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

## TL;DR

This study measures isotopic fractionation and kinetic isotope effects of a bacterial enzyme that produces nitrous oxide, a potent greenhouse gas.

## Contribution

The paper provides new isotopic data from a purified bacterial nitric oxide reductase enzyme, offering insights into the mechanism of nitrous oxide production.

## Key findings

- A normal isotope effect was observed for bulk 15N with a KIE of 1.0086 ± 0.0009.
- Position-specific KIEs for 15Nα and 15Nβ were 1.0072 ± 0.0010 and 1.0100 ± 0.0010, respectively.
- δ18O values increased with N2O production, with a slope of −19.9 ± 1.9‰.

## Abstract

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 (δ15NSP; 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, δ15Nα, δ15Nβ, and δ18O of N2O produced by a purified cytochrome c nitric oxide reductase (cNOR) from Paracoccus
denitrificans. We also calculated δ15NSP, the KIEs, and associated isotopic enrichment factors
(ε) for Nbulk, Nα, and Nβ. 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 15Nα and 15Nβ 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 δ15NSP 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 [−f ln f/(1 – f)] = −19.9 ± 1.9‰). We present implications
for the mechanism of N2O production from cNOR based on
our data.