Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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.

ABSTRACT: 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 alpha2beta2 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.

SUBMITTER: Yousafzai FK

PROVIDER: S-EPMC1218715 | biostudies-other | 1997 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Klebsiella pneumoniae nitrogenase MoFe protein: chymotryptic proteolysis affects function by limited cleavage of the beta-chain and provides high-specific-activity MoFe protein.

Project description:Proteinase treatment with chymotrypsin has been used to probe the structure of native Klebsiella pneumoniae nitrogenase MoFe protein (Kp1). Reaction with chymotrypsin did not bleach Kp1, suggesting that it did not destroy the metal centres, and the Mo and Fe contents of Kp1 were unchanged. High ratios of chymotrypsin to Kp1 (1:1 by mass) cleaved the beta-chain of Kp1 to give 44 and 14 kDa polypeptides, which N-terminal amino acid sequence analysis showed to be derived from cleavage at residue beta-Phe124. A mutant MoFe protein, Kp1Met-124, in which beta-Phe124 is replaced by methionine, was not cleaved by chymotrypsin. Under non-denaturing conditions, the 'nicked' beta-chain of the wild-type protein remained associated with the alpha-chain. The alpha-chain was not cleaved by the proteinase treatment. Fission of the wild-type beta-chain was accompanied by loss of enzyme activity, loss of intensity of the g = 3.7 e.p.r. signal derived from dithionite-reduced FeMoco and by changes in the visible spectrum. The e.p.r. spectra of potassium ferricyanide-oxidized native and digested Kp1 show differences in the signals between g = 1.6 and 2.0. After prolonged treatment, the final specific activity of Kp1 was about 25 +/- 5% of the initial activity. This corresponded to 25 +/- 5% of the beta-chain which was resistant to proteolytic action. Brief treatment of Kp1 with a lower concentration of chymotrypsin (chymotrypsin/Kp1 ratio = 1:10 by mass, for 10 min) preferentially cleaved high-molecular-mass polypeptides that routinely contaminate preparations of Kp1 prepared by standard procedures. Treatment with chymotrypsin followed by gel filtration to remove the proteinase and cleaved protein fragments can therefore be used to increase significantly the specific activity of Kp1 preparations and remove contaminating activities, such as the ATPase activity of myokinase.

| S-EPMC1132518 | biostudies-other

Nitrogenase of Klebsiella pneumoniae nifV mutants.

Project description: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.

| S-EPMC1154403 | biostudies-other

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

Project description: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.

| S-EPMC1144524 | biostudies-other

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

Project description: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.

| S-EPMC1154054 | biostudies-other

P-cluster maturation on nitrogenase MoFe protein.

Project description:Biosynthesis of nitrogenase P-cluster has attracted considerable attention because it is biologically important and chemically unprecedented. Previous studies suggest that P-cluster is formed from a precursor consisting of paired [4Fe-4S]-like clusters and that P-cluster is assembled stepwise on MoFe protein, i.e., one cluster is assembled before the other. Here, we specifically tackle the assembly of the second P-cluster by combined biochemical and spectroscopic approaches. By using a P-cluster maturation assay that is based on purified components, we show that the maturation of the second P-cluster requires the concerted action of NifZ, Fe protein, and MgATP and that the action of NifZ is required before that of Fe protein/MgATP, suggesting that NifZ may act as a chaperone that facilitates the subsequent action of Fe protein/MgATP. Furthermore, we provide spectroscopic evidence that the [4Fe-4S] cluster-like fragments can be converted to P-clusters, thereby firmly establishing the physiological relevance of the previously identified P-cluster precursor.

| S-EPMC1965529 | biostudies-literature

?-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al.

Project description:Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that ?-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-"FeMoco". Amino acid residue ?-Lys424 connects directly to ?-Ile423, and they are located in the same ?-helix (?423-431). In the present study, function of ?-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of ?-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of ?-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.

| S-EPMC6477116 | biostudies-literature

Chimeric Interaction of Nitrogenase-Like Reductases with the MoFe Protein of Nitrogenase.

Project description:The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.

| S-EPMC7317204 | biostudies-literature

Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor.

Project description: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.

| S-EPMC1153212 | biostudies-other

Molybdenum L-Edge XAS Spectra of MoFe Nitrogenase.

Project description:A molybdenum L-edge X-ray absorption spectroscopy (XAS) study is presented for native and oxidized MoFe protein of nitrogenase as well as Mo-Fe model compounds. Recently collected data on MoFe protein (in oxidized and reduced forms) is compared to previously published Mo XAS data on the isolated FeMo cofactor in NMF solution and put in context of the recent Mo K-edge XAS study, which showed a MoIII assignment for the molybdenum atom in FeMoco. The L3-edge data are interpreted within a simple ligand-field model, from which a time-dependent density functional theory (TDDFT) approach is proposed as a way to provide further insights into the analysis of the molybdenum L3-edges. The calculated results reproduce well the relative spectral trends that are observed experimentally. Ultimately, these results give further support for the MoIII assignment in protein-bound FeMoco, as well as isolated FeMoco.

| S-EPMC4510703 | biostudies-literature

The sixteenth iron in the nitrogenase MoFe protein.

Project description:Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.

| S-EPMC3891402 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data