New trends in photobiology

J. Photochern. Photobiol. B: Biol., 9 (1991) 265-280

265

New Trends in Photobiology (Invited Review)

Structures and functions of carotenoids in photosynthetic systems Yasushi Koyama Faculty of Science, Kwansei Gakuin University, Uegahara, Nishinomiya 662 (Japan) (Received December 28, 1990 ; accepted January 9, 1991) Keywords . Carotenoids, photosynthesis, energy transfer, reaction center, light-harvesting complex, singlet and triplet excited states, cis-trans configuration, isomerization . Abstract Recent achievements in the investigation of the structures and excited-state properties of carotenoids, in relation to their photoprotective and light-harvesting functions in bacterial photosynthesis, are reviewed . In purple bacteria, natural selection of the carotenoid configurations has been observed . The 15-cis configuration is selected by the reaction centers (RCs), whereas the all-trans configuration is selected by the light-harvesting complexes (LHCs) . In the photoprotective function in the RCs, involvement of one of the accessory bacteriochlorophylls in the triplet energy transfer has been demonstrated . The extremely efficient isomerization of the "15-cis" T, carotenoid in vitro, together with indications of isomerization and two forms of carotenoids in vivo, have led to the proposal of a mechanism of energy dissipation in which isomerization of the carotenoid is involved. In the energy transfer function in the LHCs, evidence for the 2A g (S,) state for carotenoids in vitro and in vivo has been obtained . Its lifetime of approximately 10 ps in vitro and an energy transfer time of 1-10 ps in vivo, determined by picosecond absorption spectroscopy, make the electron-exchange mechanism via the 2A g state likely. However, the B„ (S 2) lifetime of approximately 200 is in vitro and an energy transfer time of the same order of magnitude in vivo, determined by femtosecond absorption spectroscopy, suggest that the dipole mechanism via the B„ state is also possible . 1 . Introduction : roles of carotenoids in photosynthesis This short review attempts to highlight recent achievements (mostly within the last 5 years) in the field of carotenoids in photosynthesis . It is not intended to be comprehensive, but is focused on the reaction center (RC) and the light-harvesting complex (LHC) of purple photosynthetic bacteria, in particular, on the structures and excited-state properties of carotenoids in relation to their functions of photoprotection and light harvesting . In describing the excited-state properties, /3-carotene is chosen, because it has been extensively investigated and its number of conjugated C=C bonds (11) 1011-1344/91/$3 .50

© Elsevier Sequoia/Printed in The Netherlands



266 is in between those of neurosporene (9) and spirilloxanthin (13), which have the shortest and longest conjugated chains of the carotenoids usually found in photosynthetic bacteria . Those readers who are interested in the broader field of "carotenoids in photosynthesis" are referred to the excellent reviews by Krinsky [1], Mathis and Shenck [2], Cogdell [3, 4], Siefermann-Harms [5], Cogdell and Frank [6], Truscott [7], Mimuro and Katoh [8] and Frank et at . [9] . 1 .1 . Photoprotective function

The photoprotective reactions of carotenoids include triplet energy transfer from T, bacteriochlorophyll (3 BChl*) to the ground So carotenoid ('Car) (eqn . (1)) and energy dissipation through non-radiative relaxation (intersystem crossing) of the resultant T, carotenoid ('Car*) (eqn . (2)) 3BChl*+'Car 3 Car*

-, 'BCh1+ 3 Car*

- 1 Car+heat

(1) (2)

The energy diagram shown in Fig . 1 indicates that the relative energies of the components make the above reactions possible . The quenching of 3 BChl* prevents the generation of singlet oxygen through triplet sensitization ; this cm' 20000

S, WA,)

S2 ( ax )

5,(G,)

10000

T, l'B„)

_5,('A9) 0

Sa

h-carotene

Bchl

FSg . 1 . Energy diagram of all-trans-t:-carotene and bacteriochlorophyll a . The energy levels of fl-carotene are based on Trash et al. [10] for the S 2 and S, states and Jensen et al. [11] for the T, state . The T, energy level of bacterlochlorophyll a is based on Taldff and Boxer [12] .



267 photoprotective function is unique to the carotenoids . The mechanisms of reactions (1) and (2) need to be elucidated . Since both the 3 BChl*-*'BChI and 'Car -'Car* transitions are spin forbidden, reaction (1) must take place through the electron-exchange mechanism [13] . The mechanism of reaction (2), in particular, needs to be determined.

1 .2. Light-harvesting function The light-harvesting reactions of carotenoids include the absorption of light energy by 'Car to generate an excited singlet state ('Car*) (eqn. (3)) and its energy transfer to S 0 BChI ('BChI) (eqn . (4)) , 'Car*

(3)

'Car* +'BChl -+ 'Car+'BChI*

(4)

'Car+hv

The energy diagram (Fig . 1) shows that the energy level of the optically allowed B„ (S2) state is much higher than the Q, (S2) level of BChI . Thus, a lower level of 2Ag (S,) is expected to be involved in the energy transfer above . Hence an additional reaction of internal conversion should take place before reaction (4) 'Car * (B,,, S 2 )-

'Car* (Mg , S,)+heat

(3')

Since the 2Ag (S,) to Ag (So ) transition is optically forbidden, the singlet energy transfer reaction via the S, state must take place through the electronexchange mechanism . If the energy transfer takes place directly via the optically allowed B„ (S 2) state, the reaction can take place through the dipole mechanism [ 14] . The mechanism of reaction (4), either the electron-exchange mechanism via the 2Ag (S,) state or the dipole mechanism via the B„ (S 2) state, still needs to be determined. Nevertheless, it has been established that carotenoids function as supplementary light harvesters [15] .

2 . Structures of carotenoids in the reaction centers and lightharvesting complexes of purple photosynthetic bacteria The first important achievement in the past 5 years has been the establishment of the natural selection of the carotenoid configurations by the pigment-protein complexes, i .e . 15-cis by the RCs and all-trans by the LHCs (see Fig . 2) . Proton nuclear magnetic resonance ('H NMR) spectroscopy of carotenoids extracted from the pigment-protein complexes has been used to establish the configurational assignments, and Raman spectroscopy of the free and bound carotenoids has revealed that the 15-cis and all-trans configurations are retained in the pigment-protein complexes . However, some twisting of the conjugated chain is suggested to take place on binding .

2.1 . The structure of carotenoids in the reaction centers Raman spectroscopy has been used to correlate the configurations of both free and bound carotenoids . A unique cis configuration of spheroidene



268 Rb .

spheroides

spheroidene

RC Me0 e0

LHC all-trans

neurosporene

RC LHC

~~ all-trans

Rs . rubrum

spiritloxanthin

RC Me0

0 e

Me0 all-trans

0 e

LHC

dihydrospiritloxonthin Me0 0 e all-trans

Fig. 2 . Natural selection of carotenoid configurations in purple photosynthetic bacteria . Based on Lutz et al. [16] for spheroidene in Rhodobacter spheroides 2 .4 .1 and Koyanna et al. (17, 181 for neurosporene in Rhodobacter spheroides G1C and spirilloxanthin (3,4-dihydrospirilloxanthin) in Rhodospiriltum rubrum .

bound to the RC of Rhodobacter spheroides has been detected [19], and it has been shown that all the carotenoids bound to the RCs of Rb . spheroides (Ga and G 1 C mutant also), Rhodospiriuum rubrum and Rhodopseudomonas viridis take a common cis configuration [20] . In addition, spheroidene reconstituted into the RC of Rb . spheroides R26 (a carotenoid-less mutant) has essentially the same configuration as that in the RC of the wild type [21] . Spectral analysis using the vibrational data available has suggested a di-cis configuration with one methylated and one unmethylated double bond [20] . However, spectral comparison between RC-bound carotenoids of Rb . spheroides (neurosporene and spheroidene) and 14 different cis-trans isomers of /3-carotene has indicated that the configuration of the RC-bound carotenoids is 15-cis [22, 23] .

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Difference absorption and circular dichroism (CD) spectroscopy of the reconstituted and carotenoid-less RC preparations (for spirilloxanthin in Rs . rubrum) have also indicated a central mono-cis configuration twisted in a protohelical shape [24] . 'H NMR spectroscopy of carotenoids extracted from the RCs has provided conclusive evidence for a configurational assignment of 15-cis . A mixture of 15-cis- and all-trans-spheroidene (35 :65) was extracted from the RC of Rb . spheraides [ 16], and pure 15-cis isomers of neurosporene and spirilloxanthin were extracted from the RCs of Rb . spheroides G1C and Rs . rubrum, respectively [17, 18] . Raman spectroscopy has demonstrated that the free and bound carotenoids have essentially the same 15cis configuration (indicated by the same Raman frequencies) and that some twisting of the polyene backbone takes place (indicated by different Raman intensities) [16, 17] . Twisting in the C8 to C12 and C8' to C12' regions of spheroidene bound to the RC of Rb . spheroides [ 16] and twisting around the C15-C15' bond for neurosporene bound to the RC of Rb . spheroides G1C [17] have been suggested . Cross polarization/magnetic angle spinning (CP/MAS) NMR spectroscopy of 14' [' 3C]-substituted spheroidene incorporated into the RC of Rb . spheroides R26 reveals an upfleld shift, which indicates a 15-cis configuration [251 . X-ray crystallography has been used to determine the location of the carotenoid molecule in the RCs of Rb . spheroides [ 26] and Rps . viridis [27] and suggests a central mono-cis configuration, which is shown in Fig . 3 for Rps . viridis . However, the detailed configuration (conformation) still remains to be determined . All the experimental data now available lead to the conclusion that the carotenoids in the RCs take a 15-cis configuration with some twisting along the conjugated backbone.

2 .2. The structure of carotenoids in the light-harvesting complexes Comparison of the Raman spectra of the LHC-bound and free (all-trans) carotenoids indicates that carotenoids in the LHCs of Rb . spheroides, Rs . rubrum and Rps . viridis take an all-trans configuration [20] . (However, the possibility of a peripheral-cis (7-cis) configuration cannot be completely excluded when the assignment is simply based on the above spectral comparison ; see refs . 23 and 28 for the Raman spectrum of the 7-cis isomer .) 'H NMR spectroscopy of neurosporene and spirilloxanthin extracted from the LHCs (chromatophores) of Rb . spheroides G1C and Rs. rubram respectively provide conclusive evidence for a configurational assignment of all-trans [17, 18] . Furthermore, Raman spectroscopy detects two different types of conformation for the LHC-bound all-trans carotenoids, i .e . twisted and planar forms [29] . The higher intensity of the Raman line around 950 cm - ' (C-H out-of-plane wagging) reflects a twisting around the C11 =C12 and/or C7=C8

270

Fig. 3. Pigment organization in the RC of Rhodopseudmnonas viridis . Yellow, carotenoid; blue-green, special pair bacteriochlorophylls ; yellow-green, accessory bacteriochlorophylls ; purple, bacteriopheophytins ; white, quinones ; orange, iron . The H, M and L subunits are shown in dark blue . Based on the coordinates from the Protein Data Bank, Brookhaven National Laboratories (code number 1PRC) (see Deisenhofer and Michel [271) .

271

Fig. 4 . A model of pigment organization in the B800-850 LHC of Rhodobacter spheroides proposed by Kramer et al. [32] (modified from Fig . 8, of ref. 32) .

double bonds . The LHCs from Rs . rubrum, Chromatium vinosum and Rhodopseudomonas paistris show a twisted form, whereas those from Rb . spheroides and Rhodopseudomonas capslata show a planar form . The twisting is not ascribed to the type of carotenoid, but to the specific interaction between the carotenoid and the apoprotein [30], and it is also correlated with the efficiency of energy transfer (vide infra) [31] . Fluorescence polarization and linear dichroism spectroscopy of the B800-850 complex ofRb . spheroides lead to a model for pigment organization, which is shown in Fig. 4 [32] . It should be noted that two different types of carotenoid molecules are present in the LHC .

3 . Triplet state properties and photoprotective function of 15-cis carotenoids in the reaction center Why are 15-cis carotenoids selected by the RCs? When the electron transfer reaction is blocked under reducing conditions, charge recombination takes place at the special pair in the RC and T, BChI is generated . The main function of the RC-bound carotenoid, as described in Section 1, is to accept the triplet energy and then to dissipate it to the surroundings as heat . Therefore the 15-cis configuration must be more suited to perform this function than all the other possible cis-trans configurations, and the configurational selection can be ascribed to a T, state property . 3 .1 . Triplet state properties of /3-carotene Transient Raman spectroscopy has been applied to determine the T 1 state structure of all-traps-$-carotene, and both planar [33, 34] and

2 72 perpendicularly twisted [35] forms have been proposed . For several years, intersystem crossing from the S, to the T, state was considered to be too inefficient to be detected [36], but it has been shown to take place with a quantum yield of approximately 10 -3 [37] . The characterization of the configurational dependence of the T, state properties is another achievement of recent years . Transient Raman spectroscopy of isomeric /3-carotene has shown that the all-trans", 7-cis, 9-cis and 13-cis isomers generate their own T, species (the "all-trans", "7-cis", "9-cis" and "13-cis" T,) . However, the 15-cis isomer generates the "alltrans" T, instead of the "15-cis" T, [34] . Transient absorption spectroscopy supports the above conclusion ; T, species generated from all the isomers, with the exception of 15-cis, show their own unique T„ F T, absorption, but the T, species generated from the 15-cis isomer shows the same T„-T, absorption as that generated from the all-trans isomer . The T, lifetimes in tetrahydrofuran have been determined to be : all-trans and 15-cis, 4 .8 µs ; 7cis, 6 .3 µs ; 9-cis, 3 .7 µs ; 13-cis, 3 .5 µs [38] . The above results indicate that the "15-cis" T, isomerizes very rapidly into the "all-trans" T„ and that the "15-cis" T, is too short-lived to be detected by transient Raman and transient absorption spectroscopy with submicrosecond time resolution . The quantum yields of triplet-sensitized isomerization for isomeric /3-carotene support the concept of extremely efficient isomerization of the "15-cis" T, species . They are as follows : all-trans, 0 .04; 7-cis, 0 .12; 9-cis, 0 .15; 13-cis, 0.86; 15-cis, 0 .98 [39] . The results can be explained by the occurrence of T, potential minima at the all-trans and each cis position ; the T, potential minimum at the 15-cis position is expected to be very shallow [34, 37, 39] . 3 .2. Mechanisms of photoprotection in the reaction centers

Detailed mechanisms of the triplet energy transfer and triplet energy dissipation (see Section 1) remain to be determined . Experimental data now available indicate a route of triplet energy transfer and suggest a possible mechanism of energy dissipation in which isomerization of the "15-cis" T, carotenoid plays an essential role . The organization of pigments in the RC of Rps . viridis (Fig. 3) suggests a pathway of triplet energy transfer, i .e . (BChl)2 ->BChI(B)-.Car, since T, BChl should be generated at the special pair and the transferred energy should be dissipated by the 15-cis carotenoid to the surroundings . Involvement of the "accessory" BChI(B) in the triplet energy transfer [26] has been demonstrated by transient absorption and electron spin resonance (ESR) spectroscopy of spheroidene incorporated into the intact and BCh1(B)-deficient RCs of Rb . spheroides R26 (carotenoid-less mutant) [40]; generation of T, spheroidene is inhibited by the removal of BCh1(B), although the absorption and CD spectra indicate that the structure and the environment of the bound carotenoid are not altered . A unique T, structure of the RC-bound carotenoids has been revealed by transient Raman, transient absorption and magnetic resonance spectroscopy . Transient Raman spectroscopy of T, spheroidene and

2 73

methoxyneurosporene bound to the RCs of Rb . spheroides 2 .4 .1 and Ga reveals the generation of a T, species whose configuration (conformation) is completely different from that of T, carotenoids free in solution [411 . Transient absorption spectroscopy of the above carotenoids incorporated into the RC ofRb6 spheroides R26 reveals exactly the same T„-T,absorption . However, the carotenoids free in solution exhibit different T.. • T, absorptions, reflecting the different length of the conjugated chain [42] . ESA spectroscopy of a variety of carotenoids incorporated into the RC of Rb . spheroides R26 .1 has shown that hydroxyneurosporene, methoxyneurosporene (nine conjugated C=C bonds), spheroidene and hydroxyspheroidene (ten conjugated C=C bonds) give the same zero-field-splitting parameter IDI, and 3,4-dihydroanhydrorhodovibrin (12 conjugated C=C bonds) and spirilloxanthin (13 conjugated C=C bonds) give the same parameter IDI (but lower than the above) . On the basis of these observations, a T, state structure of the RCbound carotenoid has been proposed [43] in which conjugation is interrupted by twisting at both ends . Experimental data suggesting isomerization or two forms of RC-bound carotenoids have also been obtained . Absorption spectroscopy of spirilloxanthin in the RC of Rs . rubrum has revealed a cis to trans isomerization [44] . Resonance Raman spectroscopy of an RC preparation of Rs . rubrum has shown the presence of all-trans-spirilloxanthin in addition to 15-cisspirilloxanthin, and the contribution of the former increases under reducing conditions [45] . Absorption-detected magnetic resonance (ADMR) spectroscopy of spheroidene in the RC of Rb . spheroides 2 .4 .1 has revealed two different types of T, species at low temperature which differ in the zerofield-splitting parameters IDI [46] . Continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy of fully deuterated spheroidene in the RCs of Rb . spheroides has shown that a thermally controlled conformational change of the carotenoid occurs [47] . These results, together with the observation that the "15-cis" T, species of free (3-carotene in solution isomerizes very efficiently into "all-trans" T„ have led to the proposal of a mechanism of energy dissipation by the RCbound 15-cis carotenoids which includes isomerization in the T, state [18, 48] ; however, the proposal needs to be confirmed .

4 . Singlet state properties and light-harvesting function of all-trans carotenoids in the light-harvesting complexes The main function of carotenoids in the LHCs should be harvesting the light energy and transferring it to BCh1 . All-trans carotenoids have been selected for this function . An achievement of recent years has been the characterization of the S, (2Ag) and S 2 (B„) states of free and bound carotenoids using picosecond transient Raman and picosecond (femtosecond) transient absorption spectroscopy . The configurational dependence of the S, state properties has also been examined .

274

4.1. Singlet state properties of ,8-carotene All-traps-/3-carotene has been extensively investigated . The presence of the optically forbidden 2A g state around 17 230 ± 100cm - ' (3470 cm - 'below the optically allowed B„ state at 20 700 cm -1 ) was first proposed on the basis of a Raman excitation profile experiment in cyclohexane [101 . Recently, the above value has been questioned and a 2Ag level 5500-6500 cm - 'below the B„ level has been proposed [49] . Additional evidence for the 2Ag state as the 5, state has been obtained from transient Raman spectroscopy . Picosecond Raman spectroscopy of alltrans-$-carotene has revealed an S„ C=C stretching Raman line as high as 1777 cm - ' [50-52) . By inference from shorter polyenes, the abnormally high frequency of the Raman line can be ascribed to a vibronic coupling with the S o (Ag) state through an A,-type C=C stretching vibration ; thus the result indicates that the S, state probed by Raman spectroscopy is the 2Ag state [50] . The vibronically coupled C=C stretching Raman line has been detected for a variety of carotenoids, indicating that the 2A g state is the S, state for carotenoids in general [53-56] . The lifetime of the S, (2A g) state has been determined using the S,-S, transient absorption ; lifetimes of 10.0 ± 0 .5 ps in 3-methylpentane [57] and 12 .4±0 .5 ps in n-hexane [37] have been reported . Fluorescence from the B„ (S 2) state has recently been reported for the first time [ 58 ] . In the case of spheroidene, the low quantum yield of fluorescence (3X10 -5 ), the ground-state recovery of 15 ps, and the natural radiative lifetime of 1-10 ns suggest that non-radiative relaxation from the B„ state -5 is of the order of 30-300 fs . The quantum yield of fluorescence is 6 x 10 for /3-carotene 1581 . Femtosecond transient absorption spectroscopy (full width at half-maximum (FWHM), 240 fs) has been used to determine directly lifetimes of the B„ (S 2) state of all-trans-/3-carotene [59] . S2 lifetimes of 200 fs in ethanol and 250 fs in CS, have been reported ; the S, lifetimes are 7 .5 ps in ethanol and 11 ps in CS 2 . With regard to the configurational dependence of the S, state properties, picosecond transient absorption and transient Raman spectroscopy of alltrans-, 7-cis, 9-cis-, 13-cis- and 15-cis-/3-carotene have shown that the isomers have similar S, lifetimes (12 .4±0 .5 ps for the all-trans isomer and 14 .0±0 .5 ps for the 15-cis isomer), no isomerization takes place in the S, state, and intersystem crossing occurs with a quantum yield of approximately 0 .001 [37] .

4.2. Mechanisms of light harvesting in the light-harvesting complexes Efficient energy transfer from Car to BChI suggests a close proximity of these molecules, and the generation of T, carotenoid through singlet homofission indicates the presence of a pair of carotenoid molecules bound side-by-side . The S 2 (B„) and S, (2Ag) lifetimes of approximately 100 fs and 10 ps and the S 2 and S, energy levels of around 21 000 and 17 000 cm - ' suggest that the electron-exchange mechanism via the S, (2A g) state is

275 preferred . Furthermore, the in vivo energy transfer time of approximately 1-10 ps seems to support the above mechanism . However, in vivo energy transfer of the order of 200 fs, reported later, suggests a possible involvement of the dipole mechanism via the S 2 (B„) state . Raman spectroscopy of carotenoids and fluorescence spectroscopy of BChls in the LHCs correlate the twisted and planar forms of the all-trans carotenoids (vide supra) with the energy transfer from carotenoids to BCh1 . Carotenoids in the spirilloxanthin (spheroidene) series having 11 or 13 (nine or ten) conjugated C=C bonds take the twisted (planar) form and show lower (higher) energy transfer efficiency [31 ], which compensates the higher (lower) light-harvesting capacity, i.e. higher (lower) e value . The energy transfer efficiencies and the distortion of the polyene backbone suggest strong interaction between the Car and BCh1 molecules . The magnetic field dependence of the fluorescence from BChI and of the T„ •- T, transient absorption of carotenoids indicates three different pathways for the generation of T, carotenoids in the chromatophore membranes of Rs. rubrum and Rb. spheroides . They are : (1) charge recombination in the RC, (2) singlet homofission between carotenoids and (3) triplet energy transfer from BChI to Car in the LHC [60, 61 ] . Pathways (2) and (3) indicate close Car-to-Car and Car-to-BChI contacts in the LHC . Transient absorption spectroscopy of the chromatophores of Rs. rubrum [62] and Raman spectroscopy of the chromatophore membranes of Ch . vinosum [63] and Rs. rubrum [64] reveal singlet homofission between the carotenoid molecules . Picosecond transient Raman spectroscopy of the chromatophores of Rb. spheroides [54] and spinach thylakoid membranes [55] has shown that the 2Ag state is still the S, state in vivo and that the vibronic coupling is weakened on binding . Furthermore, the two-photon excitation profile of Chi a emission from the thylakoid membranes of Phaeodactylum tricornutum provides direct evidence for the involvement of the 2A g state in in vivo energy transfer [65] . Picosecond time-resolved absorption spectroscopy of the LHCs reveals energy transfer times in the picosecond range . This, together with the above observations, supports the electron-exchange mechanism via the 2A g state . For the B800-850 complex of Rhodopseudomonas acidophila 7750 (total instrument response function, 4 ps), the bleaching of the carotenoid absorption recovers with a 5 .6±0 .9 ps time constant and the 860 nm BChI band is bleached with a 6 .1±0 .9 ps time constant, indicating energy transfer from Car to B860 BChI [66] . For the B800-820 complex of Rps. acidophila 7050 (pulse duration, 5-7 ps), the absorption at 543 nm recovers with a time constant of 3 ps [67] . Femtosecond time-resolved absorption spectroscopy (FWHM, 240 fs) reveals much shorter transfer times . For the B800-850 complex of Rb. spheroides 2 .4 .1 (solubilized with lauryldimethylamine-N-oxide (LDAO)), the Car-to-B800 BChI and Car-to-B850 BChI energy transfer times have been determined to be 0 .34 and 0 .20 ps, respectively [68] . However, the S 2 and

276

S, lifetimes of free spheroidene in solution have been determined to be 0 .34 and 9 .1 ps. Since the S 2 lifetime of the carotenoid in vitro is of the same order of magnitude as the energy transfer time in vivo, it has been suggested that singlet energy transfer may also occur directly from the S 2 (Ba state through the dipole mechanism [691 .

5 . Conclusions A large amount of information has been accumulated in recent years on the locations and structures of carotenoids in the pigment-protein complexes (RCs and LHCs), the T„ S, and S 2 state properties in vivo and in vitro, and the triplet and singlet energy transfer in vivo . However, it is still insufficient to determine the detailed mechanisms of photoprotection and light harvesting. The following pieces of information are absolutely necessary for the determination of the mechanisms : (1) the structure of the LHC determined by X-ray crystallography; (2) the singlet-singlet energy transfer times determined by transient absorption spectroscopy with higher time resolution (both decay of the donor and rise of the acceptor should be determined to identify an energy transfer) ; (3) the energies of S, and T, states of the carotenoids determined spectroscopically ; (4) the So and T, structures of the two carotenoid components in the RCs determined by time-resolved absorption and Raman spectroscopy .

Acknowledgments The author thanks Drs . R . J . Cogdell, H . A. Frank, R . Gebhard, S . Kolaczkowski, J . Lugtenburg, M . Mimuro, T . Noguchi, T. G . Owens and M . Tasumi for sending manuscripts prior to publication and lists of publications on carotenoids . Drs . A. Angerhofer, T. Gillbro, G . Gingras and H . Hayashi are also acknowledged for sending lists of publications on carotenoids . Typing of the manuscript by Dr . H . Hashimoto, drawing of Figs . 1, 2 and 4 by Mr . M . Kuki, and computer graphic preparation of Fig . 3 by Mr. H . Nakagawa are also gratefully acknowledged .

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