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.

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 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 The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation

1984 ◽  
Vol 224 (3) ◽  
pp. 877-886 ◽  
Author(s):  
D J Lowe ◽  
R N Thorneley
Keyword(s):  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Necessary Condition ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Rapid Quench ◽  
Steady State Kinetic ◽  
H2 Formation

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.

scholarly journals Uncoupling Nitrogenase: Catalytic Reduction of Hydrazine to Ammonia by a MoFe Protein in the Absence of Fe Protein-ATP

2010 ◽  
Vol 132 (38) ◽  
pp. 13197-13199 ◽  
Author(s):  
Karamatullah Danyal ◽  
Boyd S. Inglet ◽  
Kylie A. Vincent ◽  
Brett M. Barney ◽  
Brian M. Hoffman ◽  
...  
Keyword(s):  
Catalytic Reduction ◽  
Mofe Protein ◽  
Fe Protein

scholarly journals Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange

1991 ◽  
Vol 277 (3) ◽  
pp. 735-741 ◽  
Author(s):  
R N F Thorneley ◽  
G A Ashby ◽  
C Julius ◽  
J L Hunter ◽  
M R Webb
Keyword(s):  
Klebsiella Pneumoniae ◽  
Atp Hydrolysis ◽  
Oxygen Exchange ◽  
Cleavage Reaction ◽  
Protein Ratio ◽  
Mofe Protein ◽  
Fe Protein ◽  
Water Oxygen ◽  
Atpase Reaction ◽  
Protein Dissociation

The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

scholarly journals Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre

1997 ◽  
Vol 326 (3) ◽  
pp. 637-640 ◽  
Author(s):  
Faridoon K. YOUSAFZAI ◽  
Robert R. EADY
Keyword(s):  
Klebsiella Pneumoniae ◽  
Transition State ◽  
Nitrogenase Activity ◽  
Active Species ◽  
Slow Phase ◽  
Mofe Protein ◽  
Fe Protein ◽  
Catalytically Active ◽  
Mo Content ◽  
Kinetics Of

We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per α2β2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.

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