Azotobacter vinelandii is able to synthesize three genetically different nitrogenases, which are characterized by the presence of either a molybdenum, vanadium or iron atom in their respective co-factors. Each of the nitrogenases requires an activator to express the structural genes required for the enzyme. AnfA is the activator protein for the iron only nitrogenase. The in vivo activity of the protein requires the presence of the nitrogenase Fe-protein (Joerger et al. 1991). AnfA has the characteristic three-domain structure of a aN-dependent transcriptional activator. Previous work has shown that an amino-terminally truncated ANAnfA has been shown to remain constitutively active in vivo (Frise et al. 1994) and in vitro (Austin, Lambert 1994), implying regulation of AnfA activity is via the N-tenninus. A conserved cysteine motif is present at the N-terminal domain. The cysteine residues 21 and 26 have been previously shown to be required for in vivo activity (Premakumar et al. 1994). This motif could provide potential ligands for a metallocluster, which may regulate the activity of the protein in a redox sensitive manner.
We have shown previously that the isolated N-terminal domain of AnfA (NAnfA) was a red/brown color when purified and displayed spectra characteristic of oxidized 2Fe-2S clusters identified in other proteins. A mutant version of the N-terminal domain where one of the conserved cysteines was changed to an alanine (C26A) did not display the spectral features observed with the native protein. This implicates the N-terminal cysteine motif in the formation of the Fe-S cluster. In this work we describe the effect of the C26 A mutation on the activity of the full-length protein.
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