The relationship between substrate-induced respiration and swelling in Azotobacter vinelandii

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1. When oxidizable substrates are added to a starved suspension of Azotobacter vinelandii osmotically shrunken in 0.2 M KCl, a decrease in absorbance is observed which results from a change in light scattering as the cells increase in volume.     

Nitrogenase IV. Simple method of purification to homogeneity of nitrogenase components from Azotobacter vinelandii

Extracts of Azotobacter vinelandii have been fractionated by simple techniques to obtain highly purified components of nitrogenase. The yield of each component is greater than 60%. Purified Component I has a specific activity of 1638 nmoles ethylene formed/min per mg protein. The spectrum of Component I exhibits a broad absorption between 300 and 600 nm, with no distinctive peaks or shoulders. Addition of sodium dithionite or exposure to air has no effect on the absorption spectrum. Component I, examined at 4.2 °K has EPR signals at g = 4.2, 3.65 and 2.01. Addition of sodium dithionite does not produce additional resonances nor does it alter the signals already present. Crystals of Component I are dark brown and needle-shaped.Purified Component II has a specific activity of 1815 nmoles ethylene formed/min per mg protein. The absorption spectrum has no peaks or shoulders between 390 and 650 nm. Upon exposure of Component II to air, absorption increases between 400 and 650 nm. Treatment of oxidized Component II with dithionite causes this absorption to fall below that of the native Component II. EPR spectra of Component II has signals at g values of 2.05, 1.94, and 1.88. Upon inactivation by O₂, these signals disappear.Neither component by itself has detectable acetylene-reducing or N₂-fixing activity. The ratio of acetylene reduced to N₂ fixed is 3.86 with different ratios of the components. Both components form aggregated species upon exposure to air. Dithionite does not reverse this effect.     

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.     

Purification and molecular properties of a soluble ferredoxin from Rhodopseudomonas capsulata

A soluble ferredoxin was purified from the photosynthetic bacterium Rhodopseudomonas capsulata and characterized. Unlike Rhodospirillum rubrum, where two soluble ferredoxins have been found, only a single species was found in Rps. capsulata. The amino acid composition, ultraviolet-visible spectral properties, molecular weight (12000) and biological activity were determined. The ultraviolet-visible spectrum is similar to that of other bacterial ferredoxins, with a maximum when oxidized at 380 nm (? = 26.1 · 10³ M^-1 · cm^-1). The possible roles of this ferredoxin in the cellular metabolism are discussed.     

Identification of gene products from the Azotobacter vinelandii nifBfdxNnifOQ operon

The Azotobacter vinelandii nifBfdxNnifOQ operon is required for synthesis of the nitrogenase iron-molybdenum cofactor. To further characterize the roles of its gene products, specific antibodies against NifB and NifO were generated, and the NifB, NifO and NifQ gene products were visualized and identified in nitrogen-fixing A. vinelandii cell extracts by a combination of two-dimensional gel electrophoresis of radiolabelled extracts and immunological detection methods. The three proteins showed apparent pI and M_r values similar to those expected from sequence data, except for NifO, which showed an apparent M_r of ca. 23 kDa (vs. 16 kDa expected).     

Nitrogenase V The effect of Mo,W and V on the synthesis of nitrogenase components in Azotobacter vinelandii

When Azotobacter vinelandii OP is derepressed for nitrogenase synthesis in a medium containing no added Mo, component II but not component I is detected. Derepression with W in the medium allows the cells to produce inactive component I that can be activated by addition of molybdate to the medium. This activation does not require protein synthesis. Addition of V to Mo-free media does not increase the synthesis of component I protein. It is possible that Mo is an inducer of component I synthesis and that W also is capable of inducing component I, but that this component I is inactive because it lacks Mo.     

Oxidative phosphorylation in Azotobacter vinelandii. Energy-linked pH changes and fluorescence changes of atebrin and 1-anilinonaphthalene-8-sulphonate

1. Phosphorylating particles from Azotobacter vinelandii show a rapid, respiration-induced reversible increase in pH of the suspending medium; this is not found with non-phosphorylating particles.2. The observed pH response requires the presence of low concentrations of Mg²⁺ or of higher concentrations of Na⁺ or K⁺.3. Between 40 and 10 °C the rates of proton influx and efflux have similar temperature coefficients; below 10 °C the effect of temperature is greater on proton efflux.4. The kinetics of the energy-linked enhancement of fluorescence 1-anilinonaphthalene-8-sulphonate are slower than that of the quenching of the fluorescence of atebrin.     

Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata

Two ferredoxins from nitrogen-fixing cells of the phototrophic bacterium Rhodopseudomonas capsulata, strain B10, are purified to a homogeneous state and characterized. The molecular mass of ferredoxin I is about 12 kDa and that of ferredoxin II, 18 kDa. Ferredoxin I contains 8 Fe²⁺ and 8 S^2?; ferredoxin II has 4 Fe²⁺ and 4 S^2? per molecule. The redox potential of ferredoxin I is about ?270 mV and that of ferredoxin II ?419 mV. Ferredoxin I is more labile to the action of O₂, O^?₂, H₂O₂ and heating. The ferredoxins are also different in their absorption and EPR spectra, amino acid composition and electron-transfer activity to Rps. capsulata nitrogenase: both C₂H₂ reduction and H₂ evolution by Rps. capsulata nitrogenase proceed faster in the presence of ferredoxin I than in case of ferredoxin II. Synthesis of ferredoxin I takes place only in Rps. capsulata nitrogen-fixing cells grown in light under anaerobic conditions whereas ferredoxin II formation does not depend on the source of nitrogen or the growth medium, though the amount of ferredoxin II varies with the growth conditions. Its highest level has been found in the cells grown in lactate-limited medium in the presence of CO₂ and light or in the presence of glutamate in darkness under anaerobic conditions.     

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50°C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulphur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex of nitrogenase is isolated.     

Role of ammona in regulation of nitrogenase synthesis and activity in Anabaena cylindrica

The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O₂ for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O₂ tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.     

Recent developments in the analysis of protein complexes

Recent shotgun sequencing of filtered Sargasso Sea water samples has yielded data in astounding amount and diversity. Iron–sulfur proteins, which are ancient, diverse and ubiquitous, have been implemented here to further probe the sequence diversity of the Sargasso Sea database (SSDB). Sequence searches and comparisons confirm that the SSDB by and large equals in diversity the combined currently available databases. The data thus suggest that microbial diversity has so far been underestimated by orders of magnitude.     

Role of acyclic compounds in monoterpene biosynthesis in Pinus pinaster

The biosynthesis of monoterpene hydrocarbons was studied in maritime pine needles by incorporation of ¹⁴CO₂. It was shown that the acyclic terpenes β-myrcene and trans-β-ocimene, act as transitory compounds before the biosynthesis of cyclic monoterpenes such as α- and β-pinene. The mechanisms of biosynthesis are genetically controlled.