title: The role of the Fe-S cluster in the sensory domain of nitrogenase transcriptional activator VnfA from Azotobacter vinelandii.
abstract: Transcriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobacter vinelandii. In the present study, we have purified full-length VnfA produced in E. coli as recombinant proteins (Strep-tag attached and tag-less proteins), enabling detailed characterization of VnfA for the first time. The EPR spectra of whole cells producing tag-less VnfA (VnfA) show distinctive signals assignable to a 3Fe-4S cluster in the oxidized form ([Fe(3)S(4)](+)). Although aerobically purified VnfA shows no vestiges of any Fe-S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe(3)S(4)](+) dominantly in the protein. Additional spectroscopic evidence of [Fe(3)S(4)](+)in vitro is provided by anaerobically purified Strep-tag attached VnfA. Thus, spectroscopic studies both in vivo and in vitro indicate the involvement of [Fe(3)S(4)](+) as a prosthetic group in VnfA. Molecular mass analyses reveal that VnfA is a tetramer both in the presence and absence of the Fe-S cluster. Quantitative data of iron and acid-labile sulfur in reconstituted VnfA are fitted with four 3Fe-4S clusters per a tetramer, suggesting that one subunit bears one cluster. In vivobeta-gal assays reveal that the Fe-S cluster which is presumably anchored in the GAF domain by the N-terminal cysteine residues is essential for VnfA to exert its transcription activity on the target nitrogenase genes. Unlike the NifAL system of A. vinelandii, O(2) shows no effect on the transcriptional activity of VnfA but reactive oxygen species is reactive to cause disassembly of the Fe-S cluster and turns active VnfA inactive.
authors.affiliation: Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Japan. hnakajima@mbox.chem.nagoya-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakajima
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takatani
authors.forename: Kyohei
authors.initials: K
authors.lastname: Yoshimitsu
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Itoh
authors.forename: Shigetoshi
authors.initials: S
authors.lastname: Aono
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000235:genetics*; D021122:Protein Subunits / Q000502:physiology; D017382:Reactive Oxygen Species / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D015534:Trans-Activators; C090095:VnfA protein, Azotobacter vinelandii; D009591:Nitrogenase