The 2.7 kilobase transcript of the puf operon of the photosynthetic bacterium Rhodobacter capsulatus has five open reading frames. The gene products of four of these open reading frames (pufB, A, L,and M) are well characterized as structural polypeptides of the reaction center (pufL and M) and the B870 light-harvesting antenna complex (pufB and A), The role of the pufX gene product has been unknown. By deleting the pufX gene from a plasmid carrying the puf operon and using this plasmid to reconstitute a strain of R. capsulatus which had the puf operon deleted, it was possible to characterize the pufX gene product. It was found that the pufX⁻ mutant was unable to grow photosynthetically until a secondary suppressor mutation had occurred. It appeared that either more than one type of suppressor mutation could occur or that one suppressor mutation could be accompanied by further mutations. To determine the nature of the lesion caused by the deletion of pufX, the structure of the photosynthetic unit and the ability of the subunits of the photosynthetic unit to accomplish energy and electron transfer of the mutant were compared to a pseudo-wild type. Spectrophotometric techniques, including fluorescence detection, reduced minus oxidized spectra, flash-induced absorbance change spectra, and ground state absorption spectra were used for these comparisons as well as biochemical assays. The biochemical assays measured the ability of chromatophores to transfer electrons from a quinone analog to horse-heart cytochrome c and to pump protons in response to light irradiation. The results of these comparisons indicated that the individual components of the photosynthetic unit functioned normally but that electron transfer between these components, specifically between the reaction center and the cytochrome b⁄c₁ complex, was impaired. It thus seemed likely that there was some structural defect in the photosynthetic unit. The structure of the photosynthetic units of pseudo-wild type and mutant strains was probed using sodium dodecyl sulfate-polyacrylamide gels, absorption spectroscopy, and electron microscopy. It was determined that the mutant had chromatophore vesicles that were about 50% larger than those of the pseudo-wild type and contained higher levels of reaction center and B870 light-harvesting antenna polypeptides. The suppressor mutants also had altered levels of polypeptides and showed differences in the way the expression of their B800-850 polypeptides was regulated. It was concluded that the pufX gene product plays a role in the correct assembly of the photosynthetic unit as a structural component of the unit and/or as a regulator of its assembly. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A glass electrode is described which is capable of detecting photosynthetic CO₂ uptake by monitoring the concentration of CO₂ dissolved in a small drop of algal suspension. Consideration of current theory suggests that transients should occur in the rate of CO₂ uptake which are similar to but distinct from those observed in O₂ evolution rate. These transients should be most readily observed when cells are illuminated alternately with wavelengths of 650 nm and 705 mn. The preliminary results show that C0₂ uptake by photosynthesis can be monitored by this apparatus. Although transients were not observed, this may be due to the rather low sensitivity. There is a discussion of shortcomings of the apparatus, and of improvements necessary before transients can be observed. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

In the first study presented here, the 2.5 Å crystal structure1 of the cytochrome <i>b₆f</i> complex obtained from the cyanobacterium Nostoc sp. PCC 7120 (pdb 4OGQ) was used as a guide for modification by site-directed mutagenesis in the cyanobacterium Synechococcus sp. PCC 7002 of the rate-limiting step in the central electron transport/proton translocation chain of oxygenic photosynthesis. This step is associated with the oxidation and deprotonation of plastoquinol on the electrochemically positive (p) side of the membrane. The mutagenesis strategy is based on structure studies of the <i>b₆f</i> complex in the absence and presence of quinol analogue inhibitors which bind and inhibit electron transport on the p-side of the thylakoid membrane. The strategy focused on two conserved prolines located on the p-side of the F-helix, proximal to the C-helix, in subunit IV of the seven subunit cytochrome <i>b₆f</i> complex. These prolines, residues 105 and 112 in the F-helix, are seen in the crystal structure to cause a bend in this helix away from the C-helix in the cytochrome b subunit. Thus, they are predicted to increase the portal aperture for the plastoquinol generated in the photosystem II reaction center complex that serves as the electron-proton donor to the [2Fe-2S] iron-sulfur protein and the pside b-heme. Changing the two prolines to alanine resulted in a decrease of 30-50 % in the logphase growth rate of the cell culture and reduction of photo-oxidized cytochrome f. The second study examines the binding thermodynamics of the cytochrome b6f complex and a purposed binding partner, PGRL1, using isothermal titration calorimetry. Proton Gradient Regulation-Like 1 (PGRL1) is thought to be necessary for efficient cyclic electron transfer, however, it’s mechanistic role is unknown. Here we examined for PGRL1 and cytochrome b6f complex binding and found there was no detectable interaction, indicating that PGRL1 is not a direct quinone/cyt b6f electron cofactor.<br>

This study utilizes steady state fluorescence measurements, flash-induced P680+ absorption transients, and DCIP reduction kinetics to study the inhibitory effects of linolenic acid (LA) upon Photosystem II (PSII) in whole spinach chloroplasts and insideout wheat thylakoids. It confirms the presence within PSII of LA-induced inhibition of energy trapping and/or primary charge separation (i.e., primary inhibition), in addition to donor side inhibition. The latter is diminished in the presence of 1,5-Diphenylcarbohydrazide (DPC) and probably takes place at the oxygen evolving complex. Primary inhibition, which is more controversial, probably occurs between Ph and QA, with a likely contribution at the level of PSII energy trapping. In addition, the ability of Mg2+ to delay a drop in steady state fluorescence intensity normally associated with thylakoid exposure to LA is explained by the ability of this cation to confer resistance to LA-induced destacking of thylakoid membranes. Steady state fluorescence results in the presence of DCMU, dithionite and LA also support the presence of an additional acceptor between Ph and QA. This acceptor, designated here as "R." is proposed not to be a sequential member of the transport chain, but may be accessible to it via QA when the chain blocked, such as with DCMU.R- is proposed to exert a coulombic effect upon Ph, thereby affecting the degree of primary charge recombination. It may be related to one of the several acceptors already proposed by others and the need for more study is stressed in order to confirm or refute its existence.

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

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