The transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood... Show moreThe transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood mechanism by which they interact. To better understand the effect of crosslinking on electron transfer rates, we have constructed dimers of azurin by crosslinking the monomers. The measured electron exchange rates, combined with crystal structures of the dimers, demonstrate that the length of the linker can have a dramatic effect on the structure of the dimer and the electron transfer rate. The presence of ordered water molecules in the protein protein interface may considerably influence the electronic coupling between redox centers. Show less
Amsterdam, M.C. van; Ubbink, M.; Jeuken, L.J.C.; Verbeet, M.P.; Canters, G.W.; Messerschmidt, A. 2001
The green Met121His mutant of the blue-copper protein azurin has been investigated by pulsed electron paramagnetic resonance (EPR) spectroscopy at 95 GHz on a single crystal. The axial histidine is... Show moreThe green Met121His mutant of the blue-copper protein azurin has been investigated by pulsed electron paramagnetic resonance (EPR) spectroscopy at 95 GHz on a single crystal. The axial histidine is more strongly bound to copper than the methionine for the wild-type protein. The g tensor of M121H is found to be virtually axial and the z principal axis perpendicular to the plane spanned by copper and the nitrogens of the ligating histidines 46 and 117. The direction of the x axis is close to the bond direction from copper to the nitrogen of histidine 46. Theoretical analysis of the axiality and the orientation of the principal axes shows that the wave function of the unpaired electron on copper, largely d(xy) for blue-copper proteins, acquires some d(yz) character for M121H. Comparison of these results with data for wild-type azurin and the mutant M121Q provides insight into the subtle relation between the electronic and the geometric structure of blue-copper sites. Show less
The structural details of the metal site in the [His121]azurin mutant from Alcaligenes denitrificans where the axial methionine has been replaced by a histidine have been studied after substitution... Show moreThe structural details of the metal site in the [His121]azurin mutant from Alcaligenes denitrificans where the axial methionine has been replaced by a histidine have been studied after substitution with the divalent cadmium ion and the monovalent silver ion. The studies have been carried out in solution using the technique of perturbed angular correlations of gamma-rays (PAC) of the two isotopes, Ag-111 and Cd-111m.In the pH range 6-9, the PAC spectra for cadmium-substituted [His121]azurin reveals a pH-independent equilibrium between two different metal-coordination geometries. Interpretation of the PAC data shows agreement between the dominating coordination geometry and that derived from X-ray diffraction on the Cu(II)[His121] azurin at high pH (Messerschmidt, A., unpublished results). Thus: it appears likely that cadmium for this geometry is four coordinated to the ligands His46, His117: Cys112, and His121. The other geometry is best interpreted as a substitution of His121 by a solvent water ligand. These interpretations stem from predictions of the experimentally determined nuclear quadrupole interactions (NQI) via the simple angular overlap model (AOM). At low pH (3.8), the concentration of the former species is reduced to 50% of its high pH value suggesting a pK of about 4 for His121. Two different coordination geometries have also been observed for the Cu(II) protein and assigned a type 1.5 and a type 1 copper site [Kroes, S. J., Hoitink, C. W. G., Andrew, C. R., Ai, J., Sanders-Loehr, J., Messerschmidt, A., Hagen, W. R. & Canters, G. W. (1996a) fur: J. Biochem. 240, 342-351].For silver-substituted [His121]azurin, several notable changes occur relative to the cadmium-substituted protein. At least four different metal-coordination geometries exist for silver[His121]azurin in the pH range 4-8. Changes in the population of these coordination sites occurs between pK 4 and pH 5, and pK 5 and pH 6. Furthermore, in contrast to the cadmium-substituted protein, only a single coordination geometry is present above pH 6. The change in population occurring between pH 5 and pH 6 suggests ionization of a non-coordinating histidine, here proposed as His121. The change in population at low pH could then be due to protonation of an additional coordinating histidine such as His46 or His117. The single coordination geometry existing at pK values above 6 for the silver protein cannot within our model calculations be described with His121 coordinated. However, it can be described with a coordinated water molecule but in a different angular position than for His121 in the copper protein (Messerschmidt, A., unpublished results). The reduced tendency for silver to coordinate His121 is in agreement with the general trend of lower pK values for ligands coordinating to monovalent ions relative to divalent ions.In conclusion, this work demonstrates that mutation of Met121 to other amino acid residues opens the possibility of other coordination geometries than the rigid three-coordinated structure observed for wild-type azurin, especially the possibility of increasing the coordination number by either solvent water ligands or the substituting amino acid. Furthermore, it opens up the possibility for different coordination geometries for monovalent and divalent metal ions as observed here and previously for the [Leu121]azurin mutant [Bauer, R., Danielsen, E., Kemmingsen, L., Bjerrum, M. J., Hansson, ij. & Singh, K. (1997) J. Am. Chem. Sec. 119, 157-163]. Show less
At 95 GHz, deep electron spin echo envelope modulations of ct single crystal of the blue copper protein azurin have been observed. The modulations arise from the coordinated nitrogens of the... Show moreAt 95 GHz, deep electron spin echo envelope modulations of ct single crystal of the blue copper protein azurin have been observed. The modulations arise from the coordinated nitrogens of the histidines that ligate to copper. From the ESEEM frequencies, hyperfine and quadrupole tensors of these nitrogens have been deduced. The isotropic hyperfine coupling of the copper-bound nitrogen of histidine-117 is 1.4 times larger than that of histidine-46. The anisotropic hyperfine tensors show that the wave function of the unpaired electron on both coordinated nitrogens mainly concerns the sigma bonds with copper. Show less
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 Cu ligand Met121 in azurin of Alcaligenes denitrificans was mutated to His. The spectroscopic and mechanistic properties of [M121H]azurin appear to be pH dependent with a pK(a) of 3.8 due to... Show moreThe Cu ligand Met121 in azurin of Alcaligenes denitrificans was mutated to His. The spectroscopic and mechanistic properties of [M121H]azurin appear to be pH dependent with a pK(a) of 3.8 due to the ionization of His121. The [M121H]azurin mutant exhibits two major distinct metal-site-coordination geometries which coexist in solution according to a pH-dependent equilibrium. Both species have been spectroscopically characterized by ultraviolet-visible, EPR and resonance Raman spectroscopies. At neutral DH. His121 is deprotonated and acts as the fourth ligand of the Cu; the spectroscopic characteristics of the Cu site at this pH are halfway between those of a type-1 and a type-2 Cu site, and the site is referred to as a type-1.5 or intermediate Cu site. The spectral data are compatible with a tetrahedral geometry of this site. At low pH, the spectroscopic data indicate that [M121H]azurin has a trigonal type-1 rhombic Cu site. 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
We report pulsed Electron-Nuclear-DOuble-Resonance (ENDOR) experiments at 95 GHz and 1.2 K of single crystals of the blue-copper protein azurin and its fully N-15-enriched analogue. The nitrogen... Show moreWe report pulsed Electron-Nuclear-DOuble-Resonance (ENDOR) experiments at 95 GHz and 1.2 K of single crystals of the blue-copper protein azurin and its fully N-15-enriched analogue. The nitrogen ENDOR spectra and their variation with the orientation of the magnetic field with respect to the crystals are described, and it is shown that contributions of five distant nitrogens may be distinguished. For the remote nitrogens of the copper ligands histidines-46 and -117 complete hyperfine and quadrupole tensors are presented and discussed in relation to the structure of the copper site and the extension of the wave function of the unpaired electron. Besides these nuclei three backbone nitrogens show up in the ENDOR spectra, and their hyperfine tensors are reported. One of the backbone nitrogens concerns that of cysteine-112; the others are tentatively assigned to histidine-46 and glycine-45. The wave function of the unpaired electron of oxidized azurin is found to be smeared out over both the copper ligands and parts of the protein backbone. Show less
Electron-spin-echo-detected electron-paramagnetic-resonance spectroscopy at 95 GHz (W-band) on frozen solutions and single crystals of the azurin mutant M121Q has enabled the determination of... Show moreElectron-spin-echo-detected electron-paramagnetic-resonance spectroscopy at 95 GHz (W-band) on frozen solutions and single crystals of the azurin mutant M121Q has enabled the determination of accurate principal values of the g-tensor and the orientation of the principal axes of these tensors of all molecules in the unit cell with respect to the crystallographic axes. A combination of EPR and X-ray structural data results in four possible orientations of the g-tensor axes in the copper site of M121Q. Theoretical considerations lead us to prefer one of these, in which one of the principal axes makes an angle of 10 degrees with the Cu-O epsilon(Gln121) bond; the other two are approximately parallel to the NNS plane and are rotated 55 degrees with respect to the Cu-S gamma-Cys112) bond. The description of the rhombicity of the g-tensor indicates that the wave function of the unpaired electron on copper consists of a d(xy) orbital with a small but significant admixture of d(z)2 character. 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 geometries of the metal sites in cadmium-substituted azurins have been investigated by Cd-111m perturbed angular correlation (PAC), The study includes wild type azurin as well as Met(121)... Show moreThe geometries of the metal sites in cadmium-substituted azurins have been investigated by Cd-111m perturbed angular correlation (PAC), The study includes wild type azurin as well as Met(121) mutants of azurin, where methionine has been substituted by Ala, Asn, Asp, Gin, Glu, and Leu.The nuclear quadrupole interaction of wild type azurin analyzed in the angular overlap model is well described as coordination of His(46), His(117), and Cys(112) and cannot be described by coordination of Met(121) and/or Gly(45).For most of the mutants, there exist two coordination geometries of the cadmium ion, With the exception of the Gau and Asp mutants, one of the conformations is similar to the wild type conformation. The other coordination geometries are either best described by a coordinating water molecule close to the original methionine position or by coordination by the substituting amino acid, These experiments show that even though the methionine does not coordinate it plays an important role for the geometry of the metal site.The nuclear quadrupole interaction of stellacyanin was also measured, The value resembles the most prominent nuclear quadrupole interaction of the Met(121) --> Gin mutant of Alcaligenes denitrificans azurin, indicating that the structures of the two metal sites are similar. Show less
Field-swept electron spin echo spectroscopy at 95 GHz on a frozen solution and a single crystal of azurin from Pseudomonas aeruginosa has enabled the determination of the complete g-tensor. Highly... Show moreField-swept electron spin echo spectroscopy at 95 GHz on a frozen solution and a single crystal of azurin from Pseudomonas aeruginosa has enabled the determination of the complete g-tensor. Highly accurate principal values have been obtained (g(x) = 2.0393 +/- 0.0004, g(y) = 2.0568 +/- 0.0007, g(z) = 2.273 +/- 0.004), and the orientation of the principal axes of the g-tensor with respect to the copper site has been established. One of the principal axes makes an angle of 15 degrees with the Cu-S delta(Met121) bond. The other axes lie almost in the NNS plane and are rotated by 24 degrees with respect to the Cu-S gamma(Cys112) bond. The g-tensor does not corroborate descriptions of the copper site in terms of effective C-3v, C-2v, or C-s symmetry. The observed orientation of the principal axes presents a sensitive point of reference for future quantum-chemical considerations. 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
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