| Summary: | This thesis is focused on elucidating the structure/function relationships of two copper proteins, nitrite reductase and rusticyanin using a combined approach of site-directed mutagenesis and X-ray crystallography. Dissimilatory nitrite reductase (NiR) catalyses the reduction of nitrite to nitric oxide (NO) as part of the key biological process of denitrification. A 'hydrophobic pocket' on the protein surface has been identified as the channel through which the substrate, nitrite, may be guided to the catalytic type 2 Cu site. The residues Glu133 and His313 are at the opening of this pocket and the latter was mutated to a Gin in Alcaligenes xylosoxidans NiR to investigate the role of this residue in substrate guidance. The structures of His313Gln and substrate-bound His313Gln have been determined to 1.65 A and 1.72 A, respectively. These structures confirm that His313 is the port of entry for the substrate and reveal an asymmetric bidentate oxy-co-ordinate binding of nitrite. Residue Trp138, adjacent to the type 1 Cu ligand His139 is one of the residues surrounding the small depression speculated to be important in the complex formation with the physiological redox partners, azurin I and azurin II. This Trp residue was mutated to a His and its structure determined to 1.60 A. The ability of the Trp138His mutant to reduce nitrite was investigated using both an artificial donor and a physiological partner, azurin I. These enzymatic activity experiments revealed that whilst the protein is essentially fully active using methyl viologen as the electron donor, there is a significant decrease in the activity using azurin I when compared to the native protein. These observations suggest that Trp138 is an important residue in complex formation and this with residues in the vicinity represent a likely docking surface for azurin. A simultaneous Asp92His/Met144Leu mutation in NiR was undertaken to investigate the role of redox coupling between the two copper centres and proton ii extraction. The residue Asp92 has been demonstrated to be an important residue in a water-mediated pathway for delivering protons to the T2Cu site for the reduction of nitrite. The mutation of Asp92 has a significant effect on the protein, reducing the activity to -10%. The structure determination of Asp92His/Met144Leu NiR to 1 .65 A revealed that despite the perturbation of Asp92 a route exists from the surface of the protein to deliver protons to the T2Cu centre. The blue-copper protein rusticyanin has attracted considerable interest due to its unique properties of high redox potential and stability to extremely high pH. The copper site of rusticyanin is similar to other blue-copper proteins with three strong ligands His85, Cys138, His143 and a relatively weaker Met148 ligand in an axial position. A mutation of His143 to a Met yielded a structure determination to 1.1 A from which refinement using anisotropiC displacement parameters provides an exceptional basis to examine the protein in detail. Furthermore, the metrical accuracy obtained from such refinement coupled with EXAFS analysis suggests that the Cu site alone is not wholly responsible for the properties of rusticyanin. A 2.3 A structure of His143Met demonstrates that the molecules pack in a head to head fashion thus providing further structural evidence to the hypothesis that this interaction is important for electron transfer from a partner protein. The interaction is similar to those present in Met148Leu and Met148Gln rusticyanin crystal structures although the replacement of His with Met disrupts the solvent mediated hydrogen bonding observed in those structures. In His143Met the two molecules are now associated via non-bonded interactions. In conclusion, the crystal structures of the NiR mutants presented in this thesis provide direct evidence for both the point of interaction with its proposed physiological redox partner and the probable mode of nitrite binding in blue CuNiR's as well as confirming the role of Asp92 in proton abstraction. The two crystal structures of His143Met rusticyanin have shed light on particular structural iii features that may account for the proteins unique properties which can now be examined with a future site-directed mutagenesis programme.
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