Isotopic Fractionation and Kinetic Isotope Effects of a Purified Bacterial Nitric Oxide Reductase (NOR)

Nitrous oxide (N₂O) is a serious concern due to its role in global warming and ozone destruction. Agricultural practices account for ∼80% of all anthropogenic N₂O produced in the US, due in large part to the stimulation of microbial denitrification. Stable isotopes are uniquely suited to examine both microbial N₂O sources and the mechanism of N₂O biosynthesis through the use of Site Preference (δ¹⁵N^SP; the difference in δ¹⁵N between the central and outer N atoms in N₂O) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG-IRMS), we determined the δ¹⁵N, δ¹⁵N^α, δ¹⁵N^β, and δ¹⁸O of N₂O produced by a purified cytochrome <i>c</i> nitric oxide reductase (cNOR) from <i>Paracoccus denitrificans</i>. We also calculated δ¹⁵N^SP, the KIEs, and associated isotopic enrichment factors (ε) for N^bulk, N^α, and N^β. A normal isotope effect was observed for bulk ¹⁵N, with a KIE value of 1.0086 ± 0.0009 (ε = -8.6 ± 0.9‰). The isotope effects for both ¹⁵N^α and ¹⁵N^β were also normal, with position-specific KIEs of 1.0072 ± 0.0010 (ε = -7.2 ± 1.0‰) and 1.0100 ± 0.0010 (ε = -9.9 ± 1.0‰), respectively, and δ¹⁵N^SP values ranged from 0.5 to 8.7‰ with no significant trend as the reaction proceeded. Values of δ¹⁸O increased with N₂O production (slope of δ¹⁸O against [-<i>f</i> ln <i>f</i>/(1 - <i>f</i>)] = -19.9 ± 1.9‰). We present implications for the mechanism of N₂O production from cNOR based on our data.