Molecular adaptation of photoprotection: triplet states in light-harvesting proteins

Abstract

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis.

Figure 1

Evolution-associated difference spectra (EADS) of LHCII excited at 400 nm. The EADS spectra correspond to the 100 fs (black), 1 ps (red), 14.9 ps (blue), 3.9 ns (green; Chl excited-state lifetime), and infinite (magenta) lifetimes. The Chl a singlet excited signal that is characterized by ground-state bleach at ∼675 nm within 3.9 ns is replaced by the carotenoid triplet spectrum (magenta). (Inset) Kinetic traces at 511 nm, the maximum of the T₁→T_n transition, and at 675 nm, corresponding to the maximum of the Chl a bleach signal; for clarity, the magnitude of the latter trace has been reduced 10-fold.

Figure 2

Global analysis of step-scan FTIR data of LHCII excited at 475 nm showing the lutein-Chl shared triplet state. (A) The DADS have a 20 μs component (black line, DADS1), with an amplitude of 90% (lutein-Chl shared triplet) and a nondecaying component (blue line, DADS2) that has an amplitude of 10% (unquenched Chls). For clarity, the spectra have been normalized to the keto modes. (B and C) For comparison, the FTIR of the lutein (B) and Chl a (C) triplet (redrawn from Bonetti et al. (26)) in THF are also plotted.

Figure 3

Raman spectra (800–1650cm⁻¹) of the LH2 complex from Rbl. acidophilus in resonance with the carotenoid rhodopin glucoside. (A) The subpopulation attributed to triplet carotenoid increases with laser power. (B) The deduced triplet-state Raman spectrum of rhodopin glucoside. T = 77 K, λ_ex =514.5 nm.

Figure 4

Power-induced triplet excited-state resonance Raman spectra in the v₁ (A) and v₄ (B) regions of the carotenoid molecules in LHCII trimers excited at 514.5 and 528.7 nm. The blue and black traces were obtained after excitation with 30 μW and 200 μW, respectively. The red traces are the difference spectra and are ascribed to the positions of the triplet states (these are magnified in the v₁ region). Also shown is the power-induced triplet excited-state resonance Raman spectra in the v₁ region of CP43 trimers when excited at 528.7 nm. T = 77 K.

Figure 5

Organization of the (bacterio)chlorophyll and carotenoid molecules in LH2 and LHCII. (A) A slice of the nonameric structure of the LH2 complex from Rbl. acidophilus viewed in parallel with the membrane plane and from the outside of the protein. For clarity the central outer helix from three α/β-apoprotein dimers has been removed, allowing the interaction of the carotenoid (orange) with its nearest neighbor, (B)Chl a (green) molecules to be visualized. The contacts between Car and (B)Chl 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 in parallel with the membrane plane. The colors of the two luteins (L), neoxanthin (neo), and xanthophyll (xan) cycle carotenoids are orange, purple, and magenta, respectively. The Chl a and Chl b molecules are colored 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.