title: A vanadium and iron cluster accumulates on VnfX during iron-vanadium-cofactor synthesis for the vanadium nitrogenase in Azotobacter 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 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.
authors.affiliation: Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: C
authors.initials: C
authors.lastname: Rüttimann-Johnson
authors.forename: C R
authors.initials: CR
authors.lastname: Staples
authors.forename: P
authors.initials: P
authors.lastname: Rangaraj
authors.forename: V K
authors.initials: VK
authors.lastname: Shah
authors.forename: P W
authors.initials: PW
authors.lastname: Ludden
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry; D008667:Metalloproteins / Q000096:biosynthesis* / Q000737:chemistry; D008982:Molybdenum / Q000737:chemistry; D008983:Molybdoferredoxin / Q000096:biosynthesis / Q000737:chemistry; D009591:Nitrogenase / Q000737:chemistry*; D014233:Tricarboxylic Acids / Q000032:analysis; D014639:Vanadium / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D008983:Molybdoferredoxin; D014233:Tricarboxylic Acids; C055326:iron-vanadium cofactor; D014639:Vanadium; C028143:homocitric acid; D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase