scholarly journals Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes

1978 ◽  
Vol 173 (1) ◽  
pp. 277-290 ◽  
Author(s):  
D J Lowe ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Low Concentrations ◽  
Direct Binding ◽  
Electron Paramagnetic ◽  
Single Binding Site

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo–Fe protein showed that they arose from Fe–S clusters in the Mo–Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the −1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme–product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe–S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

scholarly journals Nitrogenase from Klebsiella pneumoniae. An e.p.r. signal observed during enzyme turnover under ethylene is associated with the iron-molybdenum cofactor

1983 ◽  
Vol 211 (2) ◽  
pp. 495-497 ◽  
Author(s):  
T R Hawkes ◽  
D J Lowe ◽  
B E Smith
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Isotopic Substitution ◽  
Resonance Signal ◽  
Paramagnetic Resonance ◽  
Mofe Protein ◽  
Femo Cofactor ◽  
Enzyme Turnover ◽  
Iron Molybdenum Cofactor ◽  
Electron Paramagnetic

During turnover at 10 degrees C at pH 7.4 in the presence of ethylene, the MoFe protein of Klebsiella pneumoniae nitrogenase (Kp 1) exhibited an electron-paramagnetic-resonance signal with g-values at 2.12, 1.998 and 1.987. 57Fe isotopic substitution demonstrated that this signal arose from the Kp 1 FeMo-cofactor in an S = 1/2 spin state.

scholarly journals Protective responses of tolerant and sensitive wheat seedlings to systemic and local zearalenone application – Electron paramagnetic resonance studies

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sieprawska Apolonia ◽  
Łabanowska Maria ◽  
Kurdziel Magdalena ◽  
Filek Maria ◽  
Skórka Magdalena ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Defense Mechanisms ◽  
Protein Complexes ◽  
Protective Mechanisms ◽  
Paramagnetic Resonance ◽  
Wheat Seedlings ◽  
Fe Protein ◽  
Biochemical Analyses ◽  
Electron Paramagnetic ◽  
The Way

Abstract Background Mycotoxins are among the environmental stressors whose oxidative action is currently widely studied. The aim of this paper was to investigate the response of seedling leaves to zearalenone (ZEA) applied to the leaves (directly) and to the grains (indirectly) in tolerant and sensitive wheat cultivars. Results Biochemical analyses of antioxidant activity were performed for chloroplasts and showed a similar decrease in this activity irrespective of plant sensitivity and the way of ZEA application. On the other hand, higher amounts of superoxide radical (microscopic observations) were generated in the leaves of plants grown from the grains incubated in ZEA solution and in the sensitive cultivar. Electron paramagnetic resonance (EPR) studies showed that upon ZEA treatment greater numbers of Mn - aqua complexes were formed in the leaves of the tolerant wheat cultivar than in those of the sensitive one, whereas the degradation of Fe-protein complexes occurred independently of the cultivar sensitivity. Conclusion The changes in the quantity of stable, organic radicals formed by stabilizing reactive oxygen species on biochemical macromolecules, indicated greater potential for their generation in leaf tissues subjected to foliar ZEA treatment. This suggested an important role of these radical species in protective mechanisms mainly against direct toxin action. The way the defense mechanisms were activated depended on the method of the toxin application.

scholarly journals Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre

1987 ◽  
Vol 243 (1) ◽  
pp. 235-239 ◽  
Author(s):  
P M A Gadsby ◽  
C Greenwood ◽  
A Coddington ◽  
A J Thomson ◽  
C Godfrey
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Paramagnetic Resonance ◽  
Hyperfine Coupling ◽  
Formate Dehydrogenase ◽  
Sodium Dithionite ◽  
Paramagnetic Resonance ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Electron Paramagnetic ◽  
Fold Excess

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.

scholarly journals Complex-formation between reduced xanthine oxidase and purine substrates demonstrated by electron paramagnetic resonance

1969 ◽  
Vol 114 (4) ◽  
pp. 735-742 ◽  
Author(s):  
Frances M. Pick ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
Active Site ◽  
Paramagnetic Resonance ◽  
Freezing Method ◽  
Low Concentrations ◽  
Appearance Rate ◽  
High Concentrations ◽  
Electron Paramagnetic ◽  
Do So

The origin of the Rapid molybdenum electron-paramagnetic-resonance signals, which are obtained on reducing xanthine oxidase with purine or with xanthine, and whose parameters were measured by Bray & Vänngård (1969), was studied. It is concluded that these signals represent complexes of reduced enzyme with substrate molecules. Xanthine forms one complex at high concentrations and a different one at low concentrations. Purine forms a complex indistinguishable from the low-concentration xanthine complex. There are indications that some other substrates also form complexes, but uric acid, a reaction product, does not appear to do so. The possible significance of the complexes in the catalytic cycle of the enzyme is discussed and it is suggested that they represent substrate molecules bound at the reduced active site, waiting their turn to react there, when the enzyme has been reoxidized. Support for this role for the complexes was deduced from experiments in which frozen samples of enzyme–xanthine mixtures, prepared by the rapid-freezing method, were warmed until the signals began to change. Under these conditions an increase in amplitude of the Very Rapid signal took place. Data bearing on the origin of the Slow molybdenum signal are also discussed. This signal disappears only slowly in the presence of oxygen, and its appearance rate is unaffected by change in the concentration of dithionite. It is concluded that, like other signals from the enzyme, it is due to Mov but that a slow change of ligand takes place before it is seen. The Slow species, like the Rapid, seems capable of forming complexes with purines.

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