**6. Conclusion**

250 Infrared Spectroscopy – Life and Biomedical Sciences

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.

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

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

environment. Protein Data Bank accession number 1RWT.

LHCII.

2004). The same conclusion can be drawn for monomeric LHCII, CP29 and CP43.

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.
