Flavodoxin fromAzotobacter vinelandii

Summary A flavoprotein fromAzotobacter vinelandii, named in the literature Shethna flavoprotein or azotoflavin, has recently been shown to catalyze acetylene reduction by a cell-free nitrogenase preparation fromAzotobacter. In this communication this result is confirmed. Moreover, the Shethna flavoprotein is now shown to replace ferredoxin in the catalysis of NADP⁺-reduction by illuminated spinach chloroplasts or by molecular hydrogen and hydrogenase fromClostridium. Evidence is presented that the fully reduced form of the Shethna flavoprotein is involved in the catalysis of NADP⁺-reduction. This indicates that the Shethna flavoprotein functions as a substitute for an one electron carrier shuttling between fully reduced and semiquinoid form. From the data reported here, together with that in the literature, it is concluded that this flavoprotein belongs to the class of flavodoxins. Some preliminary results on ferredoxin fromAzotobacter are reported.A flavoprotein fromAzotobacter commonly referred in the literature as the Shethna flavoprotein or azotoflavin is found to exhibit the characteristics of a flavodoxin.     

Cloning of ferredoxin I gene fromAzotobacter vinelandii using synthetic oligonucleotide probes

Two synthetic oligonucleotide probe mixtures, whose sequences were inferred from two separate stretches of amino acids, one closer to the carboxy terminal and the other closer to the amino terminal, of ferredoxin I protein ofAzotobacter vinelandii, were used to select ferredoxin I gene clones from a cosmid gene library ofAzotobacter vinelandii. Restriction analysis revealed that 7 out of 10 selected clones were of the same type. All these clones were found to hybridize withfixABCX genes ofRhizobium meliloti.     

Solution Structure of the Lipoyl Domain of the 2-Oxoglutarate Dehydrogenase Complex fromAzotobacter vinelandii

The three-dimensional solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex fromAzotobacter vinelandiihas been determined from nuclear magnetic resonance data by using distance geometry and dynamical simulated annealing refinement. The structure determination is based on a total of 580 experimentally derived distance constraints and 65 dihedral angle constraints. The solution structure is represented by an ensemble of 25 structures with an average root-mean-square deviation between the individual structures of the ensemble and the mean coordinates of 0.71 Å for backbone atoms and 1.08 Å for all heavy atoms. The overall fold of the lipoyl domain is that of a β-barrel-sandwich hybrid. It consists of two almost parallel four-stranded anti-parallel β-sheets formed around a well-defined hydrophobic core, with a central position of the single tryptophan 21. The lipoylation site, lysine 42, is found in a β-turn at the far end of one of the sheets, and is close in space to a solvent-exposed loop comprising residues 7 to 15. The lipoyl domain displays a remarkable internal symmetry that projects one β-sheet onto the other β-sheet after rotation of approximately 180° about a 2-fold rotational symmetry axis. There is close structural similarity between the structure of this 2-oxoglutarate dehydrogenase complex lipoyl domain and the structures of the lipoyl domains of pyruvate dehydrogenase complexes fromBacillus stearothermophilusandEscherichia coli, and conformational differences occur primarily in a solvent-exposed loop close in space to the lipoylation site. The lipoyl domain structure is discussed in relation to the process of molecular recognition of lipoyl domains by their parent 2-oxo acid dehydrogenase.     

Effect of lyophilization on the nitrogenase activity of Azotobacter vinelandii]

The activity of nitrogenase in the cells of Azobacter vinelandii grown from lyophilized and non-lyophilized cultures depends on the donor of hydrogen and the concentration of oxygen in the gaseous phase. The lyophilized cells are more sensitive to oxygen (O2 optimum for nitrogen fixation is ca. 1 percent) than the non-lyophilized cells (ca. 5 percent). The determination of acetylene reduction in the course of the culture growth has shown that nitrogen fixation in the lyophilized cells takes place after a lag-period (about six hours) at a rate lower than that of the non-lyophilized cells. The results obtained suggest that lyophilization increases the sensitivity of the cells to oxygen and decreases their nitrogenase activity which is however restored after a while.     

Effect of redox mediators on nitrogenase and hydrogenase activities in Azotobacter vinelandii

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MB_o) when the concentration of this mediator in the medium is increased up to 72 M. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NR_o) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H₂ uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H₂ evolved/mg cell protein/min from the nitrogenase and 160 nM H₂-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H₂ evolution and for H₂ uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.     

Effect of Simazine on the production of lysine and methionine byAzotobacter chroococcum andAzotobacter vinelandii

Summary Production of lysine and methionine byAzotobacter chroococcum strain H23 andA. vinelandii strain ATCC 12837 was studied in chemically-defined medium and dialysed-soil medium, amended with different concentrations of Simazine. Responses on production due to Simazine were different for each strain and were fairly conditioned by culture media composition. Quantitative production of amino acids was significantly affected by the xenobiotic only at higher doses (50–100,g/ml). The effect of Simazine on methionine production by strain H23 was very pronounced when bacteria were grown in dialysed-soil medium, which was specially formulated to reproduce the natural habitat of the organisms.     

Effect of porphyrin ring ligands on the affinity of heme iron to axial ligands]

To clarify the influence of protein surrounding on the heme reactivity in heme proteins the effect of interaction between a porphyrin ring and pi-acceptor molecule, 1,2,4-trimethyl-pyridinium (TMP), on the affinity of deuteroheme to axial ligands (imidazole and cyanide) has been studied as a model system. It is shown that TMP induces the fourfold decrease in equilibrium constant of imidazole to deuteroheme. From the analysis of the two stages for cyanide binding it is concluded that TMP decreases the binding constant of the first cyanide by 40 times and does not apparently influence the second ligand binding. The effect of TMP on the reactivity of deuteroheme to axial ligands is interpreted as a result of a decrease in the electron density on the iron orbitals which is due to the altered pi-eleectron density in the porphyrin pi-system through the donor-acceptor interaction with TMP molecules. The possible significance of the contacts between the porphyrin and neighboring amino acid residues in determining heme affinity to axial ligands is discussed.     

Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii.

The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.     

Growth of Azotobacter vinelandii on Soil Nutrients

Azotobacter vinelandii cells grew well in a medium made from soil and distilled water which contained little or no carbohydrate. They utilized p-hydroxybenzoic acid and other phenolic acids, soil nitrogen, and water-soluble mineral substances. Seventeen soils which supported excellent growth of A. vinelandii contained 11 to 18 different phenolic acids each, including p-hydroxybenzoic, m-hydroxybenzoic, vanillic, p-coumeric, syringic, cis- and trans-ferrulic, and other unidentified aromatic acids. Three white, chalky “caliche” soils which were taken from areas where no plants grew failed to support the growth of A. vinelandii, and these contained no, two, and three phenolic acids, respectively. A. vinelandii did not fix nitrogen when growing in dialysates of soils which contained numerous phenolic acids. Growth was ample and rapid in most of the soils tested, but cell morphology was different from that usually seen in chemically defined, nitrogen-free media which contain glucose.     

The hydrogenase cytochrome b heme ligands of Azotobacter vinelandii are required for full H(2) oxidation capability

The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H(2) oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O(2)-dependent H(2) oxidation and H(2)-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O(2)-dependent H(2) oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H(2) oxidation. In mutant H194A/Y, imidazole (Imd) rescued H(2) oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H(2)-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H(2) oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H(2) oxidation.     

TheAzotobacter vinelandii chromosome

The chromosome ofAzotobacter vinelandii was digested with the restriction endonucleasesSpeI (5’-ACTAGT),DraI (5’-TTTAAA) andAsel (5’-ATTAAT) and the products were separated by pulsed-field gel electrophoresis. The sum of the sizes of the restriction fragments comes to around 4.5 megabasepairs. Our earlier studies had revealed the presence of about 80 copies ofnifH, nifD, nifK andleuB genes in a log-phase cell ofA. vinelandii. To determine whether there are multiple identical chromosomes inA. vinelandii or one large chromosome with identical segments joined in tandem, we have subjected gamma-irradiated DNA ofA. vinelandii andEscherichia coli to pulsed-field gel electrophoresis. The results suggest thatA. vinelandii chromosomes contain multiple identical chromosomes of about the same size as that ofE. coli.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

Effect of PEG and Salt on Crystallization of Bacterioferritin and Nitrogenase MoFe Protein from Azotobacter vinelandii

MgCl2 was added to the supernatant of the first crystallization of MoFe protein to give a final concentration of 14.6 mmol/L, followed by centrifugation. The treated supematant solution and MoFe protein could be crystallized by using method of siting drop with PEG 6000 and MgC12 as a precipitant and salt, respectively. The larger crystal from the supermatant was observed when the final concentration of PEG and MgCl2 was 4.5% and 15.6 mmol/L, respectively; but small crystal was observed when the concentration was 0 and 23.8 mmol/L, respectively. The larger crystal in brown rectangular prism of MoFe protein was also obtained using the same crystallization method when the final concentration of PEG and MgCI2 was 7.44% and 338.0 mmol/L, respectively. It suggests that the two protein crystals seem to be different, the former being bacterioferritin and the later as nitrogenase MoFe protein.     

Ultrastructure of Azotobacter vinelandii

Vegetative cells and cysts of Azotobacter vinelandii 12837 were prepared for electron microscopy by several methods assumed to preserve structural details destroyed by techniques previously reported in the literature. Examination of large numbers of cells and cysts by these methods revealed four structural details not reported previously: intine fibrils, intine vesicles, intine membrane, and microtubules. The intine fibrils form a network in the gel-like homogeneous matrix of the CC2 layer. Intine vesicles which seem to originate in the cell wall complex of the central body are seen in the intine and exine of cysts. Analogous structures are found on vegetative cells. The intine is divided into two chemically distinct areas by the two-layered intine membrane. Microtubules, previously reported only in vegetative cells, were found in cysts.     

Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction

The effect of salts on the catalytic activity of the molybdenum-containing nitrogenase complex from Azotobacter vinelandii has been investigated. NaCl was found to inhibit the reduction of the substrates, protons, acetylene, and dinitrogen by a common mechanism. The pattern of inhibition is sigmoidal, indicating a highly cooperative interaction involving multiple inhibitor sites. Sixteen other salts that were investigated also exhibited this pattern of inhibition. NaCl functions as a dead-end inhibitor without altering the number of MgATP hydrolyzed/electron transferred to substrate. The level of expressed inhibition is sensitive to MgATP concentration, the molar ratio of the MoFe-protein (Av1) to the Fe-protein (Av2), and total protein concentration. In addition, NaCl is an inhibitor of the MgATP-dependent, iron chelation of Av2. Although the inhibition is exhibited over the same salt concentration range as that for inhibition of substrate reduction, the pattern of inhibition is hyperbolic. A model based upon simple equilibrium interactions among the enzyme species, nucleotides, and inhibitor has been developed which quantitatively accounts for the observed effects of salt. In this model, the formation of the active complex between Av1 and Av2 is abolished by salts. Likewise, the apparent affinity of Av2 for MgATP is reduced. An additional prediction based upon the model is that the affinity between Av2 and Av1 is independent of nucleotide binding.