Photochemically induced dynamic nuclear polarization in the reaction center of the green sulphur bacterium Chlorobium tepidum observed by 13C MAS NMR

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by (13)C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced (13)C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.     

Purification and characterization of the photochemical reaction center of the thermophilic purple sulfur bacterium Chromatium tepidum

Pure reaction center preparations from the thermophilic species Chromatium tepidum have been obtained by lauryldimethylamine N-oxide treatment of chromatophores. The light-induced difference spectrum in presence of 10 mM sodium ascorbate revealed the presence of two high-potential cytochrome c hemes (-band, 555 nm; γ-band, 422 nm). The dithionite-minus-oxidized difference spectrum in the -band suggests the presence of additional hemes of low potential. These hemes are associated with a single polypeptide (M_r = 36 000). The reaction center pigments, probably four bacteriochorophyll a and two bacteriopheophytin a molecules, are associated with three polypeptides of apparent molecular weights equal to 33 000, 30 000 and 22 000. A carotenoid molecule is also bound to the reaction center. The three main absorption bands of this molecule are located at 480, 510 and 530 nm at liquid helium temperature. Photochemical activity is found to be stable, even after heating for 10 min at temperatures higher than 60 °C in intact chromatophore membranes. On the other hand, isolated reaction centers or chromatophores treated with 1% lauryldimethylamine N-oxide are fully inactivated after heating at temperatures higher than 50 °C. From these results, we propose that lipid-protein interactions are of prime importance in the thermal stabilization of Chromatium tepidum reaction centers.     

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c_z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c_z. It was also shown that the rereduction rate of cytochrome c_z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Efficiency of excitation energy trapping in the green photosynthetic bacterium Chlorobaculum tepidum

During the millions of years of evolution, photosynthetic organisms have adapted to almost all terrestrial and aquatic habitats, although some environments are obviously more suitable for photosynthesis than others. Photosynthetic organisms living in low-light conditions require on the one hand a large light-harvesting apparatus to absorb as many photons as possible. On the other hand, the excitation trapping time scales with the size of the light-harvesting system, and the longer the distance over which the formed excitations have to be transferred, the larger the probability to lose excitations. Therefore a compromise between photon capture efficiency and excitation trapping efficiency needs to be found. Here we report results on the whole cells of the green sulfur bacterium Chlorobaculum tepidum. Its efficiency of excitation energy transfer and charge separation enables the organism to live in environments with very low illumination. Using fluorescence measurements with picosecond resolution, we estimate that despite a rather large size and complex composition of its light-harvesting apparatus, the quantum efficiency of its photochemistry is around ~87% at 20?°C, ~83% at 45?°C, and about ~81% at 77?K when part of the excitation energy is trapped by low-energy bacteriochlorophyll a molecules. The data are evaluated using target analysis, which provides further insight into the functional organization of the low-light adapted photosynthetic apparatus.     

Determination of the degree of acetylation of chitin materials by 13C CP/MAS NMR spectroscopy

¹³C CP/MAS NMR spectroscopy has been shown to be a powerful tool to quantify the degree of acetylation of chitin and chitosan. In order to optimise the parameters which afford quantitative ¹³C cross-polarisation magic-angle spinning NMR spectra, a detailed relaxation study has been carried out on selected chitin and deacetylated chitin samples. A relaxation delay of 5 s and a contact time of 1 ms have been found to yield quantitative NMR spectra of samples with deacetylation degree values of 0.68 and 0.16. The measured spin-lattice relaxation times in the rotating frame, T_1ρH, are in the range 6.4–8.9 ms for chitin and 4.3–7.3 ms for deacetylated chitin, while T_CH values for both samples are very similar and range from 0.03 to 0.19 ms. Spin-counting experiments indicate that, within experimental error, all carbon is detected by NMR indicating that the samples studied contain no (or very few) paramagnetic centres.     

Function of the polypeptides of the photosystem II reaction center in Chlamydomonas reinhardtii. Localization of the primary reactants

The distribution of the primary quinone and of the pheophytin acceptors has been studied in PS II particles isolated from Chlamydomonas reinhardtii, with respect to the distribution of the apoproteins of the two chlorophyll-protein complexes associated with the PS II core. We show that photoreduction of the primary quinone requires the presence of the 50 and 47 kDa polypeptides. On the contrary, charge separation between P-680 and the pheophytin acceptor molecules can occur within the chlorophyll-protein complex of which the 50 kDa polypeptide is the apoprotein. Functional analysis of the PS II fractions shows that an active PS II center contains one photoreducible quinone and one photoreducible pheophytin per 45 chlorophyll molecules. Stoichiometric analysis of the PS II fractions shows that a PS II reaction center contains 45 chlorophyll molecules associated with most likely one copy of the 50 kDa and the 47 kDa polypeptides.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

A study of ordered structure in acid-modified tapioca starch by C CP/MAS solid-state NMR

Acid modification of tapioca starch earlier reported to increase the mechanical strength of tablets. The development of ordered structure (double helices) of these starches was monitored after equilibrating at 0.90 a_w (25 °C) using ¹³C CP/MAS NMR and X-ray diffraction. As the hydrolysis time increased, the intensity of the resonance for C1 and C4 amorphous fractions decreased while that for C1 and C4 double helix fractions increased. Relative crystallinity (%) obtained from ¹³C CP/MAS NMR and X-ray diffraction methods both increased sharply initially and then levelled off with hydrolysis time. The initial increase in relative double helix content and crystallinity was due to a hydrolytic destruction in the amorphous domain, retrogradation of the partially hydrolyzed amylose and crystallization of free amylopectin double helices. After 192 h, these two parameters were not significantly different (=0.05) indicating that the double helices that were not arranged into crystalline regions either had been hydrolyzed or crystallized.     

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the ‘two-color’ measurements at 820 and 835 nm upon excitation at 800 nm, whereas the ‘one-color’ kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps. This revised version was published online in June 2006 with corrections to the Cover Date.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.     

A study of chemically induced dynamic electron polarization (CIDEP) in Photosystem I of whole algal cells at ambient temperatures

Time-resolved electron paramagnetic resonance (EPR) studies were carried out at room temperature and at 273 K on whole-cell samples of the photosynthetic algae: Anacystis nidulans and Scenedesmus obliquus, the latter being 97% deuterated from the growing medium. These photosynthetic organisms show greatly enhanced EPR signals which result from the generation of nonequilibrium spin populations, a phenomenon known as chemically induced dynamic electron polarization (CIDEP). We report magnetic-field profiles of the early transient signals of Photosystem I which are very similar to those observed at low temperatures. The results suggest that one or more early reduced electron acceptors in Photosystem I are being observed at ambient physiological temperatures.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

Biosynthesis of unnatural glycolipids possessing diyne moiety in the acyl chain in the green sulfur photosynthetic bacterium Chlorobaculum tepidum grown by supplementation of 10,12-heptadecadiynic acid

Unnatural glycolipids possessing the diyne moiety in their acyl groups were successfully biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum by cultivation with supplementation of 10,12-heptadecadiynic acid. Monogalactosyldiacylglycerol (MGDG) and rhamnosylgalactosyldiacylglycerol (RGDG) esterified with one 10,12-heptadecadiynic acid were primarily formed in the cells, and small amounts of glycolipids esterified with the two unnatural fatty acids can also be detected. The relative ratio of these unnatural glycolipids occupied in the total glycolipids was estimated to be 49% based on HPLC analysis using a evaporative light scattering detector. These results indicate that the acyl groups in glycolipids, which play important roles in the formation of extramembranous antenna complexes called chlorosomes, can be modified in vivo by cultivation of green sulfur photosynthetic bacteria with exogenous synthetic fatty acids. Visible absorption and circular dichroism spectra of Cba. tepidum containing the unnatural glycolipids demonstrated the formation of chlorosomes, indicating that the unnatural glycolipids in this study did not interfere with the biogenesis of chlorosomes.     

Presence and significance of minor antenna components in the energy transfer sequence of the green photosynthetic bacterium Chloroflexus aurantiacus

Antenna components in the energy transfer processes of a green photosynthetic bacterium Chloroflexus aurantiacus were spectrally investigated by time-resolved fluorescence spectroscopy at −196°C on intact cells. Besides major antenna components so far reported, three minor components were resolved; those were Bchl c located at 785 nm, the baseplate Bchl a at 819 nm and Bchl a in the B808-866 complex at 910 nm. The last component was assigned to a longer wavelength antenna closely associated with a reaction center. An additional Bchl c fluorescence component was kinetically suggested to be present, which can be an energy donor to a major Bchl c. Presence of these minor components was signified in terms of (1) increase in the spectral overlap integral and (2) adjustment of the direction of dipole moments in the energy transfer sequence of intact cells.     

Structure of 2-C-(hydroxymethyl)-D-ribose (hamamelose) in the solid-state analyzed by CP MAS NMR and X-ray crystallography

D-Hamamelose, a branched-chain ribose (2-C-(hydroxymethyl)-D-ribose), has been synthesized and its solid-state structure analyzed by (13)C CP MAS NMR spectra and X-ray data. The presence of the complex pattern of resonances in the anomeric region, as well as in the ring carbon region, in (13)C CP MAS NMR spectrum indicated that the mixture of four cyclic forms, alpha- and beta-furanoses, as well as both alpha- and beta-pyranoses were present in the solid-state. X-ray analysis of crystals showed that D-hamamelose belongs to the monoclinic system with unit cell: a=4.790A, b=8.671A, c=8.880A and beta=98.89 degrees , space group P2(1). The furanose ring has the (2)E conformation.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P₇₀₀ recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.