A tale of two nitrous oxide reductases: a cautionary perspective

Nitrous oxide reductases (N2OR) are the sole sink of the potent greenhouse gas nitrous oxide (N₂O) in the environment. Having been studied for decades, N2OR have attracted renewed attention following the discovery of a previously unrecognized clade, now termed clade II. This clade exhibits unexpectedly widespread taxonomic distribution and prevalence across diverse environments, prompting research efforts to define and assign distinct clade-specific traits. In this perspective, we aim to critically review and evaluate dichotomous clade-based classifications, addressing oversimplifications and unresolved ambiguities in linking clade identity to physiological traits like substrate affinity, acid tolerance, and aerotolerance. Growing experimental evidence from N₂O-reducing isolates and enrichments suggests a general difference in substrate affinity between the clades. Recent discoveries of N₂O reduction at pH < 5.0 attribute the long-sought acidophilic N₂O reduction exclusively to organisms possessing clade II nosZ, and attempts have also been made to relate clade separation to aerotolerant N₂O reduction. However, it is important to note that such binary characterizations are based on limited observations and lack a solid understanding of the underlying mechanisms, exposing them to bias and oversimplification risks. We emphasize the need for a balanced research effort to establish a robust link between ecophysiology and biochemistry, enabling a more accurate evaluation of clade-based characterizations and, ultimately, a deeper understanding and effective harnessing of N₂O-reducing organisms.