Studies On The Reconstitution Of Photosystem II Pigment-protein Complexes Into Liposome And Function Of Photosystem II Reaction Center

Photosystem II (PSII) is a multisubunit chlorophyll-protein complex embedded in the thylakoid membrane, which is very important in absorbing light energy, splitting water into oxygen, protons and electrons. Its native form is the PSII-LHCII supercomplex that consists of PSII core complex and its associated light harvesting complex (LHCII), which enables efficiently energy absorption and transfer to reaction centre in which the primary photochemical reaction occurs. In order to study the interaction of PSII core complexes and LHCII in the lipid-bilayer membranes, the coarse PSII oxygen-evolving chlorophyll-protein complexes (OECC) or purified core complexes (pdOE) and the trimeric LHCII were reconstituted, both separately and together, into the preformed liposomes, which contain the main thylakoid lipids (digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)). The second part of the thesis dealt with function of the pigments in the reaction centre of PSII, studied by substituting part of the chlorophyll a in PSII RC, and the characters of energy absorption and transfer in PSII RC during the process of photodamage. The main results are as follows: 1. Studies on the characters of PSII and LHCII proteoliposomes: OECC, pdOE and LHCII preparations were incorporated into liposomes respectively. LHCII preparation showed spectroscopic properties of aggregate, and blue-shift of its absorption and fluorescence peaks after incorporated into liposomes. In addition, compared with the preparations before reconstitution, the fluorescence emission intensity increased in the proteoliposomes reconstituted with LHCII, OECC or pdOE preparations. These results suggested lipid-environment played an important role in the aggregation and interactions of protein-protein or chlorophyll-chlorophyll in pigment-protein complexes. Two types of proteoliposomes (LHCII-OECC and LHCII-pdOE proteoliposome) were obtained by reconstitution of OECC or pdOE preparations together with LHCII into liposome. It was observed by freeze-fracture electron microscopy (EM) that PSII core complexes (OECC or pdOE) and LHCII formed macromolecules, which dispersed randomly in the membranes. The formation of PSII-LHCII macromolecules prevented the formation of LHCII crystalline structure in the membrane environment. The PSII-LHCII proteoliposomes exhibited both absorption peaks of LHCII and core complex. However, the 77K fluorescence emission maximum was at peak typical of core complex (684nm-685nm) rather than that of LHCII (680 nm). The shapes of the emission spectra were nearly identical after normalization irrespective of the excitation wavelength. The results indicated the energy absorbed by LHCII was mainly transferred to PSII core complexes, and the structural and functional coupling of LHCII and PSII occurred in the proteoliposomes. LHCII-OECC (LHCII-pdOE) proteoliposomes showed a significantly higher photochemical activity than did OECC (pdOE) proteoliposomes alone both at high and low light intensity, which gives another evidence for the coupling of LHCII to OECC or pdOE complexes. The absorption cross section was enlarged because of greater antenna size in LHCII-OECC (LHCII-pdOE) proteoliposomes, so the photochemical activity of PSII was enhanced. The excitation energy transfer and trapping of proteoliposomes were investigated by 77K femtosecond time-resolved fluorescence measurement. The main decay lifetimes of LHCII, OECC and pdOE proteoliposomes were 670 ps (Max: 680 nm), 650 ps (Max: 690 nm) and 570 ps (Max: 685nm) respectively. In LHCII-OECC and LHCII-pdOE proteoliposomes, the main decay lifetimes were 940 ps (Max: 690 nm) and 840 ps (Max: 690 nm) respectively. An additional 40 ps component was observed, which was absent in proteoliposomes containing only one complex. It was presumed as the time constant of exciton equilibration between LHCII and core complex, and much faster than the mean exciton lifetime, which provided support for the trap-limited model. In addition, coupling of LHCII to OECC or pdOE increased Chl a fluorescence lifetime, which might be relevance to the photoprotection mechanisms of PSII. Both of OECC and pdOE preparations can be integrated with LHCII into liposomes and there were not evident differences of structure and function between two types of proteoliposomes. This might suggest that minor antenna and 23, 17 kDa extrinsic polypeptides were not necessary for the incorporation and energy transfer between LHCII and core complexes. 2. Effects of acceptor side photodamage and pigment exchange on PSII RC Strong light (800μmol m-2s-1) induced bleaching of the pigments in the isolated PSII reaction center under aerobic conditions (in the absence of electron donors or acceptors) was studied. The photo-bleach of chlorophyll and β-car almost occurs simultaneously. The pigments absorbing at 680 nm were the most sensitive tophotodamage and the peripheral chlorophylls absorbing at 670 nm were more stable than other pigments. After illumination the fluorescence emission intensity increased initially then decreased. At the same time, the fluorescence maximum blue shifted, showing the energy transfer was disturbed. Two groups of Resonance Raman spectra of PSII RC were gained when excited at 488.0 and 514.5 nm. It confirmed there was two spectroscopically different β-car molecules existed. The band positions and bandwidths were unchanged during light treatment indicating β-car configuration was not the parameter that regulated the photoprotection in PSII RC. Cu-Chl a was used to exchange with the natural Chl in PSII RC through a chemical exchange procedure. There was about 0.5 Cu-Chl per 2 Pheo in the RC exchanged with Cu-Chl (Cu-Chl-RC). Compared to native RC and RC treated with Chl a in the same way, Cu-Chl-RC preparation showed the appearance of Cu-Chl concomitant with partial loss of Chl. The Cu-Chl insertion caused decrease in absorption at 670 nm and the increase at 660 nm. It suggested that the peripheral chlorophyll might be displaced. Protein compositions were not changed and the P680 activity was largely retained after exchange procedure. There was only little difference in the CD spectra, suggesting that the arrangement of pigments and proteins responsible for the CD signal was not significantly affected. Compared to native RC, the fluorescence emission intensity of Cu-Chl-RC was significantly decreased, and the band maximum blue shifted and accompanied by changes of peak shape, indicating that energy transfer in modified RC was disturbed by Cu-Chl, a quencher of excited state.