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
Gorren, A.C.F.; Blaauwen, T. den; Canters, G.W.; Hopper, D.J.; Duine, J.A. 1996
The electron-transfer properties of H117G- and wild-type azurin were compared by applying both as electron accepters in the conversion of 1-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH... Show moreThe electron-transfer properties of H117G- and wild-type azurin were compared by applying both as electron accepters in the conversion of 1-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH). The reactivity of H117G-azurin was determined in the absence and presence of imidazoles, which can substitute the missing fourth ligand, In the absence of imidazoles, H117G-azurin reacted directly with 4-ethylphenol, this reaction was abolished in the presence of imidazoles, The enzymatic reduction of H117G-azurin by 4-EPMH was 40 times slower than that of wild-type azurin, The rate of this reaction was enhanced by some imidazoles, diminished by others. In all cases the reduction of H117G-azurin was irreversible. These results demonstrate that His117 is vital for electron transfer and effectively protects the copper site against aspecific reactions. Show less
Coremans, J.W.A.; Gastel, M. van; Poluektov, O.G.; Groenen, E.J.J.; Blaauwen, T. den; Pouderoyen, G. van; ... ; Messerschmidt, A. 1995
We report electron-nuclear double-resonance experiments on a single crystal of azurin at 95 GHz and electron-spin-echo envelope-modulation experiments on frozen solutions of azurin and of the H117G... Show moreWe report electron-nuclear double-resonance experiments on a single crystal of azurin at 95 GHz and electron-spin-echo envelope-modulation experiments on frozen solutions of azurin and of the H117G mutant at 9 GHz. The hyperfine and quadrupole tensors of the two remote nitrogens of the histidine ligands of copper are assigned and discussed. A third nucleus is found to contribute to the echo-modulation spectrum and this probably concerns an amide nitrogen of the peptide backbone. Show less
The development of a second generation biosensor that uses azurin as mediator for electron transport at the electrode surface is reported. Azurin is a small (14.6 kDa) bacterial protein that is... Show moreThe development of a second generation biosensor that uses azurin as mediator for electron transport at the electrode surface is reported. Azurin is a small (14.6 kDa) bacterial protein that is relatively stable and that can be expressed in high yield in a heterologous system (E. coli) making it an ideal candidate for protein engineering studies and biosensor development. This protein has a role as a natural electron transfer carrier in the bacterial anaerobic respiratory chain and is able to transfer electrons from cytochrome C-551 to nitrate reductase. The active centre of azurin is a type-1 copper centre. The copper ligand His(117) protrudes through the protein surface, and it has been mutated by site-directed mutagenesis into a glycine (His(117)Gly). The absence of the side-chain of the glycine residue creates an aperture on the protein surface that makes the copper centre accessible to external ligands. In this way the azurin His(117)Gly mutant can accept external ligands, such as 4-methylimidazole, that can be bound to the electrode surface. Attempts to establish fully reversible redox chemistry with the mutated azurin are underway. In principle, this allows the immobilization of azurin on an electrode surface for the construction of a biosensor. In this way azurin can be used as mediator in transferring electrons between the enzymatic system present in solution and the electrode. The combination of protein engineering with electrode surface modification allows further advances in biosensor technology. Show less
Blaauwen, T. den; Hoitink, C.W.G.; Canters, G.W.; Han, J.; Loehr, T.M.; Sanders-Loehr, J. 1993
The copper center of the Pseudomonas aeruginosa His117Gly azurin mutant is accessible to exogenous ligands through an aperture in its surface created by the removal of the endogenous imidazole... Show moreThe copper center of the Pseudomonas aeruginosa His117Gly azurin mutant is accessible to exogenous ligands through an aperture in its surface created by the removal of the endogenous imidazole ligand. Depending on the exogenous ligand, a surprising variety of type 1 and type 2 copper sites can be obtained that are readily distinguished by electronic, EPR, and resonance Raman (RR) spectroscopy. The RR spectrum of type 1 H117G with exogenous imidazole is nearly identical to that of wild-type azurin, indicating that the trigonal geometry and short Cu-S(Cys) bond of approximately 2.15 angstrom have been maintained. With anionic ligands (e.g., Cl-, Br-, N3-), the RR spectra show increased intensity at 370 and 400 cm-1 and a corresponding decrease in intensity at 410 cm-1, suggesting a lengthening of the Cu-S(Cys) bond as the site achieves a more tetrahedral character. An extreme example is the hydroxide adduct of H117G which is green in color and has optical and RR spectra reminiscent of the tetrahedral type 1 site in Achromobaeter cycloclastes nitrite reductase. The fact that the basic RR pattern is little changed in most of the type 1 adducts indicates that the RR spectrum is due primarily to vibrations of the Cu-cysteinate moiety and that its coplanar conformation is conserved. Type 2 H117G proteins are formed by the addition of bidentate exogenous ligands such as histidine and histamine. They have their absorption maxima blue-shifted to 400 nm and their EPR A((parallel-to) values increased to approximately 160 X 10(-4) cm-1, both of which are characteristic of tetragonal Cu sites with Cu-S(thiolate) bonds of >2.25 angstrom. The RR spectra of the type 2 H117G proteins are still dominated by multiple cysteinate-related vibrational modes. However, the vibrational modes with the greatest intensity and Cu-S(Cys) stretching character have shifted approximately 100 cm-1 to lower energy compared to the type 1 sites, consistent with a longer (Cys)S-Cu bond. It is proposed that the tetragonal type 2 character of the bidentate ligand complexes is due to the addition of a fourth strong ligand in the equatorial ligand plane. Show less
Tröger, W.; Butz, T.; Danielsen, E.; Bauer, R.; Thoenes, U.; Messerschmidt, A.; ... ; Blaauwen, T. den 1993
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
Blaauwen, T. den; Kamp, M. van de; Canters, G.W. 1991
