The role of the Fe-S cluster in the sensory domain of nitrogenase transcriptional activator VnfA from Azotobacter vinelandii.

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.