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    The expression of the gene for Azurin from Alcaligenes denitrificans in E. coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1994) Bateson, Joseph Anthony
    Azurin is a protein which funtions in electron transport and has been found to bind copper when it is expressed in its native bacterial host. In this thesis the azurin from Alcaligenes denitrificans was used. This protein is 129 amino acids long with a molecular weight of 14. 600 dalions. The azurin coding gene from Alcaligenes denitrificans had previously been cloned into a plasmid which allows an E. coli expression system to be used. Azurin was purified from the E. coli hosts using the same procedures as for purifying copper-azurin from the native hosts but was found to remain apparently impure, according to spectrophotomctric data. Efforts to increase the production of the protein by using different expression systems and by refining the existing expression system failed to increase the apparent yield of copper-azurin. Efforts to refine the purification procedure also failed to increase the amount of copper-azurin that was purified. Various experiments were performed to demonstrate that azurin was expressed and processed correctly in the E. coli host. Protein was expressed in a copper-rich and copper-sparse environment. Copper-azurin was purified from the copper-rich environment, while very little copper-azurin could be extracted from the copper-sparse environment. The results described in this thesis suggest that when azurin from A. denitrificans is expressed in an E. coli host using standard media with no copper added, the predominant form of azurin produced in zinc-azunn. As mutants are going to made of this protein, conditions where the protein would bind only copper were required. The ideal conditions for this are still to be calculated but results from this thesis would suggest that copper concentrations in the region of 0.25 mM lead to 65% incorporation of copper. compared to 17% when no copper is added to the E. coli growth medium. E. coli cells were shown to grow with no apparent inhibition of growth in 3.0 mM of CuSO4. This concentration of copper in the growth medium may allow the production of a much higher ratio of copper-azurin compared to zinc-azurin than has been achieved so far.
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    A comparison of copper(I) and copper(II) bound to thioether ligands : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University
    (Massey University, 1975) Norris, Gillian E
    The structures of Cu(II) (DTH)2(BF4)2 (where DTH = 2,5-dithiahexane) and Cu(I) (DTO)2BF4 (where DTO = 3,6-dithiaoctane) have been investigated by single crystal X-ray diffraction techniques. After full matrix least squares refinement of the structures, with anisotropic temperature factors for all non hydrogen atoms in the Cu(II) structure, and for all atoms larger than fluorine in the Cu(I) structure, the conventional R factor converged to a final value of 0.057 for the Cu(II) structure, and 0.082 for the Cu(I) complex. The dark red crystals of the Cu(II) complex belong to the centrosymmetric monoclinic space group P2 1/c. with a = 8.082(3)Å, b = 10.282(3)Å, c = 11.893(4)Å and β = 115.3 degrees. Two dithiahexane ligands and two BF4-ions were found to co-ordinate to the Cu(II) ion to form a tetragonally distorted octahedron, with four Cu(II)-S bonds averaging 2.317Å in length, and two longer Cu(II)-F bonds averaging 2.576Å. The four sulphur atoms are part of two five membered Cu(II)-S-C-C-S rings in which both carbons are on the same side of the plane containing the copper and sulphur atoms. The colourless crystals of the Cu(I) complex were obtained in the non-centric orthorhombic space group Pna2 1, with a = 14.581(2)Å, b = 13.421(2)Å and c = 10.781(2)Å. The molecules exist as discrete monomeric species, with no co-ordination of the BF4- ion to the metal ion. The two ligand molecules co-ordinate to the Cu(I) ion to form a distorted tetrahedron, with the S-Cu(I)-S angles varying between 94.0 and 121.1 degrees. The four Cu(I)-S bonds average 2.307Å in length, and hence are approximately equal to the Cu(II)-S bonds (within experimental error). The two five membered Cu(I)-S-C-C-S rings are both in a gauche conformation, with one carbon below the plane containing the Cu(I) and S atoms, and the other above. The BF4- ion was disordered and was refined using rigid group restrictions. Cu-S co-ordination is thought to occur in some copper containing oxidation-reduction proteins. The observation of similar Cu-S bond distances when Cu(I) and Cu(II) are co-ordinated to thioether ligands (resembling the side chain of the amino acid methionine) may therefore be of direct relevance to the copper co-ordination in such proteins.
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    The three dimensional structure of azurin, a blue copper protein, at 3Å resolution : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University
    (Massey University, 1982) Norris, Gillian E.
    Purifications and crystallizations of two electron transfer proteins, azurin and cytochrome c', from Alcaligenes denitrificans and Alcaligenes sp. NC1B 11015 have been carried out. The azurin crystals from Alcaligenes denitrificans were found suitable for high resolution X-ray structure analysis. They are orthorhombic, space group C2221 (with marked tetragonal pseudosymmetry), cell dimensions a = 75.0Å, b = 74.1Å, c = 99.5Å, with two molecules per asymmetric unit. A 3Å resolution electron density nap of azurin was calculated. Four isomorphous heavy atom derivatives, prepared with KAu(CN)2, uranyl acetate, Hg(NH3)2Cl2 and (KAu(CN)2 + uranyl acetate) (a double derivative) were used to calculate phases by the method of isomorphous replacement, giving an overall figure of merit of 0.614. The polypeptide chain could be followed unambiguously in both protein molecules in the asymmetric unit, with the aromatic sidechains, in particular, readily identifiable because of their distinctive appearance. Kendrew skeletal models were built for both molecules, the polypeptide chain (consisting of 129 amino acids) being found to be folded into an eight-strand β-barrel, with an additional flap containing a short helix. There is one disulphide bridge within the barrel. The topology of the molecule was found to be the same as that of plastocyanin, and a comparison of the three dimensional structures of azurin and plastocyanin allowed the sequences to be aligned on structural rather than purely statistical grounds. It also established the probability that the two proteins have evolved from a common ancestor. The copper atom has a highly-distorted tetrahedral co-ordination geometry, forming three shorter bonds (length approximately 2Å), with a cysteine thiolate sulphur (Cys 112) and two histidine imidazole nitrogens (His 46 and 117), as well as a longer bond (approximately 3Å) with a methionine thioether sulphur (Met 121). A surprising result was the closeness of a peptide carbonyl oxygen, that of Gly 45, to the copper atom. At this stage of the structure analysis it is not clear whether it should be regarded as a ligand, or not. Reduction of the protein crystals with chromous ions was attempted, and the results are discussed in terms of the possible electron transfer mechanism of the protein. The cytochrome c' crystals from both species of bacteria are hexagonal, space group P 6l22 (or P 6522), cell dimensions a = b = 54.7Å, c ~ 185Å γ = 120°, with one subunit (molecular weight 14,000) in the asymmetric unit. No structural work has been carried out on these.
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    Crystallographic analyses of apo and reduced azurins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University
    (Massey University, 1991) Shepard, William E. Botelho
    Crystals of the blue copper protein azurin, from Alc. denitrificans, have been prepared in the apo (metal-free) and reduced (Cu(I)) forms by soaking oxidised (Cu(II)) azurin crystals in solutions containing cyanide and ascorbic acid, respectively. The apo and reduced azurin crystals are isomorphous with the oxidised crystals, containing two protein molecules in the asymmetric unit. A CAD4 diffractometer was used to collect X-ray diffraction data from these crystals, to 2.2Å resolution for apo-azurin and 1.9Å for reduced azurin. Both crystal structures have been refined using restrained least-squares methods, treating the two protein molecules of the asymmetric unit independently, to give final R-factors of 0.158 for apo-azurin (1956 protein atoms and 275 solvent atoms) and 0.166 for reduced azurin (1967 protein atoms and 269 solvent atoms). Analysis and comparison of these structures with that of the oxidised form show that the protein structure is essentially identical between all three forms. However, very small changes, which are consistent between the two independent molecules of the asymmetric unit, are apparent at and near the copper binding site. Upon reduction of the oxidised form, all copper distances (left unrestrained during refinement) increase by 0.05-0.10Å, while the geometry remains unchanged. Thus the copper in reduced azurin is bound strongly by one thiolate sulphur (Cys-112) and two imidazole nitrogens (His-46 and His-117) in a trigonal arrangement, while a thioether sulphur (Met-121) and a carbonyl oxygen (Gly-45) approach the copper from axial directions to complete a distorted trigonal bipyramidal geometry. The CU...Sδ 121 distance is 3.2Å, and does not agree with the 2.7Å distance predicted by EXAFS studies despite attempts to restrain this distance during refinement. Two S...HN hydrogen bonds, between the cysteine sulphur and peptide NH groups, are shortened by ≈0.2Å upon reduction of Cu(II) to Cu(I). The configuration of the copper ligands and the surrounding structure are maintained when the copper is removed. This shows that the folding of the polypeptide chain determines the conformation of the metal ligands, enforcing a distorted geometry on the copper centre in both oxidised and reduced forms. The structure of the copper binding site in the apo form is more similar to the oxidised form than to the reduced form. The observations made in the analysis of these three forms of azurin have implications for the biological function of the protein, which is thought to be electron transfer. The distorted geometry of the copper, which is enforced upon the metal by the protein, supports the concept of the entatic state, whereby the copper is held in a geometry approximating the transition state between the coordination preferences of Cu(II) and Cu(I). Since minimal changes at the active site occur upon reduction, the protein accomplishes its function of fast electron transfer by minimising the activation energy of the process.