The X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved. In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating... Show moreThe X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved. In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating histidine residues is replaced by a glycine, creating an empty space in the coordination sphere of the copper ion. The crystal structure of His117Gly azurin at 2.4 Angstrom resolution showed that this mutant had undergone partial oxidation at the disulfide bridge between Cys3 and Cys26 and full oxidation at the copper ligand Cys112. There is no copper present in the crystallized form and the bulky group of the oxidized cysteine at position 112 causes large structural rearrangements in the protein structure, especially in the loops connecting the beta-sheets. In the structure of the wild-type holo-azurin from P. aeruginosa the hydrophobic patch is important for the packing of the azurin molecules into dimers which then arrange into tetramers. The completely different packing of the apo-His117Gly mutant can be explained by the disruption bf the hydrophobic patch area by the mutation-induced main-chain conformational change of residues 112 to 115. The structure of apo-His46Gly azurin at 2.5 Angstrom resolution is the same as the wild-type structure except for the immediate environment at the site of the mutation. In the His46Gly structure water molecules are found at positions that in the wild-type structure are occupied by the imidazole ring of His46 and the copper ion. The imidazole ring of His117 is shifted by about 1 Angstrom towards the surface of the protein, similar to that observed for 50% of the molecules in the wild-type apo-azurin structure. This shift causes a slight rearrangement of the monomers within the tetramer such that one local dyad becomes a crystallographic dyad parallel to the c-axis. This leads to a change in the space group from P2(1)2(1)2(1) to P2(1)2(1)2. (C) 1997 Academic Press Limited. Show less
The blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to... Show moreThe blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to the copper centre. This residue shows in fluorescence spectra the highest energy emission of tryptophan-containing compounds at room temperature, which is explained by its rigid and highly hydrophobic environment. In order to investigate the role of the Trp residue in electron transfer and the influence of its environment, two mutations (I7S and F11OS) were introduced that were thought to increase the polarity and the mobility in its environment. The crystal structures of these mutants were solved at 2.2 Angstrom and 2.3 Angstrom resolution, respectively These provide a structural basis for the changes observed in fluorescence spectra compared with the wild-type protein. We conclude from our results that these changes are not caused by a change in the dynamics of the Trp residue itself, but exclusively by an increased effective dielectric constant of the microenvironment of Trp48 and by changes in mobility of the mutated residues. (C) 1996 Academic Press Limited Show less
The nuclear quadrupole interaction (NQI) of Cd-111 substituted for Cu(II) on type-1 sites in blue copper proteins is characterized by high values of omega0 in the region of 300 Mrad/s, close to... Show moreThe nuclear quadrupole interaction (NQI) of Cd-111 substituted for Cu(II) on type-1 sites in blue copper proteins is characterized by high values of omega0 in the region of 300 Mrad/s, close to that for the catalytic zinc site in alcohol dehydrogenase. Type-I Cu has usually two sulfur ligands and two nitrogen ligands and in some cases an oxygen ligand in either a distorted tetrahedral geometry or in a trigonal bipyramidal geometry. The near tetrahedral arrangement together with the ligand sphere containing the same number of sulfur ligands explains the value of omega0 in the blue copper proteins. The present work determined the partial NQI for methionine using the known structure of azurin. This value was then used in the angular overlap model to calculate the NQI for ascorbate oxidase the structure of which is also known and gave good agreement with experiment. NQI data for laccase and stellacyanin the structures of which are unknown, are also given. Show less
Kamp, M. van de; Canters, G.W.; Wijmenga, S.S.; Lommen, A.; Hilbers, C.W.; Nar, H; ... ; Huber, R. 1992
Complete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14 000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH... Show moreComplete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14 000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40-degrees-C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear H-1 Hartmann-Hahn nuclear Overhauser (3D H-1 HOHAHA-NOESY) spectrum and a 3D heteronuclear H-1 nuclear Overhauser H-1{N-15} single-quantum coherence (3D H-1{N-15} NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40-degrees-C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 (NH)-H-epsilon2 resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed. Show less
Nar, H.; Huber, R.; Messerschmidt, A.; Filippou, A.C.; Barth, M.; Jaquinod, M.; ... ; Canters, G.W. 1992
Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and... Show moreAzurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions.An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45.The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative. Show less
Nar, H.; Messerschmidt, A.; Huber, R.; Kamp, M. van de; Canters, G.W. 1991
