On the difference between iron-molybdenum cofactor of wild-type and nifV- mutant molybdenum-iron proteins of Klebsiella pneumoniae: ENDOR, EXAFS, and EPR evidence

1987 ◽  
Vol 109 (3) ◽  
pp. 943-945 ◽  
Author(s):  
Paul A. McLean ◽  
Anne E. True ◽  
Mark J. Nelson ◽  
Steve Chapman ◽  
Martin R. Godfrey ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
Iron Proteins ◽  
The Difference ◽  
Iron Molybdenum Cofactor

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 Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the α- and β-subunits

1989 ◽  
Vol 264 (1) ◽  
pp. 257-264 ◽  
Author(s):  
H M Kent ◽  
I Ioannidis ◽  
C Gormal ◽  
B E Smith ◽  
M Buck
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Component ◽  
Molybdenum Cofactor ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Diazotrophic Growth ◽  
Iron Molybdenum Cofactor

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.

scholarly journals The inactive MoFe protein (NifB−Kp1) of the nitrogenase from nif B mutants of Klebsiella pneumoniae. Its interaction with FeMo-cofactor and the properties of the active MoFe protein formed

1984 ◽  
Vol 223 (3) ◽  
pp. 783-792 ◽  
Author(s):  
T R Hawkes ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Signal Intensity ◽  
Specific Activity ◽  
Maximum Activity ◽  
Gene Products ◽  
Wild Type ◽  
Activation Reaction ◽  
Mofe Protein ◽  
Femo Cofactor ◽  
Iron Molybdenum Cofactor

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.

scholarly journals Low-temperature magnetic-circular-dichroism spectroscopy of the iron-molybdenum cofactor and the complementary cofactor-less MoFe protein of Klebsiella pneumoniae nitrogenase

1984 ◽  
Vol 219 (2) ◽  
pp. 495-503 ◽  
Author(s):  
A E Robinson ◽  
A J M Richards ◽  
A J Thomson ◽  
T R Hawkes ◽  
B E Smith
Keyword(s):  
Low Temperature ◽  
Klebsiella Pneumoniae ◽  
Magnetic Circular Dichroism ◽  
Molybdenum Cofactor ◽  
Magnetization Curves ◽  
Mofe Protein ◽  
Paramagnetic Component ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Iron Molybdenum Cofactor ◽  
Circular Dichroïsm

The major metal clusters of the MoFe protein, Kpl, of Klebsiella pneumoniae nitrogenase were characterized separately by low-temperature magnetic-circular-dichroism spectroscopy. The spectra and magnetization curves of the extracted iron-molybdenum cofactor, FeMoco, and of ‘P’ clusters in NifB - Kpl, the inactive, FeMoco -less, MoFo protein from an nifB mutant, were measured and compared with those of the holoprotein. (When FeMoco and NifB - Kpl are combined, active Kpl is formed.) Reduced NifB - Kpl had a spectrum with a weak, paramagnetic, component superimposed on a diamagnetic background. The paramagnetic component was assigned to a contaminating, e.p.r.-active, species. Thionine-oxidized NifB - Kpl had a spectrum and magnetization properties very similar to those of thionine-oxidized Kpl, demonstrating that the ‘P’ clusters are not significantly affected by the absence of the FeMoco clusters. The spectra of reduced isolated FeMoco had similar magnetization curves but sharper features and higher intensities than those of this centre in dithionite-reduced Kpl . Furthermore, a shoulder near 580 nm in the Kpl spectrum was absent from that of FeMoco . This may be due to the loss of a ligand or to a change in symmetry of the FeMoco cluster on extraction.

scholarly journals Purification and characterization of the inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae

1983 ◽  
Vol 209 (1) ◽  
pp. 43-50 ◽  
Author(s):  
T R Hawkes ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Double Mutant ◽  
Specific Activity ◽  
Equimolar Amount ◽  
Wild Type ◽  
Purification And Characterization ◽  
Point Mutant ◽  
Mofe Protein ◽  
Iron Molybdenum Cofactor

The inactive MoFe protein of nitrogenase, NifB-Kp1, from two distinct nifB mutants of Klebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nifB, nifJ double mutant) has been purified and characterized. NifB-Kp1 can be activated by reaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFe protein. NifB-Kp1 purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of mol.wt. 21 000. Like active wild-type Kp1, it was an alpha 2 beta 2 tetramer, but it was far less stable than Kp1, deteriorating rapidly at temperatures above 8 degrees C or on mild oxidation. NifB-Kp1 preparations contained 0.4-0.9 Mo and 9.0 +/- 0.9 Fe atoms . mol-1 and, when activated by FeMoco, had a specific activity of approx. 500 units . mg-1. The Mo in our preparations was not associated with the e.p.r. signal normally observed from FeMoco. All preparations exhibited a weak gav. = 1.95 e.p.r. signal which was probably not associated with activatable protein.

Comparison of wild-type and nifV mutant molybdenum-iron proteins of nitrogenase from Klebsiella pneumoniae by ENDOR spectroscopy

1990 ◽  
Vol 112 (2) ◽  
pp. 651-657 ◽  
Author(s):  
Anne E. True ◽  
Paul McLean ◽  
Mark J. Nelson ◽  
W. H. Orme-Johnson ◽  
Brian M. Hoffman
Keyword(s):  
Klebsiella Pneumoniae ◽  
Wild Type ◽  
Iron Proteins ◽  
Endor Spectroscopy

The molybdenum site of nitrogenase. 2. A comparative study of molybdenum-iron proteins and the iron-molybdenum cofactor by x-ray absorption spectroscopy

1978 ◽  
Vol 100 (12) ◽  
pp. 3814-3819 ◽  
Author(s):  
Stephen P. Cramer ◽  
William O. Gillum ◽  
Keith O. Hodgson ◽  
Leonard E. Mortenson ◽  
Edward I. Stiefel ◽  
...  
Keyword(s):  
Comparative Study ◽  
Absorption Spectroscopy ◽  
Molybdenum Cofactor ◽  
X Ray ◽  
Iron Proteins ◽  
Iron Molybdenum Cofactor ◽  
X Ray Absorption
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