Student Speaker

Function of NADH-dependent Nitrite Reductase

---

Abstract

E coli uses NADH-dependent (Nir) and Formate-dependent (Nrf) Nitrite reductases in order to survive during anaerobic conditions. Previously it was proposed that the physiological function of Nir is to detoxify the buildup of Nitrite that is formed during anaerobic respiration on Nitrate. In the course of this study, it was determined that Nir does not play a role in detoxification of nitrite, but rather aids in assimilation during nitrate respiration. The results of this study are based on the analysis of cell cultures grown under anaerobic conditions with the use of 1D n15-NMR, N15-HMSQC NMR and UV-vis techniques.

Assimilation and dissimilation of Nitrite to ammonium are two distinct pathways of respiration of E coli. A series of experiments with the Nrf mutant and a wild type strains of E coli showed that the respiratory dissimilation of Nitrite to ammonium is possible only in the presence of Nrf. The presence of Nir does not aid in dissimilation, and the possible reasons for that phenomenon are mentioned in the discussion. However, it was determined that the Nir plays a crucial role in assimilation of Nitrite to ammonium, because the Nir mutant is incapable of growing on Nitrate as a sole nitrogen source. These results were further tested by the N15-HMSQC NMR, where it was shown that labelled nitrogen is incorporated in the organic matter in the case of the WT cells, but it is not in the case of the mutant. The detoxification of Nitrite suggests that ammonium should build up in the medium as a byproduct of this reaction. In the course of this study no detectable amount of ammonium were found in the medium regardless of the growth conditions. These results show that the physiological purpose of Nir is to assimilate nitrite derived from nitrate respiration. Contrary to the previous studies, no evidence of detoxification of Nitrite was found.