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 Aspergillus niger and the ¹⁵ε for NADH eukNR from cell homogenates of the marine diatom Thalassiosira weissflogii 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.