N2O is both a potent greenhouse gas, with a 100-year global warming potential approximately 300 times that of CO2, and the largest anthropogenic source of ozone depletion. Currently, the stimulation of soil microbial processes by agricultural practices account for nearly 85% of the total anthropogenic N2O production. Due in large part to the world’s growing population, atmospheric concentrations of N2O are increasing at a rate of ~0.25% per year. It is therefore imperative that we obtain a better understanding of the processes leading to microbial N2O production and the enzymatic mechanism by which this gas is formed. To address this issue, we have studied the production of N2O by two highly related Pseudomonas species as well as by purified fungal P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum and bacterial cytochrome c nitric oxide reductase (cNOR) from Paracoccus denitrificans. N2O was isolated and analyzed via isotope-ratio mass spectrometry to ascertain the isotope ratio of the oxygen atom, Nα (the central N atom in N2O), and Nβ (terminal N atom in N2O). For the fungal system, we observed normal isotope effects for δ18O and δ15Nα and inverse isotope effects for δ15Nβ. Conversely, bacterial cNOR yielded normal isotope effects for δ18O and δ15Nβ, while no isotope effect was observed for δ15Nα. Interestingly, when analyzing the net isotope effect of N2O production by the Pseudomonas species, we discovered that the isotopic fractionation (the preference for one isotope over another in the reaction pathway) changed significantly over the extent of the reaction. Mechanistic implications for the purified enzymes as well as strategies for analyzing data from whole organisms will be discussed.

Probing the mechanism of microbial N2O production

255th National Meeting (2018)

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