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1.
Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development.  相似文献   

2.
Luminescence emitted by tryptophan residues of reaction center (RC) preparations was studied. The RG preparations were isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides by treatment with lauryl dimethyl amine oxide (LDAO). After excitation at lambda 280 nm the quantum yield of luminescence is 0,02. It is shown that 60% of tryptophanyls are located inside the protein globule in the surrounding of relaxating polar groups and the rest approximately 40% on the outer surface of the globule--predominantly in the positively charged region of the LDAO-RC protein--in the surrounding of protein-bound water molecules. There is a correlation between the pH dependencies of the position of the peak of luminescence from tryptophanyls and effectivity of electron transfer from the primary (quinone) to secondary acceptor. The two parameters are invariant at pH from 7 to 9 and vary at pH less than 7 and pH greater than 9. The phenomena responsible for the observed correlation are discussed on the basis of pH-dependent changes in the RC protein which govern electron transport activity at the reaction center.  相似文献   

3.
The photochemical reaction centers from Rhodopseudomonas sphaeroides were reconstituted with soybean phospholipids into liposomes by the cholate-dialysis method. The transmembrane orientation of the reaction centers in the proteoliposomes and the morphology of the vesicles were investigated. The orientation was determined by the reduction of externally added cytochrome c after its photooxidation by a flash. The structure of the vesicles was examined by electron microscope. Discontinuous sucrose density gradient centrifugation yielded several proteoliposome fractions with different vesicular sizes and reaction-center orientations. The proportion of the reaction centers that exposed their cytochrome c reacting sites to the outside of the vesicles increased from 45 to 85% with an increase of the vesicular size. The proportion also depended on the ionic composition of the dialysis buffer. The optimal ionic environment during the dialysis (100 mM NaCl or 2.5 mM MgSO4) gave a liposome yield of 25-30% with a highly asymmetric orientation (greater than 60%). Entrapping of cytochrome c molecules into the phospholipid vesicles had little effect on the orientation of the reaction centers.  相似文献   

4.
The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, PF. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to PF, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates PF from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and PF of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of PF with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.  相似文献   

5.
Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484-501) where computer simulations of the observed triplet state spectra were employed. The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll 'special pair' lies almost entirely along one of the principal magnetic axes of the triplet state. Aso, the 870 nm transition moment makes an angle of approx. 60 degrees with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514-530).  相似文献   

6.
Linear dicroism of chromatophores and isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides strain R-26 was studied using a novel technique of orientation. The results are discussed in view of the reaction center structure and its position in the membrane. The advantages of the new orientation technique are also outlined.  相似文献   

7.
Linear dicroism of chromatophores and isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides strain R-26 was studied using a novel technique of orientation. The results are discussed in view of the reaction center structure and its position in the membrane. The advantages of the new orientation technique are also outlined.  相似文献   

8.
Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.  相似文献   

9.
Aqueous mixtures of reaction centers of Rhodopseudomonas sphaeroides and gelatin were dried to form thin films. Following hydration, these films were stretched as much as two to three times their original length. Polarized absorption spectra showing linear dichroism were obtained for both unstretched and stretched films, with the planes and stretching axes of the films mounted in various geometries relative to the electric vector of the measuring beam. These data were analyzed in terms of the following model: Reaction centers possess an axis of symmetry that is fixed in relation to the reaction center structure. In unstretched films this axis is confined to the film plane and oriented at random within the plane. In stretched films the symmetry axis is aligned with the direction of stretching. In both preparations reaction centers are distributed randomly with respect to rotation about the axis of symmetry. The data are consistent with this model when the analysis acknowledges less than perfect orientation. For perfect orientation in a stretched film the model predicts uniaxial symmetry about the axis of stretching. The approach to this condition was examined with films stretched to different extents. Extrapolation yielded dichroic ratios for the ideal case of perfect orientation, and allowed calculation of the angles between the axis of symmetry and the various optical transition dipoles in the reaction center. This treatment included the two absorption bands of the bacteriochlorophyll 'special pair' (photochemical electron donor) in the Qx region, at 600 and 630 nm, which we were able to resolve in light minus dark difference spectra.  相似文献   

10.
11.
1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect. 2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the alpha-band (lambda max at 551--551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (lambda max at 549.5 +/- 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed. 3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b50 appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b-90 was observed. 4. Difference spectra attributed to (BChl)2, (Formula: see text), c type cytochrome and cytochrome b50 were resolved in the Soret region for Rhodopseudomonas capsulata. 5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.  相似文献   

12.
Glycerol dissimilation in Rhodopseudomonas sphaeroides.   总被引:1,自引:2,他引:1       下载免费PDF全文
Rhodopseudomonas sphaeroides followed a diauxic growth curve when grown on a malate-glycerol medium, the first phase of growth being supported by malate and the second by glycerol. A soluble glycerokinase and a particulate, pyridine nucleotide-independent glycerophosphate dehydrogenase, were induced by the presence of glycerol in the medium, but neither was fully expressed nor functional until all malate had been consumed.  相似文献   

13.
Dimethylsulfide (DMS) dehydrogenase is a complex heterotrimeric enzyme that catalyzes the oxidation of DMS to DMSO and allows Rhodovulum sulfidophilum to grow under photolithotrophic conditions with DMS as the electron donor. The enzyme is a 164 kDa heterotrimer composed of an alpha-subunit that binds a bis(molybdopterin guanine dinucleotide)Mo cofactor, a polyferredoxin beta-subunit, and a gamma-subunit that contains a b-type heme. In this study, we describe the thermodynamic characterization of the redox centers within DMS dehydrogenase using EPR- and UV-visible-monitored potentiometry. Our results are compared with those of other bacterial Mo enzymes such as NarGHI nitrate reductase, selenate reductase, and ethylbenzene dehydrogenase. A remarkable similarity in the redox potentials of all Fe-S clusters is apparent.  相似文献   

14.
Ferric iron reductase of Rhodopseudomonas sphaeroides.   总被引:4,自引:1,他引:4  
Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.  相似文献   

15.
From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.  相似文献   

16.
The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (t 1/2 3-5 microseconds). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske Center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center. UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of HII(+); these may reflect additional sites of action of the inhibitor.  相似文献   

17.
Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.  相似文献   

18.
Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO 3 - under anaerobic conditions accumulated NO 2 - in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO 3 - , NO 2 - and N2O to N2 gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N2O reductase was inhibited by NaN3, C2H2 and KCNS. Cells incubated with either K15NO3 or K15NO2 produced 15N2O and 15N2. A stoichiometry of 2:1 was recorded for the reduction of either NO 3 - or NO 2 - to N2O and N2 and for N2O to N2 it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide  相似文献   

19.
Photoreduction of the two ubiquinone molecules, UQ1 and UQ2, bound to purified reaction center from Rhodopseudomonas sphaeroides induces different absorption band shifts of bacteriochlorophyll and bacteriopheophytin molecules depending on which ubiquinone is photoreduced. This allows us to study electron transfer between UQ1 and UQ2 directly by absorption spectrometry. The results support a model in which electrons are transferred one by one from UQ1 to UQ2 with a half-time 200 micro seconds, and two by two from fully reduced UQ2 to the secondary acceptor pool.  相似文献   

20.
Resonance Raman spectra of the pi-cation of bacterio-chlorophyll a in solution at 30 K are reported and discussed. Outer (formula: see text) bonds of the pyrroles and the methine bridges are weakened by the ionization, while (formula: see text) and Mg-N bonds remain essentially unaffected. Resonance Raman spectra of reaction centers suggest that the positive charge on P-870+ should be localized on a single bacteriochlorophyll molecule by the lifetime of the scattering process (approximately 10(-13)s).  相似文献   

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