We provide the first atomic resolution (<1.20 A) structure of a copper protein, nitrite reductase, and of a mutant of the catalytically important Asp92 residue (D92E). The atomic resolution where carbon-carbon bonds of the peptide become clearly resolved, remains a key goal of structural analysis. Despite much effort and technological progress, still very few structures are known at such resolution. For example, in the Protein Data Bank (PDB) there are some 200 structures of copper proteins but the highest resolution structure is that of amicyanin, a small (12 kDa) protein, which has been resolved to 1.30 A. Here, we present the structures of wild-type copper nitrite reductase (wtNiR) from Alcaligenes xylosoxidans (36.5 kDa monomer), the "half-apo" recombinant native protein and the D92E mutant at 1.04, 1.15 and 1.12A resolutions, respectively. These structures provide the basis from which to build a detailed mechanism of this important enzyme.

Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO2−) to nitric oxide (NO). Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 Å, 1.9 Å and 2.2 Å resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has ∼30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme.

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65Å resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant (∼800mV) is elevated compared to that of the native protein (∼670mV at pH 3.2) while that of the Ser86Asp mutant (∼623mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.

Copper-containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer-monomer interface, resembles the catalytic sites of a number of Zn enzymes. Nitrite reductase from Alcaligenes xylosoxidans has optimum activity at pH 5.2 which decreases to a negligible level at pH 8. The structure of this nitrite reductase has previously been determined at pH 4.6. It has now been crystallized under new conditions at pH 8.5. Its crystallographic structure provides a structural explanation for the greatly reduced activity of the enzyme at high pH. Characterization of overexpressed protein in solution by EXAFS suggested that the protein lacked Cu in the catalytic type 2 Cu site and that the site was most probably occupied by Zn. Using the anomalous signals from Cu and Zn, the crystal structure revealed that the expressed protein was devoid of Cu in the catalytic site and that only a trace amount (<10%) of Zn was present at this site in the crystal. Despite the close structural similarity of the catalytic site to a number of Zn enzymes, these data suggest that Zn, if it binds at the catalytic copper site, binds weakly in nitrite reductase.

Crystallographic structures of oxidised and reduced forms of azurin II are reported at 1.75~ resolution [1].Data were collected using one crystal in each case and by translating crystal after each oscillation range to minimise photo-reduction.Very small differences are observed at the Cu site upon reduction and that these cannot be determined with confidence at current resolution.A comparison with the 3D EXAFS reveals a good correspondence for all the ligands distances except for Cu-His46 where a larger deviation of ~0.12-0.18~ is observed indicating that this ligand is more tightly restrained in the crystallographic refinement at the current resolution.Three dimensional information is obtained for the Cu site in azurin [2] at very high resolution by combining high resolution crystallographic structures and EXAFS data for the oxidised and reduced form of the protein.This combined approach has allowed us to define the subtle structural changes (<0.1~) which take place at the Cu site during a single electron redox process.

Crystallographic structures of oxidized and reduced forms of azurin II are reported at 1.75 A resolution. Data were collected using one crystal in each case and by translating the crystal after each oscillation range to minimize photoreduction. Very small differences are observed at the Cu site upon reduction and these cannot be determined with confidence at current resolution. A comparison with the three-dimensional EXAFS reveals a good correspondence for all the ligand distances except for Cu-His46, where a larger deviation of approximately 0.12-0.18 A is observed, indicating that this ligand is more tightly restrained in the crystallographic refinement at the current resolution.

The crystallographic structures of several copper-containing nitrite reductases are now available. Despite this wealth of structural data, no definitive information is available as to whether the reaction proceeds by an ordered mechanism where nitrite binds to the oxidised type 2 site, followed by an internal electron transfer from the type 1 Cu, or whether binding occurs to the reduced type 2 Cu centre, or a random mechanism operates. We present here the first structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absorption spectroscopy. The reduced type 2 Cu site EXAFS shows striking similarity to the EXAFS data for reduced bovine superoxide dismutase (Cu2Zn2 SOD), providing strong evidence for the loss of the water molecule from the catalytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site similar to that in Cu2Zn2 SOD. The reduced type 2 Cu site of AxNiR is shown to be unable to bind inhibitory ligands such as azide, and to react very sluggishly with nitrite leading to only a slow re-oxidation of the the type 1 centre. These observations provide strong evidence that turnover of AxNiR proceeds by an ordered mechanism in which nitrite binds to the oxidised type 2 Cu centres before electron transfer from the reduced type 1 centre occurs. We propose that the two links between the Cu sites of AxNiR, namely His129-Cys130 and His89-Asp92-His94 are utilised for electron transfer and for communicating the status of the type 2 Cu site, respectively. Nitrite binding at type 2 Cu is sensed by the proton abstracting group Asp92 and the type 2 Cu ligand His94, and relayed to the type 1 Cu site via His89 thus triggering an internal electron transfer. The similarity of the type 2 Cu NiR catalytic site to the reduced Cu site of SOD is examined in some detail together with the biochemical evidence for the SOD activity of AxNiR.

Dissimilatory nitrite reductase (NiR) is a key enzyme in the anaerobic respiratory pathway of denitrifying bacteria where nitrate is sequentially reduced to the gaseous products NO, N2O or N2, leading to a significant loss of fixed nitrogen from the terrestrial environment (Payne, 1985). The NiR from Alcaligenes xylosoxidans (AxNiR) (Abraham et al., 1993) belongs to the group of NiRs which utilise copper at the redox active centres. All Cu NiRs isolated so far have a strong band near 600nm arising from a (Cys)S → Cu (II) charge transfer which is characteristic of a type 1 Cu centre. The ratio of intensity of this band to a second charge transfer absorption band at ∼460nm determines whether a Cu NiR is blue or green in colour (Han et al., 1993). AxNiR belongs to the subset of Cu-NiRs which are blue in colour and are thought to have azurin as the electron partner.

Denitrification is one of the main steps of the global nitrogen cycle that is sustained by prokaryotic organisms. Denitrifying bacteria use two entirely different enzymes in this process, one based on haem cd1 prosthetic groups and the other on type 1-type 2 Cu centres. Copper-containing nitrite reductases (NiRs) are sub-divided into blue and green NiRs, which are respectively thought to be redox partners of azurins and pseudo-azurins. Crystallographic structures of the blue nitrite reductase from Alcaligenes xylosoxidans (AxNiR) are presented in the oxidised hexagonal form and the substrate-bound orthorhombic form to 2.1 Å and 2.8 Å resolution, respectively. The complete amino acid sequence of AxNiR has been determined by conventional chemical analysis. A 3 Å structure of AxNiR has been published where the modelling was based on the sequence of another blue NiR. The higher resolution of the hexagonal form together with the correct sequence allows a detailed comparison with the crystallographic structures of the green NiRs. There is a striking difference in the overall surface charge distribution between the two sub-groups, providing a neat structural explanation for their different reactivities to pseudoazurin or azurin and supporting the view that electron transfer proceeds via complex formation. A detailed examination of the type-1 Cu site, the site responsible for the colour, reveals several subtle differences, including a lateral displacement of 0.7 Å for Smet. The structure of the type-2 Cu site, and changes that occur upon substrate binding are discussed in terms of the catalytic mechanism. The similarity of the type 2 Cu site to the catalytic Zn site in carbonic anhydrase and the catalytic Cu site of superoxide dismutase is re-examined in view of the high-resolution (2.1 Å) structure.