scholarly journals Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum

1991 ◽  
Vol 277 (2) ◽  
pp. 465-468 ◽  
Author(s):  
M J Dilworth ◽  
R R Eady
Keyword(s):  
Direct Evidence ◽  
Electron Flux ◽  
Azotobacter Chroococcum ◽  
Cross Reactions ◽  
Vanadium Nitrogenase ◽  
Dinitrogen Reduction ◽  
Enzyme Intermediate

During the enzymic reduction of N2 to NH3 by Mo-nitrogenase, free hydrazine (N2H4) is not detectable, but an enzyme-bound intermediate can be made to yield N2H4 by quenching the enzyme during turnover [Thorneley, Eady & Lowe (1978) Nature (London) 272, 557-558]. In contrast, we show here that the V-nitrogenase of Azotobacter chroococcum produces a small but significant amount of free N2H4 (up to 0.5% of the electron flux resulting in N2 reduction) as a product of the reduction of N2. The amount of N2H4 formed increased 15-fold on increasing the assay temperature from 20 degrees C to 40 degrees C. Activity cross-reactions between nitrogenase components of Mo- and V-nitrogenases showed that the formation of free N2H4 was associated with the VFe protein. These data provide the first direct evidence for an enzyme intermediate at the four-electron-reduced level during the reduction of N2 by V-nitrogenase.

scholarly journals The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane

1988 ◽  
Vol 249 (3) ◽  
pp. 745-751 ◽  
Author(s):  
M J Dilworth ◽  
R R Eady ◽  
M E Eldridge
Keyword(s):  
Electron Flux ◽  
Azotobacter Chroococcum ◽  
Ph Values ◽  
Fe Protein ◽  
C2h2 Reduction ◽  
Vanadium Nitrogenase

1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed.

scholarly journals Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase

1988 ◽  
Vol 256 (2) ◽  
pp. 429-432 ◽  
Author(s):  
R W Miller ◽  
R R Eady
Keyword(s):  
Low Temperature ◽  
Electron Flux ◽  
Specific Activity ◽  
Nitrogenase Activity ◽  
Azotobacter Chroococcum ◽  
Effect Of Temperature ◽  
High Electron ◽  
Mofe Protein ◽  
Fe Protein ◽  
Vanadium Nitrogenase

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.

scholarly journals Vanadium K-edge X-ray-absorption spectroscopy of the functioning and thionine-oxidized forms of the VFe-protein of the vanadium nitrogenase from Azotobacter chroococcum

1989 ◽  
Vol 258 (3) ◽  
pp. 733-737 ◽  
Author(s):  
J M Arber ◽  
B R Dobson ◽  
R R Eady ◽  
S S Hasnain ◽  
C D Garner ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Absorption Spectroscopy ◽  
Azotobacter Chroococcum ◽  
Vanadium Atom ◽  
Oxidation States ◽  
Edge Structure ◽  
X Ray ◽  
Vanadium Nitrogenase ◽  
Enzyme Turnover ◽  
X Ray Absorption

Vanadium K-edge X-ray-absorption spectra were collected for samples of thionine-oxidized, super-reduced (during enzyme turnover) and dithionite-reduced VFe-protein of the vanadium nitrogenase of Azotobacter chroococcum (Acl*). Both the e.x.a.f.s and the x.a.n.e.s. (X-ray-absorption near-edge structure) are consistent with the vanadium being present as part of a VFeS cluster; the environment of the vanadium is not changed significantly in different oxidation states of the protein. The vanadium atom is bound to three oxygen (or nitrogen), three sulphur and three iron atoms at 0.215(3), 0.231(3) and 0.275(3) nm respectively.

Vanadium K-edge X-ray absorption spectrum of the VFe protein of the vanadium nitrogenase of Azotobacter chroococcum

Nature ◽  
1987 ◽  
Vol 325 (6102) ◽  
pp. 372-374 ◽  
Author(s):  
Judith M. Arber ◽  
Barry R. Dobson ◽  
Robert R. Eady ◽  
Philippe Stevens ◽  
S. Samar Hasnain ◽  
...  
Keyword(s):  
Absorption Spectrum ◽  
Azotobacter Chroococcum ◽  
X Ray ◽  
Vanadium Nitrogenase ◽  
X Ray Absorption

scholarly journals Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex

1989 ◽  
Vol 257 (3) ◽  
pp. 789-794 ◽  
Author(s):  
R N F Thorneley ◽  
N H J Bergström ◽  
R R Eady ◽  
D J Lowe
Keyword(s):  
Electron Transfer ◽  
Transfer Reaction ◽  
Competitive Inhibitor ◽  
Electron Transfer Reaction ◽  
Azotobacter Chroococcum ◽  
First Order ◽  
Fe Protein ◽  
Vanadium Nitrogenase ◽  
Electron Transfer Complex ◽  
Kinetics Of

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.

scholarly journals The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein

1988 ◽  
Vol 256 (1) ◽  
pp. 189-196 ◽  
Author(s):  
R R Eady ◽  
T H Richardson ◽  
R W Miller ◽  
M Hawkins ◽  
D J Lowe
Keyword(s):  
Nitrogenase Activity ◽  
Azotobacter Chroococcum ◽  
Blue Staining ◽  
Visible Range ◽  
Coomassie Blue Staining ◽  
Reduction Activity ◽  
Fe Protein ◽  
Coomassie Blue ◽  
Purification And Properties ◽  
Vanadium Nitrogenase

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.

scholarly journals Van Allen Probes observation of plasmaspheric hiss modulated by injected energetic electrons

2018 ◽  
Vol 36 (3) ◽  
pp. 781-791 ◽  
Author(s):  
Run Shi ◽  
Wen Li ◽  
Qianli Ma ◽  
Seth G. Claudepierre ◽  
Craig A. Kletzing ◽  
...  
Keyword(s):  
Plasma Density ◽  
Direct Evidence ◽  
Electron Flux ◽  
Energetic Electron ◽  
Low Frequency ◽  
Wave Intensity ◽  
Electron Population ◽  
Ulf Wave ◽  
Van Allen Probes ◽  
First Time

Abstract. Plasmaspheric hiss was observed by Van Allen Probe B in association with energetic electron injections in the outer plasmasphere. The energy of injected electrons coincides with the minimum resonant energy calculated for the observed hiss wave frequency. Interestingly, the variations in hiss wave intensity, electron flux and ultra low frequency (ULF) wave intensity exhibit remarkable correlations, while plasma density is not correlated with any of these parameters. Our study provides direct evidence for the first time that the injected anisotropic electron population, which is modulated by ULF waves, modulates the hiss intensity in the outer plasmasphere. This also implies that the plasmaspheric hiss observed by Van Allen Probe B in the outer plasmasphere (L > ∼ 5.5) is locally amplified. Meanwhile, Van Allen Probe A observed hiss emission at lower L shells (< 5), which was not associated with electron injections but primarily modulated by the plasma density. The features observed by Van Allen Probe A suggest that the observed hiss deep inside the plasmasphere may have propagated from higher L shells.

scholarly journals Structural genes for the vanadium nitrogenase from Azotobacter chroococcum.

1989 ◽  
Vol 8 (4) ◽  
pp. 1217-1224 ◽  
Author(s):  
R. L. Robson ◽  
P. R. Woodley ◽  
R. N. Pau ◽  
R. R. Eady
Keyword(s):  
Azotobacter Chroococcum ◽  
Structural Genes ◽  
Vanadium Nitrogenase
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