Determination of the Kinetic Isotope Effects for Nitrous Oxide Reduction by Purified Form II Nitrous Oxide Reductase

Abstract. Mitigating the release of the greenhouse gas nitrous oxide (N₂O) is a major focus of sustainable agricultural practices. Currently, application of nitrogen-based fertilizers to the field drives significant microbial N₂O production. Understanding nitrogen flux through microbial metabolic processes, such as nitrification and denitrification, under different soil conditions is key to tracking sources of N₂O emissions in agricultural soils. Stable isotopes are a powerful tool for tracing sources and sinks of N₂O because isotopic preferences (e.g., isotope effects) of key enzymes in nitrogen cycling pathways allow for the differentiation of microbial sources of N₂O. Ascertaining source contributions of microbial N₂O production can be complicated, however, by the consumption of N₂O by nitrous oxide reductase (N₂OR), which itself exhibits isotopic preferences that influence the isotopic composition of the remaining N₂O. To help provide the isotopic parameters required to quantify and distinguish sources and sinks of N₂O, we determined the kinetic isotope effects (KIEs) for N₂O consumption by purified form II N₂OR (resting state: Cu_Z*) from Stutzerimonas stutzeri. The enrichment factors (ε) were –5.4 ± 0.2‰, -10.0 ± 0.4‰, -0.6 ± 0.1‰, -9.5 ± 0.4‰, and –13.5 ± 0.5‰ for ε¹⁵N^bulk, ε¹⁵N^α, ε¹⁵N^β, ε¹⁵N^SP, and ε¹⁸O, respectively. Thus, the kinetic isotope effects were normal for bulk δ¹⁵N (1.0054 ± 0.0002), δ¹⁵N^α (1.0101 ± 0.0004), and δ¹⁵N^SP (1.0095 ± 0.0004), and δ¹⁸O (1.0137 ± 0.0005). The KIE for δ¹⁵N^β was 1.0006 ± 0.0001, indicating there is essentially no isotopic preference for this position. These data improve our understanding of the isotopic fractionation associated with enzymatic N₂O consumption, which will inform modeling of nitrogen flux pathways leading to N₂O emissions in soil microbial communities.