Widespread detoxifying NO reductases impart a distinct isotopic fingerprint on N₂O under anoxia

Nitrous oxide (N₂O), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources of N₂O has the potential to improve our understanding of N₂O fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used toward this end, as bacterial and fungal nitric oxide reductases (NORs) produce N₂O with different isotopic fingerprints, spanning a large range. Here, we show that flavohemoglobin (Fhp), a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N₂O under anoxic conditions (~+10‰) that correlates with typical environmental N₂O SP measurements. Using Pseudomonas aeruginosa as a model organism, we generated strains that only contained Fhp or the dissimilatory NOR, finding that in vivo N₂O SP values imparted by these enzymes differ by over 10‰. Depending on the cellular physiological state, the ratio of Fhp:NOR varies significantly in wild-type cells and controls the net N₂O SP biosignature: When cells grow anaerobically under denitrifying conditions, NOR dominates; when cells experience rapid, increased nitric oxide concentrations under anoxic conditions but are not growing, Fhp dominates. Other bacteria that only make Fhp generate similar N₂O SP biosignatures to those measured from our P. aeruginosa Fhp-only strain. Fhp homologs in sequenced bacterial genomes currently exceed NOR homologs by nearly a factor of four. Accordingly, we suggest a different framework to guide the attribution of N₂O biological sources in nature and disease.