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

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 ironmolybdenum 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 VFe 7-8 S x 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 VFe 3 S 4 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.

Section: Methodsmentioning

confidence: 99%

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

“…The reaction-mixtures were incubated at room temperature for 20 min, and samples to be applied onto the native gels were placed on ice. The activity of the newly formed dinitrogenase 2 in the remaining vials was determined by the C 2 H 2 reduction assay for nitrogenase (31) as follows: 800 l of the ATP-regenerating mixture and 10 l (0.1 mg of protein) of dinitrogenase reductase 1 were added to the vials, which were then brought to atmospheric pressure. C 2 H 2 reduction was initiated by the addition of 0.5 ml of C 2 H 2 to the vials; following a 30-min incubation at 30°C in a rotary water-bath shaker, the reactions were terminated by the addition of 0.1 ml of 4 N NaOH.…”

Section: Methodsmentioning

confidence: 99%

“…Strain UW45 was grown and derepressed on tungsten-containing medium (molybdenum-free) as described previously (31). For derepression of the nif system, strains were grown on Mo-containing medium as described previously (32).…”

Section: Methodsmentioning

confidence: 99%

Characterization of VNFG, the δ Subunit of the vnf-Encoded Apodinitrogenase from Azotobacter vinelandii

Chatterjee

Ludden

Shah³

1997

The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an ␣ 2 ␤ 2 ␦ 2 hexamer. The ␦ subunit (the VNFG protein) has been characterized in order to further delineate its function in the nitrogenase 2 enzyme system. Two species of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG associated with the VNFDK polypeptides, and the other is a homodimer of the VNFG protein.Both species of VNFG are observed in extracts of A. vinelandii strains that accumulate dinitrogenase 2, whereas extracts of strains impaired in the biosynthetic pathway of the iron-vanadium cofactor (FeV-co) that accumulate apodinitrogenase 2 (a catalytically inactive form of dinitrogenase 2 that lacks FeV-co) exhibit only the VNFG dimer on native gels. FeV-co and nucleotide are required for the stable association of VNFG with the VNFDK polypeptides; this stable association can be correlated with the formation of active dinitrogenase 2. The iron-molybdenum cofactor was unable to replace FeV-co in promoting the stable association of VNFG with VN-FDK. FeV-co specifically associates with the VNFG dimer in vitro to form a complex of unknown stoichiometry; combination of this VNFG-FeV-co species with apodinitrogenase 2 results in its reconstitution to dinitrogenase 2. The results presented here suggest that VNFG is required for processing apodinitrogenase 2 to functional dinitrogenase 2.

“…An in vitro FeMo-co synthesis system has been described (18). It typically involves mixing of extracts from two different mutants defective in the synthesis of the cofactor or a mutant extract complemented with the purified missing component(s).…”

mentioning

confidence: 99%

Requirement of Homocitrate for the Transfer of a49V-Labeled Precursor of the Iron-Vanadium Cofactor from VnfX to nif-apodinitrogenase

Rüttimann-Johnson

Rangaraj

Shah

et al. 2001

A vanadium-and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (⌬nifHDKvn-fDGK::spc).Inthepresentstudy,weshowthehomocitratedependent transfer of 49 V label from VnfX to nifapodinitrogenase 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 (␣ 2 ␤ 2 ␥ 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.