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

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.