Nitrite is reduced by nitrite reductase NirB without small subunit NirD in Escherichia coli

The assimilatory nitrite reductase enzyme NirB and small subunit NirD genes encoded in nir operon in Escherichia coli were cloned into the pET28a vector, and the recombinant enzyme was characterized for the first time. Docking of NirB with NirD, NADH, NO₂^-, NO₃^-, and CHO₂^- was performed using docking modeling programs. Methyl viologen and sodium dithionite were used as electron couples, and the amount of reduced nitrite was measured to calculate enzyme activity. NirB is the main enzyme and shows high activity with or without NirD. However, the inclusion of NirD into the enzyme solution at a ratio of 1NirD:2NirB resulted in 10% higher nitrite reductase activity. The enzyme tends to aggregate in the absence of β-mercaptoethanol, which causes the conversion of tetrameric NirB to monomeric form, and the NirB enzyme shows its highest activity in monomeric form. The optimum temperature for enzyme activity was 37 °C and the optimum pH was found to be 7.0. K_m and V_max values of NirB were calculated as 9833 μM and 416.67 μmol NO₂^- reduced min^-1 mg^-1. Enzyme activity decreased by 55% and 50% in the presence of 100 mM nitrate and formate, respectively. The presence of 25 mM Cd²⁺ protected the enzyme at room temperature and the enzyme showed 10% higher activity in the presence of cadmium.