scholarly journals The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration

1984 ◽  
Vol 224 (3) ◽  
pp. 903-909 ◽  
Author(s):  
R N F Thorneley ◽  
D J Lowe
Keyword(s):  
Klebsiella Pneumoniae ◽  
Protein Concentration ◽  
Dilution Effect ◽  
Sodium Dithionite ◽  
Evolution Rate ◽  
H2 Evolution ◽  
Action Simulation ◽  
Mofe Protein ◽  
Fe Protein ◽  
Absolute Concentrations

The rate constants from Table 1 and Scheme 2 of Lowe & Thorneley [(1984) Biochem. J. 224, 877-886] were used to simulate the rate of H2 evolution, under various conditions, from nitrogenase isolated from Klebsiella pneumoniae. These rates depend on both the ratio and concentrations of the MoFe protein and Fe protein that comprise nitrogenase. The simulations explain the shapes of ‘protein titration’ and ‘dilution effect’ curves. The concept of an apparent Km for the reductant Na2S2O4 is shown to be invalid, since the dependence of H2-evolution rate on the square root of S2O4(2-) concentration is not hyperbolic and depends on the ratio and absolute concentrations of the MoFe protein and Fe protein.

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 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 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 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 mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution

1984 ◽  
Vol 224 (3) ◽  
pp. 895-901 ◽  
Author(s):  
D J Lowe ◽  
R N F Thorneley
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rate Constants ◽  
Competitive Inhibitor ◽  
H2 Evolution ◽  
Protein Ratio ◽  
Mofe Protein ◽  
Fe Protein ◽  
Mutual Displacement ◽  
Kinetics Of

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.

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 of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre

1987 ◽  
Vol 246 (2) ◽  
pp. 455-465 ◽  
Author(s):  
G A Ashby ◽  
R N F Thorneley
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rate Constants ◽  
Protein Conformation ◽  
Kinetic Studies ◽  
Sodium Dithionite ◽  
Conformation Change ◽  
Fe Protein ◽  
Redox Active ◽  
Dithionite Ion ◽  
Kinetics Of

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 × 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 × 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 × 10(5) less than k less than 3 × 10(6) M-1.s-1 and protein conformation change 6 × 10(2) less than k less than 6 × 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe 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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