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At least 343 records · Page 19Linked to original sources
PubMed1985

Inhibition of iron-molybdenum cofactor binding to component I of nitrogenase.

Tetrathiomolybdate inhibits iron-molybdenum cofactor (FeMo cofactor) binding to component I of nitrogenase. Molybdenum-iron cluster (a subcomponent of FeMo cofactor) and tetrathiomolybdate inhibited FeMo cofactor activation of inactive nitrogenase component I in extracts of Azotobacter vinelandii and Klebsiella pneumoniae mutant strains defective in the biosynthesis of FeMo cofactor. Addition of tetrathiotungstate, the tungsten analog of tetrathiomolybdate, to the mutant extracts had no significant inhibitory effect on subsequent activation by FeMo cofactor.

Azotobacter↗
PubMed1982

Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition.

Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.

Amino Acids↗
PubMed1978

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.

Amino Acid Sequence↗
PubMed1983

[The study of the chemical composition of nitrogenase Fe-Mo-cofactor by a new fluorimetric method of thiocompound analysis].

A purification technique for a large scale production of crystalline Mo-Fe protein of nitrogenase from Azotobacter vinelandii and of its fragment, Fe-Mo-cofactor (Fe-Mo-co) in argon atmosphere has been elaborated. The novel fluorimetric method of thiol compounds analysis has been proposed for identification of Fe-Mo-co thiol ligands; this procedure allows the determination of concentration and class of thiol compounds and of the distance between sulphur atoms in the case of dithiols. The use of this method for an analysis of Fe-Mo-co has demonstrated that it contains a thiomolybdate fragment and two atoms of inorganic sulphur. Organic thiol as a Fe ligand has not been revealed.

Azotobacter↗
PubMed1981

Isolation and sequences of the cysteinyl tryptic peptides from the MoFe-protein of Azotobacter vinelandii nitrogenase.

The cysteinyl residues in the alpha and beta subunits of the MoFe-protein from Azotobacter vinelandii nitrogenase were radiolabeled by carboxymethylation with iodo[14C]acetic acid. The tryptic peptides from the isolated subunits were separated by ion-exchange chromatography on DEAE-Sephadex and SP-Sephadex. The radiolabeled (cysteinyl) peptides were sequenced by Edman degradation. The isolation procedure and sequences of the peptides provide a method for the identification of the potential cysteinyl ligands for the protein Fe:S and Mo:Fe:S centers. Although the cysteinyl peptides account for approximately 20% of the total sequence, little sequence homology was observed between the peptides from the two subunits. In contrast to the ferredoxins, the cysteinyl residues are not grouped within the protein. Only one peptide from the beta subunit has sequence homology with other Fe:S proteins. This peptide has 15 of 22 residues identical or conservative with residues 11 to 32 of the bacterial ferredoxins. The conservation of this region suggests that it may contain an important secondary structure unique to 4Fe:4S proteins, namely, the sequence (formula, see text) where (formula, see text) represents a variable residue. One peptide from the alpha subunit has a sequence identical with the conserved residues 7 to 11 of the bacterial ferredoxins. This alpha-peptide has the correct sequence, -Glu-Pro-Val-Ser-Cys-Val-Ser-Asp-Ser-, for the glutamyl, cysteinyl, and aspartyl residues to ligand a single 4Fe:4S cluster.

Amino Acid Sequence↗
PubMed1981

Resolution of two subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.

Azotobacter↗
PubMed1982

Molecular symmetry of the MoFe protein of nitrogenase. Structural homology/nitrogen fixation/x-ray crystallography.

X-ray diffraction data to 2.4-A resolution have been collected for native monoclinic crystals of the MoFe protein of nitrogenase from Clostridium pasteurianum. The MoFe protein is an alpha 2 beta 2 tetramer of 220,000 molecular weight with 1 molecule in the crystallographic asymmetric unit. A 6-A resolution rotation function shows the orientation of the crystallographic diad and pseudo mutually perpendicular diads representing 2-fold relationships between alpha and beta chains. Hence, at least at low resolution, there exists structural homology between these two polypeptide chains.

Clostridium↗
PubMed1982

Magnetic susceptibility studies of native and thionine-oxidized molybdenum-iron protein from Azotobacter vinelandii nitrogenase.

The difference between the magnetic susceptibilities of native and thionine-oxidized molybdenum-iron protein from Azotobacter vinelandii nitrogenase was measured by the nuclear magnetic resonance method. Reversible oxidation of the MoFe protein by 4 to 8 electron eq of thionine/mol made the protein more paramagnetic than it was in the native state. The NMR susceptibility results were analyzed in terms of a model for the spin states of the iron centers in the MoFe protein based on low temperature electron paramagnetic resonance and Mössbauer spectral studies. The model proposes that the native protein contains 2 "M" centers (S = 3/2) and 4 "P" centers (S = 0)/mol and that the oxidized protein has diamagnetic M centers and paramagnetic P centers with S greater than or equal to 3/2. Assuming that this model holds at 280 K, the NMR susceptibility results show that the effective magnetic moment of the oxidized P centers is larger than that of the native M centers. Based on an analysis in terms of spin only magnetic moments, the susceptibility results suggest that the P centers in the oxidized protein are S = 5/2 systems.

Azotobacter↗
PubMed1982

The pterin of the molybdenum cofactor.

The molybdenum cofactor common to a variety of molybdoenzymes has been shown to contain a novel pterin. The pterin has been isolated from sulfite oxidase from several sources, xanthine-oxidizing enzymes from milk and chicken liver, and nitrate reductase of Chlorella vulgaris after denaturation of the proteins in the presence of I2. Investigation of the anionic nature of the isolated pterin has revealed that it is a monophosphate ester susceptible to cleavage by alkaline phosphatase. Quantitative analyses have shown that one molecule of the pterin phosphate is associated with each molybdenum atom in sulfite oxidase. Studies to date have shown that the pterin is present in a reduced form in sulfite oxidase and xanthine dehydrogenase, and that in situ oxidation of the pterin leads to inactivation of sulfite oxidase.

Animals↗
PubMed1994

Identification of a nitrogenase protein-protein interaction site defined by residues 59 through 67 within the Azotobacter vinelandii Fe protein.

During nitrogenase catalysis the Fe protein and the MoFe protein associate and dissociate in a MgATP-dependent process involving electron transfer from the Fe protein to the MoFe protein. A docking model, based primarily on the crystal structures of the separate components from Azotobacter vinelandii, was previously proposed in which the 2-fold symmetric surface of the homodimeric Fe protein interacts with the exposed surface of a MoFe protein pseudosymmetric alpha beta-unit interface. In this model, a loop, which is included within residues 59 through 67 of the Fe protein primary sequence, is likely to interact with the MoFe protein during component protein docking. In the present study, evidence supporting the component protein docking model was obtained by construction of an A. vinelandii strain that produces a hybrid Fe protein for which residues 59 through 67 have been replaced by the corresponding residues from the Fe protein of Clostridium pasteurianum. Biochemical analyses of the hybrid Fe protein revealed the following features when compared with the unaltered Fe protein. First, the hybrid Fe protein exhibited half the maximum specific activity of the normal Fe protein and was insensitive to inhibition by low levels of NaCl. Second, the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrolysis from substrate reduction. Third, stopped-flow spectrophotometry experiments showed that during catalysis the hybrid Fe protein dissociates from the MoFe protein at only half the normal rate of Fe protein-MoFe protein dissociation. Thus, the salient feature of the hybrid Fe protein is that it appears to form a relatively tighter complex with the MoFe protein. This property is in line with previous biochemical reconstitution experiments where it was shown that a heterologous mixture of Fe protein from C. pasteurianum and MoFe protein from A. vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved in component protein interaction.

Adenosine Triphosphate↗
PubMed1994

Large scale isolation and characterization of the molybdenum-iron cluster from nitrogenase.

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.

Binding Sites↗
PubMed1993

Dinitrogenase reductase- and MgATP-dependent maturation of apodinitrogenase from Azotobacter vinelandii.

The requirements for iron-molybdenum cofactor (FeMo-co) activation of apodinitrogenase from Azotobacter vinelandii strain UW97, which lacks dinitrogenase reductase activity as assayed by substrate reduction, have been examined. Activation of apodinitrogenase from strain UW97 by FeMo-co requires the addition of both dinitrogenase reductase and MgATP. When the same apodinitrogenase is pretreated with dinitrogenase reductase and MgATP and then partially purified, however, it does not require these components for activation by FeMo-co. This suggests that dinitrogenase reductase and MgATP are involved in processing apodinitrogenase to a FeMo-co activatable form. This processing step coincides with a change in the subunit composition of apodinitrogenase from alpha 2 beta 2 to a form with an additional subunit (gamma) attached. The apodinitrogenase with the associated gamma subunit is apparently the form of the protein that is competent for activation by FeMo-co.

Adenosine Triphosphate↗
PubMed1994

In vitro synthesis of the iron-molybdenum cofactor of nitrogenase. Purification and characterization of NifB cofactor, the product of NIFB protein.

The requirement of NIFB activity for the biosynthesis of iron-molybdenum cofactor (FeMo-co) can be satisfied by the addition of the low molecular weight product of NIFB, termed NifB cofactor (NifB-co). NifB-co has been purified to homogeneity by a unique one-step method. Addition of NifB-co into the FeMo-co synthesis system generated nitrogenase activity of 27-32 nmol of ethylene formed/min/nmol of iron. Iron is the only metal detected in the NifB-co. NifB-co-dependent in vitro FeMo-co synthesis is absolutely dependent on the presence of molybdate, homocitrate and active NIFNE protein in the reaction mixture. The cofactor appears to be a small Fe-S cluster synthesized by NIFB, as a precursor of FeMo-co. NifB-co did not display any EPR signal at 4 K in 0-4000 gauss range. A solution of NifB-co is greenish-brown in color, similar to FeMo-co. NifB-co exhibits a broad absorbance between 400 and 700 nm with no distinctive peaks or shoulders. NifB-co is stable to repeated freeze-thaw cycles and is also stable in N-methylformamide, the solvent used for the isolation of FeMo-co. The NifB-co is stable to a 5-min heat treatment at 60 degrees C. The cofactor is extremely O2-labile, with half-life of less then 15 s in air.

Bacterial Proteins↗
PubMed1994

Purification and characterization of nitrogenase from a delta nifW strain of Azotobacter vinelandii.

Deletion of the nifW gene in Azotobacter vinelandii yields a strain (DJ224) that grows poorly under N2 fixing conditions (Jacobson, M. R., Cash, V. L., Weiss, M. C., Laird, N. F., Newton, W. E., and Dean, D. R. (1989) Mol. & Gen. Genet. 219, 49-57). Here we report the purification of nitrogenase from DJ224. The purified Fe protein was indistinguishable from wild-type. The MoFe protein was indistinguishable from the wild-type MoFe protein by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, native gel electrophoresis, two-dimensional gel electrophoresis, metal analysis, UV/visible and EPR spectroscopies. It was different by the criteria of CD spectroscopy and specific activities. At a 5:1 molar ratio of Fe protein to MoFe protein, H2 evolution under argon was identical to wild-type, C2H2 reduction was inhibited by 27%, N2 reduction was inhibited by 38%, and CO inhibited H2 evolution by 17%. The above data show that the nifW gene product is not required for: 1) detectable alteration of the polypeptide; 2) the synthesis of the metal portion of FeMo cofactor; or 3) FeMo cofactor insertion. The MoFe protein synthesized in the absence of NifW appears to have an alteration near the FeMo cofactor site, possibly at homocitrate, which causes differential inhibition of different substrates.

Amino Acid Sequence↗
PubMed1993

Small-angle x-ray scattering studies of the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase.

The nitrogenase enzyme complex, consisting of the molybdenum-iron protein and the iron protein, plays a critical role in the biological reduction of dinitrogen to ammonia (nitrogen fixation). The nitrogen-fixing site within the molybdenum-iron protein is an iron-molybdenum-sulfur cofactor (FeMoco) of roughly 1000-2000 Dalton mass. Structural aspects of FeMoco have been determined by spectroscopic and more recently by crystallographic studies. In order to determine the radius of gyration (Rg) of isolated FeMoco, we have performed small-angle x-ray scattering studies of FeMoco in N-methylformamide solution, in the absence of the molybdenum-iron protein. Model compounds of known structure have also been examined in similar solvents, N,N-dimethylformamide and acetonitrile, as controls and for calibration purposes. The Rg values obtained for the models are in good agreement with calculations based upon their respective crystal structures. However, the Rg obtained for FeMoco clearly indicates that the cofactor is not monomeric in solution, but rather aggregated and possibly polydisperse. Further, Rg values were also measured after addition of thiol, dithionite, and thiol and dithionite, to the FeMoco samples. The results indicate, surprisingly, that oxidation state and putative thiol coordination have no detectable effect on the aggregation behavior of FeMoco in solution, as determined by these measurements.

Azotobacter vinelandii↗
PubMed1996

[Model molybdenum-sulfur and molybdenum-iron-sulfur protein complexes].

A procedure was developed for synthesizing complexes of polynuclear molybdenum sulfide and iron-molybdenum sulfide on the basis of human serum albumin. EPR showed that molybdenum and iron atoms formed clusters in the synthesized complexes. The catalytic activity of the complexes, as determined through reaction of acetylene reduction by sodium borohydride, was significantly higher than that for previously described nonbiological systems, but lower than the characteristic values of FeMo-cofactor of nitrogenase.

Catalysis↗