LAUSR

All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme‐containing molybdenum in the active site co‐factor to fix atmospheric dinitrogen to ammonia. However, in addition to the Mo‐dependent nitrogenase, some nitrogen‐fixing prokaryotes also express genetically distinct alternative nitrogenase isoenzymes, namely the V‐dependent and Fe‐only nitrogenases, respectively. Nitrogenase isoenzymes are expressed hierarchically according to metal availability and catalytic efficiency. In proteobacteria, this hierarchy is maintained via stringent transcriptional regulation of gene clusters by dedicated bacterial enhancer‐binding proteins (bEBPs). The model diazotroph Azotobacter vinelandii contains two paralogs of the vanadium nitrogenase activator VnfA (henceforth, VnfA1), designated VnfA2 and VnfA3, with unknown functions. Here we demonstrate that the VnfA1 and VnfA3 bEBPs bind to the same target promoters in the Azotobacter vinelandii genome and co‐activate a subset of genes in the absence of V, including the structural genes for the Fe‐only nitrogenase. Co‐activation is inhibited by the presence of V and is dependent on an accessory protein VnfZ that is co‐expressed with VnfA3. Our studies uncover a plethora of interactions between bEBPs required for nitrogen fixation, revealing the unprecedented potential for fine‐tuning the expression of alternative nitrogenases in response to metal availability.