- Email: [email protected]
The 2Ag− energies of all-trans-neurosporene and spheroidene as determined by fluorescence spectroscopy
29 May 1998
Chemical Physics Letters 288 Ž1998. 847–853
The 2Ayg energies of all-trans-neurosporene and spheroidene as determined by fluorescence spectroscopy Ritsuko Fujii a , Kengo Onaka b, Michitaka Kuki c , Yasushi Koyama Yasutaka Watanabe b
a,)
,
a
Department of Chemistry, Faculty of Science, Kwansei Gakuin UniÕersity, Uegahara, Nishinomiya 662-8501, Japan Department of Physics, Faculty of Science, Kwansei Gakuin UniÕersity, Uegahara, Nishinomiya 662-8501, Japan Department of Applied Chemistry, Kobe City College of Technology, Gakuen-Higashimachi, Nishi-ku, Kobe 651-2194, Japan b
c
Received 10 February 1998; in final form 24 February 1998
Abstract The fluorescence spectra of neurosporene and spheroidene were recorded in order to determine their 2Ay g levels: each y y y fluorescence spectrum was deconvoluted into two series of 1Bq ™ 1A and 2A ™ 1A vibronic transitions, and the u g g g Ž Õ s 0. state was determined to be 15300 cmy1 in neurosporene and 14200 cmy1 in spheroidene. No energy of the 2Ay g temperature dependence of these energies was seen. q 1998 Elsevier Science B.V. All rights reserved.
1. Introduction All-trans carotenoids play an important function of harvesting light energy in bacterial photosynthetic systems w1–3x. There are two pathways of singlet-energy transfer, i.e. one from the 1Bq state of u carotenoid ŽCar. to the Q x state of bacteriochlorophyll ŽBChl., and the other from the 2Ay g state of Car to the Q y state of BChl. When the conjugated chain of Car has C 2h symmetry, the transition beŽground. states is tween the 1Bq and the 1Ay u g optically allowed, whereas that between the 2Ay g and 1Ay g states is optically forbidden. Furthermore, when the length of the conjugated chain increases, ŽS 1 . fluorescence to the crossover from the 2Ay g
) C o rre s p o n d in g [email protected]
a u th o r.
E -m a il:
ŽS 2 . fluorescence takes place. These situations 1Bq u affect the mechanisms of energy transfer and make it extremely difficult to spectroscopically determine the 2Ay g energy which is one of the key factors in revealing the energy y transfer mechanisms. Recently, it was seen that the 2Ay energies of g carotenoids having a longer conjugated chain Žthe number of conjugated double bonds, n 0 10. have been predicted by the use of the 2Ay g lifetimes and the energy-gap law, the relevant parameters of which were determined by using the energy and the lifetime Ž . of the 2Ay g state for the shorter analogues n ( 9 of b-carotene w4x or spheroidene w5,6x. In order to test the applicability of this method, and also to solve the contradictions described below, it is absolutely necessary to experimentally determine the 2Ay g energy of a carotenoid for which n 0 9. Concerning carotenoids for which n s 9, DeCoster et al. w7x determined the 2Ay g energy of 3,4-dihydrospheroidene Žin methanol. to be 15300
0009-2614r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 8 . 0 0 3 7 6 - 5
848
R. Fujii et al.r Chemical Physics Letters 288 (1998) 847–853
cmy1 by fluorescence spectroscopy, although the relevant peak was not obvious in their fluorescence spectrum shown in Fig. 5 ŽRef. w7x.. On the other hand, Mimuro et al. w8x determined the 2Ay g energy of neurosporene Žin n-hexane. to be 16000–16100 cmy1 Ž623 " 2 nm. by electronic absorption spectroscopy. However, if one assumes that the 2Ay g energy is simply a function of n irrespective of the Car structure, the difference between these values, i.e. 700–800 cmy1 , is somewhat too large for a Stokes shift. Concerning a carotenoid whose n s 10, our group detected a sharp fluorescence peak at 14900 cmy1 in ‘spheroidene’ Žin n-hexane., and ascribed it to the w x 0–0 origin of the 2Ay g state 9 . This proposal was questioned by Harry Frank, because the 2Ay g fluorescence did not exhibit the vibrational structures and the spectral profile was reminiscent of a porphyrin. We actually measured the fluorescence and fluorescence-excitation spectra of 3-acetylchlorophyll a Ža gift from Hugo Scheer. which were very similar to, but not in complete agreement with, those which we claimed from spheroidene. The most difficult problem in our previous results w9x was that the fluorescence-excitation spectrum probed on that particular peak did not exhibit the vibrational structures of the 1Bq u state. On the other hand, Frank and coworkers w6x proposed that the 2Ay g energy of spheroidene is 14200" 50 cmy1 based on its 2Ay g lifetime of 8.7 " 0.1 ps and the energy-gap law mentioned above. In the present investigation, we have attempted to improve the method of purification of neurosporene and spheroidene and the setup for fluorescence measurements in order to detect the extremely weak 2Ay g vibrational structures and to determine their 0–0 origins. The results indicate that we were actually observing fluorescence and fluorescence-excitation from an impurity in the previous investigation w9x, and that the conclusions which were drawn then need to be suspended. 2. Experimental 2.1. Sample preparation Neurosporene was isolated from the cells of Rhodobacter sphaeroides G1C. Methanol was added
to the wet cells, and the mixture was centrifuged to remove water and BChl a which were dissolved in the solution. The carotenoid component was extracted from the residue with acetone and was transferred to the n-hexane layer in fractionation against warm water containing sodium chloride. Then, a 5% waterrmethanol mixture was added to the above n-hexane layer to remove the remaining BChl component. The resultant n-hexane layer was evaporated to dryness, and the residue was dissolved again in n-hexane and then subjected to two series of alumina and silica-gel chromatography; the former chromatography used alumina ŽMerck, Aluminum Oxide 90 activity II–III. as adsorbate and 1 and 3% diethyl ether in n-hexane as eluent in a stepwise gradient elution, whereas the latter used silica-gel ŽMerck, Silica gel 60. as adsorbate and 5% isopropanol in n-hexane as eluent. Neurosporene thus obtained was recrystallized from n-hexane. Spheroidene was isolated from the cells of R. sphaeroides 2.4.1 by the same method except for the following modification. In the extraction procedure, a methanol Ž2.racetone Ž7. mixture was added to the wet cells, and the solution was stirred for 30 min in the dark while bubbling through nitrogen gas. In fluorescence measurements, each sample was dissolved into n-hexane ŽDojin Chemicals, for fluorescence spectroscopy. at a concentration of 6 = 10y6 M. 2.2. Setup for fluorescence and fluorescence-excitation measurements The setup which was described previously w10x was modified as follows. Ž1. Concerning the variable-wavelength light source, the monochromator after the Xe-lamp was replaced by a double monochromator equipped with a non-aberration light-assembly system ŽJUSCO M25D., and the output was focused onto the sample tube with a pair of lens. The 488.0 nm line from a combination of an Arq-laser ŽNEC GLG 3050. and a spectrometer ŽSpectrolab, Laser spec III. was also used as an alternative light source Žwithout focusing.. Ž2. Concerning the fluorescencedetection system, the monochromator was replaced by a single monochromator ŽJUSCO CT25C. equipped with the 1200 linesrmm Ž500 nm blaze. and the 600 linesrmm Ž1000 nm blaze. gratings, and
R. Fujii et al.r Chemical Physics Letters 288 (1998) 847–853
a photomultiplier ŽHamamatsu R632-GA106. was newly introduced as an alternative PM1 for near-infrared measurements. Ž3. Concerning the input power-monitoring system, PM2 was replaced by another photomultiplier ŽHamamatsu R666.. Deconvolution of each fluorescence spectrum was first performed, by assuming Gaussian peaks, for the y district peaks and shoulders in the 1Bq u and the 2A g vibrational structures to determine the position, the intensity and the bandwidth-at-half-maximum of each peak. Then, weak peaks in-between were determined by using, as initial conditions, the average values of the spacings and the band widths obtained in the above analysis. Finally, all the parameters were adjusted again. Based on the assumptions that the spacings and the bandwidths should be similar within each spectrum, the deconvolution provided a unique solution.
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by using a laser beam. Ž2. The sensitivity of fluorescence detection in the near-infrared region Ž850–1100 nm. was increased by introducing a grating of 1000 nm blaze and a near infrared-sensitive photomultiplier. A set of electronic absorption, fluorescence and fluorescence-excitation spectra of neurosporene and spheroidene are shown in Figs. 1 and 2, respectively. The fluorescence spectra of neurosporene were recorded by using the variable-wavelength light
3. Results and discussion 3.1. ImproÕements in sample preparation and in the setup for fluorescence measurements We considered the possibility of three ‘contaminants’ of different origins: Ža. a porphyrin as a degradation product of BChl a, Žb. a shorter polyene as a degradation product of spheroidene, and Žc. an aggregate of spheroidene. In order to remove the porphyrin contaminant, we tried two series of alumina and silica-gel chromatography Žfour times in total.; to remove the shorter polyene, we tried to measure a sample immediately after purification by HPLC using a home-packed CaŽOH. 2 column and an eluent, 5.5% acetone in n-hexane; and to remove the aggregate, the sample concentration was reduced down to 6 = 10y6 M Žthis concentration was also necessary to avoid the effect of self-absorption in the fluorescence spectrum.. In order to improve the SrN ratio of fluorescence spectrum at such a low sample concentration Žthe quantum yield of fluorescence was on the order of 10y5 in neurosporene and 10y7 in spheroidene., we modified the setup for fluorescence measurements as follows. Ž1. The photon density of irradiation was increased by collimating the output from the variable-wavelength light source onto the sample tube or
Fig. 1. The molecular structure of neurosporene, and its electronic-absorption Ždotted line., fluorescence Žthicker solid line. and fluorescence-excitation Žthinner solid line. spectra at Ža. 170 and Žb. 295 K. The excitation wavelengths for the fluorescence spectra were 445 nm at 170 K and 440 nm at 295 K; the detection wavelength for the fluorescence-excitation spectra was 770 nm at both temperatures. The sharp Raman lines on the top of the fluorescence spectra are painted in black.
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ably the shorter polyene.. We are still unable to identify any of the possible contaminants, but their contribution in the fluorescence spectra is reduced to a minimum extent. 3.2. Determination of Õibronic leÕels in neurosporene and spheroidene Fig. 1a shows the results for neurosporene at 170 K. The electronic-absorption spectrum clearly exhibits four peaks which can be assigned to the 1Bq u Ž0, 1, 2 and 3. § 1Ay Ž . g 0 absorptive vibronic transitions. The fluorescence spectrum shows three peaks
Fig. 2. The molecular structure of spheroidene, and its electronicabsorption Ždotted line., fluorescence Žthicker solid line. and fluorescence-excitation Žthinner solid line. spectra at Ža. 200 and Žb. 295 K. The excitation wavelength of the fluorescence spectra was 488 nm, and the detection wavelength of the fluorescence-excitation spectra was 770 nm at both temperatures. The Raman lines are painted in black.
source, whereas those of spheroidene were recorded by using the laser beam for excitation. No such strong peaks as reported previously w9x are seen in the 670–685 nm region at all in either of the carotenoids, and the expansion of the ordinate scale gives rise to sets of vibrational structures which are ascribable to the 2Ay g state. Each fluorescence spectrum is in rough agreement with the absorption spectrum, although the higher intensity in the fluorescence-excitation spectrum in the 400 nm region still suggests a contribution of some impurity Žprob-
Fig. 3. Deconvolution of the fluorescence spectra of Ža. neurosporene at 170 K and Žb. spheroidene at 200 K. The fluorescence spectra are explained in terms of the vibronic transitions q Ž Ž . from the 2Ay broken lines. states in g dotted lines and the 1B u both cases of neurosporene and spheroidene; each transition is indicated in the figure. The weak lines indicate the sum of all the components, which is in almost complete agreement with the observed fluorescence spectra.
R. Fujii et al.r Chemical Physics Letters 288 (1998) 847–853
Ž . and a shoulder ascribable to the 1Bq u 0 ™ Ž . 1Ay 0, 1, 2 and 3 emissive vibronic transitions. Exg cept for the sharp Raman peaks Žpainted in black. on the top of it, the fluorescence and the electronic-absorption spectra are in the relation of an approximate mirror image, the Stokes shift being 300 cmy1 . Fig. 3a illustrates a part Ž10000–18000 cmy1 . of deconvolution of the entire fluorescence spectrum. The fluorescence profile is explained nicely in terms y y y of two series of the 1Bq u ™ 1A g and the 2A g ™ 1A g vibronic transitions. Most importantly, the spacing yŽ . yŽ . Ž . between the 1Bq u 0 ™ 1A g 4 and the 2A g 0 ™ y1 yŽ . 1A g 0 transitions is only 700 cm , despite of the fact that all the spacings in the rest of emissive vibronic transition are in the 1100–1300 cmy1 range. The fact indicates that these two peaks belong to different series of vibronic transitions. Thus, the y 0 ™ 0 origin of the 2Ay g ™ 1A g electronic transition is clearly identified. Fig. 4a shows an energy diagram which summarizes all the absorptive and emissive vibronic transitions.
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Fig. 1b shows the results for neurosporene at 295 K. Very similar set of spectra are obtained, and the fluorescence peaks were determined by the deconvolution procedure Žthe results not shown.. Comparison of the sets of spectra which were recorded at different temperatures reveals that the 1Bq u vibronic levels are lowered when the temperature is lowered and that the 2Ay g levels are not affected at all. Fig. 2a shows the results for spheroidene at 200 K. The electronic-absorption spectrum exhibits distinct four peaks to be assigned to the yŽ . . 1Bq u 0, 1, 2 and 3 § 1A g 0 transitions, whereas the fluorescence spectrum exhibits four peaks to be asyŽ Ž . . signed to the 1Bq u 0 ™ 1A g 0, 1, 2 and 3 transitions; the Stokes shift is again 300 cmy1 . Fig. 3b illustrates a part Ž9000–17000 cmy1 . of deconvolution of the entire fluorescence spectrum which can be explained again by two series of vibronic transiŽ . tions. Most importantly, the spacing of the 2Ay g 0 qŽ . yŽ . Ž . ™ 1Ay 0 and the 1B 0 ™ 1A 5 transitions is g u g only 400 cmy1 , a fact which leads to the assignment
Fig. 4. Energy diagrams showing the absorptive and emissive vibronic transitions for Ža. neurosporene at 170 K and Žb. spheroidene at 200 K. Spacing between each pair of vibrational levels is shown in brackets.
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of the 0–0 origin of the 2Ay g fluorescence. Fig. 4b shows an energy diagram which summarizes all the vibronic transitions. Fig. 2b shows the results for spheroidene at 295 K. Comparison of spectra recorded at different temperatures shows that the 1Bq u state is lowered at a lower temperature, and that the 2Ay g state is not affected at all. q y 3.3. The properties of the 1Ay g , 1Bu and 2A g states of neurosporene and spheroidene deduced from fluorescence spectra
The spacing of each vibronic transition is shown in bracket in Fig. 4. The spacings are in the range of 1100–1300 cmy1 in neurosporene in both the 1Bq u y y ™ 1Ay g and the 2A g ™ 1A g emissive vibronic transitions. Since these spacings reflect the vibrational levels in the 1Ay g state, the agreement of the spacings between the two electronic transitions guarantees that we are observing a pair of different electronic levels of the same molecule. The spacings are slightly smaller in spheroidene, i.e. in the range of 1000–1200 cmy1 . This difference must reflect the difference in the C5C stretching frequency Ž1532 cmy1 for neurosporene w11x and 1524 cmy1 for spheroidene w12x., because the C–C stretching frequency is almost the same Ž1160 cmy1 for neurosporene w11x and 1162 cmy1 for spheroidene w12x.. The result indicates that the in-phase C5C stretching vibration is one of the most important Franck–Condon active modes. In both cases of neurosporene and spheroidene, the 1Bq u energy was lowered when the temperature was lowered. This is ascribable to stabilization by the solute–solvent dispersive Žtransition dipole–transition dipole. interaction, which can be expressed as a term, the polarizability of the carotenoid multiplied by that of n-hexane w13,14x. Since the ionic 1Bq u state of the carotenoid is expected to have high polarizability due to the large fluctuation of the polarized electrons, and, since the solvent polarizability increases at lower temperatures, the lowering of the 1Bq u energy can be explained in terms of the above interaction. On the other hand, the 2Ay g energies are not affected by the increase in the solvent polarizability. This is most probably due to the fact that the covalent 2Ay g state of the carotenoid has much lower polarizability.
In both neurosporene and spheroidene, the yŽ . Ž . 2Ay g 0 ™ 1A g 2 transition gives rise to the highest intensity. The results indicate a large shift of the potential minimum of the 2Ay g state from that of the 1Ay state. This trend was actually reported for many g other carotenoids having the number of the conjugated double bonds, n s 5 ; 11 w4,7,15–20x. Ž . Ž The 2Ay g 0 ™ 0 energies of neurosporene n s 9. and spheroidene Ž n s 10. have been determined in the present investigation to be 15300 and 14200 cmy1 , respectively. The 2Ay energy of neug rosporene agrees completely with that of 3,4-dihydrospheroidene having the same number of conjugated double bonds Ž n s 9. w7x, and the 2Ay g energy of spheroidene agrees with the value, 14200 " 50 cmy1 , which was predicted by Frank et al. w6x based on its 2Ay g lifetime and the energy-gap law. Fig. 5 compares Ža. the 2Ay g energies of the shorter analogues of spheroidene Žclosed circles. which were determined by DeCoster et al. w7x using fluorescence spectroscopy, Žb. those of neurosporene and spheroidene Žopen circles. determined in the
Ž . Ž . Fig. 5. The 2Ay g 0 ™0 energy as a function of 1r 2 nq1 , where n is the number of conjugated double bonds. The 2Ay g energies include those of the shorter analogues of spheroidene Žclosed circles. w7x and those of neurosporene and spheroidene Žopen circles. wpresent workx, which were determined by fluorescence spectroscopy. The 2Ay g energies of the longer analogues of spheroidene Žshaded circles. which were predicted by using their w x 2Ay g lifetimes and the energy-gap law 6 are also shown for comparison.
R. Fujii et al.r Chemical Physics Letters 288 (1998) 847–853
present investigation, and Žc. those of the longer analogues of spheroidene Žshaded circles. predicted by Frank et al. w6x using their 2Ay g lifetimes and the energy-gap law. The 2Ay energies are plotted against g 1rŽ2 n q 1. where n is the number of the conjugated double bonds w21x. It is interesting to note that the carotenoids with n s 7, 8, 9 and 10 constitute a complete straight line which can be expressed as: 1 y1 2Ay q 3681 , . s 220946= g energy Ž in cm 2nq1 where n is the number of conjugated double bonds; the constant term 3681 cmy1 corresponds to the 2Ay g energy value for the infinite chain. The predicted values for the longer analogues w6x deviate only slightly from this linear relation. The results strongly support the idea that the 2Ay g energies of the spheroidene analogues must be determined by this linear relation. Acknowledgements The authors thank Dr. Harry Frank for his questioning our previous proposal, which motivated us to a further investigation. This work has been supported by a grant from Human Frontier Science Program and by a Grant-in-Aid Ža6239101. from the Ministry of Education, Science, Sports and Culture, Japan. References w1x H.A. Frank, R.J. Cogdell, in: A. Young, G. Britton ŽEds.., Carotenoids in Photosynthesis ŽChapman and Hall, London, 1993. p. 253.
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w2x Y. Koyama, J. Photochem. Photobiol. B: Biol. 9 Ž1991. 265. w3x Y. Koyama, M. Kuki, P.O. Andersson, T. Gillbro, Photochem. Photobiol. 63 Ž1996. 243. w4x P.O. Andersson, S.M. Bachilo, R.-L. Chen, T. Gillbro, J. Phys. Chem. 99 Ž1995. 16199. w5x V. Chynwat, H.A. Frank, Chem. Phys. 194 Ž1995. 237. w6x H.A. Frank, R.Z.B. Desamero, V. Chynwat, R. Gebhard, I. Hoef, F.J. Jansen, J. Lugtenburg, D. Gosztola, M.R. Wasielewski, J. Phys. Chem. A 101 Ž1997. 149. w7x B. DeCoster, R.L. Christensen, R. Gebhard, J. Lugtenburg, R. Farhoosh, H.A. Frank, Biochim. Biophys. Acta 1102 Ž1992. 107. w8x M. Mimuro, U. Nagashima, S. Nagaoka, S. Takaichi, I. Yamazaki, Y. Nishimura, T. Katoh, Chem. Phys. Lett. 204 Ž1993. 101. w9x Y. Watanabe, T. Kameyama, Y. Miki, M. Kuki, Y. Koyama, Chem. Phys. Lett. 206 Ž1993. 62. w10x Y. Miki, T. Kameyama, Y. Koyama, Y. Watanabe, J. Phys. Chem. 97 Ž1993. 6142. w11x Y. Koyama, M. Kanaji, T. Shimamura, Photochem. Photobiol. 48 Ž1988. 107. w12x Y.-S. Jiang, Y. Kurimoto, T. Shimamura, N. Ko-chi, N. Ohashi, Y. Mukai, Y. Koyama, Biospectroscopy 2 Ž1996. 47. w13x M. Kuki, H. Nagae, R.J. Cogdell, K. Shimada, Y. Koyama, Photochem. Photobiol. 59 Ž1994. 116. w14x H. Nagae, J. Chem. Phys. 106 Ž1997. 5159. w15x M. Mimuro, U. Nagashima, S. Nagaoka, Y. Nishimura, S. Takaichi, T. Katoh, I. Yamazaki, Chem. Phys. Lett. 191 Ž1992. 219. w16x M. Mimuro, U. Nagashima, S. Takaichi, Y. Nishimura, I. Yamazaki, T. Katoh, Biochim. Biophys. Acta 1098 Ž1992. 271. w17x P.O. Andersson, T. Gillbro, A.E. Asato, R.S.H. Liu, J. Luminesc. 51 Ž1992. 11. w18x T. Gillbro, P.O. Andersson, R.S.H. Liu, A.E. Asato, S. Takaishi, R.J. Cogdell, Photochem. Photobiol. 57 Ž1993. 44. w19x P.O. Andersson, T. Gillbro, A.E. Asato, R.S.H. Liu, Chem. Phys. Lett. 235 Ž1995. 76. w20x Y. Yamano, M. Mimuro, M. Ito, J. Chem. Soc. Perkin Trans. 1 Ž1997. 2713. w21x P. Tavan, K. Schulten, Phys. Rev. B 36 Ž1987. 4337.
Chemical Physics Letters 288 Ž1998. 847–853
The 2Ayg energies of all-trans-neurosporene and spheroidene as determined by fluorescence spectroscopy Ritsuko Fujii a , Kengo Onaka b, Michitaka Kuki c , Yasushi Koyama Yasutaka Watanabe b
a,)
,
a
Department of Chemistry, Faculty of Science, Kwansei Gakuin UniÕersity, Uegahara, Nishinomiya 662-8501, Japan Department of Physics, Faculty of Science, Kwansei Gakuin UniÕersity, Uegahara, Nishinomiya 662-8501, Japan Department of Applied Chemistry, Kobe City College of Technology, Gakuen-Higashimachi, Nishi-ku, Kobe 651-2194, Japan b
c
Received 10 February 1998; in final form 24 February 1998
Abstract The fluorescence spectra of neurosporene and spheroidene were recorded in order to determine their 2Ay g levels: each y y y fluorescence spectrum was deconvoluted into two series of 1Bq ™ 1A and 2A ™ 1A vibronic transitions, and the u g g g Ž Õ s 0. state was determined to be 15300 cmy1 in neurosporene and 14200 cmy1 in spheroidene. No energy of the 2Ay g temperature dependence of these energies was seen. q 1998 Elsevier Science B.V. All rights reserved.
1. Introduction All-trans carotenoids play an important function of harvesting light energy in bacterial photosynthetic systems w1–3x. There are two pathways of singlet-energy transfer, i.e. one from the 1Bq state of u carotenoid ŽCar. to the Q x state of bacteriochlorophyll ŽBChl., and the other from the 2Ay g state of Car to the Q y state of BChl. When the conjugated chain of Car has C 2h symmetry, the transition beŽground. states is tween the 1Bq and the 1Ay u g optically allowed, whereas that between the 2Ay g and 1Ay g states is optically forbidden. Furthermore, when the length of the conjugated chain increases, ŽS 1 . fluorescence to the crossover from the 2Ay g
) C o rre s p o n d in g [email protected]
a u th o r.
E -m a il:
ŽS 2 . fluorescence takes place. These situations 1Bq u affect the mechanisms of energy transfer and make it extremely difficult to spectroscopically determine the 2Ay g energy which is one of the key factors in revealing the energy y transfer mechanisms. Recently, it was seen that the 2Ay energies of g carotenoids having a longer conjugated chain Žthe number of conjugated double bonds, n 0 10. have been predicted by the use of the 2Ay g lifetimes and the energy-gap law, the relevant parameters of which were determined by using the energy and the lifetime Ž . of the 2Ay g state for the shorter analogues n ( 9 of b-carotene w4x or spheroidene w5,6x. In order to test the applicability of this method, and also to solve the contradictions described below, it is absolutely necessary to experimentally determine the 2Ay g energy of a carotenoid for which n 0 9. Concerning carotenoids for which n s 9, DeCoster et al. w7x determined the 2Ay g energy of 3,4-dihydrospheroidene Žin methanol. to be 15300
0009-2614r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 8 . 0 0 3 7 6 - 5
848
R. Fujii et al.r Chemical Physics Letters 288 (1998) 847–853
cmy1 by fluorescence spectroscopy, although the relevant peak was not obvious in their fluorescence spectrum shown in Fig. 5 ŽRef. w7x.. On the other hand, Mimuro et al. w8x determined the 2Ay g energy of neurosporene Žin n-hexane. to be 16000–16100 cmy1 Ž623 " 2 nm. by electronic absorption spectroscopy. However, if one assumes that the 2Ay g energy is simply a function of n irrespective of the Car structure, the difference between these values, i.e. 700–800 cmy1 , is somewhat too large for a Stokes shift. Concerning a carotenoid whose n s 10, our group detected a sharp fluorescence peak at 14900 cmy1 in ‘spheroidene’ Žin n-hexane., and ascribed it to the w x 0–0 origin of the 2Ay g state 9 . This proposal was questioned by Harry Frank, because the 2Ay g fluorescence did not exhibit the vibrational structures and the spectral profile was reminiscent of a porphyrin. We actually measured the fluorescence and fluorescence-excitation spectra of 3-acetylchlorophyll a Ža gift from Hugo Scheer. which were very similar to, but not in complete agreement with, those which we claimed from spheroidene. The most difficult problem in our previous results w9x was that the fluorescence-excitation spectrum probed on that particular peak did not exhibit the vibrational structures of the 1Bq u state. On the other hand, Frank and coworkers w6x proposed that the 2Ay g energy of spheroidene is 14200" 50 cmy1 based on its 2Ay g lifetime of 8.7 " 0.1 ps and the energy-gap law mentioned above. In the present investigation, we have attempted to improve the method of purification of neurosporene and spheroidene and the setup for fluorescence measurements in order to detect the extremely weak 2Ay g vibrational structures and to determine their 0–0 origins. The results indicate that we were actually observing fluorescence and fluorescence-excitation from an impurity in the previous investigation w9x, and that the conclusions which were drawn then need to be suspended. 2. Experimental 2.1. Sample preparation Neurosporene was isolated from the cells of Rhodobacter sphaeroides G1C. Methanol was added
to the wet cells, and the mixture was centrifuged to remove water and BChl a which were dissolved in the solution. The carotenoid component was extracted from the residue with acetone and was transferred to the n-hexane layer in fractionation against warm water containing sodium chloride. Then, a 5% waterrmethanol mixture was added to the above n-hexane layer to remove the remaining BChl component. The resultant n-hexane layer was evaporated to dryness, and the residue was dissolved again in n-hexane and then subjected to two series of alumina and silica-gel chromatography; the former chromatography used alumina ŽMerck, Aluminum Oxide 90 activity II–III. as adsorbate and 1 and 3% diethyl ether in n-hexane as eluent in a stepwise gradient elution, whereas the latter used silica-gel ŽMerck, Silica gel 60. as adsorbate and 5% isopropanol in n-hexane as eluent. Neurosporene thus obtained was recrystallized from n-hexane. Spheroidene was isolated from the cells of R. sphaeroides 2.4.1 by the same method except for the following modification. In the extraction procedure, a methanol Ž2.racetone Ž7. mixture was added to the wet cells, and the solution was stirred for 30 min in the dark while bubbling through nitrogen gas. In fluorescence measurements, each sample was dissolved into n-hexane ŽDojin Chemicals, for fluorescence spectroscopy. at a concentration of 6 = 10y6 M. 2.2. Setup for fluorescence and fluorescence-excitation measurements The setup which was described previously w10x was modified as follows. Ž1. Concerning the variable-wavelength light source, the monochromator after the Xe-lamp was replaced by a double monochromator equipped with a non-aberration light-assembly system ŽJUSCO M25D., and the output was focused onto the sample tube with a pair of lens. The 488.0 nm line from a combination of an Arq-laser ŽNEC GLG 3050. and a spectrometer ŽSpectrolab, Laser spec III. was also used as an alternative light source Žwithout focusing.. Ž2. Concerning the fluorescencedetection system, the monochromator was replaced by a single monochromator ŽJUSCO CT25C. equipped with the 1200 linesrmm Ž500 nm blaze. and the 600 linesrmm Ž1000 nm blaze. gratings, and
R. Fujii et al.r Chemical Physics Letters 288 (1998) 847–853
a photomultiplier ŽHamamatsu R632-GA106. was newly introduced as an alternative PM1 for near-infrared measurements. Ž3. Concerning the input power-monitoring system, PM2 was replaced by another photomultiplier ŽHamamatsu R666.. Deconvolution of each fluorescence spectrum was first performed, by assuming Gaussian peaks, for the y district peaks and shoulders in the 1Bq u and the 2A g vibrational structures to determine the position, the intensity and the bandwidth-at-half-maximum of each peak. Then, weak peaks in-between were determined by using, as initial conditions, the average values of the spacings and the band widths obtained in the above analysis. Finally, all the parameters were adjusted again. Based on the assumptions that the spacings and the bandwidths should be similar within each spectrum, the deconvolution provided a unique solution.
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by using a laser beam. Ž2. The sensitivity of fluorescence detection in the near-infrared region Ž850–1100 nm. was increased by introducing a grating of 1000 nm blaze and a near infrared-sensitive photomultiplier. A set of electronic absorption, fluorescence and fluorescence-excitation spectra of neurosporene and spheroidene are shown in Figs. 1 and 2, respectively. The fluorescence spectra of neurosporene were recorded by using the variable-wavelength light
3. Results and discussion 3.1. ImproÕements in sample preparation and in the setup for fluorescence measurements We considered the possibility of three ‘contaminants’ of different origins: Ža. a porphyrin as a degradation product of BChl a, Žb. a shorter polyene as a degradation product of spheroidene, and Žc. an aggregate of spheroidene. In order to remove the porphyrin contaminant, we tried two series of alumina and silica-gel chromatography Žfour times in total.; to remove the shorter polyene, we tried to measure a sample immediately after purification by HPLC using a home-packed CaŽOH. 2 column and an eluent, 5.5% acetone in n-hexane; and to remove the aggregate, the sample concentration was reduced down to 6 = 10y6 M Žthis concentration was also necessary to avoid the effect of self-absorption in the fluorescence spectrum.. In order to improve the SrN ratio of fluorescence spectrum at such a low sample concentration Žthe quantum yield of fluorescence was on the order of 10y5 in neurosporene and 10y7 in spheroidene., we modified the setup for fluorescence measurements as follows. Ž1. The photon density of irradiation was increased by collimating the output from the variable-wavelength light source onto the sample tube or
Fig. 1. The molecular structure of neurosporene, and its electronic-absorption Ždotted line., fluorescence Žthicker solid line. and fluorescence-excitation Žthinner solid line. spectra at Ža. 170 and Žb. 295 K. The excitation wavelengths for the fluorescence spectra were 445 nm at 170 K and 440 nm at 295 K; the detection wavelength for the fluorescence-excitation spectra was 770 nm at both temperatures. The sharp Raman lines on the top of the fluorescence spectra are painted in black.
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ably the shorter polyene.. We are still unable to identify any of the possible contaminants, but their contribution in the fluorescence spectra is reduced to a minimum extent. 3.2. Determination of Õibronic leÕels in neurosporene and spheroidene Fig. 1a shows the results for neurosporene at 170 K. The electronic-absorption spectrum clearly exhibits four peaks which can be assigned to the 1Bq u Ž0, 1, 2 and 3. § 1Ay Ž . g 0 absorptive vibronic transitions. The fluorescence spectrum shows three peaks
Fig. 2. The molecular structure of spheroidene, and its electronicabsorption Ždotted line., fluorescence Žthicker solid line. and fluorescence-excitation Žthinner solid line. spectra at Ža. 200 and Žb. 295 K. The excitation wavelength of the fluorescence spectra was 488 nm, and the detection wavelength of the fluorescence-excitation spectra was 770 nm at both temperatures. The Raman lines are painted in black.
source, whereas those of spheroidene were recorded by using the laser beam for excitation. No such strong peaks as reported previously w9x are seen in the 670–685 nm region at all in either of the carotenoids, and the expansion of the ordinate scale gives rise to sets of vibrational structures which are ascribable to the 2Ay g state. Each fluorescence spectrum is in rough agreement with the absorption spectrum, although the higher intensity in the fluorescence-excitation spectrum in the 400 nm region still suggests a contribution of some impurity Žprob-
Fig. 3. Deconvolution of the fluorescence spectra of Ža. neurosporene at 170 K and Žb. spheroidene at 200 K. The fluorescence spectra are explained in terms of the vibronic transitions q Ž Ž . from the 2Ay broken lines. states in g dotted lines and the 1B u both cases of neurosporene and spheroidene; each transition is indicated in the figure. The weak lines indicate the sum of all the components, which is in almost complete agreement with the observed fluorescence spectra.
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Ž . and a shoulder ascribable to the 1Bq u 0 ™ Ž . 1Ay 0, 1, 2 and 3 emissive vibronic transitions. Exg cept for the sharp Raman peaks Žpainted in black. on the top of it, the fluorescence and the electronic-absorption spectra are in the relation of an approximate mirror image, the Stokes shift being 300 cmy1 . Fig. 3a illustrates a part Ž10000–18000 cmy1 . of deconvolution of the entire fluorescence spectrum. The fluorescence profile is explained nicely in terms y y y of two series of the 1Bq u ™ 1A g and the 2A g ™ 1A g vibronic transitions. Most importantly, the spacing yŽ . yŽ . Ž . between the 1Bq u 0 ™ 1A g 4 and the 2A g 0 ™ y1 yŽ . 1A g 0 transitions is only 700 cm , despite of the fact that all the spacings in the rest of emissive vibronic transition are in the 1100–1300 cmy1 range. The fact indicates that these two peaks belong to different series of vibronic transitions. Thus, the y 0 ™ 0 origin of the 2Ay g ™ 1A g electronic transition is clearly identified. Fig. 4a shows an energy diagram which summarizes all the absorptive and emissive vibronic transitions.
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Fig. 1b shows the results for neurosporene at 295 K. Very similar set of spectra are obtained, and the fluorescence peaks were determined by the deconvolution procedure Žthe results not shown.. Comparison of the sets of spectra which were recorded at different temperatures reveals that the 1Bq u vibronic levels are lowered when the temperature is lowered and that the 2Ay g levels are not affected at all. Fig. 2a shows the results for spheroidene at 200 K. The electronic-absorption spectrum exhibits distinct four peaks to be assigned to the yŽ . . 1Bq u 0, 1, 2 and 3 § 1A g 0 transitions, whereas the fluorescence spectrum exhibits four peaks to be asyŽ Ž . . signed to the 1Bq u 0 ™ 1A g 0, 1, 2 and 3 transitions; the Stokes shift is again 300 cmy1 . Fig. 3b illustrates a part Ž9000–17000 cmy1 . of deconvolution of the entire fluorescence spectrum which can be explained again by two series of vibronic transiŽ . tions. Most importantly, the spacing of the 2Ay g 0 qŽ . yŽ . Ž . ™ 1Ay 0 and the 1B 0 ™ 1A 5 transitions is g u g only 400 cmy1 , a fact which leads to the assignment
Fig. 4. Energy diagrams showing the absorptive and emissive vibronic transitions for Ža. neurosporene at 170 K and Žb. spheroidene at 200 K. Spacing between each pair of vibrational levels is shown in brackets.
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of the 0–0 origin of the 2Ay g fluorescence. Fig. 4b shows an energy diagram which summarizes all the vibronic transitions. Fig. 2b shows the results for spheroidene at 295 K. Comparison of spectra recorded at different temperatures shows that the 1Bq u state is lowered at a lower temperature, and that the 2Ay g state is not affected at all. q y 3.3. The properties of the 1Ay g , 1Bu and 2A g states of neurosporene and spheroidene deduced from fluorescence spectra
The spacing of each vibronic transition is shown in bracket in Fig. 4. The spacings are in the range of 1100–1300 cmy1 in neurosporene in both the 1Bq u y y ™ 1Ay g and the 2A g ™ 1A g emissive vibronic transitions. Since these spacings reflect the vibrational levels in the 1Ay g state, the agreement of the spacings between the two electronic transitions guarantees that we are observing a pair of different electronic levels of the same molecule. The spacings are slightly smaller in spheroidene, i.e. in the range of 1000–1200 cmy1 . This difference must reflect the difference in the C5C stretching frequency Ž1532 cmy1 for neurosporene w11x and 1524 cmy1 for spheroidene w12x., because the C–C stretching frequency is almost the same Ž1160 cmy1 for neurosporene w11x and 1162 cmy1 for spheroidene w12x.. The result indicates that the in-phase C5C stretching vibration is one of the most important Franck–Condon active modes. In both cases of neurosporene and spheroidene, the 1Bq u energy was lowered when the temperature was lowered. This is ascribable to stabilization by the solute–solvent dispersive Žtransition dipole–transition dipole. interaction, which can be expressed as a term, the polarizability of the carotenoid multiplied by that of n-hexane w13,14x. Since the ionic 1Bq u state of the carotenoid is expected to have high polarizability due to the large fluctuation of the polarized electrons, and, since the solvent polarizability increases at lower temperatures, the lowering of the 1Bq u energy can be explained in terms of the above interaction. On the other hand, the 2Ay g energies are not affected by the increase in the solvent polarizability. This is most probably due to the fact that the covalent 2Ay g state of the carotenoid has much lower polarizability.
In both neurosporene and spheroidene, the yŽ . Ž . 2Ay g 0 ™ 1A g 2 transition gives rise to the highest intensity. The results indicate a large shift of the potential minimum of the 2Ay g state from that of the 1Ay state. This trend was actually reported for many g other carotenoids having the number of the conjugated double bonds, n s 5 ; 11 w4,7,15–20x. Ž . Ž The 2Ay g 0 ™ 0 energies of neurosporene n s 9. and spheroidene Ž n s 10. have been determined in the present investigation to be 15300 and 14200 cmy1 , respectively. The 2Ay energy of neug rosporene agrees completely with that of 3,4-dihydrospheroidene having the same number of conjugated double bonds Ž n s 9. w7x, and the 2Ay g energy of spheroidene agrees with the value, 14200 " 50 cmy1 , which was predicted by Frank et al. w6x based on its 2Ay g lifetime and the energy-gap law. Fig. 5 compares Ža. the 2Ay g energies of the shorter analogues of spheroidene Žclosed circles. which were determined by DeCoster et al. w7x using fluorescence spectroscopy, Žb. those of neurosporene and spheroidene Žopen circles. determined in the
Ž . Ž . Fig. 5. The 2Ay g 0 ™0 energy as a function of 1r 2 nq1 , where n is the number of conjugated double bonds. The 2Ay g energies include those of the shorter analogues of spheroidene Žclosed circles. w7x and those of neurosporene and spheroidene Žopen circles. wpresent workx, which were determined by fluorescence spectroscopy. The 2Ay g energies of the longer analogues of spheroidene Žshaded circles. which were predicted by using their w x 2Ay g lifetimes and the energy-gap law 6 are also shown for comparison.
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present investigation, and Žc. those of the longer analogues of spheroidene Žshaded circles. predicted by Frank et al. w6x using their 2Ay g lifetimes and the energy-gap law. The 2Ay energies are plotted against g 1rŽ2 n q 1. where n is the number of the conjugated double bonds w21x. It is interesting to note that the carotenoids with n s 7, 8, 9 and 10 constitute a complete straight line which can be expressed as: 1 y1 2Ay q 3681 , . s 220946= g energy Ž in cm 2nq1 where n is the number of conjugated double bonds; the constant term 3681 cmy1 corresponds to the 2Ay g energy value for the infinite chain. The predicted values for the longer analogues w6x deviate only slightly from this linear relation. The results strongly support the idea that the 2Ay g energies of the spheroidene analogues must be determined by this linear relation. Acknowledgements The authors thank Dr. Harry Frank for his questioning our previous proposal, which motivated us to a further investigation. This work has been supported by a grant from Human Frontier Science Program and by a Grant-in-Aid Ža6239101. from the Ministry of Education, Science, Sports and Culture, Japan. References w1x H.A. Frank, R.J. Cogdell, in: A. Young, G. Britton ŽEds.., Carotenoids in Photosynthesis ŽChapman and Hall, London, 1993. p. 253.
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