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PubMed1979

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.

Azotobacter↗

PubMed1979

Induction of haemorrhagic necrosis in a murine tumour by products from the culture fluid of Azotobacter.

Macromolecules in the supernatant from cultures of the nitrogen-fixing bacterium Azotobacter chroococcum when given systemically induce permanent regression of established grafts of a syngeneic murine lymphoma. The effect mimics that produced by endotoxin in that haemorrhagic necrosis is induced in the tumour, but the ratio of the toxic dose to the dose needed to cause tumour regression is greater than for this culture supernatant than for endotoxin.

Animals↗

PubMed1980

Characterization of a calcium/proton antiporter and an electrogenic calcium transporter in membrane vesicles from Azotobacter vinelandii.

Ca2+ transport across the membrane of vesicles derived from Azotobacter vinelandii was studied in the absence of respiration or functioning ATPase. Two facilitated diffusion systems were found. One, an electroneutral Ca2+/2H+ antiporter, responded to an artificially imposed deltapH, was heat-labile, and was insensitive to low concentrations of ruthenium red and lanthanides. The second, an electrogenic transporter, responded to an electrical membrane potential, was heat-stable, was inhibited by ruthenium red, lanthanides, monovalent cations, and certain anions. In vivo, when coupled to the protonmotive force, the systems would provide for the cell: (i) a mechanism to keep intracellular Ca2+ concentration low (Ca2+/2H+ antiporter); (ii) a mechanism for Ca2+ entry (electrogenic transporter).

Antiporters↗

PubMed1981

Herbicidal effect on some metabolic processes of Azotobacter chroococcum.

The effect of pyramin (1-phenyl-4-amino-5-chloro-piridazone-6) and venzar (3-cyclohexyl-5,6-trimethyleneuracil) on growth, oxygen uptake and nitrogenase activity was examined. The possibilities of herbicide accumulation in cells and binding by mucopolisaccharides were also tested. Stimulation of growth was observed in the presence of pyramin (20 and 100 ppm) but no influence of venzar was found. Venzar was found to penetrate into the cells. Pyramin did not penetrate into the cells but its content decreased in cultures of Azotobacter strains. However, no degradation products of pyramin were found. Pyramin was detected in mucopolisaccharide fractions in range 0.2-47 micrograms of Pyrazon per 100 mg. Pyramin and venzar reduced the acetylene reduction rate of crude extracts by 50% when doses of active substances were equal to the weight of crude extract protein contents. Doses required for similar effect in living cells were however more than fourteen times higher. Addition of polivinylpyrrolidone and albumin protected the N2-ase from negative effect of pyramin and to a lesser degree from that of venzar.

Azotobacter↗

PubMed1978

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.

Amino Acid Sequence↗

PubMed1984

Production of cyanocobalamine by Azotobacter chroococcum.

A strain of Azotobacter chroococcum was found to produce a considerable amount of cyanocobalamine especially when cultivated in a medium enriched with 0.3% ammonium chloride. Maximal production of the vitamin was achieved after six days of incubation in static cultures. The organism required molybdenum, iron (Fe++), cobalt and ascorbic acid for optimal production of cyanocobalamine. The initial supplementation of the medium with cyanocobalamine did not affect the formation of the vitamin. The least concentration of ascorbic acid tested greatly initiated the production of vitamin B12. Higher concentrations were not effective. In the presence of molybdenum, iron, cobalt and ascorbic acid maximal secretions of cyanocobalamine was attained in the presence of 3% sucrose or an amount of hydrolyzed rice-bran containing 2% sugar. Higher concentration of rice-bran was toxic to the organism.

Ammonium Chloride↗

PubMed1984

Preliminary crystallographic studies on bacterioferritin from Azotobacter vinelandii.

Bacterioferritin-cytochrome from Azotobacter vinelandii is an unusual protein containing haem groups as well as iron core like other ferritin. This paper reports the purification of bacterioferritin by affinity chromatography and the formation of brick-red crystals from a solution containing MgCl2. The crystals are optical isotropic with maximum dimensions of 0.4 X 0.4 X 0.1 mm3. The preliminary X-ray crystallographic studies have been performed. 1.5 degrees unscreened precession photographs show that the crystals of bacterioferritin belong to the cubic system, space group I432, with cell dimension 230 A. There are probably 8 molecules in one cubic unit cell and the molecule might have 32 symmetry. A molecular diameter of 115 A is derived from the packing of the molecules and a molecular weight of 826,000 is estimated for bacterioferritin.

Azotobacter↗

PubMed1983

Crystallization of the Azotobacter vinelandii nitrogenase iron protein.

The iron protein from Azotobacter vinelandii nitrogenase has been crystallized in the reduced form. The needle-shaped crystals are in space group P2(1)2(1)2 (a = 94.6 A, b = 179.9 A, c = 74.1 A) and diffract to at least 3.5-A resolution. Five or six Fe-protein monomers are present in the asymmetric unit.

Azotobacter↗

PubMed1983

Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii.

A procedure has been developed to examine some of the functional roles of the 14 cysteinyl residues in the nitrogenase Fe-protein (Av2) from Azotobacter vinelandii. The reduced form of Av2 was alkylated with iodo[2-14C]acetic acid under a variety of experimental conditions, e.g. reaction in the presence of nucleotides, alpha,alpha'-dipyridyl and nucleotides, or denaturants. The labeled cysteinyl residues were identified and quantified using an analytical DEAE-Sepharose ion exchange chromatography peptide mapping technique based upon the known amino acid sequence (Hausinger, R. P., and Howard, J. B. (1982) J. Biol. Chem. 257, 2483-2490). From the results of the labeling experiments, the following features of the Av2 structure have been proposed. 1) Av2 contains no disulfides, hyperreactive thiols, or surface thiols as defined by reaction with iodoacetic acid. 2) Cysteines 97 and 132 are the probable ligands for the Av2 Fe:S center which is bound symmetrically between subunits. 3) MgATP partially protects cysteine 85 from carboxymethylation by iodoacetic acid and may be part of the nucleotide-binding site. 4) Of the five nonligand thiols only cysteines 5 and 184 are completely alkylated when Av2 is denatured in hexamethylphosphoramide, whereas all five nonligand thiols appear to rapidly exchange at the Fe:S center if the protein is denatured in the absence of alkylating reagents. 5) Both Av2 and apo-Av2 appear to undergo a reversible conformational change upon binding MgATP.

Azotobacter↗

PubMed1982

[Detection of anti-Azotobacter antibodies in sera from tumor patients].

Employing the agar-gel double immunodiffusion technique, the authors describe the presence of antibodies to a protein fraction existing in the supernatant of Azotobacter chroococcum cultures in a high percentage of blood sera from cancer patients. The small number of false positive results makes them think of the convenience of increasing the sensitivity of the procedure to valorate its usefulness for the early diagnosis of malignant tumors.

Antibodies, Bacterial↗

PubMed1983

Structure of Azotobacter vinelandii 7Fe ferredoxin. Amino acid sequence and electron density maps of residues.

The complete amino acid sequence of the 7Fe ferredoxin from Azotobacter vinelandii (Av Fd) was determined by repetitive Edman degradation of the whole protein and of peptides derived from CNBr cleavage or chymotrypsin digestion. The sequence was confirmed by the 2A electron density maps for the residues calculated with difference Fourier coefficients. The density maps for all residues are included in the paper. Av Fd has several important differences with the clostridial-type ferredoxins: (i) Av Fd is 106 residues (versus 55-60 for other bacterial ferredoxins); (ii) Av Fd has 9 cysteines, one of which (residue 24) is not homologous with the bacterial ferredoxins; (iii) Av Fd has 2 extra residues between 2 cysteines (residues 11 and 16) homologous to cysteines in the bacterial ferredoxins; and (iv) Av Fd has the unique sequence -Cys-Val-Glu-Val-Cys- (residues 16-20) which are two of the ligands of the 3Fe:3S center. These sequence features are compared to the sequences of various ferredoxin groups. Structure predictions for other suspected 7Fe ferredoxins are discussed.

Amino Acid Sequence↗

PubMed1981

Isolation and sequences of the cysteinyl tryptic peptides from the MoFe-protein of Azotobacter vinelandii nitrogenase.

The cysteinyl residues in the alpha and beta subunits of the MoFe-protein from Azotobacter vinelandii nitrogenase were radiolabeled by carboxymethylation with iodo[14C]acetic acid. The tryptic peptides from the isolated subunits were separated by ion-exchange chromatography on DEAE-Sephadex and SP-Sephadex. The radiolabeled (cysteinyl) peptides were sequenced by Edman degradation. The isolation procedure and sequences of the peptides provide a method for the identification of the potential cysteinyl ligands for the protein Fe:S and Mo:Fe:S centers. Although the cysteinyl peptides account for approximately 20% of the total sequence, little sequence homology was observed between the peptides from the two subunits. In contrast to the ferredoxins, the cysteinyl residues are not grouped within the protein. Only one peptide from the beta subunit has sequence homology with other Fe:S proteins. This peptide has 15 of 22 residues identical or conservative with residues 11 to 32 of the bacterial ferredoxins. The conservation of this region suggests that it may contain an important secondary structure unique to 4Fe:4S proteins, namely, the sequence (formula, see text) where (formula, see text) represents a variable residue. One peptide from the alpha subunit has a sequence identical with the conserved residues 7 to 11 of the bacterial ferredoxins. This alpha-peptide has the correct sequence, -Glu-Pro-Val-Ser-Cys-Val-Ser-Asp-Ser-, for the glutamyl, cysteinyl, and aspartyl residues to ligand a single 4Fe:4S cluster.

Amino Acid Sequence↗

PubMed1981

Resolution of two subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.

Azotobacter↗

PubMed1982

Magnetic susceptibility studies of native and thionine-oxidized molybdenum-iron protein from Azotobacter vinelandii nitrogenase.

The difference between the magnetic susceptibilities of native and thionine-oxidized molybdenum-iron protein from Azotobacter vinelandii nitrogenase was measured by the nuclear magnetic resonance method. Reversible oxidation of the MoFe protein by 4 to 8 electron eq of thionine/mol made the protein more paramagnetic than it was in the native state. The NMR susceptibility results were analyzed in terms of a model for the spin states of the iron centers in the MoFe protein based on low temperature electron paramagnetic resonance and Mössbauer spectral studies. The model proposes that the native protein contains 2 "M" centers (S = 3/2) and 4 "P" centers (S = 0)/mol and that the oxidized protein has diamagnetic M centers and paramagnetic P centers with S greater than or equal to 3/2. Assuming that this model holds at 280 K, the NMR susceptibility results show that the effective magnetic moment of the oxidized P centers is larger than that of the native M centers. Based on an analysis in terms of spin only magnetic moments, the susceptibility results suggest that the P centers in the oxidized protein are S = 5/2 systems.

Azotobacter↗

PubMedDate not supplied

Thermoresistance of azotobacter vinelandii ATCC 12837 in defined and dialysed soil media: filtrable forms.

Thermoresistance of Azotobacter vinelandii ATCC 12837 in nitrogen-free (defined) medium and dialysed soil medium was established. These microorganisms showed thermoresistance in the defined medium, but in the dialysed soil medium they were very sensitive to heat. Thermosensitivity in the dialysed soils medium was due to the production of germinal cells (filtrable forms) and not to the presence of any kind of cytoplasmic inclusions such as poly-beta-hydroxybutyric acid, metachromatic, glycogen or unstaining granules.

Azotobacter↗

PubMed1981

Structure of a 7Fe ferredoxin from Azotobacter vinelandii.

The structure of the 7Fe ferredoxin from Azotobacter vinelandii has been solved from a 3.0-A multiple isomorphous replacement map. The crystals belong to space group P43212 with a = 55.22, c = 95.20 A, and Z = 1. Heavy-atom derivatives were prepared with K2PtCl4,K2[OsO2(OH)4], and Na3RhCl6. Anomalous scattering data were collected for native (Fe) and Pt derivative crystals. The figure of merit for 3,322 reflections to 3.0 A is 0.74. The structure consists of an NH2-terminal core of residues 1-50 which form the Fe-S cluster sites, and a COOH-terminal chain of residues 51-107 which wraps around this core. The [3Fe-3S] cluster is ligated by cysteines 8, 11, 16, 20, and 49 and a sixth ligand which is either glutamic acid 18 or an exogenous small molecule. The [4Fe-4S] cluster is ligated by cysteines 24, 39, 42, and 45. The coordination of both Fe-S centers has been confirmed by fitting of the cluster atoms and residues 1-50 to unbiased 2Fo-Fc Fourier maps at 2.5-A resolution. The structure of the 3Fe center has also been confirmed with anomalous scattering difference Fourier maps using both isomorphous replacement and refined phases. The partially refined structure at 2.5 A (3,490 reflections, 6.0 sigma(F)) has R = 35%.

Azotobacter↗

PubMed1980

Iron-sulfur clusters in Azotobacter ferredoxin at 2.5 A resolution.

X-ray crystallographic study of the ferredoxin-like protein (iron-sulfur protein III) from Azotobacter vinelandii has been extended to 2.5-A resolution. A 4.0-A resolution electron density map revealed that the molecule contains two Fe-S clusters of differing size and shape separated by 12 A (Stout, C.D. (1979) Nature 279, 83-84). Recent Mössbauer results by Emptage et al. (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796) have shown that the molecule contains 7 iron atoms as a high potential iron protein-like Fe4 center and a novel 3-Fe center. The 2.5-A electron density map shows two distinctly different Fe-S clusters. The larger cluster consists of a tetranuclear [4Fe-4S] core ligated to the protein at each iron atom. The smaller cluster is distinctly planar and cannot be modeled with [2Fe-2S] or [4Fe-4S] structures. The best model for this cluster is a [3Fe-3S] core. The protein makes six contacts with this cluster.

Azotobacter↗

PubMed1980

Presence of a cytochrome b-containing ferritin in Azotobacter vinelandii.

It has been demonstrated that a cytochrome b-containing ferritin is present in Azotobacter vinelandii. After DEAE cellulose chromatography and purification fractional precipitation by 50% of the saturated ammonium sulfate of the extract prepared from A. vinelandii cells, a hexagonal crystalline preparation is obtained. The protein contains 4--6% of nonheme iron. The protein molecule is made up of an electron dense iron core with a diameter of 70A and a protein shell with a diameter of 120A. The Fe core can be removed from the shell by the treatment with chelating and reducing agents. Electron micrographs and absorption spectra reveal that the protein shells are very similar before and after the removal of the core. The electrophoretic mobility and immunological properties of the Fe-free protein against the antibody of ferritin are very similar to those of the protein before the removal of the iron. From the above characteristics, it can be inferred that the protein belongs to ferritin. The protein part contains protoheme as prosthetic group and so it belongs to cytochrome b. Hence, the protein prepared from A. vinelandii is a kind of cytochrome b-containing ferritins. The possible role of the ferritin in biological nitrogen fixation is discussed in this paper.

Apoferritins↗