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

Abstract

The study of isotopic fingerprints in nitrate (δ15N, δ18O, Δ17O) 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 (15ε and 18ε) 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.

Deciphering the isotopic imprint of nitrate to reveal nitrogen source and transport mechanisms in a tile-drained agroecosystem

“…6). It is also consistent with the intrinsic N and O isotopic fractionations catalyzed by the Nar dissimilatory NO 3 reductase (Granger et al, 2008;Boettger et al, 2022), which is considered the dominant respiratory pathway for heterotrophic NO 3 consumption in freshwater systems (Granger and Wankel, 2016). Interestingly, if we include NO 3 isotope data measured during the corn growing season in the linear regression of δ 15 N NO3 -versus-δ 18 O NO3 , the regression slope is lowered to be around 0.6 (Fig.…”

“…This denitrification isotope effect is expressed as a linear trajectory in the biplot of δ 15 N NO3 and δ 18 O NO3 (i.e., dual isotope space; Fig. 1), reflecting the coupling of N and O isotopic fractionations during the denitrification process (Granger et al, 2008;Boettger et al, 2022). While the expression of these isotopic fractionations can be a valuable tool for quantifying the occurrence and intensity of denitrification, the large isotopic enrichment resulting from denitrification obscures the original isotopic signature of NO 3 and, therefore, requires careful corrections for N source partitioning using NO 3 isotopes.…”