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

Area of Science:

• Geochemistry
• Biogeochemistry
• Molecular enzymology

Background:

• Isotopic analysis of nitrate (δ15N, δ18O, Δ17O) provides insights into the global nitrogen cycle.
• Measuring nitrate isotopologues offers advanced understanding of nitrogen cycling, but requires new theoretical frameworks.
• The enzyme Nar (dissimilatory nitrate reductase) plays a key role in nitrate reduction.

Purpose of the Study:

• Investigate if the N-O bond-breaking step in Nar controls residual nitrate's isotopic composition.
• Develop molecular models of Nar active sites to predict kinetic isotope effects (KIEs).
• Integrate KIEs into reaction path models for nitrate reduction under different system conditions.

Main Methods:

• Utilized molecular modeling of enzyme active sites.
• Calculated intrinsic kinetic isotope effects (15ε and 18ε) for Nar.
• Employed reaction path modeling for nitrate reduction in closed and steady-state systems.

Main Results:

• Predicted intrinsic KIEs for Nar active sites align with observed fractionations in cultures and environments.
• Model results suggest Nar-mediated dissimilatory nitrate reduction could control marine nitrate isotopic composition.
• A significant negative 15N-18O clumping anomaly was predicted in residual nitrate for closed systems.

Conclusions:

• The N-O bond-breaking step in Nar is a key control on nitrate isotopic composition.
• Clumped isotope anomalies in residual nitrate can indicate nitrate consumption extent and system openness.
• Mechanistic predictions provide testable hypotheses for future experimental and field studies using advanced mass spectrometry.