**4. Comparison of higher plant and purple bacteria triplet state**

Resonance Raman is a very sensitive and selective technique which allows access to the vibrational modes of molecules via inelastic scattering. In this section we describe the use of Resonance Raman to confirm the triplet delocalisation observed by step scan spectroscopy.

In solution, upon triplet state formation, the frequency of stretching modes of the conjugated C=C's of the molecule dramatically downshifts from 1522 to 1494 cm-1 for ßcarotene in tetrahydrofuran (THF; (Hashimoto, Koyama et al. 1991), (Fujiwara, Yamauchi et al. 2008)), or from 1529 to 1500 cm-1 for *all-trans* spheroidene in hexane (Mukai-Kuroda, Fujii et al. 2002). This reflects the localization of the triplet throughout the conjugated system (triplet high spin density "hot spot" is mainly in the centre of the conjugated system; ((Mukai-Kuroda, Fujii et al. 2002), (Di Valentin, Ceola et al. 2008)), which causes a reduction in the C=C bond order. The frequencies of the 1 resonance Raman bands observed in protein-bound carotenoid triplet spectra are reported in Table 2 and compared to those observed for triplet states of other carotenoids, including ß-carotene in solution and spheroidene bound to the bacterial reaction center. The downshift depends on the carotenoid configuration, and it is always larger in *cis* configurations ((Hashimoto, Koyama et al. 1991; Mukai-Kuroda, Fujii et al. 2002)). For rhodopin glucoside in LH2, this downshift of the band from 1517 to 1493 cm-1 is very similar to that observed for all-trans ß-carotene and spheroidene in solution (24 cm-1 *vs.* 23-25 cm-1). In contrast, the observed downshifts for this band for both luteins in LHCII trimers, LHCII monomers, CP29 and CP43 are much smaller and in all cases close to 18 cm-1, *i.e.* approximately 75% of the 23-25 cm-1shift that is observed for all-*trans* carotenoid triplets in solution (see Table 2). Such a reduction of the 1

Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

**molecular adaptation of the photoprotection mechanism** 

**5. Triplet state dynamic in purple bacteria, higher plant and algae, a** 

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 249

Recent time-resolved absorption spectra show that no chlorophyll *a* triplet is accumulated in LHCII and PCP while, on the other hand, the FTIR difference spectra strongly suggest the presence of Chl *a* contributions long after the initial excitation. In order to explain this apparent contradiction, the simplest hypothesis is that the triplet could be shared between carotenoid and chlorophyll molecules. This question has already been addressed in the literature. In order to explain the influence of the carotenoid triplet state on the absorption bands of the BChl molecules in LH complexes from purple bacteria, Angerhofer *et al.* proposed 'a small delocalization of the carotenoid triplet over an adjacent BChl molecule' (10). They also noted that the apparent rate of Bchl to carotenoid triplet-triplet transfer seems to correlate with how much the carotenoid triplet state is able to influence the BChl transition. However, it should be pointed out that in LH complexes from purple bacteria, the carotenoid and BChl molecules are very closely located (an essential condition for triplet/triplet transfer), and therefore they each constitute a sizeable part of the environment (or solvation) of the other. It would thus be expected that the dielectric changes following the appearance of the carotenoid triplet state would have a measurable influence on the BChl absorption transitions. Hence, these previous results do not formally demonstrate a sharing of the triplet. In contrast, the resonance Raman spectra reported in this chapter provides a direct and unambiguous measurement of the sharing of the triplet of the carotenoid molecule. Indeed, upon sharing, the carotenoid triplet should progressively loose its pure triplet character, and the Raman signature of this state should become intermediate between ground- and triplet-state. In the case of rhodopin glucoside in LH2 from *Rbl. acidophilus*, the downshift of the 1 band is quite similar to that of ß-carotene or spheroidene in solution. We may thus safely conclude that there is very little, if any, wavefunction sharing between carotenoid and BChl triplet states in the LH2 complex from *Rbl. acidophilus*. In LHCs from higher plants, the presence of the triplet state of the lutein molecules is known to induce a net bleaching of the electronic absorption transition of the neighboring chlorophyll molecules (Peterman, Gradinaru et al. 1997). However, delocalization of the triplet was for a long time considered unlikely, due to the large energy gap between the triplet states of carotenoid and chlorophyll molecules (Peterman, Gradinaru et al. 1997). This position is challenged by time-resolved FTIR studies on PCP and LHCII, which demonstrate a co-existence of chlorophyll and carotenoid triplets throughout the entire triplet lifetime (Alexandre, Luhrs et al. 2007). In addition, resonance Raman spectroscopy provides additional information on the nature of the carotenoid triplet in these complexes. The sensitivity of the *v*1 bands of the ground and triplet states is expected to exhibit similar responses to the environment. However, in LHCII we observe a reduction of the energy gap between the *v*1 bands of the ground- and triplet states by more than 30%. This unambiguously indicates that the electronic state gained by the carotenoid has lost a fraction of its carotenoid triplet character, and consequently part of the triplet must be localized on another molecule. From the results of the step-scan time-resolved FTIR measurements (Fig. 9A), we may safely conclude that a neighbouring chlorophyll molecule is the acceptor of the carotenoid triplet. The fact that the same effect was found for both luteins to the same extent strongly substantiates this conclusion. Each lutein experiences a different protein environment (which induces the red-shift of the electronic transitions of lutein 2; (Liu, Yan et

band's downshift reflects a dramatic alteration of the nature of the triplet state, which correlates very well with a reduction in the energy gap between the S0/S2 and T1/Tn transitions observed for these carotenoids in LHCII (see Table 1; it is harder to determine this gap with precision for CP43 due to the larger number of carotenoid molecules in this complex).


Table 1. Electronic transitions of triplet carotenoid states in some LH2 complexes from purple bacteria (values obtained from (Angerhofer, Bornhaeuser et al. 1995)) and in higher plant LHCs prepared as trimers (lines 4 and 5, ((Peterman, Dukker et al. 1995), (Lampoura, Barzda et al. 2002), (Croce, Mozzo et al. 2007)) and monomers (line 6, ((Croce, Mozzo et al. 2007), (Peterman, Gradinaru et al. 1997) ), and in CP29\* (Croce, Mozzo et al. 2007).


Table 2. Comparison of the position of ν1 vibrational band in resonance Raman spectra of rhodopin glucoside in LH2, of lutein 1 (ground state of the latter from ref (Ruban, Berera et al. 2007) ) and 2 in LHCII trimers and other carotenoid-containing complexes with all-*trans*ß-carotene, and other carotenoids, in solution (taken from (Hashimoto, Koyama et al. 1991; Mukai-Kuroda, Fujii et al. 2002; Rondonuwu, Taguchi et al. 2004) ).

band's downshift reflects a dramatic alteration of the nature of the triplet state, which correlates very well with a reduction in the energy gap between the S0/S2 and T1/Tn transitions observed for these carotenoids in LHCII (see Table 1; it is harder to determine this gap with precision for CP43 due to the larger number of carotenoid molecules in this

Neurosporene (LH2) 9 495 516 822 Spheroidene (LH2) 10 514 537 833 Rhodopin(LH2) 11 529 556 918 Lutein 1 (LHCII trimers) 10 494 506 480 Lutein 2 (LHCII trimers) 10 510 525 560

monomers, CP29) 10 494,494 508,505 558,441

Table 1. Electronic transitions of triplet carotenoid states in some LH2 complexes from purple bacteria (values obtained from (Angerhofer, Bornhaeuser et al. 1995)) and in higher plant LHCs prepared as trimers (lines 4 and 5, ((Peterman, Dukker et al. 1995), (Lampoura, Barzda et al. 2002), (Croce, Mozzo et al. 2007)) and monomers (line 6, ((Croce, Mozzo et al.

2007), (Peterman, Gradinaru et al. 1997) ), and in CP29\* (Croce, Mozzo et al. 2007).

Carotenoid ν1 (cm-1) Δν1 (cm-1)

C=C S0S2 (nm) T1Tn (nm) ΔE (cm-1)

24

18

18

18

18

18

25

Number of

LH2 rhodopin glucoside ground state 1517

LH2 rhodopin glucoside triplet 1493 LHCII trimer Lutein1 ground state 1530

LHCII trimer Lutein 1 triplet 1512 LHCII trimer Lutein 2 ground state 1526

LHCII trimer Lutein 2 triplet 1508 LHCII monomer lutein ground state 1526

LHCII monomer lutein triplet 1508 CP29 lutein ground state 1526

CP29 lutein triplet 1508 CP43 lutein ground state 1522

CP43 lutein triplet 1504 all-*trans* -carotene singlet state in THF 1522

all-*trans* -carotene triplet in THF 1497 all-*trans*-spheroidene singlet state in *n*-hexane 1523

Mukai-Kuroda, Fujii et al. 2002; Rondonuwu, Taguchi et al. 2004) ).

all-*trans*-spheroidene triplet in *n*-hexane 1500 23

Table 2. Comparison of the position of ν1 vibrational band in resonance Raman spectra of rhodopin glucoside in LH2, of lutein 1 (ground state of the latter from ref (Ruban, Berera et al. 2007) ) and 2 in LHCII trimers and other carotenoid-containing complexes with all-*trans*ß-carotene, and other carotenoids, in solution (taken from (Hashimoto, Koyama et al. 1991;

complex).

Carotenoid (complex)

Lutein 1 and 2 (LHCII
