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Universiẗat, Diss., 2001--München.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20215号 / 理博第4300号 / 新制||理||1618(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 教授 長谷 あきら, 准教授 小山 時隆 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

In higher plants oxygenic photosynthesis primarily takes place in the chloroplasts of leaves. Within the chloroplasts is an intricate membrane system, the thylakoid membrane, which is the site of light harvesting and photosynthetic electron transport. Enclosed by this membrane is the lumen space, which initially was believed to only contain a few proteins, but now is known to house a distinct set of >50 proteins, many for which there is still no proposed function. The work presented in this thesis is focused on understanding the functions of the proteins in the lumen space. Using proteomic methods, we investigated first the regulation of lumenal proteins by light and secondly by dithiol-disulphide exchange, mediated by the disulphide reductase protein thioredoxin. We furthermore performed structural and functional studies of the lumenal pentapeptide repeat proteins and of the PsbP-domain protein PPD6. When studying the diurnal expression pattern of the lumen proteins, using difference gel electrophoresis, we observed an increased abundance of fifteen lumen protein in light-adapted Arabidopsis thaliana plants. Among these proteins were subunits of the oxygen evolving complex, plastocyanin and proteins of unknown function. In our analysis of putative lumenal targets of thioredoxin, we identified nineteen proteins, constituting more than 40 % of the lumen proteins observable by our methods. A subset of these putative target proteins were selected for further studies, including structure determination by x-ray crystallography. The crystal structure of the pentapeptide repeat protein TL15 was solved to 1.3 Å resolution and further biochemical characterization suggested that it may function as a novel type of redox regulated molecular chaperone in the lumen. PPD6, a member of the PsbP-family of proteins, which is unique in that it possesses a conserved disulphide bond not found in any other PsbP-family protein, was also expressed, purified and crystallized. A preliminary x-ray analysis suggests that PPD6 exists as a dimer in the crystalline state and binds zinc ions. The high representation of targets of thioredoxin among the lumen proteins, along with the characterization of the pentapeptide repeat protein family, implies that dithiol-disulphide exchange reactions play an important role in the thylakoid lumen of higher plants, regulating processes such as photoprotection, protein turnover and protein folding.

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Plants, algae and photosynthetic bacteria are able to harvest the sunlight and use its energy to transform water and carbon dioxide to carbohydrate molecules and oxygen, both important to sustain life on Earth. This process is called photosynthesis and is the route by which almost all energy enters the biosphere. As most simple things in life, the process of photosynthesis is easily explained but unfortunately not that easy to reproduce. If we could, we would be living in a much different world with almost unlimited energy. Light energy is harvested by chlorophyll molecules, bound to proteins in the chloroplast thylakoid membrane and drives the oxygen-evolving complex, to extract electrons from water. Electrons are then transferred to NADPH through photosystem II (PSII) to cytochrome b6f and photosystem I, the major photosynthetic protein complexes. The cytochrome b6f complex also transfers protons into the lumenal space of the thylakoid. These protons together with those from water oxidation create an electrochemical gradient across the thylakoid membrane, which fuels the ATP synthase to produce ATP. ATP, NADPH and carbon dioxide are used during the dark reactions to produce sugars in the chloroplast stroma. The thylakoid lumenal space where the water oxidation occurs has until recently been viewed as a proton sink with very few proteins. With the publication of the genome of Arabidopsis thaliana it seems to be a much more complex compartment housing a wide variety of biochemical processes. ATP is a nucleotide and the major energy currency, but there are also other nucleotides such as AMP, ADP, GMP, GDP and GTP. Chloroplast metabolism has mostly been associated with ATP, but GTP has been shown to have a role in integration of light harversting complexes into the thylakoid. In this work, we have demonstrated the occurrence of nucleotide-dependent processes in the lumenal space of spinach by bringing evidence first for nucleotide (ATP) transport across the thylakoid membrane, second for nucleotide inter-conversion (ATP to GTP) by a nucleoside diphosphate kinase, and third the discovery that the PsbO extrinsic subunit of PSII complex can bind and hydrolyse GTP to GDP. The active PSII complex functions as a dimer but following light-induced damage, it is monomerised allowing for repair of its reaction center D1 protein. PsbO is ubiquitous in all oxygenic photosynthetic organisms and together with other extrinsic proteins stabilises the oxygen-evolving complex. We have modelled the GTP-binding site in the PsbO structure and showed that the GTPase activity of spinach PsbO induces changes in the protein structure, dissociation from the complex and stimulates the degradation of the D1 protein, possibly by inducing momerisation of damaged PSII complexes. As compared to spinach, Arabidopsis has two isoforms of PsbO, PsbO1 and PsbO2, expressed in a 4:1 ratio. A T-DNA insertion knockout mutant of PsbO1 showed a retarded growth rate, pale green leaves and a decrease in the oxygen evolution while a PsbO2 knockout mutant did not show any visual phenotype as compared to wild type. Unexpectedly, during growth under high light conditions the turnover rate of the D1 protein was impaired in the PsbO2 knockout, whereas it occurred faster in the PsbO1 knockout as compared to wild type. We concluded that the PsbO1 protein mainly functions in stabilizing the oxygen evolving complex, whereas the PsbO2 protein regulates the turnover of the D1 protein. The two PsbO proteins also differ in their GTPase-activity (PsbO2 >> PsbO1). Although their amino acid sequences are 90% identical, they differ in the GTP-binding region which could explain the difference in their GTPase activity. Based on these data, we propose that the GTPase activity of PsbO(2) leads to structural changes in interacting loops and plays a role in the initial steps of D1 turnover such as the PSII monomerisation step. The nucleotide-dependent processes we discovered in the thylakoid lumen raise questions of transporters to facilitate these processes. As stated earlier, we provided biochemical evidence of an ATP thylakoid transporter, and most recently have identified a transporter that may be important for the export of lumenal phosphate back to the stroma. More transporters for GDP, metal ions and others solutes have still to be identified.

We present a novel thylakoid based bio-solar cell capable of generating a photoelectric current of    0.7 µA/cm2. We have introduced an electro conductive polymer, PEDOT-S, to the thylakoid membrane. PEDOT-S intervenes in the photosynthesis, captures electrons from the electron transport chain and transfers them directly across the thylakoid membrane, thus generating a current. The incorporation of the electro conductive polymer into the thylakoid membrane is therefore vital for the function of the bio-solar cell. A liposomal model system based on liposomes formed by oleic acid was used to develop and study the incorporation of PEDOT-S to fatty acid membranes. The liposomes allow for a more controllable and easily manipulated system compared to the thylakoid membrane. In the model system, PEDOT-S could successfully be incorporated to the membrane, and the developed methods were applied to the real system of thylakoid membranes. We found that a bio-compatible electrolyte and redox couple was required for this system to function. The final thylakoid based bio-solar cell was evaluated according to performance and reproducibility. We found that this bio-solar system can generate a low but reproducible current.

Photoelectrochemical cells have been intensively studied in recent years with regard to using thylakoid and photosynthesis system I/II. BBY membrane is another protein complex that has potential to be utilized to build photoelectrochemical cells. Within the BBY membrane lies the highly active photosynthesis system II complex, which upon light activation, generates electrons transported within the electron transport chain during photosynthesis in green plants. This study presents an approach of immobilizing thylakoid or BBY membrane onto gold nanoparticle modified gold plate or multi-walled carbon nanotube (MWCNT) modified indium tin oxide vi (ITO) coated glass substrate. The results show that BBY membrane has higher activity with a value of 168 ± 12 μmol DCIP/(mg Chl*hr) than the thylakoid, which has an activity of 67 ± 7 μmol DCIP/(mg Chl*hr). Further amperometric tests also show that BBY membrane generates a higher current than the thylakoid. We used gold based materials to build the cell first since gold has high electrical conductivity. However, in order to minimize the construction cost of cells, relatively cheap materials such as ITO coated glass and MWCNT were used instead. The surface morphology of cells was characterized using atomic force microscope (AFM) throughout cell modification. When comparing to the cell with gold material, the cell constructed with ITO and MWCNT generated a higher current density. The highest current density was found as 20.44 ± 1.58 μA/cm2 with a system of ITO/MWCNT/BBY. More, the stability of the system was examined and the result shows a decreasing rate of 0.78 %/hour.

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