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 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 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 Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide

1990 ◽  
Vol 272 (3) ◽  
pp. 621-625 ◽  
Author(s):  
D J Lowe ◽  
K Fisher ◽  
R N F Thorneley
Keyword(s):  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
Lag Phase ◽  
High Electron ◽  
Mofe Protein ◽  
Fe Protein ◽  
C2h2 Reduction ◽  
Electron Reduction ◽  
State Concentration

The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S2O4) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kp1) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kp1. This explanation is also consistent with a pre-steady-state lag phase for C2H4 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kp1 and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKp1C2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme.

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 Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane

1987 ◽  
Vol 247 (3) ◽  
pp. 547-554 ◽  
Author(s):  
G A Ashby ◽  
M J Dilworth ◽  
R N F Thorneley
Keyword(s):  
Electron Transfer ◽  
Transition Metal ◽  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
H2 Evolution ◽  
Total Electron ◽  
Mofe Protein ◽  
Fe Protein ◽  
Total Inhibition ◽  
Nitrogenase Inhibition

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.

scholarly journals Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein

1989 ◽  
Vol 263 (3) ◽  
pp. 725-729 ◽  
Author(s):  
R W Miller ◽  
R R Eady
Keyword(s):  
Tight Binding ◽  
Gel Filtration ◽  
Azotobacter Chroococcum ◽  
Catalytic Cycle ◽  
Elution Profile ◽  
Mofe Protein ◽  
Fe Protein ◽  
Weak Binding ◽  
Reduced Forms ◽  
Low Rate

The dye-oxidized or dithionite-reduced forms of the MoFe protein of molybdenum nitrogenase of Azotobacter chroococcum were shown to bind 2 mol of MgADP/mol of protein, as determined by column equilibrium techniques. The gel-filtration elution profile of unbound Mg[14C]ADP was not symmetrical, consistent with a low rate of dissociation from the protein. Symmetrical elution profiles were observed for the oxidized Fe protein of nitrogenase, which bound 2 mol of MgADP/mol of protein. The low rate of dissociation of MgADP from MoFe protein was shown by non-equilibrium column techniques, where 1 mol of MgADP/mol of MoFe protein remained tightly bound during chromatography. Very weak binding of MgATP (less than 0.01 mol of MgATP/mol of MoFe protein) to dye-oxidized but not to dithionite-reduced MoFe protein was observed. These results are discussed in terms of their relevance to the catalytic cycle of nitrogenase catalysis.

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