Purification and biochemical characterization of the DNA binding domain of the nitrogenase transcriptional activator NifA from Gluconacetobacter diazotrophicus

NifA is a σ ⁵⁴ activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in α-proteobacterial NifA is poorly understood. In this work, we examine if a Cys pair that is part of a C(X) ₅ C motif and located immediately upstream of NifA’s DNA binding domain is involved in redox sensing in NifA from the α-proteobacterium Gluconacetobacter diazotrophicus ( Gd ). We hypothesize that the Cys residues’ redox state may directly influence the DNA binding domain’s DNA binding affinity and/or alter the protein’s oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The K _d of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 μM. The K _d of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 μM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd -NifA does not regulate DNA binding or domain dimerization in a redox dependent manner. This suggests that other Cys residues in NifA, such as those located in the central AAA ⁺ domain, are responsible for redox sensing.