**5. Triplet state dynamic in purple bacteria, higher plant and algae, a molecular adaptation of the photoprotection mechanism**

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

Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

more favourable for triplet sharing between these molecules

Per in THF).

**6. Conclusion** 

**7. References** 

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 251

T-S spectra of A-PCP (Kleima, Hofmann et al. 2000). This estimate of a 25 and 40 % triplet sharing in A-PCP and H-PCP is also in line with the smaller observed bandshift of the carbonyl lactone for H-PCP (*i.e.* 20 cm-1 as compared to 25 cm-1 for A-PCP and 28 cm-1 for

Thus, in contrast to photosynthetic bacteria, our results provide compelling evidence of triplet sharing between carotenoid and chlorophyll molecules in plant and algal lightharvesting complexes. It is obviously tempting to try to unravel which molecular mechanisms may be at the origin of this difference. In LH2, contacts between carotenoid and bacteriochlorophyll molecules essentially occur at the very end of the C=C conjugated chain of the carotenoid (Fig. 10A and Ref. (McDermott, Prince et al. 1995)), and the minimum distance between these molecules is 3.42 Å (Prince, Papiz et al. 1997). In strong contrast, the closest contacts between Chl *a* and luteins in LHCII occur at the middle of the C=C polyenic chain (Fig. 10B), and the pigments are slightly more closely packed in these complexes. Taking into account the expected molecular structure of the carotenoid triplet state, the relative positioning of carotenoid and chlorophyll molecules in LHCII appears definitely

To summarize, our results clearly show that the nature of the triplet excited state of carotenoid molecules is fundamentally different in the LH2 from *Rbl. acidophilus* and the antenna isolated from spinach. In the former case, the triplet state is mainly (if not totally) localized on the rhodopin glucoside molecule. According to the work of Angerhofer *et al*., (Angerhofer, Bornhaeuser et al. 1995), this is the case for the vast majority of lightharvesting proteins from purple photosynthetic bacteria. This localization of the triplet state is associated with a relatively slow triplet-triplet transfer between the BChl and carotenoid molecules, in the 20-200 ns time scale. In LHCII complexes from higher plants, there is a sharing of the triplet between luteins and their neighbouring chlorophylls. As this is also the case in CP43 and CP29, as well as in PCP, it is likely that this triplet sharing exists in all light-harvesting proteins from plants and algae. This delocalization is associated with an ultrafast transfer/equilibration of the triplet between the chlorophyll and carotenoid molecules, which results in the absence of any measurable accumulation of pure triplet chlorophyll in these complexes. However, the price to pay to avoid the accumulation of this species is that the triplet state is shared between the chlorophyll and the carotenoid lasts for several microseconds. Apparently such mechanism drags the energy of the shared triplet below that of singlet oxygen. The resultant decrease in the probability of production of singlet oxygen thereby optimizes photoprotection of these complexes. It is striking that this tuning of photoprotection was found only in those organisms which perform photosynthesis in the presence of large amounts of molecular oxygen. We propose that triplet sharing represents an adaptation of the molecular mechanisms of protection against

photo-oxidative stress, associated with the evolution of oxygenic photosynthesis.

Alexandre, M. T., D. C. Luhrs, et al. (2007). "Triplet state dynamics in peridinin-chlorophylla-protein: a new pathway of photoprotection in LHCs?" Biophys J 93(6): 2118-28.

al. 2004)), but is surrounded by a number of similarly-positioned chlorophylls. The fact that both luteins exhibit the same 75/25 sharing of the triplet is thus most easily explained as a result of the pseudo-symmetry of the position of these chlorophyll molecules (Liu, Yan et al. 2004). The same conclusion can be drawn for monomeric LHCII, CP29 and CP43.

A closer analysis of the recently-obtained time-resolved FTIR data obtained for the PCP peridinin triplet supports this analysis. In FTIR, the intensity and frequency of the bands arising from the vibrational modes of the triplet state should reflect the triplet sharing. Since most of the bands are distorted by the differential method and by overlapping contributions, an accurate determination of their precise intensity and frequency is difficult except in the case of well-isolated bands. In the case of LHCII (Fig. 9), no carotenoid band can safely be used for that purpose. In the PCP spectra, the band arising from the stretching mode of the lactone carbonyl of peridinin is well isolated and contributes at *ca.* 1745 cm-1. From FTIR steady-state measurements of peridinin mixed with Chl *a* in THF (data not shown) the peridinin C=O lactone extinction coefficient was estimated to be similar to that of the Chl *a* C=O keto group. This allows us to estimate the extent of the 'triplet sharing' between peridinin and Chl *a*. From Fig. 8 the negative band area assigned to 9-keto C=O corresponds to about 25 and 40 % of the negative band area assigned to lactone C=O for A-PCP and H-PCP, respectively. Taking into account the similar C=O extinction coefficient of peridinin lactone C=O and Chl *a* 9-keto C=O, about 25 and 40 % of the 3peridinin is shared with Chl *a* in A-PCP and H-PCP, respectively. This conclusion is in good agreement with the relative amplitude of the Qy bleach as compared to the peridinin bleach of about 20 % in the

(A) A slice of the nonameric structure of the LH2 complex from *Rhodoblastus acidophilus* viewed in parallel with the membrane plane and from the outside of the protein. For clarity the central outerhelice, from three α/β-apoprotein dimers has been removed allowing the interaction of the Car (orange) with its nearest-neighbour Bchl *a* (green) molecules to be visualised. The contacts between Car and Bchl molecules essentially occur at the very end of the C=C conjugated chain of the carotenoid. Protein Data Bank accession number 1KZU. (B) View of a monomer of LHCII from *Spinacia oleracea* viewed in parallel with the membrane plane. The colours of the luteins (L1 and L2), neoxanthin (neo) and xanthophyll (xan) cycle carotenoids are orange, purple and magenta, respectively. The Chl *a* and Chl *b* molecules are coloured green and blue, respectively. The closest contacts between Chl *a* and luteins in LHCII occur at the middle of the C=C polyenic chain. Although the Chl molecules have a pseudosymmetry within the monomer lutein 1 (L1) and lutein 2 (L2) experience a different protein environment. Protein Data Bank accession number 1RWT.

Fig. 10. The organisation of the (bacterio)chlorophyll and carotenoid molecules in LH2 and LHCII.

T-S spectra of A-PCP (Kleima, Hofmann et al. 2000). This estimate of a 25 and 40 % triplet sharing in A-PCP and H-PCP is also in line with the smaller observed bandshift of the carbonyl lactone for H-PCP (*i.e.* 20 cm-1 as compared to 25 cm-1 for A-PCP and 28 cm-1 for Per in THF).

Thus, in contrast to photosynthetic bacteria, our results provide compelling evidence of triplet sharing between carotenoid and chlorophyll molecules in plant and algal lightharvesting complexes. It is obviously tempting to try to unravel which molecular mechanisms may be at the origin of this difference. In LH2, contacts between carotenoid and bacteriochlorophyll molecules essentially occur at the very end of the C=C conjugated chain of the carotenoid (Fig. 10A and Ref. (McDermott, Prince et al. 1995)), and the minimum distance between these molecules is 3.42 Å (Prince, Papiz et al. 1997). In strong contrast, the closest contacts between Chl *a* and luteins in LHCII occur at the middle of the C=C polyenic chain (Fig. 10B), and the pigments are slightly more closely packed in these complexes. Taking into account the expected molecular structure of the carotenoid triplet state, the relative positioning of carotenoid and chlorophyll molecules in LHCII appears definitely more favourable for triplet sharing between these molecules
