scholarly journals Reduction of cyclopropene by NifV- and wild-type nitrogenases from Klebsiella pneumoniae

1989 ◽  
Vol 258 (2) ◽  
pp. 487-491 ◽  
Author(s):  
J P Gemoets ◽  
M Bravo ◽  
C E McKenna ◽  
G J Leigh ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Azotobacter Vinelandii ◽  
Electron Flux ◽  
H2 Production ◽  
Relative Increase ◽  
Analogous Reaction ◽  
Wild Type ◽  
H2 Evolution ◽  
Product Ratio ◽  
In The Wild

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.

scholarly journals Nitrogenase of Klebsiella pneumoniae nifV mutants. Investigation of the novel carbon monoxide-sensitivity of hydrogen evolution by the mutant enzyme

1983 ◽  
Vol 211 (3) ◽  
pp. 589-597 ◽  
Author(s):  
P A McLean ◽  
B E Smith ◽  
R A Dixon
Keyword(s):  
Klebsiella Pneumoniae ◽  
Acetylene Reduction ◽  
Competitive Inhibitor ◽  
H2 Production ◽  
Mutant Enzyme ◽  
Wild Type ◽  
H2 Evolution ◽  
Ph Optimum ◽  
Mofe Protein ◽  
Fe Protein

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.

scholarly journals The Basis of Ammonium Release in nifLMutants of Azotobacter vinelandii

1999 ◽  
Vol 181 (23) ◽  
pp. 7356-7362 ◽  
Author(s):  
Brett Brewin ◽  
Paul Woodley ◽  
Martin Drummond
Keyword(s):  
Azotobacter Vinelandii ◽  
Transcriptional Level ◽  
Uptake System ◽  
Wild Type ◽  
Two Component System ◽  
Two Component ◽  
In The Wild ◽  
Reducing Activity ◽  
State Growth ◽  
Acetylene Reducing Activity

ABSTRACT In Azotobacter vinelandii, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by nifLA. Certain mutations innifL result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator ofnif gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination ofnifL or overexpression of nifA, resulted in ammonium release, correlating with enhanced levels of nifHmRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH4 + uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen.

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 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 Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction

1981 ◽  
Vol 193 (3) ◽  
pp. 971-983 ◽  
Author(s):  
M J Dilworth ◽  
R N F Thorneley
Keyword(s):  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
Competitive Inhibitor ◽  
Spectrometric Analysis ◽  
H2 Evolution ◽  
Total Electron ◽  
Steady State Kinetic ◽  
Nitrogen Bonds ◽  
State Kinetic ◽  
Nitrogen Bond

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3.

scholarly journals Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein

1991 ◽  
Vol 279 (1) ◽  
pp. 81-85 ◽  
Author(s):  
K Fisher ◽  
D J Lowe ◽  
R N F Thorneley
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
High Electron ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Kinetics Of

The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the ‘Fe-protein cycle’ are independent of the level of reduction of the MoFe-protein.

scholarly journals Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata

1984 ◽  
Vol 219 (2) ◽  
pp. 593-600 ◽  
Author(s):  
J C Willison ◽  
D Madern ◽  
P M Vignais
Keyword(s):  
Carbon Source ◽  
H2 Production ◽  
Rhodopseudomonas Capsulata ◽  
Hydrogenase Activity ◽  
Organic Substrates ◽  
Uptake Hydrogenase ◽  
Wild Type ◽  
Benzyl Viologen ◽  
In The Wild ◽  
Different Strains

Non-autotrophic (Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants (IR3, IR4 and IR5) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in ‘uptake’ hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source.

Molybdenum enhancement of nitrogen fixation in a Mo-starved Azotobacter vinelandii Nif− mutant

1982 ◽  
Vol 28 (10) ◽  
pp. 1173-1180 ◽  
Author(s):  
William J. Page ◽  
S. Karen Collinson
Keyword(s):  
Molecular Weight ◽  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Cell Extracts ◽  
In The Wild ◽  
Dinitrogenase Reductase

Molybdenum (Mo)-starved wild-type and Nif− strains of Azotobacter vinelandii reduced acetylene (fixed nitrogen) in Mo-limited nitrogen-free medium. Vanadate enhanced this activity in all of the strains. Molybdate caused repression of nitrogenase activity in the Nif− mutants and enhanced the nitrogenase activity in the wild type. The nitrogenase activity in the Nif− mutant UW3, however, was enhanced by Mo, became maximal after 3 h, and then declined to zero after 10 h of incubation. The activation of nitrogenase by Mo followed a 5- to 10-min lag and was inhibited when streptomycin or rifampin was added with Mo. Examination of Mo-starved nitrogen-fixing UW3 cell extracts by two-dimensional polyacrylamide gel electrophoresis revealed molecular weight 57 000, 50 000, and 30 000 proteins that were Mo and NH4+ repressive. The molecular weight 30 000 protein appeared in the same position on the gel as the wild-type dinitrogenase reductase, although UW3 did not produce this protein under Mo-sufficient nitrogen-fixing conditions. Cell extracts prepared 3 h after Mo addition lacked the molecular weight 57 000 and 50 000 proteins but contained a new protein corresponding to the β subunit of dinitrogenase. When UW3 nitrogenase activity was lost, the dinitrogenase reductase-like protein also was absent. The results suggest that a complex active in nitrogen fixation may form between components of the traditional Mo-sufficient and alternative Mo-starved cell nitrogen fixation systems.

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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