X-ray Structure and Site-Directed Mutagenesis of a Nitrite Reductase from Alcaligenes Faecalis S-6: Roles of Two Copper Atoms in Nitrite Reduction

Abstract

Nitrite reductase (NIR) from the denitrifying bacterium Alcaligenes faecalis S-6 is a copper-containing enzyme which requires pseudoazurin, a low molecular weight protein containing a single type I copper atom, as a direct electron donor in vivo. Crystallographic analysis shows that NIR is a trimer composed of three identical subunits, each of which contains one atom of type I copper and one atom of type II copper, and that the ligands to the type I and type II copper atoms are the same as those of the Achromobacter cycloclastes NIR. An efficient NIR expression-secretion system in Escherichia coli was constructed and used for site-directed mutagenesis. An NIR mutant with a replacement of the type II copper ligand, His135, by Lys still retained a type II copper site as well as a type I copper atom, but it completely lost nitrite-reducing activity as measured with methyl viologen as an electron donor. On the other hand, another mutant with a replacement of the type I copper ligand, Met150, by Glu contained only a type II copper atom, but it still retained significant nitrite-reducing activity with methyl viologen. When pseudoazurin was used as an electron donor for the reaction, however, Met150Glu failed to catalyze the reduction of nitrite. Kinetic analysis of the electron transfer between NIR and pseudoazurin revealed that the electron-transfer rate between Met150Glu and pseudoazurin was reduced 1000-fold relative to that of wild-type NIR.(ABSTRACT TRUNCATED AT 250 WORDS)

Site‐directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron‐transfer complex with a copper‐containing nitrite reductase from Alcaligenes faecalis S‐6

“…Purity of each protein was analyzed by SDS-PAGE [15]. A. faecalis NIR was produced in E. coli and purified as described previously [16].…”

“…Kinetic analysis of electron transfer from wild-type or mutated azurins to NIR was carried out as described previously [16]. Each reaction mixture contains 20 mM potassium phosphate buffer, pH 7.0, 2 mM KNO2 and reduced azurin with concentrations of 0.2 1.3 raM.…”

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

“…1NRD) [9], Alcaligenes faecalis S-6 (AfNiR, pdb ref. 1AFN) [10] and Alcaligenes xylosoxidans (AxNiR, pdb ref. 1NDS, R1IDSSF) [11] have been determined.…”

pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

“…NiR reduces nitrite to produce NO (g) , and NO reductase reduces NO to N 2 O, where NO is the first gaseous compound in the pathway. There are two main categories of NiRs; those that are heme-containing (cd1-NiRs) (3,4) and those that are copper-containing (Cu-NiRs) (5)(6)(7)(8). The overall enzymatic reaction performed by NiRs is NO 2 Ϫ ϩ 2H ϩ ϩ e Ϫ 3 NO (g) ϩ H 2 O. Cu-NiRs are blue or green in color, showing visible absorption at 460 and 600 nm (1).…”

“…As with all other Cu-NiRs, the protein is a homotrimer with two copper atoms per monomer, ϳ12 Å apart. The type 1 copper site has four ligands (His-126, His-177, Cys-167, and Met-182) and is located a few angstroms beneath a hydrophobic surface (8), which is the docking site for an electron donor protein (10,11). The type 2 copper site is situated at the monomer-monomer interfaces and has ligands from both monomers (His A131, His A166, His B338), forming a propeller-shaped structure with the copper at its center.…”