Molecular and kinetic properties of copper nitrite reductase from Sinorhizobium meliloti 2011 upon substituting the interfacial histidine ligand coordinated to the type 2 copper active site for glycine.

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a âˆ¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

Copper nitrite reductase from Sinorhizobium meliloti 2011: Crystal structure and interaction with the physiological versus a nonmetabolically related cupredoxin-like mediator.

We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 Å, showed a striking resemblance with the overall structure of the well-known Class I NirKs composed of two Greek key ß-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.