Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction

Abstract

To provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ( NO3−), 15N/14N and 18O/16O, in the environment, we measured the enzymatic NO3− N and O isotope effects (15ε and 18ε) during its reduction by NO3− reductase enzymes, including (1) a prokaryotic respiratory NO3− reductase, Nar, from the heterotrophic denitrifier Paracoccus denitrificans, (2) eukaryotic assimilatory NO3− reductases, eukNR, from Pichia angusta and from Arabidopsis thaliana, and (3) a prokaryotic periplasmic NO3− reductase, Nap, from the photoheterotroph Rhodobacter sphaeroides. Enzymatic Nar and eukNR assays with artificial viologen electron donors yielded identical 18ε and 15ε of ∼28‰, regardless of [ NO3−] or assay temperature, suggesting analogous kinetic mechanisms with viologen reductants. Nar assays fuelled with the physiological reductant hydroquinone (HQ) also yielded 18ε ≈ 15ε, but variable amplitudes from 21‰ to 33.0‰ in association with [ NO3−], suggesting analogous substrate sensitivity in vivo. Nap assays fuelled by viologen revealed 18ε:15ε of 0.50, where 18ε ≈ 19‰ and 15ε ≈ 38‰, indicating a distinct catalytic mechanism than Nar and eukNR. Nap isotope effects measured in vivo showed a similar 18ε:15ε of 0.57, but reduced 18ε ≈ 11‰ and 15ε ≈ 19‰. Together, the results confirm identical enzymatic 18ε and 15ε during NO3− assimilation and denitrification, reinforcing the reliability of this benchmark to identify NO3− consumption in the environment. However, the amplitude of enzymatic isotope effects is apt to vary in vivo. The distinctive signature of Nap is of interest for deciphering catalytic mechanisms but may be negligible in most environments given its physiological role.

How Important is Denitrification in Riparian Zones? Combining End‐Member Mixing and Isotope Modeling to Quantify Nitrate Removal from Riparian Groundwater

“…Similarly, in agreement with Dhondt et al (2003) who measured enrichment factors of ε N = −4.4‰ ± 0.3‰ in hydroponics, plant uptake has been generally associated with minor isotope fractionation compared to denitrification (Denk et al, 2017;Högberg et al, 1999;Lund et al, 1999). In contrast, recent enzymatic assays using eukaryotic NO 3 − assimilatory reductases (Karsh et al, 2012;Treibergs & Granger, 2017) have reported ε N and ε O values as large as ≈30‰. Nonetheless, we are not able to assess either the transferability of such enzyme-level isotope effects to field conditions or the role of the associated eukaryotes for NO 3 − removal at the field site.…”

Isotopic overprinting of nitrification on denitrification as a ubiquitous and unifying feature of environmental nitrogen cycling

“…However, following the advent of the denitrifier method (18,19), measurements in cultures of both freshwater and marine denitrifying bacteria revealed dual isotope enrichments associated with assimilatory and dissimilatory NO 3 − consumption systematically following linear trajectories of ∼1 (9,10,12,19,20), contrasting with the lower values widely observed in freshwater systems. This invariant coupling of N and O isotopic enrichments has been shown to originate from fractionation during enzymatic bond-breakage (8,21,22), confirmed directly from in vitro enzyme studies of eukaryotic assimilatory and prokaryotic dissimilatory NO 3 − reductases (11,23). Interpretation schemes conventionally ascribed to nitrification are also at odds with isotope systematics uncovered in culture observations.…”

High nitrogen isotope fractionation of nitrate during denitrification in four forest soils and its implications for denitrification rate estimates

“…However, the use of 15 N natural abundance to quantify denitrification rates and to constrain N transformation relies heavily on detailed knowledge of the denitrification process and the isotope fractionation involved. The N isotope effect ( 15 ) varies greatly with environmental and experimental conditions and has been reported to range from 5‰ to 40‰ in pure culture studies of heterotrophic denitrifying bacteria (Barford et al, 1999;Dabundo, 2014;Delwiche and Steyn, 1970;Frey et al, 2014;Granger et al, 2008;Hosono et al, 2015;Karsh et al, 2012;Knöeller et al, 2011;Kritee et al, 2012;Treibergs and Granger, 2016;Wunderlich et al, 2012) and in open ocean systems (Brandes et al, 1998;Cline and Kaplan, 1975;Sigman et al, 2005;Sigman et al, 2003;Voss et al, 2001), from 0‰ to 18‰ in continental sediments (Brandes and Devol, 1997;Brandes and Devol, 2002;Dähnke and Thamdrup, 2015;Kessler et al, 2014), from 5‰ to 30‰ in groundwater (Aravena and Robertson, 1998;Böttcher et al, 1990;Fukada et al, 2003;Lehmann et al, 2003;Mariotti et al, 1988;Mengis et al, 1999;Smith et al, 1991;Vogel et al, 1981;Wenk et al, 2014), and from 2‰ to 50‰ in agricultural soils (Blackmer and Bremner, 1977;Chien et al, 1977;Grabb et al, 2017;Lewicka-Szczebak et al, 2014;Lewicka-Szczebak et al, 2015;Mariotti et al, 1981;Mariotti et al, 1982;…”

“…Despite numerous studies of the N isotope effect performed using pure cultures of heterotrophic denitrifying bacteria (Granger et al, 2008;Treibergs and Granger, 2016, and references therein), groundwater (Lehmann et al, 2003;Wenk et al, 2014, and references therein), sediments (Dähnke and Thamdrup, 2015;Kessler et al, 2014, and references therein), and agricultural soils (Grabb et al, 2017;Lewicka-Szczebak et al, 2014;Lewicka-Szczebak et al, 2015;Mariotti et al, 1982;Mathieu et al, 2007;Well and Flessa, 2009), only four studies have examined forest soils (Houlton et al, 2006;Menyailo and Hungate, 2006;Perez et al, 2006;Snider et al, 2009).…”

“…18 O: 15 N; when plotting 18 O over 15 N of the residual NO3 -, a range from 0.47 to 0.89 was found in terrestrial environments and freshwater systems (Böttcher et al, 1990;Fukada et al, 2003;Lehmann et al, 2003;Wenk et al, 2014), a value of 1.25 in marine environments (Sigman et al, 2005), and a range from 0.33 to 1.02 in pure cultures of heterotrophic denitrifying bacteria (Dabundo, 2014;Frey et al, 2014;Granger et al, 2008;Hosono et al, 2015;Karsh et al, 2012;Knöeller et al, 2011;Kritee et al, 2012;Treibergs and Granger, 2016;Wunderlich et al, 2012). Thus, it is critical to determine the coupled N-and O-isotope discrimination in forest soils during denitrification.…”