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Under conditions of excess sunlight the efficient light-harvesting antenna found in the chloroplast membranes of plants is rapidly and reversibly switched into a photoprotected quenched state in which potentially harmful absorbed energy is dissipated as heat, a process measured as the non-photochemical quenching of chlorophyll fluorescence or qE. Although the biological significance of qE is established, the molecular mechanisms involved are not. LHCII, the main light-harvesting complex, has an inbuilt capability to undergo transformation into a dissipative state by conformational change and it was suggested that this provides a molecular basis for qE, but it is not known if such events occur in vivo or how energy is dissipated in this state. The transition into the dissipative state is associated with a twist in the configuration of the LHCII-bound carotenoid neoxanthin, identified using resonance Raman spectroscopy. Applying this technique to study isolated chloroplasts and whole leaves, we show here that the same change in neoxanthin configuration occurs in vivo, to an extent consistent with the magnitude of energy dissipation. Femtosecond transient absorption spectroscopy, performed on purified LHCII in the dissipative state, shows that energy is transferred from chlorophyll a to a low-lying carotenoid excited state, identified as one of the two luteins (lutein 1) in LHCII. Hence, it is experimentally demonstrated that a change in conformation of LHCII occurs in vivo, which opens a channel for energy dissipation by transfer to a bound carotenoid. We suggest that this is the principal mechanism of photoprotection.

Photosynthetic organisms are crucial for life on Earth as they provide food and oxygen and are at the basis of most energy resources. They have a large variety of light-harvesting strategies that allow them to live nearly everywhere where sunlight can penetrate. They have adapted their pigmentation to the spectral composition of light in their habitat, they acclimate to slowly varying light intensities and they rapidly respond to fast changes in light quality and quantity. This is particularly important for oxygen-producing organisms because an overdose of light in combination with oxygen can be lethal. Rapid progress is being made in understanding how different organisms maximize light harvesting and minimize deleterious effects. Here we summarize the latest findings and explain the main design principles used in nature. The available knowledge can be used for optimizing light harvesting in both natural and artificial photosynthesis to improve light-driven production processes.

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat. This is essential for plant survival, because it provides protection against the potential photo-damage of the photosynthetic membrane. But whereas the features that establish high photosynthetic efficiency have been highlighted, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvesting.

We have modeled steady-state spectra and energy-transfer dynamics in the peripheral plant light-harvesting complex LHCII using new structural data. The dynamics of the chlorophyll (Chl) b-->Chl a transfer and decay of selectively excited "bottleneck" Chl a and b states have been studied by femtosecond pump-probe spectroscopy. We propose an exciton model of the LHCII trimer (with specific site energies) which allows a simultaneous quantitative fit of the absorption, linear-dichroism, steady-state fluorescence spectra, and transient absorption kinetics upon excitation at different wavelengths. In the modeling we use the experimental exciton-phonon spectral density and modified Redfield theory. We have found that fast b-->a transfer is determined by a good connection of the Chls b to strongly coupled Chl a clusters, i.e., a610-a611-a612 trimer and a602-a603 and a613-a614 dimers. Long-lived components of the energy-transfer kinetics are determined by a quick population of red-shifted Chl b605 and blue-shifted Chl a604 followed by a very slow (3 ps for b605 and 12 ps for a604) flow of energy from these monomeric bottleneck sites to the Chl a clusters. The dynamics within the Chl a region is determined by fast (with time constants down to sub-100 fs) exciton relaxation within the a610-a611-a612 trimer, slower 200-300 fs relaxation within the a602-a603 and a613-a614 dimers, even slower 300-800 fs migration between these clusters, and very slow transfer from a604 to the quasi-equilibrated a sites. The final equilibrium is characterized by predominant population of the a610-a611-a612 cluster (mostly the a610 site). The location of this cluster on the outer side of the LHCII trimer probably provides a good connection with the other subunits of PSII.

Spectral and kinetic information on energy transfer from carotenoids (Cars) to chlorophylls (Chls) within light-harvesting complex II (LHCII) and CP29 was obtained from femtosecond transient absorption study by using selective Car excitation (489 and 506 nm) and detecting the induced changes over a wide spectral interval (460-720 nm). By examining the evolution of entire spectral bands rather than looking at a few single traces, we were able to identify the species (pigments and/or electronic states) which participate in the energy flow, as well as the lifetimes and quantum yields of individual processes. Hence, it was found that the initially excited Car S 2 state decays very fast, with lifetimes of 70-90 fs in CP29 and 100 ( 20 fs in LHCII, via two competing channels: energy transfer to Chls (60-65%) and internal conversion to the lower, optically forbidden S 1 state (35-40%). In CP29, the energy acceptors are exclusively Chls a, while in LHCII, this is only valid for lutein and violaxanthin. In the latter case, neoxanthin transfers energy mostly to Chls b. In both complexes, ca. 15-20% of the initial Car excitations are transferred to Chls a via the S 1 level, with a time constant of around 1 ps, thus bringing the total Car-Chl transfer efficiency to ca. 80%. Given the yield of this process and the large difference between the transfer time and the intrinsic S 1 lifetime (∼20 ps), it seems that lutein is the only species active on this pathway. From the measured transfer rates, we estimated that a coupling of 280-330 cm -1 drives the transfer via the S 2 route, while a coupling value of around 100 cm -1 was estimated for the S 1 transfer. The Car S 2 state is coupled to both Q x and Q y states of the Chl through a Coulombic mechanism; from the available structural information, we estimated the dipole-dipole contribution to be 450-500 cm -1 . The S 1 state is coupled to the Chl a Q y transition via an exchange and/or a Coulombic mechanism.

Since the crystal structure of the major light-harvesting complex II (LHCII) of green plants was obtained by Ku ¨hlbrandt, Wang and Fujiyoshi (Nature 1994, 367, 614-621), this chlorophyll-containing trimeric membrane protein has been the subject of intensive investigation. The complex contains between 36 and 42 chlorophyll molecules per trimer (Chl a and Chl b) and 10 to 12 xanthophyll molecules (lutein, neoxanthin and violaxanthin). The protein displays a rich spectrum of interactions, both between pigments and between the pigments and the protein, and these interactions have been studied with a multitude of different techniques. In this article we present an overview of the most important experimental results that have become available over the past decade and relate these to the structural knowledge. Emphasis will be put on the pigment identities, their spectroscopic features, and the interactions between the pigments, which determine both steady-state (polarized) properties and singlet and triplet energy transfer dynamics. Remaining questions will be pinpointed and hopefully they can help direct research in the near future.

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