Date of Completion


Embargo Period



Dr. Craig Tobias, Dr. Pieter Visscher

Field of Study



Master of Science

Open Access

Open Access


Nitrate N and O isotope distributions in the environment can be used to elucidate salient biogeochemical N transformations. In order to do so, it is necessary to know the isotopic imprints characteristic of respective N transformations, and to understand the underlying mechanisms that determine these patterns. In order to provide mechanistic constraints on the isotopic imprints associated with nitrate consuming processes, we measured the enzymatic N and O isotope effects (15ε and 18ε) imparted on nitrate by three types of nitrate reductase enzymes, including (a) a prokaryotic respiratory nitrate reductase, Nar, from the heterotrophic denitrifier Paracoccus denitrificans, (b) a prokaryotic periplasmic nitrate reductase, Nap, from the photoheterotroph Rhodobacter sphaeroides, and (c) two commercially purified extracts of eukaryotic assimilatory nitrate reductases (EukNR) from Pichia angusta and from Arabidopsis thaliana. Enzymatic Nar assays fuelled with the artificial electron donors methyl and benzyl viologen yielded identical N and O isotope effects (Δδ18O:Δδ15N ≈ 1) of ~27‰, regardless of the initial nitrate concentration (200 µM vs. 1000 µM) or assay temperature (20˚C vs. 4˚C). Enzymatic assays with EukNR fuelled by methyl viologen yielded strikingly identical results to Nar, namely a Δδ18O:Δδ15N ≈ 1 and isotope effect magnitudes of ~27‰. Nar assays fuelled with the physiological reductant hydroquinone also yielded a consistent Δδ18O:Δδ15N ≈ 1, but showed more variable isotope effect amplitudes, from 22.9 ± 1.5‰ to 33.0 ± 4.3‰. This suggests that isotope effect amplitudes may be sensitive to the rate of internal electron transfer to the enzyme’s catalytic site. Nap assays showed unique fractionation patterns, including a Δδ18O:Δδ15N ≈ 0.5, N isotope effect of ~38‰, and O isotope effect of ~19‰, which portends a different catalytic mechanism than that of the closely related Nar and distantly related EukNR enzyme types. These results confirm that dominant nitrate consuming processes in the environment fractionate with a Δδ18O:Δδ15N ≈ 1, providing a reliable benchmark from which to identify their specific signature from environmental isotope distributions. The distinctive isotopic signature of the auxiliary Nap enzyme is of interest with respect to deciphering catalytic mechanisms, but is unlikely to account for imprints on nitrate in the environment given the auxiliary role of Nap in bacterial physiology.

Major Advisor

Dr. Julie Granger