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

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide Nitrous oxide (N 2 O) is a serious concern due to its role in global warming and ozone destruction. Agricultural practices account for ∼80% of all anthropogenic N 2 O produced in the US, due in large part to the stimulation of microbial denitrification. Stable isotopes are uniquely suited to examine both microbial N 2 O sources and the mechanism of N 2 O biosynthesis through the use of Site Preference (δ 15 N SP; the difference in δ 15 N between the central and outer N atoms in N 2 O) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG-IRMS), we determined the δ 15 N, δ 15 N α, δ 15 N β, and δ 18 O of N 2 O produced by a purified cytochrome c nitric oxide reductase (cNOR) from Paracoccus denitrificans . We also calculated δ 15 N 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 15 N, with a KIE value of 1.0086 ± 0.0009 (ε = −8.6 ± 0.9‰). The isotope effects for both 15 N α and 15 N 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 δ 15 N SP values ranged from 0.5 to 8.7‰ with no significant trend as the reaction proceeded. Values of δ 18 O increased with N 2 O production (slope of δ 18 O against [− f ln f /(1 – f )] = −19.9 ± 1.9‰). We present implications for the mechanism of N 2 O production from cNOR based on our data.

Source: Biochemistry

DOI: 10.1021/acs.biochem.5c00417