Nitrate reductase (15)N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli

Stable (15)N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves the reduction of nitrate (NO(−)(3)) to nitrite (NO(−)(2)) catalyzed by nitrate reductase (NR). Discrimination against (15)N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro (15)N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic (15)N discrimination by the enzyme and to evaluate the validity of measurements made using (15)N-enriched NO(−)(3). Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022–1.032) among the different isozymes at natural abundance (15)N (0.37%). As the fractional (15)N content of substrate NO(−)(3) increased from natural abundance, the product (15)N fraction deviated significantly from that expected based on substrate enrichment and (15)N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO(−)(3) and NO(−)(2) in some protocols became a greater source of error as (15)N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched (15)N and recommend against the use of highly enriched (15)N tracers to study N discrimination in plants or soils.