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 ([Fe3S4]+). Although aerobically purified VnfA shows no vestiges of any Fe‐S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe3S4]+ dominantly in the protein. Additional spectroscopic evidence of [Fe3S4]+in vitro is provided by anaerobically purified Strep‐tag attached VnfA. Thus, spectroscopic studies both in vivo and in vitro indicate the involvement of [Fe3S4]+ 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 vivoβ‐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, O2 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. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7311946: VnfA (uniprotkb:http://www.uniprot.org/uniprot/C1DI41?format=text&ascii) and VnfA (uniprotkb:http://www.uniprot.org/uniprot/C1DI41?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7311931: VnfA (uniprotkb:http://www.uniprot.org/uniprot/C1DI41?format=text&ascii) and VnfA (uniprotkb:http://www.uniprot.org/uniprot/C1DI41?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by blue native page (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0276)

VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase-2 in Azotobacter vinelandii. VnfA consists of three domains: an N-terminal regulatory domain termed GAF, including a Cys-rich motif; a central domain from the AAA+ family; and a C-terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe-S cluster (presumably of the 3Fe-4S type) as a prosthetic group and the Cys-rich motif were possibly associated with coordination of the Fe-S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: DN15 VnfA and DGAF VnfA that lack the N-terminal 15 residues and whole GAF domain, respectively, and GAF VnfA consisting of only the GAF domain. DN15 VnfA and DGAF VnfA lost the ability to bind the Fe-S cluster, whereas GAF VnfA was still able to bind to the cluster, consistent with the hypothesis that the Cysrich motif is essential for Fe-S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe-S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain. Structured digital abstractl VnfA binds to VnfA by molecular sieving (View Interaction 1, 2) Database VnfA has been submitted to the Swiss-Prot database under accession number: C1DI41.

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The transcriptional activator, VnfA, is necessary for the expression of the structural genes encoding vanadium-dependent nitrogenase in Azotobacter vinelandii. We have previously reported that VnfA harbours a Fe-S cluster as a prosthetic group, presumably a 3Fe-4S type, which is vital for the transcriptionally active VnfA. A plausible effector molecule is a reactive oxygen species (ROS), which disassembles the Fe-S cluster switching the active VnfA to become fully inactive. This finding prompted us to investigate the effect of nitric oxide (NO), another physiologically important radical species on the VnfA activity. Unlike ROS, the VnfA activity was moderately inhibited and converged to 70% of the maximum by NO irrespective of its concentration. The Fe-S cluster of VnfA was found to react with NO to form a dinitrosyl-iron complex, either in the dimeric or monomeric form, dependent on the relative stoichiometry of NO to the Fe-S cluster. The VnfA species harbouring the dinitrosyl-iron complexes in each form exhibited 50% ATPase activity compared to the active VnfA. The findings of this study would open an argument about a biological effect of NO on nitrogenase in light of its transcriptional regulatory system.