Isotopic fractionation by plant nitrate reductase, twenty years later

Plant nitrate reductase, the enzyme that reduces nitrate (NO ₃^-) to nitrite (NO₂^-), is known to fractionate N isotopes, depleting nitrite in ¹⁵N compared with substrate nitrate. Nearly 20 years ago, the nitrogen isotope effect associated with this reaction was found to be around 1.015. However, the relationships between the isotope effect and the mechanism of the reaction have not yet been examined in the light of recent advances regarding the catalytic cycle and enzyme structure. We thus give here the mathematical bases of the ¹⁴N / ¹⁵N and also the ¹⁶O / ¹⁸O isotope effects as a function of reaction rates. Enzymatic nitrate reduction involves steps other than NO₃^- reduction itself, in which the oxidation number of N changes from +V (nitrate) to +III (nitrite). Using some approximations, we give numerical estimates of the intrinsic N and O isotope effects and this leads us to challenge the assumptions of nitrate reduction itself as being a rate-limiting step within the nitrate reductase reaction, and of the formation of a bridging oxygen as a reaction intermediate.