Overexpression, purification, and biochemical and spectroscopic characterization of copper-containing nitrite reductase from Sinorhizobium meliloti 2011. Study of the interaction of the catalytic copper center with nitrite and NO

The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis–Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs.

SmNir is a green-type Cu-containing nitrite reductase from Sinorhizobium meliloti, which contains two Cu sites, one of type 1 and the other of type 2. EPR reveals the formation of a radical-type Cu-NO species not detected before in other Nirs.Figure optionsDownload as PowerPoint slideHighlights
► SmNir is a Cu-nitrite reductase codified in the genome of Sinorhizobium meliloti.
► UV–vis, EPR, and molecular properties indicate that SmNir is a green-type Nir.
► SmNir contains two Cu sites, one of type 1 and the other of type 2.
► EPR reveals the formation of a Cu-NO species not detected before.
► SmNir originates a distinct radical species during nitrite reduction.