Isotopic Fractionation by a Fungal P450 Nitric Oxide Reductase during the Production of N2O

Nitrous oxide (N2O) is a potent greenhouse gas with a 100-year global warming potential approximately 300 times that of CO2. Because microbes account for over 75% of the N2O released in the U.S., understanding the biochemical processes by which N2O is produced is critical to our efforts to mitigate climate change. In the current study, we used gas chromatography–isotope ratio mass spectrometry (GC–IRMS) to measure the δ15N, δ18O, δ15Nα, and δ15Nβ of N2O generated by purified fungal nitric oxide reductase (P450nor) from Histoplasma capsulatum. The isotope values were used to calculate site preference (SP) values (difference in δ15N between the central (α) and terminal (β) N atoms in N2O), enrichment factors (ε), and kinetic isotope effects (KIEs). Both oxygen and Nα displayed normal isotope effects during enzymatic NO reduction with ε values of −25.7‰ (KIE = 1.0264) and −12.6‰ (KIE = 1.0127), respectively. However, bulk nitrogen (average δ15N of Nα and Nβ) and Nβ exhibited inverse isotope effects with ε values of 14.0‰ (KIE = 0.9862) and 36.1‰ (KIE = 0.9651), respectively. The observed inverse isotope effect in δ15Nβ is consistent with reversible binding of the first NO in the P450nor reaction mechanism. In contrast to the constant SP observed during NO reduction in microbial cultures, the site preference measured for purified H. capsulatum P450nor was not constant, increasing from ∼15‰ to ∼29‰ during the course of the reaction. This indicates that SP for microbial cultures can vary depending on the growth conditions, which may complicate source tracing during microbial denitrification.