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At least 217 records · Page 12Linked to original sources
PubMed1995

Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT.

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.

Amino Acid Sequence↗
PubMed1987

[Oxidation by nitrite of azurin and cytochrome c-551 from Pseudomonas aeruginosa].

The nitrite oxidizes reduced azurin and cytochrome c-551 from Pseudomonas aeruginosa. The effects of pH, ionic strength and concentrations of nitrite, EDTA and the protein on the oxidation were investigated. The results obtained indicate that nitrite interacts not only with the terminal electron carrier of the nitrite reducing chain (nitrite reductase, cytochrome cd1) but also with the intermediate electron carrier components of the chain (azurin and cytochrome c-551).

Azurin↗
PubMed1990

Optical absorption spectra of azurin and stellacyanin in glycerol/water and ethylene glycol/water solutions in the temperature range 290-20 K.

We have measured the optical absorption spectra of azurin and stellacyanin in the wavelength range 1100-350 nm and in the temperature interval 290-20 K. Samples are protein aqueous solutions containing 65% (v/v) glycerol or ethylene glycol as cryoprotectants and remain homogeneous and transparent throughout the whole temperature range investigated. Spectra are deconvoluted into Gaussian components and the temperature dependence of the zeroth, first and second moments of the various bands is analyzed, within the harmonic Franck-Condon approximation, to obtain information on the stereodynamic properties of the active sites of these proteins. Sizable differences of the integrated intensities of all the bands with temperature are observed and are attributed to variations of the metal-ligand relative positions (i.e., deformations of the active site) that occur as the temperature is lowered. The mean effective frequency of the nuclear vibrations coupled to all the observed bands is about 150 cm(-1) for both proteins in both solvents used; this indicates that the electronic transitions from which the optical spectrum originates are substantially coupled with low-frequency vibrational modes, likely ligand-metal-ligand deformations. The relevance of the stereodynamic properties of azurin and stellacyanin, investigated in this work, to their functional behavior is also suggested.

Journal Article↗
PubMed1997

Resolving multiple protein conformers in equilibrium unfolding reactions: a time-resolved emission spectroscopic (TRES) study of Azurin.

Unlike steady-state spectrofluorimetry, time-resolved emission spectroscopy (TRES) can resolve emissions from fluorophores with similar quantum yields and overlapping steady-state emission spectra. Time-resolved emission studies of the protein-intrinsic fluorophore, tryptophan (Trp), can thus potentially be used to examine protein conformational heterogeneity in solution, as well as to investigate the existence of populated intermediate structural states in equilibrium unfolding reactions of single-tryptophan proteins. Here, the single-Trp copper protein, azurin, is examined in various concentrations of guanidine hydrochloride (GdnCl) with its disulphide bond in an intact state. Interestingly, multiple envelopes of Trp emission are observed in all TRES spectra acquired, instead of just two emission envelopes (corresponding to the native and unfolded states) expected from two-state unfolding. These envelopes appear to be centred around the same set of emission wavelengths in different TRES spectra, and only intensities and decay rates vary with the concentration of denaturant used. This suggests that structural states representing different levels of exposure of Trp to the aqueous solvent might, in fact, be populated at equilibrium during the unfolding of azurin by GdnCl.

Journal Article↗
PubMed1997

An EPR investigation on the structural heterogeneity in copper azurin and plastocyanin.

The effects of cooling rate and of solvent properties on the active site heterogeneity of two copper proteins, azurin and plastocyanin, have been investigated at low temperature by electron paramagnetic resonance spectroscopy. The spectra of theses proteins have been analyzed, by an accurate computer simulation, in terms of a distribution of some relevant spin-Hamiltonian parameters. The results show that the structural heterogeneity of both proteins, quantified by the width of the distribution in the g and A tensors, is affected by both the freezing procedure and the solvent composition. In particular, the g distribution width is found to be reduced in the slow cooling regime; such a reduction appearing more significant when glycerol is added to the protein solutions. Despite of the similarity in the copper ion microenvironments of the two proteins, the effects are more pronounced in azurin. The results are discussed also in connection with the role played by the solvent and the rate of freezing in featuring the conformational substate landscape.

Journal Article↗
PubMed1999

Long-term molecular dynamics simulation of copper azurin: structure, dynamics and functionality.

A long-term molecular dynamics simulation (1.1 ns), at 300 K, of fully hydrated azurin has been performed to put into relationship the protein dynamics to functional properties with particular attention to those structural elements involved in the electron transfer process. A detailed analysis of the root mean square deviations and fluctuations and of the intraprotein H-bonding pattern has allowed us to demonstrate that a rigid arrangement of the beta-stranded protein skeleton is maintained during the simulation run, while a large mobility is registered in the solvent-exposed connecting regions (turns) and in the alpha-helix. Moreover, the structural elements, likely involved in the electron transfer path, show a stable H-bonding arrangement and low fluctuations. Analysis of the dynamical cross-correlation map has revealed the existence of correlated motions among residues connected by hydrogen bonds and of correlated and anti-correlated motions between regions which are supposed to be involved in the functional process, namely the hydrophobic patch and the regions close to the copper reaction center. The results are briefly discussed also in connection to the current through-bond tunneling model for the electron transfer process. Finally, a comparison with the structural and the dynamical behaviour of plastocyanin, whose structure and functional role are very similar to those of azurin, has been performed.

Journal Article↗
PubMed1997

Paramagnetic NMR Spectroscopy of Cobalt(II) and Copper(II) Derivatives of Pseudomonas aeruginosa His46Asp Azurin.

NMR spectra of paramagnetic Co(II) and Cu(II) derivatives of Pseudomonas aeruginosa His46Asp azurin have been investigated. In each derivative, assignment of hyperfine-shifted resonances outside the diamagnetic envelope of spectra recorded at 200 and 500 MHz confirms that the Asp carboxylate is coordinated to the paramagnetic metal center. The reduced paramagnetic shifts of the Cys112 proton resonances in Cu(II) and Co(II) His46Asp azurins compared to those of the corresponding wild type proteins indicate that metal-S(Cys) bonding is weakened in this mutant. The downfield shifts of the gamma-CH(2) of Met121 suggest a stronger interaction between the metal and the Met thioether group than is present in the wild type protein. Molecular modeling of the metal site structure indicates a distorted tetrahedral geometry with Asp46 (monodentate carboxylate), Cys112, and His117 equatorial ligands. In this structure, the metal ion is shifted 0.3 Å out of the O(Asp)S(Cys)N(His) trigonal plane toward Met121.

Journal Article↗
PubMed2004

Ambipolar transistors based on azurin proteins.

A new type of transistor is presented. It is realised by using a metalloprotein; namely, azurin. Thanks to their natural functional characteristics, which involve inter- and intramolecular electron transfer, metalloproteins are good candidates for biomolecular nanoelectronics. The implementation of a prototype of protein transistor operating in air and in the solid state based on self-organised films of azurins is reported. Experimental current-voltage characteristics are shown. The new device presents an ambipolar behaviour as the gate bias voltage is changed. Exploiting this peculiar characteristic, a fully integrated logic gate which can be a good starting point for a new class of nanoelectronics devices has been realised.

Journal Article↗
PubMed1995

Structure of the azurin mutant Phe114Ala from Pseudomonas aeruginosa at 2.6 A resolution.

The crystal structure of azurin mutant Phe114Ala from Pseudomonas aeruginosa has been solved by molecular replacement. The final crystallographic R value is 0.185 for 9832 reflections to a resolution of 2.6 A. The root-mean-square deviation for main-chain atom positions is 0.020 A between the four independent monomers in the asymmetric unit. The mutant Ala114 crystallized from PEG 4000 in a new crystal form and the crystals are monoclinic, P2(1), a= 51.0, b = 83.6, c= 66.4 A and beta = 110.5 degrees. The four molecules in the asymmetric unit are packed as a dimer of dimers and are related by an approximate twofold axis. The dimer packing and the dimer contact region are very similar to that of the Alcaligenes denitrificans azurin dimer. The mutation was performed at residue Phe114, which exhibits a pi-electron overlap with the copper ligand His117, to investigate its suggested role in the electron self-exchange reaction. Removal of steric constrains from the phenylalanine side chain created a somewhat different geometry around the copper site with an increased mobility of His117 resulting in an enlarged Cu-N length which may be responsible for the slight differences obtained in the spectral properties of the mutant versus the wild-type protein.

Journal Article↗
PubMed1996

Mutant Met121Ala of Pseudomonas aeruginosa azurin and its azide derivative: crystal structures and spectral properties.

The crystal structures of the azurin mutant Met121Ala and its azide derivative Met121Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met121Ala mutant, the distance between the copper ion and His117 increases by 0.34 A compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74 A. In the Met121Ala-azide structure, the azide anion occupies the cavity created by replacing the Met121 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met121Ala mutant still maintains the properties of an ordinary type 1 copper site while the Met121Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

Journal Article↗
PubMed2000

Molecular dynamics simulation of solvated azurin: correlation between surface solvent accessibility and water residence times.

A system containing the globular protein azurin and 3,658 water molecules has been simulated to investigate the influence on water dynamics exerted by a protein surface. Evaluation of water mean residence time for elements having different secondary structure did not show any correlation. Identically, comparison of solvent residence time for atoms having different charge and polarity did not show any clear trend. The main factor influencing water residence time in proximity to a specific site was found to be its solvent accessibility. In detail for atoms belonging to lateral chains and having solvent-accessible surface lower than approximately 16 A(2)a relation is found for which charged and polar atoms are surrounded by water molecules characterized by residence times longer than the non polar ones. The involvement of the low accessible protein atom in an intraprotein hydrogen bond further modulates the length of the water residence time. On the other hand for surfaces having high solvent accessibility, all atoms, independently of their character, are surrounded by water molecules which rapidly exchange with the bulk solvent. Proteins 2000;39:56-67.

Azurin↗
PubMed2005

Calculation of the redox potential of the protein azurin and some mutants.

Azurin from Pseudomonas aeruginosa is a small 128-residue, copper-containing protein. Its redox potential can be modified by mutating the protein. Free-energy calculations based on classical molecular-dynamics simulations of the protein and from mutants in aqueous solution at different pH values were used to compute relative redox potentials. The precision of the free-energy calculations with the lambda coupling-parameter approach is evaluated as function of the number and sequence of lambda values, the sampling time and initial conditions. It is found that the precision is critically dependent on the relaxation of hydrogen-bonding networks when changing the atomic-charge distribution due to a change of redox state or pH value. The errors in the free energies range from 1 to 10 k(B)T, depending on the type of process. Only qualitative estimates of the change in redox potential by protein mutation can be obtained.

Amino Acid Substitution↗
PubMed2006

Spin-density distribution in the copper site of azurin.

A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur.

Anisotropy↗
PubMed2003

Free energy perturbation and molecular dynamics calculations of copper binding to azurin.

Free energy perturbation/molecular dynamics simulations have been carried out on copper/azurin systems calculating the binding affinities of copper (II) ion to azurin either in the native or in the unfolded state. In order to test the validity of the strategy adopted for the calculations and to establish what force field is suitable for these kinds of calculations, three different force fields, AMBER, CVFF, and CFF, have been alternatively used for the calculations and the results have been compared with experimental data obtained by spectroscopic titrations of copper (II)/azurin solutions and denaturation experiments. Our findings have pointed out that only CFF gives satisfactory results, thus providing a reliable tool for copper binding simulations in copper protein.

Algorithms↗
PubMed2006

A computational protocol to probe the role of solvation effects on the reduction potential of azurin mutants.

Semiquantitative relationships between thermodynamic parameters of Cu2+ reduction experimentally measured for a series of azurin mutants and the solvation free energy of the oxidized state of the proteins were derived. Solvation free energy calculations were carried out within an ONIOM/PCM scheme specifically adapted to this protein series. The method proved to be able to capture the main determinants of the measured reduction parameters, providing satisfactory predictions of the E degrees '.

Algorithms↗
PubMed1996

X-ray crystal structure of the two site-specific mutants Ile7Ser and Phe110Ser of azurin from Pseudomonas aeruginosa.

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 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 (17S and F110S) 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 A and 2.3 A 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.

Azurin↗
PubMed2006

Coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification.

The denitrifying bacterium Alcaligenes xylosoxidans synthesises two azurins (Az), which are termed Az I and Az 2. Both function as effective electron donors to copper nitrite reductase (NiR) in vitro. As a first step towards identifying the physiological relevance of these electron transfer proteins in the denitrification process, the gene (azuA) encoding Az I was characterised and its expression with respect to denitrification determined. We show that the azuA gene from A. xylosoxidans is monocistronic and its expression is increased when cells are grown under denitrifying conditions in the presence of nitrate or nitrite. The expression pattern of azuA was similar, though not identical, to that of the monocistronic nirK gene, which encodes copper NiR, and is in accord with both gene products being synthesised when the bacterium denitrifies. Recombinant Az I was exported to the periplasm of the heterologous host Escherichia coli, was synthesised at very high levels (80 mg purified protein per litre) and was fully loaded with copper. Electron donation from reduced recombinant Az to NiR was indistinguishable from the activity determined with the native protein. Taken together, these findings indicate that in A. xylosoxidans azuA expression is coordinated with denitrification and recombinant Az I is processed and matured in the periplasm of E. coli in the same way it is in A. xylosoxidans.

Alcaligenes↗