Haem is involved in the NO-mediated regulation by Bradyrhizobium diazoefficiens NnrR transcription factor

Nitric oxide (NO) and the greenhouse gas (GHG) nitrous oxide (N₂O) contribute significantly to climate change. In rhizobia, the denitrifying enzyme c-type nitric oxide reductase (cNor), encoded by norCBQD genes, is crucial for maintaining a delicate balance of NO and N₂O levels. In the soybean endosymbiont Bradyrhizobium diazoefficiens, maximal expression of norCBQD genes in response to NO is controlled by NnrR, which belongs to a distinct clade of the CRP/FNR family of bacterial transcription factors. This protein participates in the FixLJ-FixK₂-NnrR regulatory cascade that induces denitrification genes expression in response to oxygen limitation and nitrogen oxides. However, the molecular mechanism underpinning NO sensing by B. diazoefficiens NnrR has remained elusive. Here, we revealed that NnrR induces norCBQD gene expression in response to NO uncoupled from the superimposed FixK₂ control. Moreover, NO-mediated induction by NnrR is dependent on haem, as the expression of a norC-lacZ fusion was impaired in a hemN₂ mutant defective in haem biosynthesis. In vitro studies showed that NnrR bound haem with a 1:1 stoichiometry (monomer:haem), according to titration experiments of recombinant NnrR protein with hemin performed under anaerobic conditions. Furthermore, the full UV-Visible spectra of haem-reconstituted NnrR showed a peak at 411 nm (ferric form), and at 425 nm (ferrous derivative). This latter complex was able to bind NO under anaerobic conditions. Finally, we performed a functional mutagenesis of specific residues in NnrR predicted as putative ligands for haem binding. While H11 was important for norC expression and Nor activity, a H11A-H56A protein variant showed a reduced affinity for haem binding. Taken together, our results identify haem as the cofactor for NnrR-mediated NO sensing in B. diazoefficiens denitrification, with H11 as a key residue for NnrR function, providing the first insight into the mechanism of an NnrR-type protein. These findings advance our understanding of how bacterial systems orchestrate the denitrification process and respond to environmental cues such as NO.