means of a rhodamine 10 1 quantum counter. The fluorescence was collected at right angles to the excitation beam. No correction of the fluorescence spectra for photomultiplier sensitivity was made in this work. The fluorescence anisotropy, r, was calculated according to
All measurements ture (23-25°C).
9 June 1989
were made at room tempera-
3. Results In figs. lA-1C fluorescence emission, excitation, and absorption spectra of the carotenoids p-carotene, rhodopin and spheroidenone in carbon disulphide are shown. There is good agreement between the position of the peak maxima in the absorption spectrum and the corresponding peaks in the excitation spectrum. There is also a considerable overlap between the corresponding fluorescence and excitation spectra. We calculated the fluorescence quan-
F\,- F,
r=F,, +2FI
’
where F,, and F, denote fluorescence polarized parallel and perpendicular to the excitation polarization, respectively. Correction to F,, and F, was made for the apparatus sensitivity [ 61. The excitation anisotropy was calculated in an equivalent way. Fluorescence yield measurements were made with rhodamine 6G in methanol with a quantum yield of 0.95 as a standard [ 71. In the quantum yield measurements as well as in the fluorescence and excitation spectra presented in fig. 1 we have subtracted the background signal from a pure solution of carbon disulfide. In the anisotropy measurements this correction was not made. The main reason for choosing carbon disulfide as a solvent was that other solvents such as petroleum ether, acetone, toluene and chloroform all gave interfering Raman lines z 3000 cm- ’ from the excitation wavelength. The picosecond absorption-recovery measurements were performed with the pump-probe technique [ 81. A train of 8 ps long pulses from a cavity-dumped dye laser (laser dye rhodamine 110) were divided by a beam splitter. The most intense part (90%) of the pulse was used to excite the sample, while the weak reflected part (10%) was used to monitor the absorbance change in the sample. The time resolution was obtained by changing the optical pathlength of the analyzing light relative to that of the excitation light. Absorption spectra of the purified carotenoids were recorded on a Beckman DW 70 spectrophotometer.
Fig. I. Absorption (---), fluorescence excitation (-) and emission (-) spectra of [A) p-carotene, (B) rhodopin and (C) spheroidenone in carbon disulfide. The excitation and emission wavelengths are indicated in the figure.
313
Volume 158,number 3,4
9 June 1989
CHEMICALPHYSICSLETTERS
turn yields for p-carotene and spheroidenone in carbon disulfide to be about 6 x 10m5 and 3x IO-‘, respectively. In figs. 2 and 3 the fluorescence excitation and emission anisotropies of p-carotene and spheroidenone are shown. For both compounds the anisotropy was found to be high (r=0.35-0.36) over the main absorption and emission bands, while it drops at shorter excitation wavelengths, e.g. at 400 nm r= 0.16 for p-carotene. This indicates that there are two states with different polarization in the main absorption band of p-carotene. For the sake of completeness we also show the absorption recovery of spheroidcnonc in CS, at 555 nm in fig. 4. The recovery follows a single exponential with a lifetime of 15 ps. Similar measurements on the other carotenoids at the available wavelengths above 535 nm normally gave a very
I 0
I
I
25
50
75
I
I
100
125
TlME,p*
Fig. 4. Picosecond absorption recovery kinetics for spheroidenone in carbon disulfide at 555 nm.
weak signal, probably due to excited state absorption.
4. Discussion
1
001 400
435
470
Fig. 2. Fluorescence excitation spectrum and amsotropy otene in carbon &sulfide. Emission at 570 nm.
00
1 550
ofa-car-
I
635
Fig. 3. Fluorescence emission spectrum and anisotropy oidenone in carbon disultide. Excitation at 480 nm.
314
640
505
720
of spher-
From the spectra in fig. 1 there is no doubt that we have observed the fluorescence of several carotenoids. The possibility that we have observed the fluorescence of an impurity instead is excluded for two reasons. Firstly, as can be seen in fig. 1 the excitation spectra show great similarity with the corresponding absorption spectra. The occurrence of impurities with absorption spectra shifting in exactly the same manner as for the carotenoids is highly unlikely. Secondly, the carotenoids were prepared from different starting materials and the purity checked by chromatography. The occurrence of similar impurities in different preparations also seems highly unlikely. The origin of the carotenoid emission is believed to be from the first cxcitcd singlet IB, state, which is populated directly by light absorption by the allowed transition 1B,+- l$. The main reason for this interpretation is the small Stokes shift between the highest energy vibronic transition of the ground state and the lowest energy vibronic transition of the excited state. This shift is of the order of a few hundred cm-’ as can be seen clearly in figs. lb and lc. This is less than the observed linewidths in the absorption spectra.
CHEMICAL PHYSICS LETTERS
Volume 158, number 3,4
If the origin of the emission had been from the forbidden singlet 2A, state, situated below the allowed 1B, state, we would have expected a large Stokes shift similar to that which has been observed in several shorter polyenes [ 9 1. The quantum yield of the fluorescence in CS, is low, i.e. 6x 10P5 for p-carotene and 3 x 10P5 for spheroidenone:
this indicates
that non-radiative
pro-
cesses from
lB, are very fast and compete efficiently with the radiative process. The latter has been calculated to have a lifetime of l-10 ns from the p-carotene absorption spectrum [ 3,4]. Since the extinction coefficient at the absorption maximum as well as the vibrational structure of the absorption spectrum is similar for the bacterial carotenoids used in the present study, i.e. e(max) x 1.5x 10’ M-’ cm-’ [lo], we can safely assume that their natural radiative lifetime, ro, is in the range 1O-8-1O-9 s. Forexamplewithzo=10-9sand@P,=3x10-5we obtain a non-radiative lifetime of 3 X lo-l4 s for the spheroidenone 1B, state, while the measured ground state recovery lifetime, 7g, is 1.5 x 1O- ’ ’ s in the same solvent. Clearly we have to consider a third state in our relaxation scheme (fig. 5 ) in order to explain this large difference. It is now generally accepted that polyenes with more than two conjugated double bonds exhibit a forbidden excited state 2A,, whose energy is lower that the allowed lB, state 191. It
’ B”
w Tnr
2 *g
Fig. 5. Schematic energy level diagram for carotenoids. Here 1A, is the ground state and IB, the allowed excited state. 2A, is a forbidden excited electronic state populated by non-radiative relaxation from the 1B. state. In the figure different radiative and non-radiative processes are indicated.
9 June 198Y
therefore seems likely that the low fluorescence quantum yield is caused by a fast non-radiative relaxation 1B,-,2A,, whose lifetime, r,,, is of the order of 30-300 fs. The lifetime of the 2A, state then determines the rate of ground state recovery, which is about 10 ps for the carotenoids studied thus far [ 4 1. The high anisotropy (r=0.35-0.36) for both excitation and emission of the main transition shows that the emitting state has a polarization close to that of the allowed lA,ulB, transition. It is unlikely that the deviation of the anisotropy from the theoretical optimum value of 0.40 is due to rotation of the carotenoid in CS2 since this is expected to be about two order of magnitude slower than the calculated lifetime of the excited 1B, state [ 111. A possible explanation is that the direction of the excited state transition is changed due to interaction with solventinduced dipoles. From the anisotropy measurements we cannot exclude the possibility that 1A,contributes to the emission, The reason for this is that this transition is forbidden and is induced by intensity borrowing from the strongly allowed lB,-tlA, transition and thus the 2A,-+ lA, transition should have the same polarization as the allowed transition [ 91. As can be clearly seen in fig. 2 there is a decrease of the P-carotene excitation anisotropy at shorter wavelengths. At about 420 nm there is a plateau with an anisotropy of about 0.16. This indicates the presence of a higher excited state with a polarization differing from the 1A,-1 lB, transition. A similar drop in the linear dichroism has been observed for p-carotene in lipid bilaycrs [ 121. There are in our opinion two main reasons why carotenoid fluorescence has escaped detection by conventional spectrofluorometry. These are both due to the choice of solvent. Firstly, we have noted that solvents containing C-H bonds, for instance toluene, acetone or ethanol, all give rise to Raman scattering of the same order of magnitude as the fluorescence. These peaks will occur at N 3000 cm-’ from the excitation wavelength. No such interfering Raman scattering was observed for CSZ over the wavelength interval studied in this work. Secondly, we have observed (to be published) that there is a redshift of the carotenoid absorption maxima with increasing solvent polarizability. This is in accordance with earlier findings for other polyenes [ 13,141. Since 315
Volume 158. number
3,4
CHEMICAL
PHYSICS LETTERS
according to the current theory [ 151 the energy shift of the allowed transition should be larger than that of the forbidden lA,-+2A, transition, one might expect that the coupling between the lB,, and 2A, levels should change (see fig. 5 ) . This would lead to a different rate for the non-radiative relaxation from lB,. We believe that this mechanism might be the reason why B-carotene has a quantum yield of < 10s5 in, e.g., toluene and ethanol [ 3,4] while it is 6x 10m5 in CS,. In the carotenoids studied by us (see fig. 1) the long wavelength (lowest) vibronic transition exhibits higher intensity in the fluorescence excitation spectrum than in the absorption spectrum. A possible explanation for this effect is that the rate of transfer from 1B, to 2A, is of the same order of magnitude as the vibrational relaxation within the lB, state (see fig. 5 ). This seems to be a reasonable assumption since we have calculated the transfer rate from the quantum yield of fluorescence to be 30-300 fs for spheroidenone and 60-600 fs for B-carotene. This should indeed be fast enough to compete with vibrational relaxation, which has been found to be about 80 fs for some dye molecules in liquid solution [ 161. It should also be mentioned that the laser excitation profile of B-carotene fluorescence was shown recently to exhibit similar deviations from the absorption spectrum as found in this work [ 171, We believe that the same explanation holds here as well. Finally, our results provide further support for previous suggestions [ 18-2 1 ] that the energy transfer from excited carotenoids to chlorophylls actually proceeds through a forbidden state below the lB, state. The very short lifetime of the lB, state seems to exclude the Fijrster mechanism [22] as a candidate for efficient energy transfer from carotenoids in some light-harvesting antennae. The most likely mechanism seems to be a short range electron exchange interaction as suggested by Dexter [ 231.
Acknowledgement We are grateful to the Swedish Natural Science Research Council (TG) and the Science and Engi-
316
9 June 1989
neering Research Council (RJC) for financial support. RJC also thanks the British Council for a travel grant which allowed him to visit Sweden. Ms. Linda Ferguson provided expert technical assistance.
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