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Site-selection spectroscopy of tetraphenylporphyrin aminoacid model photosynthetic systems
CHEMICAL PHYSICS LETTERS
Volume 12 3. number 5
SITE-SELECTION SPECTROSCOPY OF TETRAPHENYLPORPHYRIN AMINOACID
MODEL PHOTOSYNTHETIC
J. HALA,
and K. VACEK
I. PELANT,
M. AMBROZ,
P. DOUSA’
24 January 1986
SYSTEMS
Department of Chemrcal Phyxs, Faculty of Mathemarrcs and Physrcs. Charles Unroersra: Ke Karlouu 3, I21 16 Prague 2, C:echoslouakla
Received 8 November 1985
Site-selection spectroscopy of carboxytrimethyl tetraphenylporphyrin and its phenylalanine complexes m n-octane at 10 K are reported. The frequencies of normal vibrations and the site distribution functions were determined from the fluorescence spectra. Pigment-pigment and pigment-short-chain-protein interactions are discussed.
1. Introduction Tetraphenylporphyrins phyrin-like
molecules.
(TPP) are very stable porA systematic investigation of
TPP optical A recent
spectra has been made in many papers. review can be found in refs. [ 1,2]. The vibra-
structure of TPP low-temperature fluorescence spectra has been studied under different experimental conditions. The supersonic jet expansion technique has been applied to H2-TPP in ref. [3]. Further valuable results have been obtained from Shpolskii spectra of H2-TPP in nitrobenzene [4] and site-selection spectra of Zn-TPP in polystyrene [5]. Hole-burning experiments performed on the same material in frozen ethanol solution are presented in ref. [6]. An analysis of site-selection spectra provides frequencies of normal vibrations (FNV) [ 7,8] and site distribution functions (SDF) [ 9, lo]. The FNV bring information about molecular vibrations. SDF, especially their full widths at half maximum (fwhm) and spectral positions, give new insight into the role of interactions occurring in low-temperature matrices. Recently, several photosynthetic systems have been studied by this technique; chlorophyll-b in membranes of lecithin vesicles [ 111, greening and ethlolated barley leaves [ 121 and pheophorbide-a (PHEO) bonded tional
’ Present
address: Institute of Macromolecular Czechoslovak Academy of Science. 162 06 Czechoslovakia.
Chemistry, Prague
6.
0 009-2614/86/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
to a long polypeptide chain [ IO]. The aim of this paper is to present site-selection spectra of carboxytrimethyl TPP (M-TPP), orthophenylalanine trimethyl TPP (ORTHO-TPP) and paraphenylalanine trimethyl TPP (PARA-TPP) model photosynthetic systems, with the view of investigating pigment-pigment and pigment-shortchain-aminoacid interactions.
2. Experimental The M-TPP, ORTHO-TPP and PARA-TPP molecules were used as obtained in n-octane solution with a small excess of ethanol (C= 0.1-5 @vf). The quality of the solutions was checked using absorption spectra recorded on a Specord M40 spectiophotometer. Polycrystalline samples were obtained by a half hour cooling to the temperature 10 K in 0.2-1.0 mm quartz cells in a gas flow cryostat, Leybold-Heraeus VSK 3-300. A Lambda Physik M 100 pulsed nitrogen laser at 15-30 Hz was used to pump an FL 1000 T/IR tunable dye laser with rhodamine dyes having 2 ns pulses with nearly 100 kW peak power. The 0.05 nm bandwidth dye laser output served as a light source for selective excitation of the fluorescence. The sample emission was resolved in a Jobin-Yvon HRD 1 doublegrating monochromator having a linear dispersion of 0.6 &mm. The fluorescence signal was detected by 437
Volume 123, number 5
CHEMICAL PHYSICS LETTERS
24 January 1986
an RCA C3 1034 A photomul~plier along with a PAR 1621165 due-channel boxcar integrator (2 ns aperture). The aperture position of the boxcar was held in temporal coincidence with the laser pulse. A more detailed description of the apparatus and the whole procedure can be found in ref. [ 131.
r
3. Results The fluorescence site-selection spectra of all investigated TPP systems in n-octane matrix exhibit sharp vibronic structure on a broad-band fluorescence background in the 640-670 mn spectral region. The wavenumbers of particular fluorescence vibronic lines change as the dye laser is tuned (575-615 nm); however, the wavenumber differences between vibronic lines and laser excitation remain constant within ex-
,
I
640 Table 1 Frequencies (cm-‘)
Fig. 1. Site-selection fluorescence spectra of tetraphenylporphyrin molecules in n-octane with a small excess of ethanol. Laser excitation at 596.86 nm, T = 10 K.
I
660
nm
of normal vibrations rj of tetraphenylporph~n
M-TPP
ORTHO-TI’P
1680.8 f 1.9
PARA-TPP
molecules in the region 950-1700
cm-’
-
H2-TPP
HZ-TPP
Zn-TPP
[31
I41
I51
1686.0 f 4.7
1675
1649.0 f 3.9 1607.0 * 2.5 1540.1 f 1.5
438
1524.3 1515.4 1500.2 1468.3 1422.8 1359.9 1340.3 1316.2
+ 1.3 f 0.8 f 1.3 -I 1.5 i 1.1 + 1.7 f 1.2 + 2.7
1295.2 1273.1 1246.8 1230.5
f f f f
2.5 1.5 *1.4 1.0
1190.1 1121.9 1068.4 999.1 978.3 963.8
i f i f i i
0.4 0.7 0.7 0.5 0.7 0.9
1536.6 f. 1.6
1536.3 f 1.4
1503.3 1: 1.9 1469.1 t 1.0
1503.8 1469.6 1432.7 1358.6 1341.8 1315.7
* f * + c f
2.1 1.5 2.1 1.6 1.5 1.8
1290.9 1275.1 1248.4 1230.5 1209.8 1189.3 1121.2 1072.5 1000.1 974.5 958.4
i f f f + i f f f f t
1.4 2.2 1.7 0.6 1.6 2.0 1.0 1.3 0.4 2.0 1.5
1357.6 f 1.6 1324.1 f 2.7 1289.0 f 1.8 1246.0 1229.9 1207.8 1191.2 1120.3 1073.8 1001.2 969.8 956.4
4 f f i f f i f f
1.9 0.2 1.8 0.6 1.2 1.4 0.6 1.9 0.9
1544 1515 1511
1513 1504
1365
1367 1341
1543
1507 1471 1433 1358 1321
1258 1197 1188
1000 950
1002 978
1248 1227 1207 1165 1124 1080 1001 969 955
Volume 123, number 5
perimental accuracy (2 cm-‘). These differences represent FNV in the first excited singlet state [7,8] (see fig. 1). The FNV of M-TPP, ORTHO- and PARATPP molecules are collected in table 1 together with the FNV of H2-TPP and Zn-TPP summarized from the literature. SDF for selected FNV have been determined using a procedure described in the literature [9,10]. Fig. 2 shows three SDF for FNV of 1190, 1360 and 1503 cm-l for all three investigated TPP systems. The laser excitation corresponding to the fluorescence spectra in fig. 1 is depicted by an arrow. Further, one can find in fig. 1 sharp vibronic fluorescence lines denoted by the same wavenumbers as those crossed by the laser excitation arrow in fig. 2. A detailed picture of SDF for FNV of 1190 cm-I is shown in fig. 3. The FNV collected in table 1 show good agreement for all kinds of TPP molecules. This implies that the abovementioned FNV are connected with vibrations in the TPP skeleton. Neither the addition of the carboxy group, phenylalanine chain and Zn central atom, nor any possible aggregation of M-TPP and PARA-TPP molecules influence the FNV. The aggregation of TPP molecules was investigated by studying
600
24 January 1986
CHEMICAL PHYSICS LETTERS
590
nm
1n (5 +119Oci’l
_
~“-Tpp I
1
I
16500
I
16800
I
cm-’
Fig. 3. Detailed picture of site distribution functions ni(‘; + 1190 cm-‘) of tetraphenylporphyrin model systems.
concentration effects in fluorescence, absorption and CD spectra [ 141. M-TPP and PARA-TPP molecules strongly aggregate; on the other hand, ORTHO-TPP molecules behave like monomers. The SDF of M-TPP and PARA-TPP are of very similar shape (fig. 2). Their fwhm of SDF corresponding to FNV of 1190 cm-l are 230-240 cm-l. The observed mutual spectral shift is below 25 cm-l. The SDF of ORTHO-TPP exhibits significantly different features. Its fwhm is only 100 cm-l and the whole SDF is about 115140 cm-l blue-shifted from SDF of M-TPP and PARA-TPP.
4. Discussion
Fig. 2. Site distribution function ni(i; + i;i) of tetraphenylporphyrin molecules in n octane with small excess of ethanol (zj = 1190, 1360 and 1500 cm-‘). Laser excitation corresponding to the fluorescence spectra in fig. 1 is depicted by an arrow.
Conclusions can be drawn especially clearly by comparing the present results with those obtained previously on PHEO bonded to a long-chain polypeptide (Llysyl-Lalanyl-Galanine) [lo], (i) Experimentally determined FNV have been found, in both cases, to be characteristic for the vibration of the skeleton of the relevant porphyrin molecule: The FNV of free PHEO and PHEO bonded to long-chain polypeptide systems were found in ref. [IO] to be equivalent to FNV of chlorophyll-a, protochlorophyll-a, pheophytin-a and protopheophy tin-a [8] (these FNV are common vibrations of the chlorophyll-a skeleton). In the present work, the 439
Volume 123, number 5
CHEMICAL PHYSICS LETTERS
determined FNV belong to the TPP skeleton. (ii) Novel features: The attachment of a shortchain aminoacid causes only negligible changes of both the shape and spectral position of SDF (compare SDF of M-TPP, i.e. free TPP, to that of PARA-TPP in fig. 3), in contrast to a long-chain polypeptide attachment (the SDF of PHEO bonded to a long-chain polypeptide was found to be 460 cm-l broad and 150 cm -l red-shifted with respect to the SDF of free PHEO which is only 280 cm-l broad [lo]). On the other hand, similar spectral behaviour, i.e. significant broadening and red-shift of SDF, has been found in the present work to be due to aggregation (pigmentpigment interaction), as can be seen by comparing in fig. 3 the SDF of M-TPP and PARA-TPP (strongly aggregated state) to that of ORTHO-TPP (monomer). (iii) Our results confirm that FNV of SDF give information on the number of sites in the matrix: The fwhm of TPP SDF are approximately a factor of two narrower than fwhm of PHEO SDF. This fact can easily be understood due to the relatively more complex chemical structure of PHEO systems in dimethylformamide matrix and consequently the higher number of mutual positions of pigment and matrix molecules.
440
24 January 1986
Acknowledgement The authors would like to thank to Dr. A. Harriman for supplying the TPP pigments and Dr. P. Pancoska for stimulating discussions.
References
111L. Benthem, Thesis, Agriculture University, Wageningen (1984).
121P. Duosa, Thesis, Charles University, Prague (1985). [31 V. Even, J. Magen and J. Jortner, J. Chem. Phys. 77 (1982) 4374. [41 R. Tamkivi, I. Renge and R. Avarmaa, Chem. Phys. Letters 103 (1983) 103. 151 L.A. Bykovskaya, RI. Personov and Y.V. Romanovskii, Zh. Prikl. Spectroskopiya 31 (1979) 910. 161 B.M. Kharlamov, L.A. Bykovskaya and RI. Personov, Chem. Phys. Letters 50 (1977) 407. I71 J. Hala, I. Pelant, L. Parma and K. Vacek, J. Luminescence 26 (1981) 117. PI K.K. Rebane and R.A. Avarmaa, Chem. Phys. 68 (1982) 191. [91 J. Fiinfschihing and I. Zschokke-Grinacher, Chem. Phys. Letters 91 (1982) 122. 1101 J. Hala,I. Pelant, M. Ambroz, P. Pancoska and K. Vacek, Photochem. Photobiol. 41 (1985) 643. [Ill J. Fiinfschilling and D. Walz, Photochem. Photobiol. 38 (1983) 389. [la R.A. Avarmaa, I. Renge and K.K. Mauring, FEBS Letters 167 (1984) 180. 1131 L. Parma, I. Pelant and J. Hala, Czech. J. Phys. A30 (1980) 134. iI41 L. Souckova, Thesis, Charles University, Prague (1985).
Volume 12 3. number 5
SITE-SELECTION SPECTROSCOPY OF TETRAPHENYLPORPHYRIN AMINOACID
MODEL PHOTOSYNTHETIC
J. HALA,
and K. VACEK
I. PELANT,
M. AMBROZ,
P. DOUSA’
24 January 1986
SYSTEMS
Department of Chemrcal Phyxs, Faculty of Mathemarrcs and Physrcs. Charles Unroersra: Ke Karlouu 3, I21 16 Prague 2, C:echoslouakla
Received 8 November 1985
Site-selection spectroscopy of carboxytrimethyl tetraphenylporphyrin and its phenylalanine complexes m n-octane at 10 K are reported. The frequencies of normal vibrations and the site distribution functions were determined from the fluorescence spectra. Pigment-pigment and pigment-short-chain-protein interactions are discussed.
1. Introduction Tetraphenylporphyrins phyrin-like
molecules.
(TPP) are very stable porA systematic investigation of
TPP optical A recent
spectra has been made in many papers. review can be found in refs. [ 1,2]. The vibra-
structure of TPP low-temperature fluorescence spectra has been studied under different experimental conditions. The supersonic jet expansion technique has been applied to H2-TPP in ref. [3]. Further valuable results have been obtained from Shpolskii spectra of H2-TPP in nitrobenzene [4] and site-selection spectra of Zn-TPP in polystyrene [5]. Hole-burning experiments performed on the same material in frozen ethanol solution are presented in ref. [6]. An analysis of site-selection spectra provides frequencies of normal vibrations (FNV) [ 7,8] and site distribution functions (SDF) [ 9, lo]. The FNV bring information about molecular vibrations. SDF, especially their full widths at half maximum (fwhm) and spectral positions, give new insight into the role of interactions occurring in low-temperature matrices. Recently, several photosynthetic systems have been studied by this technique; chlorophyll-b in membranes of lecithin vesicles [ 111, greening and ethlolated barley leaves [ 121 and pheophorbide-a (PHEO) bonded tional
’ Present
address: Institute of Macromolecular Czechoslovak Academy of Science. 162 06 Czechoslovakia.
Chemistry, Prague
6.
0 009-2614/86/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
to a long polypeptide chain [ IO]. The aim of this paper is to present site-selection spectra of carboxytrimethyl TPP (M-TPP), orthophenylalanine trimethyl TPP (ORTHO-TPP) and paraphenylalanine trimethyl TPP (PARA-TPP) model photosynthetic systems, with the view of investigating pigment-pigment and pigment-shortchain-aminoacid interactions.
2. Experimental The M-TPP, ORTHO-TPP and PARA-TPP molecules were used as obtained in n-octane solution with a small excess of ethanol (C= 0.1-5 @vf). The quality of the solutions was checked using absorption spectra recorded on a Specord M40 spectiophotometer. Polycrystalline samples were obtained by a half hour cooling to the temperature 10 K in 0.2-1.0 mm quartz cells in a gas flow cryostat, Leybold-Heraeus VSK 3-300. A Lambda Physik M 100 pulsed nitrogen laser at 15-30 Hz was used to pump an FL 1000 T/IR tunable dye laser with rhodamine dyes having 2 ns pulses with nearly 100 kW peak power. The 0.05 nm bandwidth dye laser output served as a light source for selective excitation of the fluorescence. The sample emission was resolved in a Jobin-Yvon HRD 1 doublegrating monochromator having a linear dispersion of 0.6 &mm. The fluorescence signal was detected by 437
Volume 123, number 5
CHEMICAL PHYSICS LETTERS
24 January 1986
an RCA C3 1034 A photomul~plier along with a PAR 1621165 due-channel boxcar integrator (2 ns aperture). The aperture position of the boxcar was held in temporal coincidence with the laser pulse. A more detailed description of the apparatus and the whole procedure can be found in ref. [ 131.
r
3. Results The fluorescence site-selection spectra of all investigated TPP systems in n-octane matrix exhibit sharp vibronic structure on a broad-band fluorescence background in the 640-670 mn spectral region. The wavenumbers of particular fluorescence vibronic lines change as the dye laser is tuned (575-615 nm); however, the wavenumber differences between vibronic lines and laser excitation remain constant within ex-
,
I
640 Table 1 Frequencies (cm-‘)
Fig. 1. Site-selection fluorescence spectra of tetraphenylporphyrin molecules in n-octane with a small excess of ethanol. Laser excitation at 596.86 nm, T = 10 K.
I
660
nm
of normal vibrations rj of tetraphenylporph~n
M-TPP
ORTHO-TI’P
1680.8 f 1.9
PARA-TPP
molecules in the region 950-1700
cm-’
-
H2-TPP
HZ-TPP
Zn-TPP
[31
I41
I51
1686.0 f 4.7
1675
1649.0 f 3.9 1607.0 * 2.5 1540.1 f 1.5
438
1524.3 1515.4 1500.2 1468.3 1422.8 1359.9 1340.3 1316.2
+ 1.3 f 0.8 f 1.3 -I 1.5 i 1.1 + 1.7 f 1.2 + 2.7
1295.2 1273.1 1246.8 1230.5
f f f f
2.5 1.5 *1.4 1.0
1190.1 1121.9 1068.4 999.1 978.3 963.8
i f i f i i
0.4 0.7 0.7 0.5 0.7 0.9
1536.6 f. 1.6
1536.3 f 1.4
1503.3 1: 1.9 1469.1 t 1.0
1503.8 1469.6 1432.7 1358.6 1341.8 1315.7
* f * + c f
2.1 1.5 2.1 1.6 1.5 1.8
1290.9 1275.1 1248.4 1230.5 1209.8 1189.3 1121.2 1072.5 1000.1 974.5 958.4
i f f f + i f f f f t
1.4 2.2 1.7 0.6 1.6 2.0 1.0 1.3 0.4 2.0 1.5
1357.6 f 1.6 1324.1 f 2.7 1289.0 f 1.8 1246.0 1229.9 1207.8 1191.2 1120.3 1073.8 1001.2 969.8 956.4
4 f f i f f i f f
1.9 0.2 1.8 0.6 1.2 1.4 0.6 1.9 0.9
1544 1515 1511
1513 1504
1365
1367 1341
1543
1507 1471 1433 1358 1321
1258 1197 1188
1000 950
1002 978
1248 1227 1207 1165 1124 1080 1001 969 955
Volume 123, number 5
perimental accuracy (2 cm-‘). These differences represent FNV in the first excited singlet state [7,8] (see fig. 1). The FNV of M-TPP, ORTHO- and PARATPP molecules are collected in table 1 together with the FNV of H2-TPP and Zn-TPP summarized from the literature. SDF for selected FNV have been determined using a procedure described in the literature [9,10]. Fig. 2 shows three SDF for FNV of 1190, 1360 and 1503 cm-l for all three investigated TPP systems. The laser excitation corresponding to the fluorescence spectra in fig. 1 is depicted by an arrow. Further, one can find in fig. 1 sharp vibronic fluorescence lines denoted by the same wavenumbers as those crossed by the laser excitation arrow in fig. 2. A detailed picture of SDF for FNV of 1190 cm-I is shown in fig. 3. The FNV collected in table 1 show good agreement for all kinds of TPP molecules. This implies that the abovementioned FNV are connected with vibrations in the TPP skeleton. Neither the addition of the carboxy group, phenylalanine chain and Zn central atom, nor any possible aggregation of M-TPP and PARA-TPP molecules influence the FNV. The aggregation of TPP molecules was investigated by studying
600
24 January 1986
CHEMICAL PHYSICS LETTERS
590
nm
1n (5 +119Oci’l
_
~“-Tpp I
1
I
16500
I
16800
I
cm-’
Fig. 3. Detailed picture of site distribution functions ni(‘; + 1190 cm-‘) of tetraphenylporphyrin model systems.
concentration effects in fluorescence, absorption and CD spectra [ 141. M-TPP and PARA-TPP molecules strongly aggregate; on the other hand, ORTHO-TPP molecules behave like monomers. The SDF of M-TPP and PARA-TPP are of very similar shape (fig. 2). Their fwhm of SDF corresponding to FNV of 1190 cm-l are 230-240 cm-l. The observed mutual spectral shift is below 25 cm-l. The SDF of ORTHO-TPP exhibits significantly different features. Its fwhm is only 100 cm-l and the whole SDF is about 115140 cm-l blue-shifted from SDF of M-TPP and PARA-TPP.
4. Discussion
Fig. 2. Site distribution function ni(i; + i;i) of tetraphenylporphyrin molecules in n octane with small excess of ethanol (zj = 1190, 1360 and 1500 cm-‘). Laser excitation corresponding to the fluorescence spectra in fig. 1 is depicted by an arrow.
Conclusions can be drawn especially clearly by comparing the present results with those obtained previously on PHEO bonded to a long-chain polypeptide (Llysyl-Lalanyl-Galanine) [lo], (i) Experimentally determined FNV have been found, in both cases, to be characteristic for the vibration of the skeleton of the relevant porphyrin molecule: The FNV of free PHEO and PHEO bonded to long-chain polypeptide systems were found in ref. [IO] to be equivalent to FNV of chlorophyll-a, protochlorophyll-a, pheophytin-a and protopheophy tin-a [8] (these FNV are common vibrations of the chlorophyll-a skeleton). In the present work, the 439
Volume 123, number 5
CHEMICAL PHYSICS LETTERS
determined FNV belong to the TPP skeleton. (ii) Novel features: The attachment of a shortchain aminoacid causes only negligible changes of both the shape and spectral position of SDF (compare SDF of M-TPP, i.e. free TPP, to that of PARA-TPP in fig. 3), in contrast to a long-chain polypeptide attachment (the SDF of PHEO bonded to a long-chain polypeptide was found to be 460 cm-l broad and 150 cm -l red-shifted with respect to the SDF of free PHEO which is only 280 cm-l broad [lo]). On the other hand, similar spectral behaviour, i.e. significant broadening and red-shift of SDF, has been found in the present work to be due to aggregation (pigmentpigment interaction), as can be seen by comparing in fig. 3 the SDF of M-TPP and PARA-TPP (strongly aggregated state) to that of ORTHO-TPP (monomer). (iii) Our results confirm that FNV of SDF give information on the number of sites in the matrix: The fwhm of TPP SDF are approximately a factor of two narrower than fwhm of PHEO SDF. This fact can easily be understood due to the relatively more complex chemical structure of PHEO systems in dimethylformamide matrix and consequently the higher number of mutual positions of pigment and matrix molecules.
440
24 January 1986
Acknowledgement The authors would like to thank to Dr. A. Harriman for supplying the TPP pigments and Dr. P. Pancoska for stimulating discussions.
References
111L. Benthem, Thesis, Agriculture University, Wageningen (1984).
121P. Duosa, Thesis, Charles University, Prague (1985). [31 V. Even, J. Magen and J. Jortner, J. Chem. Phys. 77 (1982) 4374. [41 R. Tamkivi, I. Renge and R. Avarmaa, Chem. Phys. Letters 103 (1983) 103. 151 L.A. Bykovskaya, RI. Personov and Y.V. Romanovskii, Zh. Prikl. Spectroskopiya 31 (1979) 910. 161 B.M. Kharlamov, L.A. Bykovskaya and RI. Personov, Chem. Phys. Letters 50 (1977) 407. I71 J. Hala, I. Pelant, L. Parma and K. Vacek, J. Luminescence 26 (1981) 117. PI K.K. Rebane and R.A. Avarmaa, Chem. Phys. 68 (1982) 191. [91 J. Fiinfschihing and I. Zschokke-Grinacher, Chem. Phys. Letters 91 (1982) 122. 1101 J. Hala,I. Pelant, M. Ambroz, P. Pancoska and K. Vacek, Photochem. Photobiol. 41 (1985) 643. [Ill J. Fiinfschilling and D. Walz, Photochem. Photobiol. 38 (1983) 389. [la R.A. Avarmaa, I. Renge and K.K. Mauring, FEBS Letters 167 (1984) 180. 1131 L. Parma, I. Pelant and J. Hala, Czech. J. Phys. A30 (1980) 134. iI41 L. Souckova, Thesis, Charles University, Prague (1985).