Natural-abundance stable isotopes can provide information on the redox reactions involved in nitrogen cycling. Molecular models employing density functional theory are used to predict the impact of denitrification (nitrate to nitrite) by enzyme Nap and Nar active sites on isotopic fractionation (δ15N and δ18O) and clumping (Δ15N-18O and Δ18O-18O) in residual nitrate. Predicted Nar intrinsic isotope effects match observed fractionations in both microbial culture and environmental samples. An anti-clumping signal develops during closed-system denitrification, which is counterintuitive given the accumulation of heavy isotopes by Rayleigh fractionation. Steady-state behavior differs; thus clumped isotopes could provide information on system closure to nitrate addition, or initial compositions and concentrations of nitrate. The predicted signal magnitude should be measurable by electrospray Orbitrap mass spectrometry. In contrast, predicted Nap intrinsic isotope effects do not match observed fractionations when employing presumed monodentate binding of nitrate to the active site. However, bidentate binding models do match observed fractionations, suggesting a mechanism for observed differences in isotope fractionation produced by Nap and Nar. Molecular models can predict new position-specific and clumped isotope signals, which themselves provide information on mechanistic parameters of geochemical reactions.

Microbial denitrification: Active site models predict isotopic fingerprints of enzyme binding and system closure

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