EGUsphere EGUsphere EGUsphere EGUsphere Copernicus Publications Göttingen, Germany 10.5194/egusphere-2026-6015 Determination of the Kinetic Isotope Effects for Nitrous Oxide Reduction by Purified Form II Nitrous Oxide Reductase Ellis Louisa R. 1 2 Moran James J. https://orcid.org/0000-0001-9081-9017 3 4 Hegg Eric L. https://orcid.org/0000-0003-2055-5495 1 2 Rivett Elise D. https://orcid.org/0000-0002-5118-2612 1 2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, 48824, United States of America DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, 48824, United States of America Department of Integrative Biology, Michigan State University, East Lansing, 48824, United States of America Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, 48824, United States of America 20 10 2026 2026 1 41 Copyright: © 2026 Louisa R. Ellis et al. 2026 This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/ This article is available from https://egusphere.copernicus.org/preprints/2026/egusphere-2026-6015/ The full text article is available as a PDF file from https://egusphere.copernicus.org/preprints/2026/egusphere-2026-6015/egusphere-2026-6015.pdf

Mitigating the release of the greenhouse gas nitrous oxide (N<sub>2</sub>O) is a major focus of sustainable agricultural practices. Currently, application of nitrogen-based fertilizers to the field drives significant microbial N<sub>2</sub>O production. Understanding nitrogen flux through microbial metabolic processes, such as nitrification and denitrification, under different soil conditions is key to tracking sources of N<sub>2</sub>O emissions in agricultural soils. Stable isotopes are a powerful tool for tracing sources and sinks of N<sub>2</sub>O because isotopic preferences (<em>e.g.</em>, isotope effects) of key enzymes in nitrogen cycling pathways allow for the differentiation of microbial sources of N<sub>2</sub>O. Ascertaining source contributions of microbial N<sub>2</sub>O production can be complicated, however, by the consumption of N<sub>2</sub>O by nitrous oxide reductase (N<sub>2</sub>OR), which itself exhibits isotopic preferences that influence the isotopic composition of the remaining N<sub>2</sub>O. To help provide the isotopic parameters required to quantify and distinguish sources and sinks of N<sub>2</sub>O, we determined the kinetic isotope effects (KIEs) for N<sub>2</sub>O consumption by purified form II N<sub>2</sub>OR (resting state: Cu<sub>Z</sub>*) from<em> Stutzerimonas stutzeri</em>. The enrichment factors (&epsilon;) were &ndash;5.4 &plusmn; 0.2&permil;, -10.0 &plusmn; 0.4&permil;, -0.6 &plusmn; 0.1&permil;, -9.5 &plusmn; 0.4&permil;, and &ndash;13.5 &plusmn; 0.5&permil; for &epsilon;<sup>15</sup>N<sup>bulk</sup>, &epsilon;<sup>15</sup>N<sup>&alpha;</sup>, &epsilon;<sup>15</sup>N<sup>&beta;</sup>, &epsilon;<sup>15</sup>N<sup>SP</sup>, and &epsilon;<sup>18</sup>O, respectively. Thus, the kinetic isotope effects were normal for bulk &delta;<sup>15</sup>N (1.0054 &plusmn; 0.0002), &delta;<sup>15</sup>N<sup>&alpha; </sup>(1.0101 &plusmn; 0.0004), and &delta;<sup>15</sup>N<sup>SP </sup>(1.0095 &plusmn; 0.0004), and &delta;<sup>18</sup>O (1.0137 &plusmn; 0.0005). The KIE for &delta;<sup>15</sup>N<sup>&beta; </sup>was 1.0006 &plusmn; 0.0001, indicating there is essentially no isotopic preference for this position. These data improve our understanding of the isotopic fractionation associated with enzymatic N<sub>2</sub>O consumption, which will inform modeling of nitrogen flux pathways leading to N<sub>2</sub>O emissions in soil microbial communities.

 Great Lakes Bioenergy Research Center DE-SC0018409