We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.
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