Time-Resolved Fluorescence Analysis of the Photosystem II Antenna Proteins in Detergent Micelles and Liposomes

Abstract

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.The photosynthetic apparatus of higher plants is composed of two photosystems (PS), 1 PSI and PSII, each consisting of a core complex moiety, binding chlorophyll (Chl) a and -carotene, and a peripheral antenna system, binding Chl a, Chl b, and xanthophylls (Cars). The major component of the PSII antenna system is LHCII, a heterotrimer of the Lhcb1, -2, and -3 gene products (also called LHCIIb) binding half of the thylakoid pigments. Minor components are CP29 (LHCIIa), CP26 (LHCIIc), and CP24 (LHCIId), respectively the products of the Lhcb4, -5, and -6 genes, which are monomeric and connect the PSII core complex to the peripheral LHCII trimers.The large absorption cross-section of the antenna systems allows plants to grow in dim light. However, the light intensity experienced by a plant varies largely over ...

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

“…A decrease of chlorophyll fluorescence lifetimes was found when the number of LHCs in the membrane was increased. 4 Comparable to the results presented here that study indicated a correlation of chlorophyll fluorescence quenching in a membrane and the corresponding protein density. Figure 5 summarizes the results of this study.…”

“…It was already shown that the chlorophyll fluorescence lifetime and thus also the fluorescence quenching of LHCs incorporated in liposomes are affected by the density of LHCs in membranes. 4 Furthermore, research on isolated LHCII trimers demonstrated a dependency between aggregation of LHCII and energy quenching. 6,10,26 Additionally, it was found that aggregation of LHCII trimers in vitro also causes an increase of the abovementioned coupling parameter.…”

A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements

“…The two shortest components might be correlated with a dissipative conformation of the protein backbone, as suggested before (Moya et al 2001).…”

“…For native LHC II complexes only two components (ns) were found (see Table 2). Recently, Moya et al have correlated the existence of two decay components in the ns regime to the existence of two conformations of the LHC II backbone (Moya et al 2001). It was suggested that one of them might dissipate excess energy and be correlated with the shortest component, whereas the other conformation, associated with the longest component, would participate in light-harvesting and energy transfer.…”

“…However, whereas in the spectra reported by Caffarri et al Lhcb1 exhibits more fluorescence than Lhcb2 and Lhcb3 at wavelengths between 685 and 700 nm, that is not the case in the spectra reported by Standfuss and Ku¨hlbrandt, where Lhcb2 appears to be the one fluorescing more at those wavelengths. Time-resolved fluorescence measurements have only been reported for reconstituted Lhcb1 and for native LHC II complexes (Ide et al 1987;Bittner et al 1994;Connelly et al 1997;Vasile´v et al 1997;Barzda et al 2001;Moya et al 2001;Palacios et al 2002). Whereas for the former complexes two components were reported (Moya et al 2001), for the latter a single component or two have been found (Ide et al 1987;Bittner et al 1994;Connelly et al 1997;Vasil'ev et al 1997;Barzda et al 2001;Palacios et al 2002).…”