Activation of Nitrite Reductase from Escherichia coli

Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction ofNO2by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2(2mM) the Michaelis constant for NADH is about 16,UM. The Michaelis constant for NO2is about 5AM and is largely independent of the NAD+ concentration. Similar but more pronounced effects ofNAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate ofNADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.