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PubMed1991

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa.

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.

Amino Acid Sequence↗

PubMed1992

Complex formation between the copper protein, azurin and the cytochrome c peroxidase of Pseudomonas aeruginosa.

Reduced azurin reacts with the resting, oxidized cytochrome c peroxidase of Pseudomonas aeruginosa to yield time courses observed at 420 nm, which consist of the sum of two exponential processes. Each process exhibits a hyperbolic dependence of the observed rate constant on the reduced azurin concentration. The fraction of the total optical density change which each process contributes is found to be dependent on the reduced azurin concentration. This pattern of reactivity is maintained at pH values between 5.5 and 8.0. The data has been analyzed in terms of a complex formation between the two proteins followed by an intramolecular electron exchange reaction. This analysis yields values for the binding constants at each pH value. The intramolecular exchange reaction is independent of pH, whilst the pH dependence of the binding reaction suggests the involvement of a histidine residue in this process.

Azurin↗

PubMed1986

The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS.

The EXAFS of the K-edge of copper in azurin from Pseudomonas aeruginosa has been measured in solutions of the oxidized and reduced protein, at both low and high pH. Model compounds of known molecular structure, exhibiting Cu-N and Cu-S bonds of varying length, were studied as well. The major shell of the high-pH oxidized azurin EXAFS contains contributions of two N(His) at 1.95 +/- 0.03 A, and one S(Cys) at 2.23 +/- 0.03 A. Some minor contributions from the carbon atoms of the histidine residues and the distal sulfur atom are observed in the 3-4 A region. Upon reduction a decrease is seen in amplitude of the main peak in the Fourier transform, due to a lengthening of one of the Cu-N(His) bonds (2.05 +/- 0.03 A), and a shortening of the other (1.89 +/- 0.03 A), both by approx. 0.1 A. Indications for a Cu-S(Met) bond are found in the reduced azurin data (2.70 +/- 0.05 A). However, in the oxidized protein, this bond could not be determined unambiguously, in line with results of a model compound featuring weak Cu-thioether coordination. The effect of pH is only slight for both the oxidized and the reduced protein, and no significant changes in bond lengths are found upon a change of pH from 4.1 to 9.1. The relevance of these findings for the interpretation of the existing data on the redox activity of the protein is discussed.

Azurin↗

PubMed2004

Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins.

Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins. Cu(I) to "Re(II)" electron tunneling in Re(H107) azurin occurs in the microsecond range. This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep ("hopping") mechanism might be involved. Although a Y108 radical can be generated by flash-quenching a Re(H107)M(II) (M=Cu, Zn) protein, the evidence suggests that it is not an active intermediate in the enhanced Cu(I) oxidation. Rather, the likely explanation is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radical), followed by electron tunneling from Cu(I) to the hole in the imidazole ligand.

Azurin↗

PubMed2006

Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide.

A novel biosensor for superoxide radical (O(2)(*-)) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O(2)(*-) was found to be 10(5)M(-1)s(-1) in solution and five times lower, i.e., 0.2 x 10(5)M(-1)s(-1), for azurin coupled to gold by 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6s(-1). The sensitivity of this biosensor to O(2)(*-) was 6.8 x 10(2)Am(-2)M(-1). The response to the interference substances, such as uric acid, H(2)O(2), and dimethylsulfoxide was negligible below 10 microM. The electrode was applied in three O(2)(*-) generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO(2)), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O(2)(*-) in all three environments, and the signals were antagonized by superoxide dismutase.

Azurin↗

PubMed2006

The alkaline transition of blue copper proteins, Cucumis sativus plastocyanin and Pseudomonas aeruginosa azurin.

Autoreduction of Cucumis sativus plastocyanin and Pseudomonas aeruginosa azurin took place at alkaline pHs, having been accompanied by the decrease in the intensities of the charge transfer band, Cys-S- (pi)-->Cu(II) at 597 and 626 nm, and the Cu(II)-EPR signals with small AII values of 6.5 x 10(-3) and 5.3 x 10(-3) cm(-1) for plastocyanin and azurin, respectively. Further, an extra Cu(II)-EPR signal with a large AII value of 21 x 10(-3) cm(-1) also reversibly emerged with increasing pH. Plastocyanin and azurin are in an equilibrium of the three forms at alkaline pHs.

Azurin↗

PubMed1996

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.

Apoproteins↗

PubMed1999

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives.

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.

Azurin↗

PubMed1983

Structure of azurin from Alcaligenes denitrificans at 2.5 A resolution.

The structure of the blue copper protein, azurin, from Alcaligenes denitrificans has been determined from an electron density map at a nominal resolution of 3.0 A. 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. The overall figure of merit was 0.61. The two molecules in the asymmetric unit are related by an approximate 2-fold axis. Independent interpretations of the density were made for the two molecules, and the structures have since been partially refined. After 12 refinement cycles, using the Hendrickson-Konnert restrained least-squares program, the R factor is 0.318 for data to 2.5 A resolution and there are no major conformational differences between the two molecules. Refinement is continuing. Eight extended strands of the polypeptide chain form a beta-barrel structure whose topology is the same as that of plastocyanin and the alternative folding proposed for Pseudomonas aeruginosa azurin. As in the latter two proteins, the copper atom forms three short bonds, with His-46 N delta 1, His117 N delta 1 and Cys112 S gamma, and one longer bond, with Met121 S delta, these four ligands forming a very distorted tetrahedron. A possible additional interaction, between copper and the carbonyl oxygen of Gly45, cannot be discounted at the present stage of the analysis. A surface hydrophobic patch, around the edge of the imidazole ring of His117 appears the most likely electron transfer locus. The sequences of azurin and plastocyanin have been aligned and the homology between the two proteins is discussed.

Alcaligenes↗

PubMed1979

Characteristics of azurin from Pseudomonas aeruginosa via 270-MHz 1H nuclear magnetic resonance spectroscopy.

Assignments of resonances in the 1H nmr spectra of Cu(I) azurin to proton groups in the protein are discussed in detail. Comparisons are drawn between Cu(I), Cu(II), apo, Hg(II), and Co(II) azurin samples. Redox titration of Cu(I) azurin with K3Fe(CN)6, is used to correlate Cu(I) and Cu(II) 1H nmr spectral features, and observed line broadenings deriving from Cu(II) paramagnetic effects are used to deduce the distances of assigned proton groups from the copper center. Histidine residues are characterized in terms of pK values, rates of acid-base exchange near the the pK, and rates of C2H exchange with solvent deuterium. The possibility of histidine involvement in the azurincytochrome 551 electron exchange mechanism is discussed. A small number of NH protons observed to be distinctively inert to 2H exchange with solvent 2H2O, in the Cu(I) protein, are found to show increased lability on removal of the metal.

Azurin↗

PubMed2002

Effects of chaotropic anions on the distribution of conformational substates of amicyanin, wild type and Cys3Ala/Cys26Ala azurin mutant.

The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric <--> g and <--> A tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins. Such a reduction, quantified by the standard deviations sigma(g axially) and sigma(A axially) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(g axially) and sigma(A axially) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N(-)(3) and SCN(-) addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions.

Amino Acid Substitution↗

PubMed1997

The effect of the metal ion on the folding energetics of azurin: a comparison of the native, zinc and apoprotein.

The unfolding by guanidine hydrochloride (GuHCl) and the refolding on dilution of zinc and apoazurin have been monitored by far-UV circular dichroism (CD). With the native protein, the unfolding was followed by CD and optical absorption in the visible spectral region. With the zinc protein, the reversible unfolding has also been followed by tryptophan fluorescence and NMR. The zinc and Cu2+ metal ions remain associated with the protein in the unfolded state. When the unfolding of the native protein is followed by CD, the initial, reversible transition due to unfolding is followed by a slow change associated with the reduction of Cu2+ by the thiol group of the ligand Cys112. The unfolding of apoazurin displays two CD transitions, which evidence suggests represent different folding domains, the least stable one including the metal-binding site and the other one the rest of the beta-sheet structure. Both occur at a lower GuHCl concentration than the unfolding of the native protein. The CD titrations also demonstrate that zinc azurin has a lower stability than the copper protein. Unfolding of zinc azurin followed by tryptophan fluorescence occurs at a much lower GuHCl concentration than the CD changes, and NMR spectra show that there is no loss of secondary and tertiary structure at this concentration, whereas the CD-detected loss of secondary structure correlates with the NMR changes. Thus, the fluorescence change is ascribed to a small local perturbation of the structure around the single tryptophan residue. The differences in stability of the three forms of azurin are discussed in terms of the rack mechanism. A bound metal ion stabilizes the native fold, and this stabilization is larger for Cu(II) than for Zn(II), reflecting the higher affinity of the protein for Cu(II).

Apoproteins↗

PubMed1975

Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.

The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.

Amides↗

PubMed2001

Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin.

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.

Acid-Base Equilibrium↗

PubMed2002

Effects of metal ligation and oxygen on the reversibility of the thermal denaturation of Pseudomonas aeruginosa azurin.

Thermodynamic equilibrium transition models in DSC are only applicable to reversible processes, but reversibility of the thermal transitions of proteins is comparatively rare because of intermolecular aggregation of denatured proteins and the degradation that occurs at high temperatures. The cupredoxin azurin from Pseudomonas aeruginosa has previously been found to exhibit irreversible thermal denaturation, both as holo- and apoprotein [Engeseth, H. R., and McMillin, D. R. (1986) Biochemistry 25, 2448-2455]. In this study, however, we demonstrate that this beta-barrel protein of Greek key topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligated to the protein. We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusively oxidized in a transition metal catalyzed oxidation reaction with dissolved O(2) at high temperatures. Both Cu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as well as the Zn(II)-coordinating disulfide-deficient C3A/C26A mutant. Correspondingly, apoazurin mutants C112A and C112S unfold reversibly, even in aerobic solutions, and exhibit nearly perfect two-state transitions. Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due to autoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.

Amino Acid Substitution↗

PubMed2004

The early steps in the unfolding of azurin.

High-temperature molecular dynamics simulations were used to gain insight into the early steps in the unfolding pathway of azurin, a blue copper protein with a beta-barrel structure formed by two sheets arranged in a Greek key folding topology. The results reveal that unfolding of the beta-barrel in azurin is associated with dislocation of its unique alpha-helix with respect to the protein scaffold. Exposure of the hydrophobic core to solvent precedes complete disruption of secondary and tertiary structure, and modifications in the region around the active site are directly connected with this event. Denaturation of the protein initiates from the sheet coordinating the copper ion, and the other sheet maintains its topology. Results derived from the simulation were compared with experimental data obtained with different techniques, showing excellent agreement and providing a framework to understand the process of disruption and formation of the beta-barrel in azurin.

Azurin↗

PubMed2003

Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids.

Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.

Absorptiometry, Photon↗

PubMed2004

Mimicking protein-protein electron transfer: voltammetry of Pseudomonas aeruginosa azurin and the Thermus thermophilus Cu(A) domain at omega-derivatized self-assembled-monolayer gold electrodes.

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.6) and subunit II (Cu(A) domain) of ba(3)-type cytochrome c oxidase (T. thermophilus) (pI = 6.0) exhibit optimal voltammetric responses on 1:1 mixtures of [H(3)C(CH(2))(n)()SH + HO(CH(2))(n)()SH] SAMs. The electron transfer (ET) rate vs distance behavior of azurin and Cu(A) is independent of the omega-derivatized alkanethiol SAM headgroups. Strikingly, only wild-type azurin and mutants containing Trp48 give voltammetric responses: based on modeling, we suggest that electronic coupling with the SAM headgroup (H(3)C- and/or HO-) occurs at the Asn47 side chain carbonyl oxygen and that an Asn47-Cys112 hydrogen bond promotes intramolecular ET to the copper. Inspection of models also indicates that the Cu(A) domain of ba(3)-type cytochrome c oxidase is coupled to the SAM headgroup (H(3)C- and/or HO-) near the main chain carbonyl oxygen of Cys153 and that Phe88 (analogous to Trp143 in subunit II of cytochrome c oxidase from R. sphaeroides) is not involved in the dominant tunneling pathway. Our work suggests that hydrogen bonds from hydroxyl or other proton-donor groups to carbonyl oxygens potentially can facilitate intermolecular ET between physiological redox partners.

Azurin↗