Replacement of the essential catalytic aspartate with serine leads to an active form of copper-containing nitrite reductase from the denitrifier Sinorhizobium meliloti 2011.

We report the molecular, biochemical and spectroscopic characterization and computational calculations of a variant of the copper-containing nitrite reductase from the rhizobial microorganism S. meliloti (SmNirK), in which the catalytic aspartate residue (Asp) has been replaced with serine (Ser, D134S) by site-directed mutagenesis. Like the wild-type enzyme, D134S is a homotrimer with the typical catalytic pocket of two-domain NirK containing two copper centers, one of type 1 (T1) and another of type 2 (T2). The T1 electron transfer center is similar to that of the wild-type enzyme but the electronic and covalent properties of T2 active site are altered by the mutation. As for the wild-type enzyme, the enzymatic activity of D134S is pH-dependent, i.e. it is higher at lower pH values, but the k is an order of magnitude lower. EPR studies showed a decrease in g and an increase in A of D134S relative to wild-type enzyme. This indicates changes in the electronic and covalent properties of T2 upon mutation, which affects the reduction potential of T2 and the T1-T2 reduction potential gap. Taken together, this evidence points to the importance of the ligands of the second coordination sphere of T2 in controlling critical parameters in catalysis. The possibility that Asp/Ser is the switch that triggers T1 → T2 electron transfer upon T2 nitrite binding and the importance of His for the pH-dependent catalytic activity of NirK are discussed.