A new, unquenched intermediate of LHCII

Abstract

When plants are exposed to high-light conditions, the potentially harmful excess energy is dissipated as heat, a process called non-photochemical quenching. Efficient energy dissipation can also be induced in the major light-harvesting complex of photosystem II (LHCII) in vitro, by altering the structure and interactions of several bound cofactors. In both cases, the extent of quenching has been correlated with conformational changes (twisting) affecting two bound carotenoids, neoxanthin, and one of the two luteins (in site L1). This lutein is directly involved in the quenching process, whereas neoxanthin senses the overall change in state without playing a direct role in energy dissipation. Here we describe the isolation of an intermediate state of LHCII, using the detergent n-dodecyl-α-D-maltoside, which exhibits the twisting of neoxanthin (along with changes in chlorophyll-protein interactions), in the absence of the L1 change or corresponding quenching. We demonstrate that neoxanthin is actually a reporter of the LHCII environment-probably reflecting a large-scale conformational change in the protein-whereas the appearance of excitation energy quenching is concomitant with the configuration change of the L1 carotenoid only, reflecting changes on a smaller scale. This unquenched LHCII intermediate, described here for the first time, provides for a deeper understanding of the molecular mechanism of quenching.

Keywords: LHCII; NPQ; light-harvesting complex; photoprotection; resonance Raman.

Figure 1

Molecular structures of the LHCII tightly bound pigments and the detergents used for LHCII purification. Molecular structures of chlorophyll a, chlorophyll b, 9′-cis neoxanthin, all-trans lutein, α-dodecyl-D-maltoside (α-DM), and β-dodecyl-D-maltoside (β-DM).

Figure 2

Absorption and time-resolved fluorescence spectra of LHCII.A, absorption spectra at 4.2 K of LHCII in α-DM and β-DM (blue and red, respectively). B, difference spectrum “α-DM-LHCII minus β-DM-LHCII.” C, time-resolved fluorescence of α-DM-LHCII (blue) and β-DM-LHCII (red), excited at 405 nm with emission recorded at 680 nm. The grayed region represents instrumental response function in time domain.

Figure 3

High-frequency region of resonance Raman spectra of LHCII. Resonance Raman spectra at 77 K in the 1540 to 1720 cm⁻¹ region for α-DM-LHCII (blue), β-DM-LHCII (red), and LHCII aggregates (black) excited at (A) 413.1 and (B) 441.6 nm.

Figure 4

ν₃and ν₄regions of resonance Raman spectra of LHCII. Resonance Raman spectra at 77 K in the 930 to 1050 cm⁻¹ region for α-DM-LHCII (blue), β-DM-LHCII (red), and LHCII aggregates (black) excited at 488.0 nm.

Figure 5

ν₃region of LHCII resonance Raman spectra of LHCII. Resonance Raman spectra at 77 K in the ν₄ region for LHCII in α-DM (red), β-DM (blue), and in the aggregated form (black), for excitation at 488.0 (A) and 496.5 nm (B).