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Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts
Spectrochimica Acta Part A 56 (1999) 211 – 214 www.elsevier.nl/locate/saa
Letter
Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts T. Ja´vorfi a, G. Garab a, K. Razi Naqvi b,* a
Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary b Department of Physics, Norwegian Uni6ersity of Science and Technology, N-7491 Trondheim, Norway Received 28 June 1999; received in revised form 28 September 1999; accepted 28 September 1999
Abstract A comparison of the triplet-minus-singlet (TmS) absorption spectrum of spinach chloroplasts, recorded some thirty years ago, with the more recently published TmS spectrum of isolated Chla/b LHCII (light-harvesting complexes associated with photosystem II of higher plants) shows that the two spectra are very similar, which is to be expected, since only the carotenoid pigments contribute to each spectrum. Be that as it may, the comparison also reveals a dissimilarity: photoexcitation of the sample does, or does not, affect the absorbance in the Qy region (650 – 700 nm), depending on whether the sample is a suspension of chloroplasts or of isolated LHCII. The Qy-signal in the TmS spectrum of LHCII decays, it should be noted, at the same rate as the rest of the difference spectrum, and its most prominent feature is a negative peak. As the carotenoids do not absorb in the Qy region, the presence of a signal in this region calls for an explanation: van der Vos, Carbonera and Hoff, the first to find as well as fathom the phenomenon, attributed the Qy-signal to a change, in the absorption spectrum of a chlorophyll a (Chla) molecule, brought about by the presence of triplet excitation on a neighbouring carotenoid (Car). The difference in the behaviours of chloroplasts and LHCII, if reproducible, would imply that the Car triplets which give rise to the TmS spectrum of chloroplasts do not influence the absorption spectra of their Chla neighbours. With a view to reaching a firm conclusion about this vexed issue, spinach chloroplasts and thylakoids have been examined with the aid of the same kinetic spectrometer as that used for investigating LHCII; the TmS spectra of both chloroplasts and thylakoids contain prominent bleaching signals centred at 680 nm, and the triplet decay time in each case is comparable to that of the Chla/b LHCII triplets. Results pertaining to other closely related systems are recalled, and it is concluded that, so far as the overall appearance of the TmS spectrum is concerned, spinach chloroplasts are by no means abnormal. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Carotenoids; Photosynthesis; Triplet–triplet transfer; Chloroplasts
* Corresponding author. Tel.: + 47-73-591853; fax: +47-73-591852. E-mail address: [email protected] (K. Razi Naqvi) 1386-1425/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 9 9 ) 0 0 2 2 6 - 7
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T. Ja´6orfi et al. / Spectrochimica Acta Part A 56 (2000) 211–214
1. Introduction One often comes across systems, natural as well as man-made, where a carotenoid (Car) pigment is in the vicinity of a chlorophyll (Chl) or a Chl-like tetrapyrrole pigment [1]. The ordinary singlet–singlet (Sn S0) absorption spectrum of such a system [2,3] gives little indication of the interactions between the Car and its tetrapyrrole (Pyr) neighbour, but other photophysical data — derived, for example, from fluorescence excitation or kinetic absorption spectroscopy — have shown that the two chromophores do interact with each other [4–10]. The particular aspect which we wish to scrutinise here is the appearance, in the tripletminus-singlet (TmS) spectra, of a signal in the Qy region of Pyr, under conditions when the Pyr chromophores are unexcited; this signal, whose dominant feature is a negative peak, was noticed first by van der Vos, Carbonera and Hoff [4], when they examined Chla/b-LHCII (the Chla/b light-harvesting complex associated with the photosystem II of higher plants); analogous signals have since been found in several bacterial antenna complexes [5], in peridinin – Chla – protein (PCP) complexes isolated from two dinoflagellates [6], in thylakoids and Chla/c-LHCII of the yellow-green alga Pleurochloris meiringensis [7], and — very recently — in a synthetic bichromophoric compound [8]. Since a Car does not absorb significantly at wavelengths longer than 600 nm, van der Vos and co-authors concluded that the absorbance change in the Qy region, which will henceforth be represented by the symbol DA (Qy), reflects the perturbation, by the presence of triplet excitation on the Car, in the singlet-singlet absorption spectrum of nearby Pyr molecules; given this interpretation, the fact that DA (Qy) decays at the same rate as the rest of the TmS spectrum [7–10], requires no further explanation. It would not be too rash to expect, after examining the observations summarised above, that DA (Qy) will be present in the TmS spectrum of all photosynthetic samples, where Car chromophores are in close contact with Chl pigments. However, Wolf and Witt [11], who were the first to report the TmS spectrum of chloroplasts, emphasised the absence of such a signal through the following
statement: ‘‘No absorption changes are observed in the red region!’’. This result, if reproducible, would imply that the TmS signal in chloroplasts originates from a group of Car’s whose photophysical behaviour is appreciably different from the Car’s which give rise to the TmS spectrum of Chla/b-LHCII. Accordingly, we decided to re-investigate the TmS spectrum of chloroplasts under the same experimental conditions as those employed earlier for recording the TmS spectra of Chla/b-LHCII, Chla/c-LHCII, and of other systems where DA (Qy) was observed. It also seemed worthwhile to examine the TmS spectra of thylakoid membranes isolated from the same leaves as those used for preparing the chloroplasts. The results of these investigations are described below, and compared with previously published TmS spectra of natural and artificial antenna systems.
2. Materials and methods Chloroplasts were isolated from fresh spinach obtained in the local supermarket. Leaves were homogenised in a buffer containing 0.35 M sorbitol, 20 mM Tricine/NaOH, pH 7.6 and 0.2 mM MgCl2. The suspension was filtered through four layers of cheesecloth and centrifuged 20–30 s at 2000× g. The supernatant was centrifuged for 4 min at 4000 × g. The pellet was resuspended in a medium containing 1 mM Tricine/NaOH, pH 7.6 and 0.35 M sorbitol and centrifuged again at 4000× g for 4 min. Finally the pellet was resuspended in 20 mM Tricine/NaOH, pH 7.6 supplemented with 0.35 M sorbitol, 5 mM MgCl2 and 10 mM KCl, and was stored at 4°C. For thylakoid isolation the chloroplast pellet was resuspended in hypotonic buffer, containing 10 mM Tricine/NaOH, pH 7.6, 2 mM Na-EDTA and centrifuged at 27 000× g for 5 min. The thylakoid pellet was resuspended and stored in the same buffer as the chloroplasts [12]. The TmS spectra reported here were recorded by means of a nanosecond laser flash photolysis equipment with right angle geometry, which has been described earlier in some detail [9]. Briefly, the excitation source was an optical parametric oscillator pumped by the third harmonic of
T. Ja´6orfi et al. / Spectrochimica Acta Part A 56 (2000) 211–214
Nd:YAG laser; the duration of the excitation pulse was about 7 ns and its energy around 10 mJ. A 250 W xenon arc lamp and a mechanical chopper provided the analysing beam. A gated, multichannel spectrum analyser (with a linear dispersion of 0.6 nm per channel) served as the spectrophotometer; for the spectra reported here, the excitation wavelength was tuned to 670 nm, close to the maximum of the Qy peak (678 nm), the gate width was held at 50 ns, and the delay between the excitation pulse and the opening of the observation window was either 100 ns or 5 ms. For approaching oxygen-free conditions, high-purity argon was bubbled through the sample for 10 min prior to the measurements; during the measurements, the tip of the hypodermic needle bringing the argon to the sample was kept near the top
Fig. 1. Triplet-minus-singlet spectra of chloroplasts at two delays.
Fig. 2. Triplet-minus-singlet spectra of thylakoid membranes at two delays.
213
of the sample. All measurements were carried out at ambient temperature (ca. 295 K).
3. Results and discussion The technique of flash spectroscopy entails two measurements of the intensity of the monitoring beam after it has traversed the sample: one, denoted by I(l), refers to an unirradiated sample; the other, denoted by I0 (l;t), is the intensity at time t after the irradiation of the sample by a sufficiently narrow exictation pulse. Let A(l) and A0 (l;t) be the corresponding values of the absorbance. The resulting change in absorbance can then be calculated by means of the relation DA(l;t) A0 (l;t)− A(l)= lg[I(l)/I0 (l;t)]. When the change is caused by the population of the triplet state, the difference spectrum represents the triplet-minus-singlet (TmS) spectrum of the sample. Fig. 1 shows the TmS spectra obtained by irradiating a suspension of chloroplasts under anaerobic conditions; it is apparent that the halflife of the triplets is close to 5 ms. The spectra obtained by using aerated samples (not shown), though very similar, showed signs of contamination from free Chl triplets (probably released as a result of photodamage in the presence of oxygen); the half-life of the triplets in this case was estimated to be 2.2 ms. The TmS spectra of argonbubbled suspensions of thylakoid membranes are displayed in Fig. 2. All the spectra shown in Figs. 1 and 2 contain a negative signal centred at 680 nm, and their overall shape bears a great resemblance to the recently published TmS spectra of the following systems: Chla/b-LHCII [4,9,10,13], PCP complexes [6] LHCII and thylakoids of P. meirengensis [7]. Furthermore, our estimates of the half-life of Car triplets in chloroplasts (ca. 2.2 ms in aerobic conditions and ca. 5 ms in argon-bubbled samples) are in excellent agreement with those reported by Mathis, Butler and Satoh [14]. The half-life of Car triplets in deoxygenated suspensions of thylakoid membranes, estimated from an examination of the two spectra in Fig. 2, is slightly longer than 5 ms; since no attempt was
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T. Ja´6orfi et al. / Spectrochimica Acta Part A 56 (2000) 211–214
made to measure the final oxygen concentration in the samples, no significance is attached to the differences between the decay times in chloroplasts and thylakoids. Essentially the same spectra and lifetimes were obtained when DCMU or gramicidin was added to suspensions of thylakoids. The failure, on the part of Wolf and Witt [11], who used a long-pass filter for removing photons of wavelengths shorter than 610 nm from their pump source (a conventional flash lamp of sub-ms duration), to detect DA (Qy) might have been caused by insufficient protection, resulting in an overload, of the detector from scattered exciting light. Interference by scattered light ceases to be a problem in modern instruments, which use a tunable laser as the pump source. We conclude, on the basis of the spectra presented here and elsewhere [4 – 10,13], that the TmS spectrum of chloroplasts is similar to the TmS spectra of other natural and artificial systems, where Car pigments are in intimate contact with Pyr pigments, and that the presence of DA (Qy) is a telltale sign of Car – Pyr intimacy. Apart from PCP complexes [6], for which time-resolved TmS spectra are not yet available, the Car – Pyr contiguity also gives rise, in all systems examined so far, to efficient (close to 100%) and rapid (within a 100 ns or so) transfer of triplet excitation from a Pyr pigment to a neighbouring Car pigment. Since the magnitude of DA (Qy) and the rate of triplet–triplet transfer are both determined by the strength of Pyr – Car interaction, it seems likely that when time-resolved TmS spectra of PCP complexes are recorded, triplet transfer would be found to be as rapid as in the other systems mentioned above.
Acknowledgements TJ thanks the ESF programme Biophysics of Photosynthesis for financing a visit to Trondheim during which the investigation described above was started. The authors are also grateful to the Research Council of Norway (NFR).
References [1] H.A. Frank, R.J. Cogdell, Photochem. Photobiol. 63 (1996) 257. [2] D. Gust, T.A. Moore, A.L. Moore, C. Devadoss, P.A. Liddell, R. Hermant, R.A. Nieman, L.J. Demanche, J.M. DeGraziano, I. Gouni, J. Am. Chem. Soc. 114 (1992) 3590 (and references cited therein). [3] A. Osuka, T. Kume, Tetrahedron Lett. 39 (1998) 655 (and previous papers cited therein). [4] R. van der Vos, D. Carbonera, A.J. Hoff, Appl. Magn. Res. 2 (1991) 179. [5] A. Angerhofer, F. Bornha¨user, A. Gall, R.J. Cogdell, Chem. Phys. 194 (1995) 259. [6] D. Carbonera, G. Giacometti, U. Segre, J. Chem. Soc. Faraday Trans. 92 (1996) 989. [7] C. Bu¨chel, K. Razi Naqvi, T.B. Melø, Spectrochim. Acta Part A 54 (1998) 719. [8] A. Osuka, T. Kume, G. Haggquist, T. Ja´vorfi, J.C. Lima, E. Melo, K. Razi Naqvi, Chem. Phys. Lett. 313 (1999) 499. [9] E.J.G. Peterman, F.M. Dukker, R. van Grondelle, H. van Amerongen, Biophys. J. 69 (1995) 2670. [10] K. Razi Naqvi, T.B. Melø, B. Bangar Raju, T. Ja´vorfi, I. Simidjiev, G. Garab, Spectrochim. Acta Part A 53 (1997) 2659. [11] Ch. Wolf, H.T. Witt, Z. Naturfor. 24b (1969) 1031. [12] G. Peter, J.P. Thornber, J. Biol. Chem. 266 (25) (1991) 16745. [13] R. Scho¨del, K.-D. Irrgang, J. Voigt, G. Renger, Biophs. J. 75 (1998) 3143. [14] P. Mathis, W.L. Butler, K. Satoh, Photochem. Photobiol. 30 (1979) 603.
Letter
Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts T. Ja´vorfi a, G. Garab a, K. Razi Naqvi b,* a
Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, PO Box 521, H-6701 Szeged, Hungary b Department of Physics, Norwegian Uni6ersity of Science and Technology, N-7491 Trondheim, Norway Received 28 June 1999; received in revised form 28 September 1999; accepted 28 September 1999
Abstract A comparison of the triplet-minus-singlet (TmS) absorption spectrum of spinach chloroplasts, recorded some thirty years ago, with the more recently published TmS spectrum of isolated Chla/b LHCII (light-harvesting complexes associated with photosystem II of higher plants) shows that the two spectra are very similar, which is to be expected, since only the carotenoid pigments contribute to each spectrum. Be that as it may, the comparison also reveals a dissimilarity: photoexcitation of the sample does, or does not, affect the absorbance in the Qy region (650 – 700 nm), depending on whether the sample is a suspension of chloroplasts or of isolated LHCII. The Qy-signal in the TmS spectrum of LHCII decays, it should be noted, at the same rate as the rest of the difference spectrum, and its most prominent feature is a negative peak. As the carotenoids do not absorb in the Qy region, the presence of a signal in this region calls for an explanation: van der Vos, Carbonera and Hoff, the first to find as well as fathom the phenomenon, attributed the Qy-signal to a change, in the absorption spectrum of a chlorophyll a (Chla) molecule, brought about by the presence of triplet excitation on a neighbouring carotenoid (Car). The difference in the behaviours of chloroplasts and LHCII, if reproducible, would imply that the Car triplets which give rise to the TmS spectrum of chloroplasts do not influence the absorption spectra of their Chla neighbours. With a view to reaching a firm conclusion about this vexed issue, spinach chloroplasts and thylakoids have been examined with the aid of the same kinetic spectrometer as that used for investigating LHCII; the TmS spectra of both chloroplasts and thylakoids contain prominent bleaching signals centred at 680 nm, and the triplet decay time in each case is comparable to that of the Chla/b LHCII triplets. Results pertaining to other closely related systems are recalled, and it is concluded that, so far as the overall appearance of the TmS spectrum is concerned, spinach chloroplasts are by no means abnormal. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Carotenoids; Photosynthesis; Triplet–triplet transfer; Chloroplasts
* Corresponding author. Tel.: + 47-73-591853; fax: +47-73-591852. E-mail address: [email protected] (K. Razi Naqvi) 1386-1425/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 9 9 ) 0 0 2 2 6 - 7
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T. Ja´6orfi et al. / Spectrochimica Acta Part A 56 (2000) 211–214
1. Introduction One often comes across systems, natural as well as man-made, where a carotenoid (Car) pigment is in the vicinity of a chlorophyll (Chl) or a Chl-like tetrapyrrole pigment [1]. The ordinary singlet–singlet (Sn S0) absorption spectrum of such a system [2,3] gives little indication of the interactions between the Car and its tetrapyrrole (Pyr) neighbour, but other photophysical data — derived, for example, from fluorescence excitation or kinetic absorption spectroscopy — have shown that the two chromophores do interact with each other [4–10]. The particular aspect which we wish to scrutinise here is the appearance, in the tripletminus-singlet (TmS) spectra, of a signal in the Qy region of Pyr, under conditions when the Pyr chromophores are unexcited; this signal, whose dominant feature is a negative peak, was noticed first by van der Vos, Carbonera and Hoff [4], when they examined Chla/b-LHCII (the Chla/b light-harvesting complex associated with the photosystem II of higher plants); analogous signals have since been found in several bacterial antenna complexes [5], in peridinin – Chla – protein (PCP) complexes isolated from two dinoflagellates [6], in thylakoids and Chla/c-LHCII of the yellow-green alga Pleurochloris meiringensis [7], and — very recently — in a synthetic bichromophoric compound [8]. Since a Car does not absorb significantly at wavelengths longer than 600 nm, van der Vos and co-authors concluded that the absorbance change in the Qy region, which will henceforth be represented by the symbol DA (Qy), reflects the perturbation, by the presence of triplet excitation on the Car, in the singlet-singlet absorption spectrum of nearby Pyr molecules; given this interpretation, the fact that DA (Qy) decays at the same rate as the rest of the TmS spectrum [7–10], requires no further explanation. It would not be too rash to expect, after examining the observations summarised above, that DA (Qy) will be present in the TmS spectrum of all photosynthetic samples, where Car chromophores are in close contact with Chl pigments. However, Wolf and Witt [11], who were the first to report the TmS spectrum of chloroplasts, emphasised the absence of such a signal through the following
statement: ‘‘No absorption changes are observed in the red region!’’. This result, if reproducible, would imply that the TmS signal in chloroplasts originates from a group of Car’s whose photophysical behaviour is appreciably different from the Car’s which give rise to the TmS spectrum of Chla/b-LHCII. Accordingly, we decided to re-investigate the TmS spectrum of chloroplasts under the same experimental conditions as those employed earlier for recording the TmS spectra of Chla/b-LHCII, Chla/c-LHCII, and of other systems where DA (Qy) was observed. It also seemed worthwhile to examine the TmS spectra of thylakoid membranes isolated from the same leaves as those used for preparing the chloroplasts. The results of these investigations are described below, and compared with previously published TmS spectra of natural and artificial antenna systems.
2. Materials and methods Chloroplasts were isolated from fresh spinach obtained in the local supermarket. Leaves were homogenised in a buffer containing 0.35 M sorbitol, 20 mM Tricine/NaOH, pH 7.6 and 0.2 mM MgCl2. The suspension was filtered through four layers of cheesecloth and centrifuged 20–30 s at 2000× g. The supernatant was centrifuged for 4 min at 4000 × g. The pellet was resuspended in a medium containing 1 mM Tricine/NaOH, pH 7.6 and 0.35 M sorbitol and centrifuged again at 4000× g for 4 min. Finally the pellet was resuspended in 20 mM Tricine/NaOH, pH 7.6 supplemented with 0.35 M sorbitol, 5 mM MgCl2 and 10 mM KCl, and was stored at 4°C. For thylakoid isolation the chloroplast pellet was resuspended in hypotonic buffer, containing 10 mM Tricine/NaOH, pH 7.6, 2 mM Na-EDTA and centrifuged at 27 000× g for 5 min. The thylakoid pellet was resuspended and stored in the same buffer as the chloroplasts [12]. The TmS spectra reported here were recorded by means of a nanosecond laser flash photolysis equipment with right angle geometry, which has been described earlier in some detail [9]. Briefly, the excitation source was an optical parametric oscillator pumped by the third harmonic of
T. Ja´6orfi et al. / Spectrochimica Acta Part A 56 (2000) 211–214
Nd:YAG laser; the duration of the excitation pulse was about 7 ns and its energy around 10 mJ. A 250 W xenon arc lamp and a mechanical chopper provided the analysing beam. A gated, multichannel spectrum analyser (with a linear dispersion of 0.6 nm per channel) served as the spectrophotometer; for the spectra reported here, the excitation wavelength was tuned to 670 nm, close to the maximum of the Qy peak (678 nm), the gate width was held at 50 ns, and the delay between the excitation pulse and the opening of the observation window was either 100 ns or 5 ms. For approaching oxygen-free conditions, high-purity argon was bubbled through the sample for 10 min prior to the measurements; during the measurements, the tip of the hypodermic needle bringing the argon to the sample was kept near the top
Fig. 1. Triplet-minus-singlet spectra of chloroplasts at two delays.
Fig. 2. Triplet-minus-singlet spectra of thylakoid membranes at two delays.
213
of the sample. All measurements were carried out at ambient temperature (ca. 295 K).
3. Results and discussion The technique of flash spectroscopy entails two measurements of the intensity of the monitoring beam after it has traversed the sample: one, denoted by I(l), refers to an unirradiated sample; the other, denoted by I0 (l;t), is the intensity at time t after the irradiation of the sample by a sufficiently narrow exictation pulse. Let A(l) and A0 (l;t) be the corresponding values of the absorbance. The resulting change in absorbance can then be calculated by means of the relation DA(l;t) A0 (l;t)− A(l)= lg[I(l)/I0 (l;t)]. When the change is caused by the population of the triplet state, the difference spectrum represents the triplet-minus-singlet (TmS) spectrum of the sample. Fig. 1 shows the TmS spectra obtained by irradiating a suspension of chloroplasts under anaerobic conditions; it is apparent that the halflife of the triplets is close to 5 ms. The spectra obtained by using aerated samples (not shown), though very similar, showed signs of contamination from free Chl triplets (probably released as a result of photodamage in the presence of oxygen); the half-life of the triplets in this case was estimated to be 2.2 ms. The TmS spectra of argonbubbled suspensions of thylakoid membranes are displayed in Fig. 2. All the spectra shown in Figs. 1 and 2 contain a negative signal centred at 680 nm, and their overall shape bears a great resemblance to the recently published TmS spectra of the following systems: Chla/b-LHCII [4,9,10,13], PCP complexes [6] LHCII and thylakoids of P. meirengensis [7]. Furthermore, our estimates of the half-life of Car triplets in chloroplasts (ca. 2.2 ms in aerobic conditions and ca. 5 ms in argon-bubbled samples) are in excellent agreement with those reported by Mathis, Butler and Satoh [14]. The half-life of Car triplets in deoxygenated suspensions of thylakoid membranes, estimated from an examination of the two spectra in Fig. 2, is slightly longer than 5 ms; since no attempt was
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T. Ja´6orfi et al. / Spectrochimica Acta Part A 56 (2000) 211–214
made to measure the final oxygen concentration in the samples, no significance is attached to the differences between the decay times in chloroplasts and thylakoids. Essentially the same spectra and lifetimes were obtained when DCMU or gramicidin was added to suspensions of thylakoids. The failure, on the part of Wolf and Witt [11], who used a long-pass filter for removing photons of wavelengths shorter than 610 nm from their pump source (a conventional flash lamp of sub-ms duration), to detect DA (Qy) might have been caused by insufficient protection, resulting in an overload, of the detector from scattered exciting light. Interference by scattered light ceases to be a problem in modern instruments, which use a tunable laser as the pump source. We conclude, on the basis of the spectra presented here and elsewhere [4 – 10,13], that the TmS spectrum of chloroplasts is similar to the TmS spectra of other natural and artificial systems, where Car pigments are in intimate contact with Pyr pigments, and that the presence of DA (Qy) is a telltale sign of Car – Pyr intimacy. Apart from PCP complexes [6], for which time-resolved TmS spectra are not yet available, the Car – Pyr contiguity also gives rise, in all systems examined so far, to efficient (close to 100%) and rapid (within a 100 ns or so) transfer of triplet excitation from a Pyr pigment to a neighbouring Car pigment. Since the magnitude of DA (Qy) and the rate of triplet–triplet transfer are both determined by the strength of Pyr – Car interaction, it seems likely that when time-resolved TmS spectra of PCP complexes are recorded, triplet transfer would be found to be as rapid as in the other systems mentioned above.
Acknowledgements TJ thanks the ESF programme Biophysics of Photosynthesis for financing a visit to Trondheim during which the investigation described above was started. The authors are also grateful to the Research Council of Norway (NFR).
References [1] H.A. Frank, R.J. Cogdell, Photochem. Photobiol. 63 (1996) 257. [2] D. Gust, T.A. Moore, A.L. Moore, C. Devadoss, P.A. Liddell, R. Hermant, R.A. Nieman, L.J. Demanche, J.M. DeGraziano, I. Gouni, J. Am. Chem. Soc. 114 (1992) 3590 (and references cited therein). [3] A. Osuka, T. Kume, Tetrahedron Lett. 39 (1998) 655 (and previous papers cited therein). [4] R. van der Vos, D. Carbonera, A.J. Hoff, Appl. Magn. Res. 2 (1991) 179. [5] A. Angerhofer, F. Bornha¨user, A. Gall, R.J. Cogdell, Chem. Phys. 194 (1995) 259. [6] D. Carbonera, G. Giacometti, U. Segre, J. Chem. Soc. Faraday Trans. 92 (1996) 989. [7] C. Bu¨chel, K. Razi Naqvi, T.B. Melø, Spectrochim. Acta Part A 54 (1998) 719. [8] A. Osuka, T. Kume, G. Haggquist, T. Ja´vorfi, J.C. Lima, E. Melo, K. Razi Naqvi, Chem. Phys. Lett. 313 (1999) 499. [9] E.J.G. Peterman, F.M. Dukker, R. van Grondelle, H. van Amerongen, Biophys. J. 69 (1995) 2670. [10] K. Razi Naqvi, T.B. Melø, B. Bangar Raju, T. Ja´vorfi, I. Simidjiev, G. Garab, Spectrochim. Acta Part A 53 (1997) 2659. [11] Ch. Wolf, H.T. Witt, Z. Naturfor. 24b (1969) 1031. [12] G. Peter, J.P. Thornber, J. Biol. Chem. 266 (25) (1991) 16745. [13] R. Scho¨del, K.-D. Irrgang, J. Voigt, G. Renger, Biophs. J. 75 (1998) 3143. [14] P. Mathis, W.L. Butler, K. Satoh, Photochem. Photobiol. 30 (1979) 603.