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)

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1.9-Å resolution. In the native structure, His-255 N⑀2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.

The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin.

Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wild-type pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.