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

Abstract

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.

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

“…It has been described previously that in A. vinelandii CA11.1 most of the 49 V accumulates on VnfX, as seen in Fig. 1, lane 1, in the form of a vanadium-containing Fe-S cluster (16).…”

“…It has been reported that the V-Fe-S cluster accumulating on VnfX in extracts of A. vinelandii CA11.1 contains no or very low levels of homocitrate (16). To test whether homocitrate incorporation into the unfinished cluster occurs in vitro, we studied the effect of homocitrate analogues on the reaction.…”

“…Extracts of A. vinelandii DJ42.48 grown in vanadium medium were complemented either with purified NifNE and VnfX (in the presence of nif-apodinitrogenase) (Table IV, lines 7 and 8) or with an extract of A. vinelandii UW45 grown in tungsten medium and VnfX (Table IV, lines 5 and 6). The VnfX used in these assays was purified from a nifB Ϫ strain and does not have a cluster bound to it (16). The acetylene reduction activities observed in these conditions were not significantly above background.…”

“…NifX stimulates the synthesis of FeMo-co in an in vitro system (17). Both proteins are able to bind NifB-co (16). 2 The complete biosynthetic pathways of FeMo-co and FeV-co have not yet been elucidated, and other not yet identified components have been shown to stimulate the synthesis of FeMo-co in vitro (17).…”

“…The products of nifX and its homologue vnfX have also been shown to be involved in FeMo-co and FeV-co synthesis, respectively, although their exact role remains to be established. A vanadium-and iron-containing cluster having similar EPR characteristics to FeV-co accumulates on VnfX during FeV-co synthesis (16). NifX stimulates the synthesis of FeMo-co in an in vitro system (17).…”