10 Apr 2024
 | 10 Apr 2024
Status: this preprint is open for discussion.

Position-specific kinetic isotope effects for nitrous oxide: A new expansion of the Rayleigh model

Elise D. Rivett, Wenjuan Ma, Nathaniel E. Ostrom, and Eric L. Hegg

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and the most significant anthropogenic ozone-depleting substance currently being emitted. A major source of anthropogenic N2O emissions is the microbial conversion of fixed nitrogen species from fertilizers in agricultural soils. Thus, understanding the enzymatic mechanisms by which microbes produce N2O has environmental significance. Measurement of the 15N/14N isotope ratios of N2O produced by purified enzymes or axenic microbial cultures is a promising technique for studying N2O biosynthesis. Typically, N2O-producing enzymes combine nitrogen atoms from two identical substrate molecules (NO or NH2OH). Position-specific isotope analysis of the central (Nα) and outer (Nβ) nitrogen atoms in N2O enables the determination of the individual kinetic isotope effects (KIEs) for Nα and Nβ, providing mechanistic insight into the incorporation of each nitrogen atom. Previously, position-specific KIEs (and fractionation factors) were quantified using the Rayleigh distillation equation, i.e., via linear regression of δ15Nα or δ15Nβ against [-flnf/(1-f)], where f is the fraction of substrate remaining in a closed system. This approach, however, is inaccurate for Nα and Nβ because it does not account for fractionation at Nα affecting the isotopic composition of substrate available for incorporation into the β position (and vice versa). Therefore, we developed a new expansion of the Rayleigh model that includes specific terms for fractionation at the individual N2O nitrogen atoms. By applying this Expanded Rayleigh model to a variety of simulated N2O synthesis reactions with different combinations of normal/inverse/no KIEs at Nα and Nβ, we demonstrate that our new model is both accurate and robust. We also applied this new model to two previously published datasets describing N2O production from NH2OH oxidation in a methanotroph culture (Methylosinus trichosporium) and N2O production from NO by a purified Histoplasma capsulatum (fungal) P450 NOR, demonstrating that the Expanded Rayleigh model is a useful tool in calculating position-specific fractionation for N2O synthesis.

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Elise D. Rivett, Wenjuan Ma, Nathaniel E. Ostrom, and Eric L. Hegg

Status: open (until 21 Jun 2024)

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Elise D. Rivett, Wenjuan Ma, Nathaniel E. Ostrom, and Eric L. Hegg

Data sets

Results for datasets with simulated error (derived from Datasets 1-5) Elise Rivett

Model code and software

position-specific-kie Elise Rivett

Elise D. Rivett, Wenjuan Ma, Nathaniel E. Ostrom, and Eric L. Hegg


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Short summary
Many different processes produce nitrous oxide (N2O), a potent greenhouse gas. Measuring the ratio of heavy and light nitrogen isotopes (15N/14N) for the non-exchangeable central and outer N atoms of N2O helps to distinguish sources of N2O. To accurately calculate the position-specific isotopic preference, we developed an expansion of the widely-used Rayleigh model. Application of our new model to simulated and experimental data demonstrates its improved accuracy for analyzing N2O synthesis.