Iron-molybdenum Cofactor Of Nitrogenase Research Articles

A facile method of isolation for the iron molybdenum cofactor of nitrogenase and bacterial ferritin from extracts of azotobacter vinelandii

An alternative method has been developed for the isolation of both the iron molybdenum cofactor of nitrogenase (FeMoco), a small molecular weight FeMoS cluster which is the putative nitrogen- reducing site of the enzyme, and bacterioferritin, an iron storage protein similar to other ferritins, but containing heme prosthetic groups. Previously the isolation of these two species, the characterization of which is of significant current interest, has been dependent on the purification of the nitrogenase enzyme from Azotobacter vinelandii. Out new procedure eliminates the use of the anaerobic column chromatography necessary to obtain pure nitrogenase components, involving instead the heat and RNAase/ DNAase treatment of crude extracts of ruptured cells followed by sedimentation (150000 × g for 18 h) of both the 'nitrogenase complex' and bacterioferritin. The redissolved pellet from this centrifugation yields the pure crystalline bacterioferritin on addition of Mg 2+. and cooling, the iron content of the protein being higher by this method than in previous reports. Likewise, denaturation by acid/base treatment of this protein mixture yields a precipitate which can be extracted with either N-methylformamide or N,N-dimethylformamide containing dithionite ion to yield solutions of FeMoco, as evidenced by UW 45 reconstitution and EPR spectral criteria. Unfortunately, preparations of FeMoco obtained by this method have a variable, but consistently low, Fe/Mo ratio and additional visible spectral features, indicating that they are significantly less pure than that those generated from purified nitrogenase. The aqueous supernatant from the denaturation also yields bacterioferritin, but with a lower iron content than that from the direct crystallization method.

Identification of the V factor needed for synthesis of the iron-molybdenum cofactor of nitrogenase as homocitrate.

Nitrogenase catalyses the ATP-dependent reduction of N2 to NH3, and is composed of two proteins, dinitrogenase (MoFe protein or component I) and dinitrogenase reductase (Fe protein or component II). Dinitrogenase contains a unique prosthetic group (iron-molybdenum cofactor, FeMoco) comprised of Fe, Mo and S, which has been proposed as the site of N2 reduction. Biochemical and genetic studies of Nif- (nitrogen fixation) mutants of Klebsiella pneumoniae which are defective in nitrogen fixation, have shown that the nifB, nifQ, nifN, nifE and nifV genes are required for the biosynthesis of FeMo-co. Recently, a system for in vitro synthesis of FeMoco was described. The assay requires at least the nifB, nifN and nifE gene products, and a low-molecular-weight factor (V factor) produced in the presence of the nifV gene product. We have used this system to study FeMoco biosynthesis. We report here the isolation of V factor and identify it as homocitric acid ([R]2-hydroxy-1,2,4-butanetricarboxylic acid).

The nifH gene product is required for the synthesis or stability of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae.

The MoFe protein of nitrogenase from Klebsiella pneumoniae contains an iron-molybdenum cofactor, FeMoco, the synthesis or processing of which involves the products of at least five genes, nifQ, nifB, nifN, nifE and nifV. We have detected FeMoco activity in extracts of strains which synthesise neither of the MoFe protein subunits, indicating that FeMoco can be synthesised prior to combination with the MoFe protein polypeptides. Expression of the nifH gene (or a large part of it), was essential for FeMoco activity to be observed either in the presence or in the absence of the MoFe protein subunits. The nifH gene product was not involved in the control of the transcription of other nif gene products known to be involved in FeMoco synthesis or processing, nor was it essential for the stability of performed FeMoco before its combination with the MoFe protein polypeptides.

Solubilization of the iron molybdenum cofactor of Azotobacter vinelandii nitrogenase in dimethylformamide and acetonitrile

The iron molybdenum cofactor of Azotobacter vinelandii nitrogenase has been solubilized for the first time in dimethylformamide and acetonitrile. These solutions have the ability to reconstitute the inactive nitrogenase of the UW 45 mutant of A. vinelandii and exhibit an S = 3/2 EPR signal similar to that for the cofactor in N-methylformamide. Our ability to obtain solutions of FeMoco in these solvents seemingly refutes a previous hypothesis concerning the necessity of solvents with a dissociable proton for iron molybdenum cofactor solubility and should facilitate the spectroscopic characterization of this important species.

NifV-dependent, low-molecular-weight factor required for in vitro synthesis of iron-molybdenum cofactor of nitrogenase.

The molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase requires a low-molecular-weight factor. The factor is present in extracts of nitrogen fixation-derepressed cultures of Klebsiella pneumoniae and Azotobacter vinelandii, but not in extracts of repressed cultures of these bacteria. Analysis of K. pneumoniae Nif mutants has indicated that the nifV gene product is the only nif protein (besides nifA) necessary for the synthesis and accumulation of the factor. The factor is stable to oxygen, temperatures below 120 degrees C, and extremely acidic and basic conditions. The activity of the factor was completely destroyed by dry ashing or digestion with sulfuric acid. The factor has been partially purified by filtration through an Amicon PM-10 DIAFLO membrane and chromatography on DEAE-cellulose, hydroxylapatite, silica gel, and Sephadex G-25.

Elicitation of thiomolybdates from the iron-molybdenum cofactor of nitrogenase. Comparison with synthetic Fe-Mo-S complexes

Aerial oxidation of the iron-molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4]2-) or the oxotrithiomolybdate ion ([MoOS3]2-), depending on the reaction conditions. Thus, when N-methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air-saturated NMF, [MoOS3]2- was found to be the predominant product while dilution of NMF solutions of FeMoco with air-saturated methanol produced [MoS4]2- almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe-Mo-S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X-ray absorption spectroscopic data which suggest the presence of an 'MoS3' core in FeMoco. A possible mechanism whereby [MoS4]2- might be rapidly formed from this core is presented.

In vitro synthesis of the iron-molybdenum cofactor of nitrogenase.

Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen-reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae.

Role of the nifQ gene product in the incorporation of molybdenum into nitrogenase in Klebsiella pneumoniae.

NifQ- mutants of Klebsiella pneumoniae are defective in nitrogen fixation due to an elevated requirement for molybdenum. When millimolar concentrations of molybdate were added to the medium, the effects of the nifQ mutations were suppressed. NifQ- mutants were not impaired in the uptake of molybdate, but molybdate accumulation was defective in these mutants. All of the nif-coded proteins were present in NifQ- cells derepressed in the absence of molybdenum. Molybdenum-activatable nitrogenase component I was found at the same level observed in the wild type. Molybdenum, thus, does not play a role in nif expression or in the short-term stability of nif-coded proteins. The defect in NifQ- mutants was in the incorporation of molybdenum into nitrogenase component I. The nifQ gene product acts together with the products of nifB, nifN, and nifE in the biosynthesis of the iron-molybdenum cofactor of nitrogenase.

ChemInform Abstract: EXAFS MODEL FOR THE IRON-MOLYBDENUM COFACTOR OF NITROGENASE: MOLYBDENUM, TUNGSTEN, AND IRON EXAFS OF THE (CL2FES2MS2FECL2)2- (M = MOLYBDENUM OR TUNGSTEN) DIANIONS. A COMPARISON WITH THE MO EXAFS OF NITROGENASE

Chemischer InformationsdienstVolume 14, Issue 49 Physical Organic Chemistry ChemInform Abstract: EXAFS MODEL FOR THE IRON-MOLYBDENUM COFACTOR OF NITROGENASE: MOLYBDENUM, TUNGSTEN, AND IRON EXAFS OF THE (CL2FES2MS2FECL2)2- (M = MOLYBDENUM OR TUNGSTEN) DIANIONS. A COMPARISON WITH THE MO EXAFS OF NITROGENASE B. K. TEO, B. K. TEOSearch for more papers by this authorM. R. ANTONIO, M. R. ANTONIOSearch for more papers by this authorD. COUCOUVANIS, D. COUCOUVANISSearch for more papers by this authorE. D. SIMHON, E. D. SIMHONSearch for more papers by this authorP. P. STREMPLE, P. P. STREMPLESearch for more papers by this author B. K. TEO, B. K. TEOSearch for more papers by this authorM. R. ANTONIO, M. R. ANTONIOSearch for more papers by this authorD. COUCOUVANIS, D. COUCOUVANISSearch for more papers by this authorE. D. SIMHON, E. D. SIMHONSearch for more papers by this authorP. P. STREMPLE, P. P. STREMPLESearch for more papers by this author First published: December 6, 1983 https://doi.org/10.1002/chin.198349039AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume14, Issue49December 6, 1983 RelatedInformation

EXAFS model for the iron-molybdenum cofactor of nitrogenase: molybdenum, tungsten, and iron EXAFS of the [Cl2FeS2MS2FeCl2]2- (M = molybdenum or tungsten) dianions. A comparison with the Mo EXAFS of nitrogenase

EXAFS model for the iron-molybdenum cofactor of nitrogenase: molybdenum, tungsten, and iron EXAFS of the [Cl2FeS2MS2FeCl2]2- (M = molybdenum or tungsten) dianions. A comparison with the Mo EXAFS of nitrogenase

Iron EXAFS studies of the ironmolybdenum cofactor of nitrogenase and the 3Fe ferredoxin II of desulfovibrio gigas

Chemical and physical analyses indicate that the ironmolybdenum cofactor (FeMo(co)) of nitrogenase contains 6–8 mol or iron and 4–6 mol of sulfur per mol of molybdenum. The physical properties of this cofactor suggest that it contains a novel MoFeS cluster. Extended X-ray absorption fine structure (EXAFS) data taken at the Mo edge indicate that the molybdenum has two or three iron atoms and four or five sulfur atoms as nearest neighbors. Several models are consistent with these data. More information concerning the iron environment is needed to better define the structure of the FeMo(co). In this talk, we will present our recent results [1] on the iron edge EXAFS of the FeMo(co) from Azotobacter vinelandii and relate structural information about the iron sites in the cluster. In a related study, we have obtained the iron K-edge EXAFS of the 3Fe ferredoxin II of Desulfovibrio gigas in the oxidized and reduced states [2]. For both states, interpretation of the EXAFS data suggests that the FeS distance is near 2.25 Å, in agreement with crystallographic studies of model compounds and proteins containing 2Fe2S and 4Fe4S centers, as well as with a recent crystallographic study of Azotobacter vinelandii ferredoxin I (D. Ghosh, W. Furey, Jr., S. O'Donnell and C.D. Stout, J. Biol. Chem. 256, 4185 (1981).). The FeFe distance of 2.7 Å, however, agrees with similar distances observed in other FeS centers, but disagrees with the 3Fe cluster in the Azotobacter vinelandii ferredoxin I structure, for which an FeFe distance of 4.2 Å was reported. We conclude that the two 3Fe ferredoxins may have substantially different core dimensions, a possibility apparently unique to 3Fe centers among known FeS systems in proteins. The implication of such structural variation of 3Fe centers in the nitrogenase problem will be discussed.

Iron EXAFS of the iron-molybdenum cofactor of nitrogenase

Iron EXAFS of the iron-molybdenum cofactor of nitrogenase

Oxidation-reduction properties and complexation reactions of the iron-molybdenum cofactor of nitrogenase.

The interactions of the iron-molybdenum cofactor, FeMoco, isolated from acid-treated Azotobacter vinelandii molybdenum-iron protein (Av1) with EDTA and thiophenol in N-methylformamide solution have been reinvestigated. Our studies show that EDTA alone is sufficient to eliminate the EPR signal of dithionite-reduced FeMoco. Neither light/5-deazaflavin nor carbon monoxide are required, which implies that this EPR-silent form of FeMoco does not correspond to the EPR-silent, substrate-reducing state of Av1. As EDTA-treated FeMoco does not regain EPR activity on addition of sodium dithionite or thiophenol, it is apparently distinct from the EPR-silent form of either dye-oxidized FeMoco or dye-oxidized Av1. Thiophenol sharpens the EPR signal of dithionite-reduced FeMoco and shifts the g = 3.3 feature to g = 3.6. This shift is complete at 1:1 ratio of thiophenol/Mo atom, while the EDTA effect requires about 40 molecules/Mo atom. Thiophenol and EDTA probably affect different sites of FeMoco. The binding of either reactant does not affect the activity of FeMoco as measured by its ability to reconstitute extracts of A. vinelandii mutant UW45.

Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence

Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence