Stable isotopic signature of dissimilatory nitrate reduction is robust against enzyme mutation

The proportionality of oxygen-to-nitrogen isotope effects (¹⁸ε/¹⁵ε) is used as a key isotopic signature of nitrogen cycling processes in the environment. Dissimilatory nitrate reduction is observed to have an ¹⁸ε/¹⁵ε proportionality of ~0.9 in marine and ~0.6 in freshwater/terrestrial ecosystems. The origins of this difference are uncertain, with both geochemical and biological factors conceivably at play. One potential factor is variation in the isotope effect of nitrate reduction among different forms of the nitrate reductase enzyme. NarG nitrate reductases are observed to typically have an ¹⁸ε/¹⁵ε of ~0.9. However, a recent study uncovered an exception, with Bacillus NarG enzymes having an ¹⁸ε/¹⁵ε proportionality of ~0.6. This provides an opportunity to investigate genetic controls on ¹⁸ε/¹⁵ε. Furthermore, this atypical NarG signature also raises the question of whether intrinsic isotope signatures can evolve as the enzymes that produce them accumulate mutations through time. Here, we present data from site-directed mutagenesis experiments of key NarG residues, which suggest that the distinct Bacillus ¹⁸ε/¹⁵ε cannot be caused by single mutations alone and is potentially uncommon in nature. Variation in the intrinsic isotope effects of an enzyme through time may thus require more extensive evolutionary changes.