Microbial Denitrification: Active Site and Reaction Path Models Predict New Isotopic Fingerprints

The study of isotopic fingerprints in nitrate (delta 15N, delta 18O, Delta 17O) has enabled pivotal insights into the global nitrogen cycle and revealed new knowledge gaps. Measuring populations of isotopic homologs of intact NO3- ions (isotopologues) shows promise to advance the understanding of nitrogen cycling processes; however, we need new theory and predictions to guide laboratory experiments and field studies. We investigated the hypothesis that the isotopic composition of the residual nitrate pool is controlled by the N-O bond-breaking step in Nar dissimilatory nitrate reductase using molecular models of the enzyme active sites and associated kinetic isotope effects (KIEs). We integrated the molecular model results into reaction path models representing the reduction of nitrate under either closed-system or steady-state conditions. The predicted intrinsic KIE (15 epsilon and 18 epsilon) of the Nar active site matches observed fractionations in both culture and environmental studies. This is what would be expected if the isotopic composition of marine nitrate were controlled by dissimilatory nitrate reduction by Nar. For a closed system, the molecular models predict a pronounced negative 15N-18O clumping anomaly in residual nitrate. This signal could encode information about the amount of nitrate consumed in a closed system and thus constrain initial nitrate concentration and its isotopic composition. Similar clumped isotope anomalies can potentially be used to distinguish whether a system is open or closed to new nitrate addition. These mechanistic predictions can be tested and refined in combination with emerging ESI-Orbitrap measurements.

Automated summary

The study of isotopic fingerprints in nitrate has provided important insights into the global nitrogen cycle and uncovered new knowledge gaps. Measuring the populations of isotopic homologs of intact NO3- ions shows promise for advancing the understanding of nitrogen cycling processes. The research also found that the isotopic composition of residual nitrate is controlled by the N-O bond-breaking step in Nar dissimilatory nitrate reductase.