title: Requirement of homocitrate for the transfer of a 49V-labeled precursor of the iron-vanadium cofactor from VnfX to nif-apodinitrogenase.
abstract: A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (DeltanifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of (49)V label from VnfX to nif-apodinitrogenase 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 (alpha(2)beta(2)gamma(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.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: C
authors.initials: C
authors.lastname: Ruttimann-Johnson
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 / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D014233:Tricarboxylic Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D014233:Tricarboxylic Acids; C055326:iron-vanadium cofactor; C028143:homocitric acid; D009591:Nitrogenase