Time-resolved fluorescence analysis of the photosystem II antenna proteinsin detergent micelles and liposomes

We have studied the time-resolved fluorescence properties of the light-harv esting complexes (Lhe) of photosystem II (Lhcb) in order to obtain informat ion on the mechanism of energy dissipation (non-photochemical quenching) wh ich is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCI I, CP29, CP26, and CP24 in detergent solution is mostly determined by two l ifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the fa ster 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 relativ e amplitude of the faster component is increased and, consequently, the chl orophyll fluorescence quenching is enhanced. Analysis of quenching in mutan ts of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxan thin 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 co mponent corresponds to a distinct conformation of the Lhc proteins. Upon in corporation of Lhc proteins into liposomes, a quenching of chlorophyll fluo rescence was observed due to shortening of all their lifetime components: t his indicates that the equilibrium between the two conformations of Lhcb pr oteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein de nsity in the liposomes, and therefore the probability of protein-protein in teractions, a further decrease of fluorescence lifetimes takes place down t o values typical of quenched leaves. We conclude that at least two major fa ctors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions wit hin the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.