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The energies and kinetics of triplet carotenoids in the LH2 antenna complexes as determined by phosphorescence spectroscopy
Chemical Physics Letters 384 (2004) 364–371 www.elsevier.com/locate/cplett
The energies and kinetics of triplet carotenoids in the LH2 antenna complexes as determined by phosphorescence spectroscopy Ferdy S. Rondonuwu a, Tokio Taguchi a, Ritsuko Fujii b, Kyosuke Yokoyama a, Yasushi Koyama b,*, Yasutaka Watanabe a a b
Department of Physics, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan Department of Chemistry, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan Received 4 September 2003; in final form 4 September 2003 Published online: 9 January 2004
Abstract The triplet (T1 ) states of carotenoids (Cars) and bacteriochlorophyll a (BChl) in the LH2 antenna complexes from Rhodobacter sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rhodospirillum molischianum, containing neurosporene, spheroidene and lycopene, respectively, were examined by stationary-state and time-resolved phosphorescence spectroscopy. The T1 energies of Cars were determined, irrespective of the Car or BChl excitation, to be 7030 cm1 (neurosporene), 6920 cm1 (spheroidene) and 6870 cm1 (lycopene), respectively, whereas that of BChl to be 7590 cm1 . In the Rba. sphaeroides G1C, the Car and BChl triplet states decayed in similar time constant as the BChl Qy state, a fact which indicates that the pair of triplet states decays through the triplet–triplet annihilation mechanism. Ó 2003 Elsevier B.V. All rights reserved.
1. Introduction Carotenoids (Cars) in bacterial photosynthetic systems have two different functions of light-harvesting and photo-protection [1,2]. In the former function, Cars collect the light energy and transfer it to bacteriochlorophyll a (BChl), whereas in the latter function, Cars quench triplet BChl to prevent sensitized generation of harmful singlet oxygen. Depending on the major function, two different kinds of configurations have been selected by the pigment–protein complexes: all-trans Cars are selectively bound to the antenna complexes, while 15-cis Cars to the reaction center [2,3]. In order to reveal the mechanisms of the light-harvesting and the photoprotective functions, the energies and kinetics of excitedstate Cars are key factors to be determined: Concerning the singlet-excited states, a set of four different low-lying 1 1 singlet-excited states, including 11 Bþ u , 1 Bu , 3 Ag and 1 2 Ag , were identified [4], and their relaxation pathways
*
Corresponding author. Fax: +81-795-65-9077. E-mail address: [email protected] (Y. Koyama).
0009-2614/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2003.12.024
and decay time constants have been determined [5,6]. However, the energies of the triplet-excited states remain to be determined, although the lifetimes of the lowest triplet (T1 13 Bu ) state have been determined free in solution and bound to the pigment–protein complexes [7]. The direct observation of the T1 state of Cars by phosphorescence spectroscopy has been hampered by the very low quantum yield of intersystem crossing, 103 [8], and by extremely low efficiency of the spinforbidden T1 ! So transition (phosphorescence). However, very recently, Martson et al. [9] determined the T1 energy of all-trans b-carotene to be 7360 250 cm1 by phosphorescence spectroscopy using sensitive, Fourier transform-based interferometry. There have been no other applications of phosphorescence spectroscopy to photosynthetic Cars as far as the authors know (see also [1,10] for reviewers). In order to estimate the Car T1 energies, the T1 energies of shorter polyenes with the number of conjugated double bonds n ¼ 1–4, which were determined by O2 -perturbed electronic-absorption spectroscopy [11–13], were extrapolated, by the use of an empirical equation, until n ¼ 13 [1]. The T1 energies were also evaluated by the use of triplet sensitizers [14].
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Theoretically, the Pariser–Parr–Pople calculations for shorter polyenes with n ¼ 2–8 including multi-reference, doubly-excited configurational interactions [15] have predicted that the T1 (13 Bu ) energy should be approximately one-half of the S1 (21 A g ) energy. Extrapolation 1 1 1 of the 11 Bþ u , 1 Bu , 3 Ag and 2 Ag energies that were calculated by this refined method [16] correctly predicted the ordering of those singlet states in Cars [4]. It is important to examine phosphorescence from alltrans Cars that are bound to the LH2 antenna complexes. Here, the BChl and Cars molecules are in van der Waals contact with each other, and therefore, BChl can function as an efficient triplet sensitizer for Cars. This attempt is important not only for the spectroscopic determination of the T1 -state energies of Car, in reference to that of BChl, but also for probing the kinetics of T1 Car and T1 BChl in the LH2 complexes. The energies and kinetics might be closely related to the light-harvesting and/or the photo-protective function(s). We built a setup for phosphorescence spectroscopy with extremely-high sensitivity, exhibiting an S=N ratio as high as 3000/200 around the triplet signal, by improving the performance of our setup that was used for detecting weak fluorescence from the 21 A g state [17]. Then, we examined the LH2 complexes from Rhodobacter (Rba.) sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rhodospirillum (Rsp.) molischianum, containing neurosporene (n ¼ 9), spheroidene (n ¼ 10) and lycopene (n ¼ 11), respectively. We have addressed the following three specific questions: (1) How do the T1 energies of those Cars compare with the T1 energy of BChl? (2) How fast do those T1 Cars and T1 BChl decay? (3) How can those energies and kinetics be correlated with the light-harvesting and/or the photo-protective function(s) in the particular antenna complexes?
2. Experimental 2.1. Sample preparation The LH2 complexes from Rba. sphaeroides G1C and 2.4.1 were prepared with some modifications of the method described previously [18,19]. The preparation of the LH2 complex from Rsp. molischianum (DSM 199) was described elsewhere [20]. The LH2 preparations exhibited the A850/A270 absorption ratio of 2.4, 2.9 and 2.3 for Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rsp. molischianum, respectively. The LH2 complexes were suspended in 20 mM Tris–HCl (pH 8.0) buffer containing 0.2% sucrose monocholate (SMC). 2.2. Setup for emission spectroscopy Each sample solution after degassing was sealed into a quartz tube having inner diameter of 3 mm, and
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mounted on a stage in a closed-cycle helium cryostat (Leybold-Heraeus, ROK 10-300 & RW2). Then, the temperature was set to 25 K by the use of a thermostat (Scientific Instrument, model 9650). For stationary-state emission spectroscopy, two different light sources were used for excitation; one, an Arþ laser (NEC, GLG 3050, 10–15 mW) for exciting Cars at 488 nm (no focusing), and the other, a diode laser (SHARP, LT024MD, 10 mW) for exciting BChl at 780 nm (a f ¼ 10 mm lens was used for correction of beam divergence). For the Arþ laser, the UV radiation and the 515 nm line were removed by the used of a prism spectrometer (Spectrolab, model Laserspec III). For emission spectroscopy in the longer-wavelength region, 90o emission was collected after passing through a glass filter that is transparent above 930 nm (PIP, F93), and focused onto the entrance slit (1 mm) of a monochromator (JASCO, M10-TP) equipped with a 600 lines/mm grating (blaze wavelength, 1000 nm). The output from the monochromator was detected by an InP/InGaAs (photocathode) NIR photo-multiplier (PMT) (Hamamatsu, R5509-71). By the use of a home-made liquid-nitrogen cooling chamber, the PMT was cooled down to 140 K (at the PMT surface) reducing the dark count down to about 700. (Note that at the lowest temperature recommended by the manufacturer of the PMT, i.e. 190 K, the dark current was more than 5000 counts, which was not sufficient for detecting weak phosphorescence signals.) The output signal from the PMT was lead to a set of amplifier and discriminator (PARC 1121), and then to a photon counter (PARC 1109). Here, emission was recorded in a mode compensating the above background; the dark counts during the sector of a mechanical chopper (170 Hz) was closed were subtracted from the signal counts during it was open. For time-resolved emission spectroscopy, time-correlated single photon-counting was used. As a light source, the output from a regenerative amplifier (Spectra Physics, Spitfire) that was seeded with a mode-locked Tisapphire laser (Spectra Physics, Tsunami) was used. It delivered 800 nm, 120 fs and 1 kHz pulses; the power was attenuated down to 0.8 lJ/pulse. The laser beam was split into two components; the major component was sent to the sample tube for the excitation of BChl, while the minor component was sent to a photodiode for synchronization of the detecting system. In order to minimize Ôthe dead timeÕ, we adopted Ôa reverse start/stopassignment configurationÕ for triggering a time-to-amplitude converter (TAC) unit (ORTEC, model 467). In this configuration Ôthe start signalÕ was driven by reaction product i.e., the phosphorescence signal from the PMT, through a constant fraction discriminator (CFD) (ORTEC, model 473A), while Ôthe stop signalÕ was picked up from the excitation pulse detected by the photodiode through another CFD and a delay unit (EG&G, model 425 Delay). The output signal from CFD was then ana-
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lyzed by a multichannel analyzer unit (ORTEC, model 5600 Multichannel analyzer) having 4096 channels, corresponding to the time interval of 12.2 ps, when the TAC was set to the 50 ns time range.
3. Results and discussion 3.1. Electronic absorption and fluorescence spectra: effect of intermolecular interaction between Car and BChl on the Qy fluorescence Table 1 lists the energies of the 11 Bþ u absorptions of Cars and the Qx and Qy absorptions of BChls bound to the LH2 complexes as determined by electronic absorption spectroscopy. The Car absorption systematically shifts to the lower energies when n increases, whereas the BChl absorptions are the same within the experimental error, 2 nm (30 cm1 ). The large red-shift of the Car 11 Bþ u (0) absorptions of the three Cars from, i.e., neurosporene 467 nm (21,410 cm1 ), spheroidene 484 nm (20,660 cm1 ) and lycopene 501 nm (19,960 cm1 ) free in n-hexane solution to the values listed in the table when bound to the LH2 complexes, as well as the large red-shift of the BChl Qy absorption from 769 nm (13,000 cm1 ) in acetone solution to 800 and 848 nm in the LH2 complexes reflect, at least partially, van der Waals (dispersive) interaction between the Car and BChl molecules. Table 2 lists the energies of the BChl Qy fluorescence; it shifts to the lower energies slightly on going from n ¼ 9–10, and more drastically on going from n ¼ 10–11. On the other hand, Table 1 shows that the Qy absorption of B850 BChl is the same within the experimental error, irrespective of the kind of Cars bound to the LH2 complex. Close examination of the chemical structures of the three Cars and the X-ray structures of
their binding sites, i.e., rhodopin glucoside and lycopene in the LH2 complexes from Rps. acidophilla 10,050 [21] and Rsp. molischianum [22], respectively, leads us to suggest that one end of the conjugated chain of Car and the conjugated macrocycle of B800a BChl start to interact with each other on going from spheroidene (n ¼ 10) to lycopene (n ¼ 11). The result strongly supports the idea that the particular pair of the Car and BChl molecules in the ring aggregate should be responsible for the observed down-shift of the Qy fluorescence, in the other words, functions as an outlet of the Qy singlet energy. Fig. 1 shows the entire spectral profile of the Qy fluorescence of the LH2 complex from Rba. sphaeroides G1C; the ordinate scale is expanded stepwise from the higher to the lower energies. As guided by the smooth background line, it is found that the Qy fluorescence exhibits a series of peaks due to vibrational progression (0 ! 0, 0 ! 1, 0 ! 2, and 0 ! 3) with an equal interval of 1040 cm1 . The peak intensity decreases systematically toward the lower energies. Close examination of the entire profile revealed the presence of an additional weak Qy progression, whose origin is shifted by 520 cm1 to the lower energy. 3.2. Phosphorescence spectra: the T1 energies of Cars and BChl Fig. 2a–c exhibit the emission spectra in the 6500– 9000 cm1 region for the LH2 complexes from (a) Rba. sphaeroides G1C, (b) Rba. sphaeroides 2.4.1, and (c) Rsp. molischianum both on excitation of Cars at 488 nm (solid line) and on excitation of BChl at 780 nm (broken line). The spectral profiles are similar to each other, irrespective of the Car or the BChl excitation. The pair of prominent peaks that appear below 8000 cm1 , in other words, at the end of the 0 ! 3 peak of the major Qy
Table 1 Electronic absorption peaks of carotenoids and bacteriochlorophyll a in the LH2 antenna complexes in nm (cm1 ) LH2 complex
Carotenoid (n)
Car 11 Bþ u 2
Rba. sphaeroides G1C Rba. sphaeroides 2.4.1 Rsp. molischianum
Neurosporene (9) Spheroidene (10) Lycopene (11)
0
432 (23,150) 450 (22,220) 467 (21,410)
BChl 1
0
459 (21,790) 476 (21,010) 494 (20,240)
0
0
491 (20,370) 510 (19,610) 529 (18,900)
Qx
Qy (B800)
Qy (B850)
589 (16,980) 589 (16,980) 589 (16,980)
800 (12,500) 800 (12,500) 800 (12,500)
8 48 (11,790) 849 (11,780) 848 (11,790)
Table 2 Phosphorescence and fluorescence peaks of carotenoids and bacteriochlorophyll a in the LH2 antenna complexes in nm (cm1 ) LH2 complex
Rba. sphaeroides G1C Rba. sphaeroides 2.4.1 Rsp. molischianum
Carotenoid (n)
Neurosporene (9) Spheroidene (10) Lycopene (11)
Fluorescence
Phosphorescence
Qy BChl
T1 Car
T1 BChl
D
884 (11,310) 888 (11,260) 903 (11,070)
1422 (7030) 1445 (6920) 1456 (6870)
1316 (7600) 1319 (7580) 1319 (7580)
(570) (660) (710)
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Fig. 1. An emission spectrum of the LH2 complex from Rba. sphaeroides G1C. The spectrum is expanded along the ordinate step-bystep toward the lower energies, and smooth background lines are drawn to indicate the progression peaks of the Qy BChl fluorescence and the phosphorescence peaks of the T1 BChl and T1 Car.
fluorescence progression (see Fig. 1 also), will be ascribed to phosphorescence from T1 BChl and T1 Cars as follows: The apparent peaks actually turned out to be a composite of the T1 BChl and the T1 Car phosphores-
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cence and the above-mentioned pair of the Qy BChl fluorescence progressions. Therefore, the decomposition of each entire spectral profile was necessary to correctly locate the phosphorescence peaks in this region. Here, we assumed, for all the three LH2 complexes, the pair of Qy fluorescence progressions mentioned above is present, i.e., one starting from the strongest peak of Qy (0) and the other starting from a shoulder shifted to the lower energy by 520 cm1 . Fig. 2d–f show the results of the decomposition of an averaged emission profiles of the Car excitation and the BChl excitation: The energies of the peaks on the higherenergy side were determined to be 7600, 7580 and 7580 cm1 in the LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rsp. molischianum, respectively (an average, 7590 cm1 ). On the other hand, the energies of the peak on the lower-energy side were 7030, 6920 and 6870 cm1 in those LH2 complexes containing neurosporene, spheroidene and lycopene, respectively. The energies of the former peaks are similar to one another, and in good agreement with that of BChl in the reaction center, 7590 cm1 , reported previously [23]. Therefore, this peak can be definitely assigned to T1 BChl. On the other hand, the energy of the latter peak systematically decreases with increasing n and it can be assigned to T1 Cars.
Fig. 2. Emission spectra of the LH2 complexes from (a) Rba. sphaeroides G1C, (b) Rba. sphaeroides 2.4.1 and (c) Rsp. molischianum for the Car excitation at 488 nm (solid lines) and for the BChl excitation at 780 nm (broken lines). Decomposition of the emission profiles (an average of the emission spectra on the BChl and Car excitation) into the T1 BChl and T1 Car phosphorescence peaks (shadowed) and a pair of Qy progressions (dotted lines); for the LH2 complexes from (d) Rba. sphaeroides G1C, (e) Rba. sphaeroides 2.4.1 and (f) Rsp. molischianum, composite spectra are shown by the use of smooth solid lines.
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Fig. 3 shows an energy diagram including the 11 Bþ u, 1 1 3 3 A g , 1 Bu and 2 Ag singlet states [4] and the 1 Bu (T1 ) state of Cars; the one-half of the 21 A g -state energy is also shown. The energy diagram also show the Qx and Qy singlet states and the T1 states of the B800 and B850 BChls for comparison. The energy of T1 BChl is basically the same. On the other hand, the energy of T1 Car decreases slightly but systematically as a function of 1=ð2n þ 1Þ. Interestingly, the slope for the T1 -state energy of Cars bound to the LH2 complexes are much gentler than that for one-half of the 21 A g -state energy, which was predicted by Tavan and Schulten [16]. The energies of T1 Cars in the LH2 complexes may be so tuned by the intermolecular interactions among the Car, BChl and peptide molecules as to facilitate an equilibrium between T1 Car and T1 BChl (vide infra). 1
up to the Qy state of B800-BChl, a pair of peaks due to T1 BChl and T1 Car was always seen in close proximity and with comparative intensity. The present observation may originate from the fact that the energy difference between T1 BChl and T1 Car is in the range of 570– 710 cm1 . This energy difference can be readily supplemented by heat that was generated in the processes of internal conversion, intersystem crossing and exciton reactions which should be followed by vibrational relaxation. Thus, both T1 BChl and T1 Car can coexist in almost equal amounts in the stationary state. Fig. 4 shows the decay time profiles of (a) T1 Car, (b) T1 BChl and (c) Qy BChl in the LH2 complex from Rba. sphaeroides G1C. For simplicity, we have tried to fit all the decay time profiles by a single exponential function, introducing a Gaussian-type instrumental response function with a full width at half maximum of 1.88 ns; the
3.3. Decay of phosphorescence: the triplet–triplet annihilation mechanism In the stationary-state emission spectroscopy using the CW excitation either up to the 11 Bþ u state of Cars or n 13
12
11
10
9
20000 11Bu+ _
11Bu
Qx
Energy / cm −1
_ 31Ag
15000
B800 B850
_
21Ag
Qy
B800 B850
10000
T1
1/
_
2
21Ag
B800 B850
13Bu
5000 0.04
0.05 1 / (2n +1)
Fig. 3. An energy diagram showing the dependence of the energies of 1 1 1 the singlet (11 Bþ u , 3 Ag , 1 Bu and 2 Ag ) states and that of the triplet 3 (1 BuT1 ) state on the number of conjugated double bonds, n (solid lines). The one-half of the 21 A g energy is also shown (dotted broken line). The energies of the Qx and Qy singlet states and that of the T1 state of B800 and B850 BChl are shown for comparison (dotted lines).
Fig. 4. Time profiles for (a) T1 Car, (b) T1 BChl and (c) Qy BChl in the LH2 complex from Rba. sphaeroides G1C. The decay time constants are indicated in each panel.
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following decay time constants were obtained: (a) 2.0 ns, (b) 1.8 ns and (c) 1.8 ns with an estimated error 0.3 ns. Interestingly, the decay time constants are equal, within the limit of experimental error, for all the T1 Car, T1 BChl and Qy BChl excited-state species. In solution, the decay time constant of T1 Car is on the order of microseconds [7,24] and that of T1 BChl is on the order of decamicroseconds [25,26]. Therefore, such short decay time constants presently observed can be explained only in terms of the triplet–triplet (T–T) annihilation reaction between them. Fig. 5 shows an energy diagram for the lowest singlet-excited (Qy ) state of BChl and triplet-excited (T1 ) states of both Cars and BChl. In BChl, all the energy levels are the same among the three antenna complexes. By the use of this energy diagram, we propose that the T–T annihilation reaction takes place as follows: Starting from the pair of T1 Car and T1 BChl, a T–T annihilation reaction to generate Qy BChl, i.e., T1 Car þ T1 BChl ! G Car þ Qy BChl ;
ð1Þ
can take place because the pair of reactions is exothermic in total in this Car, neurosporene (in Eq. (1), G stands for the ground state and asterisk indicates excited states). The mechanisms of the reaction for the LH2 complex from Rba. sphaeroides G1C is depicted in Fig. 5 (see the pair of solid arrows). However, the other T–T
Fig. 5. An energy diagram showing the triplet-excited states of both Cars and BChl and the Qy singlet-excited state of BChl in LH2 complex from Rba. sphaeroides G1C. The T–T annihilation reactions generating Qy BChl take place through a pair of electronic-exchange transitions shown by solid arrows (Car ! BChl and BChl ! Car ). Higher vibrational levels of BChl Qy need to be used to realize a pair of resonant, electron-exchanges transitions. Wavy arrow indicates delay fluorescence from BChl Qy state.
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1 annihilation reaction to generate 21 A g or 1 Bu Car (scheme not shown), 1 T1 Car þ T1 BChl ! 21 A g =1 Bu Car þ G BChl;
ð2Þ
cannot take place because the pair of reactions becomes endothermic in total. Assuming that only the former T–T annihilation reaction to generate Qy BChl is feasible, the same decay time constants observed for all the three excited states in Eq. (1) (shown by asterisks) can be easily understood when the particular T–T annihilation reaction is fast enough and the decay of Qy BChl is the ratedetermining process. 3.4. The roles of T1 Cars in the LH2 complexes Two different routes are now known for the generation of T1 Cars in the LH2 complexes: (1) Direct singletto-triplet conversion from a higher singlet state. Singletto-triplet conversion from a higher singlet state, branching from the singlet internal conversion, was first proposed, for spirilloxanthin and spheroidene in the LH1 and LH2 antenna complexes, respectively [27,28]. The branching singlet state was named Ôthe S* stateÕ. Most recently, we showed the presence of (1) the singlet inter1 1 1 nal conversion of 11 Bþ u ! 1 Bu ! 2 Ag ! 1 Ag (G), and (2) the singlet-to-triplet internal conversion of 11 B u ! 13 Ag (T2 ) followed by the triplet internal conversion of 13 Ag (T2 ) ! 13 Bu (T1 ) [6]. The 11 B u ! T2 branching ratio increased from 3% to 26% on going from n ¼ 9 to 13. Thus, T1 Cars can be generated by the singlet-to-triplet conversion from the 11 B u state. (2) Triplet-energy transfer from BChl. The energy diagram in Fig. 3 shows that plural channels of Carto-BChl singlet-energy transfer are feasible. Since the internal conversion within the Car molecule takes place very rapidly (in the 1014 –1011 s range), strong Coulombic coupling due to close contacts between the Car and BChl molecules in the LH2 complexes [29] is absolutely necessary to facilitate the efficient singlet-energy transfer reaction. The resultant BChl Qy state efficiently intersystem cross into the T1 state, the quantum yield being P40% [30]. Then, T1 Car can be generated by triplet-energy transfer from T1 BChl. The arrangement of their energy levels shown in Fig. 3 as well as the above-mentioned close contacts between the BChl and Car molecules facilitate this BChl-to-Car triplet-energy transfer reaction through the electron exchange mechanism. It is to be noted that T1 Car and T1 BChl can reach an equilibrium by the use of thermal energy. This equilibrium also facilitates the T–T annihilation reaction to take place. Based on the above considerations, the possible roles of T1 Cars in the LH2 complex can be proposed as follows: (a) An energy sink. When too much light energy is absorbed by Cars, the 11 Bþ u state can be densely
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populated. During the process of internal conversion, the 11 B u state can be relaxed eventually to the T1 state through the singlet-to-triplet conversion to the T2 state followed by the internal-conversion down to the T1 state. When too much light energy is absorbed by BChl, or transferred from Car, the Qy state can be highly populated. Then, the Qy state can be relaxed to the T1 state through efficient intersystem crossing. In those processes of internal conversion, intersystem crossing and vibrational relaxation, the excess energy can be dissipated as heat. Since T1 Cars are energetically lower than T1 BChl, the BChl to Car triplet-energy transfer takes place, and the T1 energy is eventually dissipated through Cars. Thus, T1 Car can function as an energy sink. (b) An energy reservoir and an energy supplier. The residual energy can be stored in T1 Car within its lifetime. As seen in the present investigation, Cars do not quench T1 BChl completely, most probably due to their delicately tuned T1 energies and to achieve the thermal equilibrium condition. As a result, both T1 Car and T1 BChl co-exists in the LH2 complexes to facilitate the T–T annihilation reaction to generate Qy BChl, whose energy can then be transferred to the BChl molecules in the LH1 complex, and eventually to the special-pair BChls in the reaction center. This type of delayed singlet-energy transfer can continue within the lifetime of T1 Car, i.e., on the order of microseconds.
4. Conclusion The following answers to the questions addressed in Section 1 have been obtained: (1) In the LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rsp. molischianum, the T1 energies of neurosporene (n ¼ 9), spheroidene (n ¼ 10) and lycopene (n ¼ 11) have been determined to be 7030, 6920 and 6870 cm1 , respectively, whereas the T1 energy of BChl a to be 7590 cm1 . The Car T1 energy decreases slightly but systematically when n increases, but the energy difference between T1 BChl and T1 Car is small enough to allow those species co-exist in the stationary state. (2) T1 Car and T1 BChl decay simultaneously in LH2 complex from Rba. sphaeroides G1C. Their decay time constants were the same as the decay time constant of the BChl Qy fluorescence. Therefore, the T–T annihilation generating the BChl Qy state must be in operation. (3) The T1 Car can function as an energy sink when excess energy is present in both Car and BChl singlet states in LH2 complexes. However, Car does not quench T1 BChl completely but allows T1 BChl to remain in equilibrium. Then, the T–T annihilation reaction between the pair of triplet species converts the triplet energy into Qy BChl for delay singlet-energy transfer. Thus, T1 Car performs both the photo-protective and the light-harvesting functions.
Acknowledgements This work has been supported by a grant from Ministry of Education, Science, Sport and Culture (Open Research Center Project) and a grant from NEDO (New Energy and Industrial Technology Development Organization, International Joint Research Grant).
References [1] H.A. Frank, R.J. Cogdell, in: A. Young, G. Britton (Eds.), Carotenoids in Photosynthesis, Chapman & Hall, London, 1993, p. 252. [2] Y. Koyama, R. Fujii, in: H.A. Frank, A.J. Young, G. Britton, R.J. Cogdell (Eds.), Advances in Photosynthesis, The Photochemistry of Carotenoids, vol. 8, Kluwer Academic Publishers, London, 1999, p. 161. [3] Y. Koyama, J. Photochem. Photobiol. B: Biology 9 (1991) 265. [4] K. Furuichi, T. Sashima, Y. Koyama, Chem. Phys. Lett. 356 (2002) 547. [5] R. Fujii, T. Inaba, Y. Watanabe, Y. Koyama, J.-P. Zhang, Chem. Phys. Lett. 369 (2003) 165. [6] F.S. Rondonuwu, Y. Watanabe, R. Fujii, Y. Koyama, Chem. Phys. Lett. 376 (2003) 292. [7] Y. Koyama, H. Hashimoto, in: A. Young, Britton (Eds.), Carotenoids in Photosynthesis, Chapman & Hall, London, 1993, p. 327. [8] H. Hashimoto, Y. Koyama, Y. Hirata, N. Mataga, J. Phys. Chem. 95 (1991) 3072. [9] G. Martson, T.G. Truscott, R.P. Wayne, J. Chem. Soc. Faraday Trans. 91 (1995) 4059. [10] R.L. Christensen, in: H.A. Frank, A.J. Young, G. Britton, R.J. Cogdell (Eds.), Advances in Photosynthesis, The Photochemistry of Carotenoids, vol. 8, Kluwer Academic Publishers, London, 1999, p. 137. [11] D.F. Evans, J. Chem. Soc. (1960) 1735. [12] D.F. Evans, J. Chem. Soc. (1961) 2566. [13] D.F. Evans, J.N. Tucker, J. Chem. Soc. Faraday Trans. II 54 (1972) 174. [14] R. Bensasson, E.J. Land, B. Maudinas, Photochem. Photobiol. 23 (1976) 189. [15] P. Tavan, K. Schulten, J. Chem. Phys. 85 (1986) 6602. [16] P. Tavan, K. Schulten, Phys. Rev. B 36 (1987) 4337. [17] R. Fujii, K. Onaka, H. Nagae, Y. Koyama, Y. Watanabe, J. Luminescence 92 (2001) 213. [18] R.J. Cogdell, I. Durant, J. Valentine, J.G. Lindsay, K. Schmidt, Biochim. Biophys. Acta 722 (1983) 427. [19] M.B. Evans, R.J. Cogdell, G. Britton, Biochim. Biophys. Acta 935 (1988) 292. [20] J.-P. Zhang, R. Fujii, P. Qian, T. Inaba, T. Mizoguchi, Y. Koyama, K. Onaka, Y. Watanabe, H. Nagae, J. Phys. Chem. B 104 (2000) 3683. [21] G. McDermott, S.M. Prince, A.A. Freer, A.M. HawthornthwaiteLawless, M.Z. Papiz, R.J. Cogdell, N.W. Isaacs, Nature 374 (1995) 517. [22] J. Koepke, X. Hu, C. Muenke, K. Schulten, H. Michel, Structure 4 (1996) 581. [23] L. Takiff, S.G. Boxer, J. Am. Chem. Soc. 110 (1988) 4425. [24] R. Fujii, K. Furuichi, J.-P. Zhang, H. Nagae, H. Hashimoto, Y. Koyama, J. Phys. Chem. A 106 (2002) 2410. [25] Y. Koyama, Y. Mukai, M. Kuki, SPIE Laser Spectrosc. Biomolecules 1921 (1992) 191.
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The energies and kinetics of triplet carotenoids in the LH2 antenna complexes as determined by phosphorescence spectroscopy Ferdy S. Rondonuwu a, Tokio Taguchi a, Ritsuko Fujii b, Kyosuke Yokoyama a, Yasushi Koyama b,*, Yasutaka Watanabe a a b
Department of Physics, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan Department of Chemistry, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan Received 4 September 2003; in final form 4 September 2003 Published online: 9 January 2004
Abstract The triplet (T1 ) states of carotenoids (Cars) and bacteriochlorophyll a (BChl) in the LH2 antenna complexes from Rhodobacter sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rhodospirillum molischianum, containing neurosporene, spheroidene and lycopene, respectively, were examined by stationary-state and time-resolved phosphorescence spectroscopy. The T1 energies of Cars were determined, irrespective of the Car or BChl excitation, to be 7030 cm1 (neurosporene), 6920 cm1 (spheroidene) and 6870 cm1 (lycopene), respectively, whereas that of BChl to be 7590 cm1 . In the Rba. sphaeroides G1C, the Car and BChl triplet states decayed in similar time constant as the BChl Qy state, a fact which indicates that the pair of triplet states decays through the triplet–triplet annihilation mechanism. Ó 2003 Elsevier B.V. All rights reserved.
1. Introduction Carotenoids (Cars) in bacterial photosynthetic systems have two different functions of light-harvesting and photo-protection [1,2]. In the former function, Cars collect the light energy and transfer it to bacteriochlorophyll a (BChl), whereas in the latter function, Cars quench triplet BChl to prevent sensitized generation of harmful singlet oxygen. Depending on the major function, two different kinds of configurations have been selected by the pigment–protein complexes: all-trans Cars are selectively bound to the antenna complexes, while 15-cis Cars to the reaction center [2,3]. In order to reveal the mechanisms of the light-harvesting and the photoprotective functions, the energies and kinetics of excitedstate Cars are key factors to be determined: Concerning the singlet-excited states, a set of four different low-lying 1 1 singlet-excited states, including 11 Bþ u , 1 Bu , 3 Ag and 1 2 Ag , were identified [4], and their relaxation pathways
*
Corresponding author. Fax: +81-795-65-9077. E-mail address: [email protected] (Y. Koyama).
0009-2614/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2003.12.024
and decay time constants have been determined [5,6]. However, the energies of the triplet-excited states remain to be determined, although the lifetimes of the lowest triplet (T1 13 Bu ) state have been determined free in solution and bound to the pigment–protein complexes [7]. The direct observation of the T1 state of Cars by phosphorescence spectroscopy has been hampered by the very low quantum yield of intersystem crossing, 103 [8], and by extremely low efficiency of the spinforbidden T1 ! So transition (phosphorescence). However, very recently, Martson et al. [9] determined the T1 energy of all-trans b-carotene to be 7360 250 cm1 by phosphorescence spectroscopy using sensitive, Fourier transform-based interferometry. There have been no other applications of phosphorescence spectroscopy to photosynthetic Cars as far as the authors know (see also [1,10] for reviewers). In order to estimate the Car T1 energies, the T1 energies of shorter polyenes with the number of conjugated double bonds n ¼ 1–4, which were determined by O2 -perturbed electronic-absorption spectroscopy [11–13], were extrapolated, by the use of an empirical equation, until n ¼ 13 [1]. The T1 energies were also evaluated by the use of triplet sensitizers [14].
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Theoretically, the Pariser–Parr–Pople calculations for shorter polyenes with n ¼ 2–8 including multi-reference, doubly-excited configurational interactions [15] have predicted that the T1 (13 Bu ) energy should be approximately one-half of the S1 (21 A g ) energy. Extrapolation 1 1 1 of the 11 Bþ u , 1 Bu , 3 Ag and 2 Ag energies that were calculated by this refined method [16] correctly predicted the ordering of those singlet states in Cars [4]. It is important to examine phosphorescence from alltrans Cars that are bound to the LH2 antenna complexes. Here, the BChl and Cars molecules are in van der Waals contact with each other, and therefore, BChl can function as an efficient triplet sensitizer for Cars. This attempt is important not only for the spectroscopic determination of the T1 -state energies of Car, in reference to that of BChl, but also for probing the kinetics of T1 Car and T1 BChl in the LH2 complexes. The energies and kinetics might be closely related to the light-harvesting and/or the photo-protective function(s). We built a setup for phosphorescence spectroscopy with extremely-high sensitivity, exhibiting an S=N ratio as high as 3000/200 around the triplet signal, by improving the performance of our setup that was used for detecting weak fluorescence from the 21 A g state [17]. Then, we examined the LH2 complexes from Rhodobacter (Rba.) sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rhodospirillum (Rsp.) molischianum, containing neurosporene (n ¼ 9), spheroidene (n ¼ 10) and lycopene (n ¼ 11), respectively. We have addressed the following three specific questions: (1) How do the T1 energies of those Cars compare with the T1 energy of BChl? (2) How fast do those T1 Cars and T1 BChl decay? (3) How can those energies and kinetics be correlated with the light-harvesting and/or the photo-protective function(s) in the particular antenna complexes?
2. Experimental 2.1. Sample preparation The LH2 complexes from Rba. sphaeroides G1C and 2.4.1 were prepared with some modifications of the method described previously [18,19]. The preparation of the LH2 complex from Rsp. molischianum (DSM 199) was described elsewhere [20]. The LH2 preparations exhibited the A850/A270 absorption ratio of 2.4, 2.9 and 2.3 for Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rsp. molischianum, respectively. The LH2 complexes were suspended in 20 mM Tris–HCl (pH 8.0) buffer containing 0.2% sucrose monocholate (SMC). 2.2. Setup for emission spectroscopy Each sample solution after degassing was sealed into a quartz tube having inner diameter of 3 mm, and
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mounted on a stage in a closed-cycle helium cryostat (Leybold-Heraeus, ROK 10-300 & RW2). Then, the temperature was set to 25 K by the use of a thermostat (Scientific Instrument, model 9650). For stationary-state emission spectroscopy, two different light sources were used for excitation; one, an Arþ laser (NEC, GLG 3050, 10–15 mW) for exciting Cars at 488 nm (no focusing), and the other, a diode laser (SHARP, LT024MD, 10 mW) for exciting BChl at 780 nm (a f ¼ 10 mm lens was used for correction of beam divergence). For the Arþ laser, the UV radiation and the 515 nm line were removed by the used of a prism spectrometer (Spectrolab, model Laserspec III). For emission spectroscopy in the longer-wavelength region, 90o emission was collected after passing through a glass filter that is transparent above 930 nm (PIP, F93), and focused onto the entrance slit (1 mm) of a monochromator (JASCO, M10-TP) equipped with a 600 lines/mm grating (blaze wavelength, 1000 nm). The output from the monochromator was detected by an InP/InGaAs (photocathode) NIR photo-multiplier (PMT) (Hamamatsu, R5509-71). By the use of a home-made liquid-nitrogen cooling chamber, the PMT was cooled down to 140 K (at the PMT surface) reducing the dark count down to about 700. (Note that at the lowest temperature recommended by the manufacturer of the PMT, i.e. 190 K, the dark current was more than 5000 counts, which was not sufficient for detecting weak phosphorescence signals.) The output signal from the PMT was lead to a set of amplifier and discriminator (PARC 1121), and then to a photon counter (PARC 1109). Here, emission was recorded in a mode compensating the above background; the dark counts during the sector of a mechanical chopper (170 Hz) was closed were subtracted from the signal counts during it was open. For time-resolved emission spectroscopy, time-correlated single photon-counting was used. As a light source, the output from a regenerative amplifier (Spectra Physics, Spitfire) that was seeded with a mode-locked Tisapphire laser (Spectra Physics, Tsunami) was used. It delivered 800 nm, 120 fs and 1 kHz pulses; the power was attenuated down to 0.8 lJ/pulse. The laser beam was split into two components; the major component was sent to the sample tube for the excitation of BChl, while the minor component was sent to a photodiode for synchronization of the detecting system. In order to minimize Ôthe dead timeÕ, we adopted Ôa reverse start/stopassignment configurationÕ for triggering a time-to-amplitude converter (TAC) unit (ORTEC, model 467). In this configuration Ôthe start signalÕ was driven by reaction product i.e., the phosphorescence signal from the PMT, through a constant fraction discriminator (CFD) (ORTEC, model 473A), while Ôthe stop signalÕ was picked up from the excitation pulse detected by the photodiode through another CFD and a delay unit (EG&G, model 425 Delay). The output signal from CFD was then ana-
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lyzed by a multichannel analyzer unit (ORTEC, model 5600 Multichannel analyzer) having 4096 channels, corresponding to the time interval of 12.2 ps, when the TAC was set to the 50 ns time range.
3. Results and discussion 3.1. Electronic absorption and fluorescence spectra: effect of intermolecular interaction between Car and BChl on the Qy fluorescence Table 1 lists the energies of the 11 Bþ u absorptions of Cars and the Qx and Qy absorptions of BChls bound to the LH2 complexes as determined by electronic absorption spectroscopy. The Car absorption systematically shifts to the lower energies when n increases, whereas the BChl absorptions are the same within the experimental error, 2 nm (30 cm1 ). The large red-shift of the Car 11 Bþ u (0) absorptions of the three Cars from, i.e., neurosporene 467 nm (21,410 cm1 ), spheroidene 484 nm (20,660 cm1 ) and lycopene 501 nm (19,960 cm1 ) free in n-hexane solution to the values listed in the table when bound to the LH2 complexes, as well as the large red-shift of the BChl Qy absorption from 769 nm (13,000 cm1 ) in acetone solution to 800 and 848 nm in the LH2 complexes reflect, at least partially, van der Waals (dispersive) interaction between the Car and BChl molecules. Table 2 lists the energies of the BChl Qy fluorescence; it shifts to the lower energies slightly on going from n ¼ 9–10, and more drastically on going from n ¼ 10–11. On the other hand, Table 1 shows that the Qy absorption of B850 BChl is the same within the experimental error, irrespective of the kind of Cars bound to the LH2 complex. Close examination of the chemical structures of the three Cars and the X-ray structures of
their binding sites, i.e., rhodopin glucoside and lycopene in the LH2 complexes from Rps. acidophilla 10,050 [21] and Rsp. molischianum [22], respectively, leads us to suggest that one end of the conjugated chain of Car and the conjugated macrocycle of B800a BChl start to interact with each other on going from spheroidene (n ¼ 10) to lycopene (n ¼ 11). The result strongly supports the idea that the particular pair of the Car and BChl molecules in the ring aggregate should be responsible for the observed down-shift of the Qy fluorescence, in the other words, functions as an outlet of the Qy singlet energy. Fig. 1 shows the entire spectral profile of the Qy fluorescence of the LH2 complex from Rba. sphaeroides G1C; the ordinate scale is expanded stepwise from the higher to the lower energies. As guided by the smooth background line, it is found that the Qy fluorescence exhibits a series of peaks due to vibrational progression (0 ! 0, 0 ! 1, 0 ! 2, and 0 ! 3) with an equal interval of 1040 cm1 . The peak intensity decreases systematically toward the lower energies. Close examination of the entire profile revealed the presence of an additional weak Qy progression, whose origin is shifted by 520 cm1 to the lower energy. 3.2. Phosphorescence spectra: the T1 energies of Cars and BChl Fig. 2a–c exhibit the emission spectra in the 6500– 9000 cm1 region for the LH2 complexes from (a) Rba. sphaeroides G1C, (b) Rba. sphaeroides 2.4.1, and (c) Rsp. molischianum both on excitation of Cars at 488 nm (solid line) and on excitation of BChl at 780 nm (broken line). The spectral profiles are similar to each other, irrespective of the Car or the BChl excitation. The pair of prominent peaks that appear below 8000 cm1 , in other words, at the end of the 0 ! 3 peak of the major Qy
Table 1 Electronic absorption peaks of carotenoids and bacteriochlorophyll a in the LH2 antenna complexes in nm (cm1 ) LH2 complex
Carotenoid (n)
Car 11 Bþ u 2
Rba. sphaeroides G1C Rba. sphaeroides 2.4.1 Rsp. molischianum
Neurosporene (9) Spheroidene (10) Lycopene (11)
0
432 (23,150) 450 (22,220) 467 (21,410)
BChl 1
0
459 (21,790) 476 (21,010) 494 (20,240)
0
0
491 (20,370) 510 (19,610) 529 (18,900)
Qx
Qy (B800)
Qy (B850)
589 (16,980) 589 (16,980) 589 (16,980)
800 (12,500) 800 (12,500) 800 (12,500)
8 48 (11,790) 849 (11,780) 848 (11,790)
Table 2 Phosphorescence and fluorescence peaks of carotenoids and bacteriochlorophyll a in the LH2 antenna complexes in nm (cm1 ) LH2 complex
Rba. sphaeroides G1C Rba. sphaeroides 2.4.1 Rsp. molischianum
Carotenoid (n)
Neurosporene (9) Spheroidene (10) Lycopene (11)
Fluorescence
Phosphorescence
Qy BChl
T1 Car
T1 BChl
D
884 (11,310) 888 (11,260) 903 (11,070)
1422 (7030) 1445 (6920) 1456 (6870)
1316 (7600) 1319 (7580) 1319 (7580)
(570) (660) (710)
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Fig. 1. An emission spectrum of the LH2 complex from Rba. sphaeroides G1C. The spectrum is expanded along the ordinate step-bystep toward the lower energies, and smooth background lines are drawn to indicate the progression peaks of the Qy BChl fluorescence and the phosphorescence peaks of the T1 BChl and T1 Car.
fluorescence progression (see Fig. 1 also), will be ascribed to phosphorescence from T1 BChl and T1 Cars as follows: The apparent peaks actually turned out to be a composite of the T1 BChl and the T1 Car phosphores-
367
cence and the above-mentioned pair of the Qy BChl fluorescence progressions. Therefore, the decomposition of each entire spectral profile was necessary to correctly locate the phosphorescence peaks in this region. Here, we assumed, for all the three LH2 complexes, the pair of Qy fluorescence progressions mentioned above is present, i.e., one starting from the strongest peak of Qy (0) and the other starting from a shoulder shifted to the lower energy by 520 cm1 . Fig. 2d–f show the results of the decomposition of an averaged emission profiles of the Car excitation and the BChl excitation: The energies of the peaks on the higherenergy side were determined to be 7600, 7580 and 7580 cm1 in the LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rsp. molischianum, respectively (an average, 7590 cm1 ). On the other hand, the energies of the peak on the lower-energy side were 7030, 6920 and 6870 cm1 in those LH2 complexes containing neurosporene, spheroidene and lycopene, respectively. The energies of the former peaks are similar to one another, and in good agreement with that of BChl in the reaction center, 7590 cm1 , reported previously [23]. Therefore, this peak can be definitely assigned to T1 BChl. On the other hand, the energy of the latter peak systematically decreases with increasing n and it can be assigned to T1 Cars.
Fig. 2. Emission spectra of the LH2 complexes from (a) Rba. sphaeroides G1C, (b) Rba. sphaeroides 2.4.1 and (c) Rsp. molischianum for the Car excitation at 488 nm (solid lines) and for the BChl excitation at 780 nm (broken lines). Decomposition of the emission profiles (an average of the emission spectra on the BChl and Car excitation) into the T1 BChl and T1 Car phosphorescence peaks (shadowed) and a pair of Qy progressions (dotted lines); for the LH2 complexes from (d) Rba. sphaeroides G1C, (e) Rba. sphaeroides 2.4.1 and (f) Rsp. molischianum, composite spectra are shown by the use of smooth solid lines.
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Fig. 3 shows an energy diagram including the 11 Bþ u, 1 1 3 3 A g , 1 Bu and 2 Ag singlet states [4] and the 1 Bu (T1 ) state of Cars; the one-half of the 21 A g -state energy is also shown. The energy diagram also show the Qx and Qy singlet states and the T1 states of the B800 and B850 BChls for comparison. The energy of T1 BChl is basically the same. On the other hand, the energy of T1 Car decreases slightly but systematically as a function of 1=ð2n þ 1Þ. Interestingly, the slope for the T1 -state energy of Cars bound to the LH2 complexes are much gentler than that for one-half of the 21 A g -state energy, which was predicted by Tavan and Schulten [16]. The energies of T1 Cars in the LH2 complexes may be so tuned by the intermolecular interactions among the Car, BChl and peptide molecules as to facilitate an equilibrium between T1 Car and T1 BChl (vide infra). 1
up to the Qy state of B800-BChl, a pair of peaks due to T1 BChl and T1 Car was always seen in close proximity and with comparative intensity. The present observation may originate from the fact that the energy difference between T1 BChl and T1 Car is in the range of 570– 710 cm1 . This energy difference can be readily supplemented by heat that was generated in the processes of internal conversion, intersystem crossing and exciton reactions which should be followed by vibrational relaxation. Thus, both T1 BChl and T1 Car can coexist in almost equal amounts in the stationary state. Fig. 4 shows the decay time profiles of (a) T1 Car, (b) T1 BChl and (c) Qy BChl in the LH2 complex from Rba. sphaeroides G1C. For simplicity, we have tried to fit all the decay time profiles by a single exponential function, introducing a Gaussian-type instrumental response function with a full width at half maximum of 1.88 ns; the
3.3. Decay of phosphorescence: the triplet–triplet annihilation mechanism In the stationary-state emission spectroscopy using the CW excitation either up to the 11 Bþ u state of Cars or n 13
12
11
10
9
20000 11Bu+ _
11Bu
Qx
Energy / cm −1
_ 31Ag
15000
B800 B850
_
21Ag
Qy
B800 B850
10000
T1
1/
_
2
21Ag
B800 B850
13Bu
5000 0.04
0.05 1 / (2n +1)
Fig. 3. An energy diagram showing the dependence of the energies of 1 1 1 the singlet (11 Bþ u , 3 Ag , 1 Bu and 2 Ag ) states and that of the triplet 3 (1 BuT1 ) state on the number of conjugated double bonds, n (solid lines). The one-half of the 21 A g energy is also shown (dotted broken line). The energies of the Qx and Qy singlet states and that of the T1 state of B800 and B850 BChl are shown for comparison (dotted lines).
Fig. 4. Time profiles for (a) T1 Car, (b) T1 BChl and (c) Qy BChl in the LH2 complex from Rba. sphaeroides G1C. The decay time constants are indicated in each panel.
F.S. Rondonuwu et al. / Chemical Physics Letters 384 (2004) 364–371
following decay time constants were obtained: (a) 2.0 ns, (b) 1.8 ns and (c) 1.8 ns with an estimated error 0.3 ns. Interestingly, the decay time constants are equal, within the limit of experimental error, for all the T1 Car, T1 BChl and Qy BChl excited-state species. In solution, the decay time constant of T1 Car is on the order of microseconds [7,24] and that of T1 BChl is on the order of decamicroseconds [25,26]. Therefore, such short decay time constants presently observed can be explained only in terms of the triplet–triplet (T–T) annihilation reaction between them. Fig. 5 shows an energy diagram for the lowest singlet-excited (Qy ) state of BChl and triplet-excited (T1 ) states of both Cars and BChl. In BChl, all the energy levels are the same among the three antenna complexes. By the use of this energy diagram, we propose that the T–T annihilation reaction takes place as follows: Starting from the pair of T1 Car and T1 BChl, a T–T annihilation reaction to generate Qy BChl, i.e., T1 Car þ T1 BChl ! G Car þ Qy BChl ;
ð1Þ
can take place because the pair of reactions is exothermic in total in this Car, neurosporene (in Eq. (1), G stands for the ground state and asterisk indicates excited states). The mechanisms of the reaction for the LH2 complex from Rba. sphaeroides G1C is depicted in Fig. 5 (see the pair of solid arrows). However, the other T–T
Fig. 5. An energy diagram showing the triplet-excited states of both Cars and BChl and the Qy singlet-excited state of BChl in LH2 complex from Rba. sphaeroides G1C. The T–T annihilation reactions generating Qy BChl take place through a pair of electronic-exchange transitions shown by solid arrows (Car ! BChl and BChl ! Car ). Higher vibrational levels of BChl Qy need to be used to realize a pair of resonant, electron-exchanges transitions. Wavy arrow indicates delay fluorescence from BChl Qy state.
369
1 annihilation reaction to generate 21 A g or 1 Bu Car (scheme not shown), 1 T1 Car þ T1 BChl ! 21 A g =1 Bu Car þ G BChl;
ð2Þ
cannot take place because the pair of reactions becomes endothermic in total. Assuming that only the former T–T annihilation reaction to generate Qy BChl is feasible, the same decay time constants observed for all the three excited states in Eq. (1) (shown by asterisks) can be easily understood when the particular T–T annihilation reaction is fast enough and the decay of Qy BChl is the ratedetermining process. 3.4. The roles of T1 Cars in the LH2 complexes Two different routes are now known for the generation of T1 Cars in the LH2 complexes: (1) Direct singletto-triplet conversion from a higher singlet state. Singletto-triplet conversion from a higher singlet state, branching from the singlet internal conversion, was first proposed, for spirilloxanthin and spheroidene in the LH1 and LH2 antenna complexes, respectively [27,28]. The branching singlet state was named Ôthe S* stateÕ. Most recently, we showed the presence of (1) the singlet inter1 1 1 nal conversion of 11 Bþ u ! 1 Bu ! 2 Ag ! 1 Ag (G), and (2) the singlet-to-triplet internal conversion of 11 B u ! 13 Ag (T2 ) followed by the triplet internal conversion of 13 Ag (T2 ) ! 13 Bu (T1 ) [6]. The 11 B u ! T2 branching ratio increased from 3% to 26% on going from n ¼ 9 to 13. Thus, T1 Cars can be generated by the singlet-to-triplet conversion from the 11 B u state. (2) Triplet-energy transfer from BChl. The energy diagram in Fig. 3 shows that plural channels of Carto-BChl singlet-energy transfer are feasible. Since the internal conversion within the Car molecule takes place very rapidly (in the 1014 –1011 s range), strong Coulombic coupling due to close contacts between the Car and BChl molecules in the LH2 complexes [29] is absolutely necessary to facilitate the efficient singlet-energy transfer reaction. The resultant BChl Qy state efficiently intersystem cross into the T1 state, the quantum yield being P40% [30]. Then, T1 Car can be generated by triplet-energy transfer from T1 BChl. The arrangement of their energy levels shown in Fig. 3 as well as the above-mentioned close contacts between the BChl and Car molecules facilitate this BChl-to-Car triplet-energy transfer reaction through the electron exchange mechanism. It is to be noted that T1 Car and T1 BChl can reach an equilibrium by the use of thermal energy. This equilibrium also facilitates the T–T annihilation reaction to take place. Based on the above considerations, the possible roles of T1 Cars in the LH2 complex can be proposed as follows: (a) An energy sink. When too much light energy is absorbed by Cars, the 11 Bþ u state can be densely
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populated. During the process of internal conversion, the 11 B u state can be relaxed eventually to the T1 state through the singlet-to-triplet conversion to the T2 state followed by the internal-conversion down to the T1 state. When too much light energy is absorbed by BChl, or transferred from Car, the Qy state can be highly populated. Then, the Qy state can be relaxed to the T1 state through efficient intersystem crossing. In those processes of internal conversion, intersystem crossing and vibrational relaxation, the excess energy can be dissipated as heat. Since T1 Cars are energetically lower than T1 BChl, the BChl to Car triplet-energy transfer takes place, and the T1 energy is eventually dissipated through Cars. Thus, T1 Car can function as an energy sink. (b) An energy reservoir and an energy supplier. The residual energy can be stored in T1 Car within its lifetime. As seen in the present investigation, Cars do not quench T1 BChl completely, most probably due to their delicately tuned T1 energies and to achieve the thermal equilibrium condition. As a result, both T1 Car and T1 BChl co-exists in the LH2 complexes to facilitate the T–T annihilation reaction to generate Qy BChl, whose energy can then be transferred to the BChl molecules in the LH1 complex, and eventually to the special-pair BChls in the reaction center. This type of delayed singlet-energy transfer can continue within the lifetime of T1 Car, i.e., on the order of microseconds.
4. Conclusion The following answers to the questions addressed in Section 1 have been obtained: (1) In the LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rsp. molischianum, the T1 energies of neurosporene (n ¼ 9), spheroidene (n ¼ 10) and lycopene (n ¼ 11) have been determined to be 7030, 6920 and 6870 cm1 , respectively, whereas the T1 energy of BChl a to be 7590 cm1 . The Car T1 energy decreases slightly but systematically when n increases, but the energy difference between T1 BChl and T1 Car is small enough to allow those species co-exist in the stationary state. (2) T1 Car and T1 BChl decay simultaneously in LH2 complex from Rba. sphaeroides G1C. Their decay time constants were the same as the decay time constant of the BChl Qy fluorescence. Therefore, the T–T annihilation generating the BChl Qy state must be in operation. (3) The T1 Car can function as an energy sink when excess energy is present in both Car and BChl singlet states in LH2 complexes. However, Car does not quench T1 BChl completely but allows T1 BChl to remain in equilibrium. Then, the T–T annihilation reaction between the pair of triplet species converts the triplet energy into Qy BChl for delay singlet-energy transfer. Thus, T1 Car performs both the photo-protective and the light-harvesting functions.
Acknowledgements This work has been supported by a grant from Ministry of Education, Science, Sport and Culture (Open Research Center Project) and a grant from NEDO (New Energy and Industrial Technology Development Organization, International Joint Research Grant).
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