Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O Isotopes with a Ratio near Unity

In order to (i) establish the biological systematics necessary to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate (¹⁵N/¹⁴N and ¹⁸O/¹⁶O) in the environment and (ii) investigate the potential for isotopes to elucidate the mechanism of a key N cycle enzyme, we measured the nitrate N and O isotope effects (¹⁵ε and ¹⁸ε) for nitrate reduction by two assimilatory eukaryotic nitrate reductase (eukNR) enzymes. The ¹⁵ε for purified extracts of NADPH eukNR from the fungus <i>Aspergillus niger</i> and the ¹⁵ε for NADH eukNR from cell homogenates of the marine diatom <i>Thalassiosira weissflogii</i> were indistinguishable, yielding a mean ¹⁵ε for the enzyme of 26.6 ± 0.2‰. Both forms of eukNR imparted near equivalent fractionation on N and O isotopes. The increase in ¹⁸O/¹⁶O versus the increase in ¹⁵N/¹⁴N (relative to their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and 1.09 ± 0.03 for NADH eukNR. These results are the first reliable measurements of the coupled N and O isotope effects for any form of eukNR. They support the prevailing view that intracellular reduction by eukNR is the dominant step in isotope fractionation during nitrate assimilation and that it drives the ¹⁸ε:¹⁵ε ≈ 1 observed in phytoplankton cultures, suggesting that this O-to-N isotope signature will apply broadly in the environment. Our measured ¹⁵ε and ¹⁸ε may represent the intrinsic isotope effects for eukNR-mediated N–O bond rupture, a potential constraint on the nature of the enzyme’s transition state

The contamination of commercial 15N2 gas with 15N-labeled nitrate/nitrite and ammonium and consequences for nitrogen fixation measurements

<p>The data contained herein are associated with the manuscript, "The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements", which is available at PLOS ONE.</p> <p>We report on the presence of 15N-labeled nitrate/nitrite, ammonium, and nitrous oxide in commercial 15N2 gas and the enrichment of particulate nitrogen as a result of the biological assimilation of 15N labeled contaminants.</p

The δ¹⁵N of particulate nitrogen (δ¹⁵N_PN) of <i>D. tertiolecta</i> harvested in stationary phase following growth in media containing sodium nitrate (and no ammonium) and equilibrated with ¹⁵N₂ gas from Sigma Aldrich or Cambridge Isotopes.

<p>n = the number of experimental replicates.</p

(a) δ¹⁵N_NH4 (log scale) of 5 µmol L⁻¹ ammonium solutions after equilibration with 0.1 mL ¹⁵N₂ gas from respective Sigma-Aldrich and Cambridge Isotopes lecture bottles <i>vs.</i> control solutions.

<p>Sigma-Aldrich treatments utilized 40 mL ammonium solutions, whereas Cambridge Isotopes treatments utilized 100 mL ammonium solutions. (<b>b</b>) δ¹⁵N_NH4 of higher sensitivity equilibrations of 5 µmol L⁻¹ ammonium solutions (10 mL) with 2.0 mL ¹⁵N₂ gas from Cambridge Isotopes lecture bottles <i>vs.</i> control solutions. n = the number of experimental replicates.</p

(<b>a</b>) δ¹⁵N_NO3+NO2 (log scale) of nitrate solutions (10–300 µmol L⁻¹) following equilibration with 0.1 mL ¹⁵N₂ gas from lecture bottles procured from three distributors.

<p>Solutions were 40 mL for Sigma-Aldrich and Campro Scientific equilibrations, and 100 mL for Cambridge Isotopes equilibrations. The solid line corresponds to the δ¹⁵N_NO3 of the control solutions for Sigma-Aldrich and Cambridge Isotopes experiments (δ¹⁵N_NO3 = 23.5±0.5‰); the dashed line corresponds to controls for Campro Scientific experiments (δ¹⁵N_NO3 = 14.15±0.1‰). Paired symbols identify replicate experimental treatments. (<b>b</b>) Corresponding apparent δ¹⁸O_NO3+NO2 of the experimental nitrate solutions. The solid line corresponds to the δ¹⁸O_NO3 of control solutions for the Sigma-Aldrich and Cambridge Isotope experiments (δ¹⁸O_NO3 = 18.9±0.3‰); the dashed line corresponds to controls for Campro Scientific experiments (25.4±0.3‰).</p

(a) δ¹⁵N_NO3+NO2 (log scale) of higher sensitivity equilibrations of 10 µmol L⁻¹ nitrate solutions (10 mL) with 2 mL of ¹⁵N₂ gas from a Cambridge Isotopes or a Sigma-Aldrich bottle.

<p>(<b>b</b>) Corresponding apparent δ¹⁸O_NO3 (log scale) of higher sensitivity equilibrations of the two stocks. n = the number of experimental replicates.</p