scholarly journals Nitrogenase of Klebsiella pneumoniae: electron-paramagnetic-resonance studies on the catalytic mechanism

1972 ◽  
Vol 130 (2) ◽  
pp. 641-643 ◽  
Author(s):  
B E Smith ◽  
D J Lowe ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Catalytic Mechanism ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

scholarly journals Studies by electron paramagnetic resonance on the catalytic mechanism of nitrogenase of Klebsiella pneumoniae

1973 ◽  
Vol 135 (2) ◽  
pp. 331-341 ◽  
Author(s):  
Barry E. Smith ◽  
David J. Lowe ◽  
Robert C. Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Catalytic Mechanism ◽  
Rapid Freezing ◽  
Paramagnetic Resonance ◽  
Iron Protein ◽  
Axial Form ◽  
Electron Paramagnetic ◽  
Generating System

The properties and catalytic reactions of the enzyme nitrogenase purified from Klebsiella pneumoniae were studied by electron-paramagnetic-resonance (e.p.r.) spectroscopy at temperatures down to 8°K. The two protein fractions, Kp1 (the iron–molybdenum protein) and Kp2 (the iron protein), were examined alone and in steady-state mixtures and also in pre-steady-state experiments, by using the rapid-freezing method. Kp1 protein in dithionite solution shows a rhombic type of spectrum with g1 4.32, g2 3.63, g3 2.009 at pH6.8 (0°C). Small changes in the spectrum produced by protons (pK=8.7 at 0°C) or by acetylene indicate binding of these oxidizing substrates to this protein fraction. Kp2 protein shows a rhombic spectrum with g1 2.053, g2 1.942, g3 1.865, which integrates to about 0.45 electron/molecule. Binding of ATP, with a dissociation constant of 4×10-4m, changes the spectrum to an axial form with g∥ 2.036, g⊥ 1.929, thus indicating a conformation change of Kp2 protein. The Kp2 protein spectrum disappears reversibly on cautious oxidation. The signals of both proteins are diminished in their steady-state mixtures, obtained in the presence of ATP and dithionite (with an ATP-generating system and Mg2+ ions) and with protons, N2 or acetylene as oxidizing substrate. At the same time as dithionite is consumed in such reactions, the Kp1 protein signal is gradually restored and the Kp2 protein signal diminishes to zero. In rapid-freezing experiments the signals from the two proteins decreased at indistinguishable rates (t½ about 10ms), then they remained constant. Results are interpreted in terms of a scheme in which reducing equivalents pass from dithionite to Kp2 protein, then, in an ATP-dependent reaction to Kp1 protein, this being finally reoxidized by N2 or another oxidizing substrate. In this scheme Kp1 protein cycles between its signal-giving state and a very highly reduced signal-free state.

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.

ELECTRON PARAMAGNETIC RESONANCE STUDIES OF PHASE TRANSITIONS IN KCN

1976 ◽  
Vol 37 (C7) ◽  
pp. C7-241-C7-246 ◽  
Author(s):  
J.P. VON DER WEID ◽  
L.C. SCAVARDA DO CARMO ◽  
R. R. DO SANTOS ◽  
B. KOILLER ◽  
S. COSTA RIBEIRO ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Phase Transitions ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic
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