Herbaspirillum seropedicae is an endophytic diazotroph of the b subclass of the Proteobacteria that associates with important agricultural crops. The NifA protein activates the transcription of nif genes, which are responsible for nitrogen fixation in bacteria. The activity of H. seropedicae NifA is negatively controlled by oxygen and ammonium. The NifA protein of H. seropedicae has a conserved motif of 4 cysteines located within the central and interdomain between the central and C-terminal regions. These cysteines may be involved in the formation of an iron-sulfur cluster probably involved in NifA activity and in oxygen sensing, and thus modulating NifA activity. In this work, we investigated the effect of substituting each of cysteines for serines in the NifA protein of H. seropedicae. To achieve this change, the codons of the cysteines of an 5’ truncated nifA gene were individually mutagenized, and the mutant genes cloned into the expression vector pET28a generating a series of pMAO plasmids. Plasmids pMAO1, pMAO2, pMAO3 and pMAO4 express respectively N-truncated NifA proteins with cysteine to serine substitutions in the positions 414, 426, 446 and 451. These plasmids were introduced into Escherichia coli strains carrying a nifH::lacZ fusion to determine the activity of the NifA mutant proteins.  A mutant NifA protein having a substitution of the Cys 451 to serine failed to activate the nifH::lacZ fusion, indicating that this cysteine is essential for NifA activity. The effect of mutations in the other three cysteines will be presented.