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.

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 Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur ‘P’ clusters

1993 ◽  
Vol 291 (3) ◽  
pp. 709-711 ◽  
Author(s):  
R W Miller ◽  
B E Smith ◽  
R R Eady
Keyword(s):  
Gel Filtration ◽  
Good Evidence ◽  
Published Data ◽  
Midpoint Potential ◽  
Mofe Protein ◽  
Fe Protein ◽  
The Press ◽  
Pattern Characteristic ◽  
Iron Molybdenum Cofactor ◽  
Hydrolysis Of

Hydrolysis of MgATP to MgADP is essential for nitrogenase action. There is good evidence for binding of both nucleotides to the Fe protein of nitrogenase, but data indicating their binding to the MoFe protein have been controversial [see Miller and Eady (1989) Biochem. J. 263, 725-729]. The binding of MgADP to the MoFe protein of nitrogenase of Klebsiella pneumoniae was investigated by non-equilibrium gel-filtration column methods. No binding of MgADP to the dithionite-reduced protein could be detected. Treatment of the MoFe protein with phenosafranine [midpoint potential (Em) -270 mV] did not affect the activity, and oxidized the ‘P’ clusters but not the iron-molybdenum cofactor (FeMoco) centres. This oxidized species bound 3.9 mol of MgADP with a binding pattern characteristic of low rates of ligand dissociation. These observations suggest that the variability in published data on nucleotide binding to the MoFe protein is related to poor control of the protein oxidation level. Our data, coupled with the observation that ‘P’ clusters become oxidized during reduction of N2 [Lowe, Fisher and Thorneley (1993) Biochem. J., in the press], led us to propose that the ADP binding sites are transiently filled during enzyme turnover by hydrolysis of ATP originally bound to the Fe protein, and that hydrolysis occurs on a bridging site on the MoFe-Fe-protein complex.

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.

Electron transfer and half-reactivity in nitrogenase

2011 ◽  
Vol 39 (1) ◽  
pp. 201-206 ◽  
Author(s):  
Thomas A. Clarke ◽  
Shirley Fairhurst ◽  
David J. Lowe ◽  
Nicholas J. Watmough ◽  
Robert R. Eady
Keyword(s):  
Electron Transfer ◽  
Protein Molecule ◽  
Stopped Flow ◽  
Protein Binding Sites ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Mofe Protein ◽  
Fe Protein ◽  
Rapid Quench ◽  
Important Enzyme

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum–iron protein (MoFe protein) and a protein containing an [Fe4–S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

Biological nitrogen fixation: fundamentals

1982 ◽  
Vol 296 (1082) ◽  
pp. 375-385 ◽  
Keyword(s):  
Nitrogen Fixation ◽  
Klebsiella Pneumoniae ◽  
Biological Nitrogen Fixation ◽  
Mofe Protein ◽  
Fe Protein ◽  
Metal Contents ◽  
Physiological Constraint ◽  
Biological Nitrogen

The enzyme responsible for N 2 fixation, nitrogenase, is only found in prokaryotes. It consists of two metalloproteins, both irreversibly destroyed by exposure to the O 2 of air. The MoFe-protein binds N 2 and the Fe-protein, after activation by MgATP, supplies electrons. H 2 is evolved during the reduction of N 2 to NH 3 and can become the sole reaction in the absence of N 2 ; valuable information has been obtained by exploiting the ability of nitrogenase to reduce substrates such as acetylene, azides and cyanides. Substrate quantities of MgATP are required for all such reactions. The sensitivity of nitrogenase to oxygen is an important physiological constraint on its use and distribution; the ATP requirement and metal contents are less serious constraints. O 2 and NH 3 regulate synthesis and sometimes function of nitrogenase. Nitrogen fixation by Klebsiella pneumoniae is genetically encoded by 17 genes (the nif genes) in a cluster of seven or eight operons. The functions of several of these genes are known and the outlines of their regulation can be discerned. The nif cluster can be transferred to new prokaryotic genera, sometimes yielding new diazotrophic strains or species; they have been transferred to yeast and are silent. They have been cloned and alien DNA ( lac ) has been fused into nif Transfer of expressible nif to new genetic backgrounds has probably occurred in Nature and may be exploitable for agriculture.

scholarly journals Guanosine triphosphate cyclohydrolase activity in rat tissues

1984 ◽  
Vol 217 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Z Bellahsene ◽  
J L Dhondt ◽  
J P Farriaux
Keyword(s):  
Formic Acid ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Rat Tissues ◽  
Synthetase Activity ◽  
Guanosine Triphosphate ◽  
Heat Treated ◽  
Liver Homogenates ◽  
Acid Release ◽  
Reduced Forms

The GTP cyclohydrolase activity of rat tissues has been studied by means of the measurement of formic acid release and neopterin synthesis from GTP. After gel filtration of a 45%-satd.-(NH4)2SO4 fraction of liver homogenates, three enzyme fractions were separated and named A1, A2 and A3 according to the order of their elution. Fractions A1 and A3 displayed an 8-formyl-GTP deformylase activity; no proof of cyclized product has yet been established. This activity was heat-labile and required Mg2+ for maximal activity. Fraction A2 displayed a ‘neopterin-synthetase’ activity, with dihydroneopterin triphosphate and formic acid formed in stochiometric amounts. Fraction A1 isolated from heat-treated homogenates also produced dihydroneopterin triphosphate. Neopterin synthetase activity in fractions A1 and A2 was heat-resistant and inhibited by Mg2+. In liver the A2 fraction represented 70-75% of the neopterin synthetase capacity and was inhibited by reduced pterines (sepiapterin, dihydrobiopterin and tetrahydrobiopterin) and to a lesser extent by reduced forms of folic acid. In kidney and brain, fraction A1 and A3 GTP 8-formylhydrolase activities were found in significant amounts, in contrast with the neopterin synthetase activity, which was low and appeared to be confined to the A1 fraction.

scholarly journals Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor

1984 ◽  
Vol 217 (1) ◽  
pp. 317-321 ◽  
Author(s):  
T R Hawkes ◽  
P A McLean ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Metal Contents ◽  
Altered Form ◽  
Iron Molybdenum Cofactor

When the iron-molybdenum cofactor (FeMoco) was extracted from the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae and combined with the FeMoco-deficient MoFe protein from a nifB mutant, the resultant MoFe protein exhibited the NifV phenotype, i.e. in combination with wild-type Fe protein it exhibited poor N2-fixation activity and its H2-evolution activity was inhibited by CO. These data provide strong evidence that FeMoco contains the active site of nitrogenase. The metal contents and e.p.r. properties of FeMoco from wild-type and nifV mutants of K. pneumoniae are very similar.

scholarly journals Nitrogenase bioelectrocatalysis: heterogeneous ammonia and hydrogen production by MoFe protein

2016 ◽  
Vol 9 (8) ◽  
pp. 2550-2554 ◽  
Author(s):  
Ross D. Milton ◽  
Sofiene Abdellaoui ◽  
Nimesh Khadka ◽  
Dennis R. Dean ◽  
Dónal Leech ◽  
...  
Keyword(s):  
Hydrogen Production ◽  
Protein Immobilization ◽  
Mofe Protein ◽  
Fe Protein

Nitrogenase MoFe protein immobilization yields a bioelectrode capable of producing H2 and NH3 independent of the ATP-hydrolyzing Fe protein.

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