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

ABSTRACT: 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.

SUBMITTER: Hawkes TR

PROVIDER: S-EPMC1154054 | biostudies-other | 1983 Jan

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1144363 | 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.

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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

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

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

A substrate channel in the nitrogenase MoFe protein.

Project description:Nitrogenase catalyzes the six electron/six proton reduction of N2 to two ammonia molecules at a complex organometallocluster called "FeMo cofactor." This cofactor is buried within the alpha-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N2, acetylene, azide, and propyne. For each MoFe protein, V (max) values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the K(m) values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.

| S-EPMC2819195 | 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

?-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

Nitrogenase of Klebsiella pneumoniae. Rhodanese-catalysed restoration of activity of the inactive 2Fe species of the Fe protein.

Project description:The inactive 2Fe species of the Fe protein of the nitrogenase of Klebsiella pneumoniae was generated by treating oxidized Fe protein (Kp2) with MgATP and chelator. Incubation of the 2Fe species of Kp2 with the sulphurtransferase rhodanese in the presence of thiosulphate, ferric citrate and reduced lipoate reproducibly restored activity. The extent of restoration of activity depended on the molar ratio of 2Fe Kp2 to rhodanese and was time-dependent. Re-activation did not occur in the reaction mixture lacking rhodanese.

| S-EPMC1148016 | biostudies-other

Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 A resolution: comparison with the Azotobacter vinelandii MoFe protein.

Project description:The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

| S-EPMC4321486 | biostudies-literature

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