nitrogenase and homocitric-acid

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.

Topics: Azotobacter vinelandii; Molybdoferredoxin; Nitrogenase; Proteomics; Tricarboxylic Acids

Nitrogen (N

Topics: Biocatalysis; Carbon; Iron; Molybdenum; Nitrogen; Nitrogenase; Sodium; Sulfur; Tricarboxylic Acids; Trimethylsilyl Compounds

Mo-containing nitrogenase is the main enzyme that is able to take N

Topics: Coordination Complexes; Iron; Kinetics; Ligands; Models, Molecular; Molecular Structure; Molybdenum; Nitrogen; Nitrogenase; Tricarboxylic Acids

Glycolato and R,S-lactato imidazole molybdenum(iv) complexes [Mo3SO3(glyc)2(im)5]·im·H2O (1), Na2[Mo3SO3(R,S-lact)3(im)3]·10H2O (2), and [Mo6O10(R,S-lact)2(im)10]·16H2O (3) have been isolated and characterized (H2glyc = glycolic acid, H2lact = lactic acid, im = imidazole). α-Alkoxy coordination with molybdenum [Mo-Oα-alkoxy 1.993(7)av Å] in 1 and 2 showed obvious differences to their counterpart with α-hydroxy coordination [MoIV3S4(PPh3)3(Hlact)2(lact)] [2.204(4)av Å] as shown in M. N. Sokolov, S. A. Adonin, A. V. Virovets, P. A. Abramov, C. Vicent, R. Llusar and V. P. Fedin, Inorg. Chim. Acta, 2013, 395, 11-18. This was also true for the 36 reported structures of FeMo-cofactors in the RCSB protein data bank (Mo-Oav 2.272 Å), which can serve as indirect evidence for the protonation of homocitrate in FeMo-co. The C-OHα-hydroxy bonds were longer than the short C-Oα-alkoxy bonds. Trinuclear Mo3SO3 cores were stabilized by imidazoles and/or α-hydroxycarboxylates, whereas only two glycolates were present in 1. α-Hydroxycarboxylates in 1 and 2 acted as bidentate ligands of Mo(iv) atoms through α-alkoxy and α-carboxy groups, while the imidazoles coordinated monodentately with nitrogen atoms. The lactates in 3 coordinated with Mo(iv) atoms through two oxygen atoms of α-carboxy groups, leaving the α-hydroxy group free. Furthermore, novel hexanuclear oxomolybdenum(v) malate Na6[(Mo2O4)3(mal)4]·5H2O (4) was also isolated (H3mal = malic acid). Solid-state and solution 13C NMR resonances of carbon atoms in α-alkoxy groups appeared in a high-field region (71.6, 77.4 ppm), indicating that α-alkoxy groups were easy to protonate.

Topics: Carbonates; Glycolates; Imidazoles; Iron; Lactic Acid; Ligands; Malates; Molybdenum; Molybdoferredoxin; Nitrogen; Nitrogenase; Protein Conformation; Protons; Tricarboxylic Acids

Topics: Molecular Dynamics Simulation; Nitrogenase; Protons; Quantum Theory; Thermodynamics; Tricarboxylic Acids

Nitrogenase naturally converts N2 to NH3, but it also hydrogenates a variety of small molecules, in many cases requiring multiple electrons plus protons for each catalytic cycle. A general mechanism, arising from many density functional calculations and simulations, is proposed to account for all of these reactions. Protons, supplied serially in conjunction with electrons to the active site FeMo-co (a CFe7MoS9 (homocitrate) cluster), generate H atoms that migrate over and populate two S and two Fe atoms in the reaction domain. The mechanistic paradigm is conceptually straightforward: substrate (on Fe) and H atoms (on S and Fe) are bound contiguously in the reaction zone, and H atoms transfer (probably with some quantum tunneling) to the substrate to form product. Details and justifications of the mechanisms for N2 and other key substrates are summarised, and the unusual structure of FeMo-co as a general hydrogenation catalyst is rationalised. Testing experiments are suggested.

Topics: Ammonia; Biocatalysis; Catalytic Domain; Electrons; Hydrogen; Hydrogenation; Nitrogen; Nitrogenase; Protons; Quantum Theory; Tricarboxylic Acids

Direct reaction of potassium molybdate (with natural abundance Mo or enriched with (92)Mo or (100)Mo) with excess hydrolyzed homocitric acid-γ-lactone in acidic solution resulted in the isolation of a cis-dioxo bis-homocitrato molybdenum(VI) complex, K(2)[*MoO(2)(R,S-H(2)homocit)(2)]·2H(2)O (1) (*Mo=Mo, 1; (92)Mo, 2; (100)Mo, 3; H(4)homocit=homocitric acid-γ-lactone·H(2)O) and K(2)[MoO(2)((18)O-R,S-H(2)homocit)(2)]·2H(2)O (4). The complex has been characterized by elemental analysis, FT-IR, solid and solution (13)C NMR, and single crystal x-ray diffraction analysis. The molybdenum atom in (1) is quasi-octahedrally coordinated by two cis oxo groups and two bidentate homocitrate ligands. The latter coordinates via its α-alkoxy and α-carboxy groups, while the β- and γ-carboxylic acid groups remain uncomplexed, similar to the coordination mode of homocitrate in the Mo-cofactor of nitrogenase. In the IR spectra, the MoO stretching modes near 900 cm(-1) show 2-3 cm(-1) red- and blue-shifts for the (92)Mo-complex (2) and (100)Mo-complex (3) respectively compared with the natural abundance version (1). At lower frequencies, bands at 553 and 540 cm(-1) are assigned to ν(Mo-O) vibrations involving the alkoxide ligand. At higher frequencies, bands in the 1700-1730 cm(-1) region are assigned to stretching modes of protonated carboxylates. In addition, a band at 1675 cm(-1) was observed that may be analogous to a band seen at 1677 cm(-1) in nitrogenase photolysis studies. The solution behavior of (1) in D(2)O was probed with (1)H and (13)C NMR spectra. An obvious dissociation of homocitrate was found, even though bound to the high valent Mo(VI). This suggests the likely lability of coordinated homocitrate in the FeMo-cofactor with its lower valence Mo(IV).

Topics: Bacterial Proteins; Coenzymes; Coordination Complexes; Crystallography, X-Ray; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Molecular Mimicry; Molybdenum; Nitrogenase; Spectroscopy, Fourier Transform Infrared; Tricarboxylic Acids

The iron-molybdenum cofactor (FeMo-co), which is the catalytic center for the enzymatic conversion of N(2) to NH(3), has the composition [NFe(7)MoS(9)(homocitrate)], and, with a cluster of eight transition metal atoms and nine sulfur atoms, has a complex delocalized electronic structure. The electronic dimensions of FeMo-co and of each of its derivatives appear as sets of electronic states lying close in energy. These electronic dimensions naturally partner the geometrical changes and the reactivity patterns during the catalytic cycle, and also connect with spectroscopic investigations of the mechanism. This paper describes straightforward computational procedures for the determination and management of the low-lying electronic states of FeMo-co and of its coordinated intermediates and transition states during density functional simulations of steps in the catalytic mechanism. General principles for the distribution of electron spin density over all atoms are presented, using several proposed intermediates as examples. A tough general irony arises in the distribution of spin density over FeMo-co and its derivatives: the less interesting atoms get the spin, and the most interesting atoms do not.

Topics: Binding Sites; Catalysis; Catalytic Domain; Computer Simulation; Hydrogen; Iron; Models, Molecular; Molybdenum; Molybdoferredoxin; Nitrogen; Nitrogenase; Sulfur; Tricarboxylic Acids

Molybdenum (Mo)-dependent nitrogenase is a complex metalloprotein that catalyzes the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) at the molybdenum-iron cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Here we report the formation of a homocitrate-free, iron-molybdenum ("FeMo") cluster on the biosynthetic scaffold of FeMoco, NifEN. Such a NifEN-associated "FeMo" cluster exhibits EPR features similar to those of the NifEN-associated, fully-complemented "FeMoco", which originate from the presence of Mo in both cluster species; however, "FeMo" cluster and "FeMoco" display different temperature-dependent changes in the line shape and the signal intensity of their respective EPR features, which reflect the impact of homocitrate on the redox properties of these clusters. XAS/EXAFS analysis reveals that the Mo centers in both "FeMo" cluster and "FeMoco" are present in a similar coordination environment, although Mo in "FeMo" cluster is more loosely coordinated as compared to that in "FeMoco" with respect to the Mo-O distances in the cluster, likely due to the absence of homocitrate that normally serves as an additional ligand for the Mo in the cluster. Subsequent biochemical investigation of the "FeMo" cluster not only facilitates the determination of the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation, but also permits the examination of the role of homocitrate in the transfer of FeMoco between NifEN and MoFe protein. Combined outcome of these studies establishes a platform for future structural analysis of the interactions between NifEN and MoFe protein, which will provide useful insights into the mechanism of cluster transfer between the two proteins.

Topics: Bacterial Proteins; Electron Spin Resonance Spectroscopy; Iron; Molybdenum; Molybdoferredoxin; Nitrogen Fixation; Nitrogenase; Tricarboxylic Acids; X-Ray Absorption Spectroscopy

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe(2+), S(2-), MoO4(2-), and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe(2+), S(2-), MoO4(2-), R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.

Topics: Azotobacter vinelandii; Bacterial Proteins; Indicators and Reagents; Iron; Klebsiella pneumoniae; Molybdenum; Molybdoferredoxin; Nitrogen Fixation; Nitrogenase; Sulfur; Tricarboxylic Acids

A combined broken-symmetry density functional and electrostatics approach has been used to model the one-electron reduced and protonated state of the iron-molybdenum cofactor active site of nitrogenase. The active site of the protein contains Fe, Mo, S, N, and O atoms, and many possible sites for protonation have been examined. A novel hydridic proton asymmetrically located in the central cavity created by six Fe sites is most favored from the calculations. Under physiological turnover conditions of low electron flux, the formation of this iron-hydride intermediate may represent a first step towards cofactor liberation of dihydrogen in the absence of dinitrogen.

Topics: Binding Sites; Electrons; Iron; Molybdenum; Molybdoferredoxin; Nitrogenase; Protons; Thermodynamics; Tricarboxylic Acids

A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (DeltanifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of (49)V label from VnfX to nif-apodinitrogenase in vitro. This transfer of radiolabel correlates with acquisition of acetylene reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of alternative nitrogenases. Structural analogues of homocitrate prevent the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB cofactor (-co) or a source of vanadium (Na(3)VO(4) or VCl(3)) does not increase nitrogenase activity. Our results suggest that there is in vitro incorporation of homocitrate into the V-Fe-S cluster associated with VnfX and that the completed cluster can be inserted into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into nif-apodinitrogenase (alpha(2)beta(2)gamma(2)) do not require MgATP. Attempts to achieve FeV-co synthesis using extracts of other FeV-co-negative mutants were unsuccessful, showing that earlier steps in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not occur in vitro.

Topics: Azotobacter vinelandii; Bacterial Proteins; Metalloproteins; Nitrogenase; Tricarboxylic Acids

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [Sørlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.

Topics: Amino Acid Substitution; Azotobacter vinelandii; Binding Sites; Crystallography, X-Ray; Electron Spin Resonance Spectroscopy; Ethylenes; Glutamine; Histidine; Hydrogen Bonding; Iron; Molybdenum; Molybdoferredoxin; Nitrogenase; Structure-Activity Relationship; Tricarboxylic Acids

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aluminum Compounds; Argon; Catalytic Domain; Dithionite; Electron Spin Resonance Spectroscopy; Electrons; Fluorides; Hydrogen; Iron; Iron-Sulfur Proteins; Kinetics; Klebsiella pneumoniae; Molybdoferredoxin; Nitrogenase; Oxidation-Reduction; Protons; Reducing Agents; Tricarboxylic Acids

The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the iron-molybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe7-8Sx precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe3S4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

Topics: Azotobacter vinelandii; Bacterial Proteins; Binding Sites; Electron Spin Resonance Spectroscopy; Iron; Metalloproteins; Molybdenum; Molybdoferredoxin; Nitrogenase; Tricarboxylic Acids; Vanadium

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.

Topics: Acetylene; Apoenzymes; Bacterial Proteins; Dinitrogenase Reductase; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Ethane; Iron; Iron-Sulfur Proteins; Isoenzymes; Metals; Mutation; Nitrogenase; Oxidation-Reduction; Oxidoreductases; Protein Conformation; Quaternary Ammonium Compounds; Rhodospirillum rubrum; Substrate Specificity; Tricarboxylic Acids

Here we report the large scale isolation and characterization of a species, designated MoFe cluster, that exhibits an S = 3/2 EPR signal, and the comparison of this entity to isolated FeMo cofactor in N-methylformamide and to the active site of the enzyme nitrogenase. MoFe cluster is isolated from purified nitrogenase by extraction into acidic methyl ethyl ketone and it is stable in that solvent in the absence of thiols. As initially isolated, MoFe cluster solutions exhibit an S = 1/2 EPR signal that arises from an oxidized species that can be reduced by dithionite or thiols to an EPR silent state and then to a state that exhibits an S = 3/2 EPR signal. The S = 3/2 signal is as sharp as the signal exhibited by the protein and much sharper than the signal exhibited by isolated FeMo cofactor. Circular dichroism experiments indicate that unlike the last two species, MoFe cluster does not contain the endogenous ligand R-homocitrate and thus, the sharpness of the S = 3/2 signal is an intrinsic property of the metal center and does not depend upon specific interactions with this organic ligand or with the protein. Metal analyses indicate that the metal core responsible for the S = 3/2 signal contains 6 Fe atoms per molybdenum. X-ray absorption spectroscopy experiments show that although the molybdenum atom in MoFe cluster retains its pseudo-octahedral geometry, its first coordination shell has one less iron atom than that of FeMo cofactor and there has been a significant change in the long range order of the cluster.

Topics: Binding Sites; Butanones; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Molybdoferredoxin; Nitrogenase; Oxidation-Reduction; Tricarboxylic Acids

A plausible structure of the iron-molybdenum cofactor of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1] is presented based on altered substrate reduction properties of dinitrogenase containing homocitrate analogs within the cofactor. Alterations on each carbon of the four-carbon homocitrate backbone were correlated with altered substrate reduction properties of dinitrogenase containing these analogs. Altered substrate reduction properties are the basis for a model in which homocitrate is oriented about two cubane metal clusters.

Topics: Formates; In Vitro Techniques; Molecular Structure; Molybdoferredoxin; Nitrogenase; Oxidation-Reduction; Structure-Activity Relationship; Substrate Specificity; Tricarboxylic Acids

Azotobacter vinelandii DJ71, which contains a mutation in the nifV gene, was derepressed for nitrogenase in the presence of homocitrate. When dinitrogenase was isolated from this culture, it was found to be identical to the wild-type dinitrogenase. However, when the same NifV- strain was derepressed in the presence of erythrofluorohomocitrate, a homocitrate analog which produces a nitrogenase with wild-type properties in vitro, the isolated dinitrogenase was characteristic of the NifV- enzyme. These data show that homocitrate, but not fluorohomocitrate, is utilized by NifV- mutant cells. Fluorohomocitrate does not inhibit the uptake of homocitrate because the wild-type phenotype resulted when both compounds were added to the medium during nitrogenase derepression. Homocitrate lactone failed to cure the NifV- phenotype.

Topics: Azotobacter; Genes, Bacterial; Lactones; Mutation; Nitrogen Fixation; Nitrogenase; Phenotype; Tricarboxylic Acids

An organic acid extracted from purified dinitrogenase isolated from a nifV mutant of Klebsiella pneumoniae has been identified as citric acid. H2 evolution by the citrate-containing dinitrogenase is partially inhibited by CO, and by some substrates for nitrogenase. The response of maximum velocities to changes in pH for both the wild-type and the NifV- dinitrogenase was compared. No substantial differences between the enzymes were observed, but there are minor differences. Both enzymes are stable in the pH range 4.8-10, but the enzyme activities dropped dramatically below pH 6.2.

Topics: Citrates; Citric Acid; Genes, Bacterial; Hydrogen-Ion Concentration; Klebsiella pneumoniae; Molybdoferredoxin; Nitrogen Fixation; Nitrogenase; Tricarboxylic Acids

When apodinitrogenase (lacking FeMo-co) was activated with FeMo-co synthesized in vitro in the presence of 3H-labeled homocitrate, label was incorporated into dinitrogenase. The physical association of the label with FeMo-co was demonstrated by reisolation and purification of the cofactor from dinitrogenase. The presence of homocitrate in FeMo-co was established by NMR analysis of the organic acid extracted from dinitrogenase. Quantitation of homocitrate in dinitrogenase showed it to be present at a 1:1 ratio with molybdenum.

Topics: Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Ferredoxins; Kinetics; Klebsiella pneumoniae; Magnetic Resonance Spectroscopy; Molybdoferredoxin; Nitrogenase; Tricarboxylic Acids

The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C2H2 reduction assays and the lack of 99Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of 99Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C2H2 or N2. Citrate also promoted the incorporation of 99Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C2H2 effectively but not N2. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV- dinitrogenase. We also observed low rates of HD formation from NifV- dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. We propose that in vivo NifV- mutants utilize citrate for FeMo-co synthesis.

Topics: Enzyme Activation; Ferredoxins; Kinetics; Klebsiella pneumoniae; Molybdoferredoxin; Nitrogenase; Substrate Specificity; Tricarboxylic Acids

Dinitrogenase was isolated from a culture of a Klebsiella pneumoniae NifV- strain derepressed for nitrogenase in the presence of homocitrate. The enzyme isolated from this culture was identical to the wild-type dinitrogenase. These data provide in vivo evidence that the absence of homocitrate is responsible for the NifV- phenotype.

Topics: Dinitrogenase Reductase; Enzyme Repression; Ferredoxins; Genes, Bacterial; Klebsiella pneumoniae; Mutation; Nitrogen Fixation; Nitrogenase; Phenotype; Tricarboxylic Acids