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PubMed1996

Effect of pH and ligand binding on the structure of the Cu site of the Met121Glu mutant of azurin from Pseudomonas aeruginosa.

A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins. As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two [Karlsson, B.G., et al. (1991) Protein Eng. 4 (3), 343-349]. These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G. X-ray absorption a the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 A) and Cu-Cys (by 0.06 A) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 A. The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique. The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand. Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G. This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.

Absorptiometry, Photon↗

PubMed1997

X-ray structure determination and characterization of the Pseudomonas aeruginosa azurin mutant Met121Glu.

The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.

Azurin↗

PubMed1998

Determination of the magnetic axes of cobalt(II) and nickel(II) azurins from 1H NMR data: influence of the metal and axial ligands on the origin of magnetic anisotropy in blue copper proteins.

The orientation and the axial, Deltachiax, and rhombic, Deltachirh, components of the magnetic susceptibility tensor anisotropy for the cobalt(II) and nickel(II) derivatives of azurin from Pseudomonas aeruginosa have been determined from 1H NMR data. For both derivatives, the axial geometry of the system determines the orientation of the chi-tensor, whose z-axis forms an angle of 18.6 and 20.1 degrees with the Cu-OGly45 axial bond in the cobalt(II) and nickel(II) derivatives, respectively. For protons close to this axis, large negative pseudocontact shifts are observed, while those close to the NNS plane of the equatorial ligands experience lower and positive pseudocontact shifts for the same distance. Dipolar shifts are larger in the cobalt derivative, not only because of the larger spin number but also due to its intrinsically higher anisotropy. The contact contribution to the hyperfine shifts for the coordinated residues has been evaluated and analyzed in terms of unpaired spin delocalization mechanisms and geometry considerations. The results are extended to other blue copper proteins whose cobalt derivatives have been studied by 1H NMR. The electronic structure and its implications in the redox properties of the native copper proteins are also commented.

Anisotropy↗

PubMed2002

An ab initio quantum-chemical study of the blue-copper site of azurin.

The electronic structure of the blue-copper site of Pseudomonas aeruginosa azurin has been investigated by ab initio multireference determinantal configuration interaction (MRD-CI) calculations. A truncated site consisting of copper and its three equatorial ligands has been studied with emphasis on the g tensor and the nitrogen hyperfine tensors of the coordinating histidines. In the ground state the singly occupied molecular orbital (SOMO) involves a copper 3d orbital pi antibonded to the cysteine sulfur and sigma antibonded to the histidine nitrogens. A proper description of the electron-paramagnetic-resonance parameters has been achieved through the use of an effective core potential for copper up to and including the 3s electrons. Both the complete g tensor and the anisotropic hyperfine tensors at the nitrogens are essentially reproduced. Mulliken spin densities of 35 and 59% on copper and sulfur, respectively, and 2.1 and 1.7% on the respective coordinating nitrogens reflect the delocalized character of the SOMO and the inequivalence of the histidines.

Azurin↗

PubMed2001

Deconvolution and assignment of different optical transitions of the blue copper protein azurin from optically detected electron paramagnetic resonance spectroscopy.

Magnetic circular dichroism is a powerful spectroscopic tool for the assignment of optical resonance lines. An extension of this technique, microwave-modulated circular dichroism, provides additional details, in particular information about the orientation of optical transition moments. It arises from magnetization precessing around the static magnetic field, excited by a microwave field, in close analogy to electron paramagnetic resonance (EPR). In this paper we investigate the visible and near-infrared spectrum of the blue copper protein Pseudomonas aeruginosa azurin. Using a nonoriented sample (frozen solution), we apply this technique to measure the variation of the optical anisotropy with the wavelength. A comparison with the optical anisotropies of the possible ligand-field and charge-transfer transitions allows us to identify individual resonance lines in the strongly overlapping spectrum and assign them to specific electronic transitions. The technique is readily applicable to other proteins with transition metal centers.

Anisotropy↗

PubMed2001

Electron tunneling in single crystals of Pseudomonas aeruginosa azurins.

Rates of reduction of Os(III), Ru(III), and Re(I) by Cu(I) in His83-modified Pseudomonas aeruginosa azurins (M-Cu distance approximately 17 A) have been measured in single crystals, where protein conformation and surface solvation are precisely defined by high-resolution X-ray structure determinations: 1.7(8) x 10(6) s(-1) (298 K), 1.8(8) x 10(6) s(-1) (140 K), [Ru(bpy)2(im)(3+)-]; 3.0(15) x 10(6) s(-1) (298 K), [Ru(tpy)(bpy)(3+)-]; 3.0(15) x 10(6) s(-1) (298 K), [Ru(tpy)(phen)(3+)-]; 9.0(50) x 10(2) s(-1) (298 K), [Os(bpy)2(im)(3+)-]; 4.4(20) x 10(6) s(-1) (298 K), [Re(CO)3(phen)(+)] (bpy = 2,2'-bipyridine; im = imidazole; tpy = 2,2':6',2' '-terpyridine; phen = 1,10-phenanthroline). The time constants for electron tunneling in crystals are roughly the same as those measured in solution, indicating very similar protein structures in the two states. High-resolution structures of the oxidized (1.5 A) and reduced (1.4 A) states of Ru(II)(tpy)(phen)(His83)Az establish that very small changes in copper coordination accompany reduction but reveal a shorter axial interaction between copper and the Gly45 peptide carbonyl oxygen [2.6 A for Cu(II)] than had been recognized previously. Although Ru(bpy)2(im)(His83)Az is less solvated in the crystal, the reorganization energy for Cu(I) --> Ru(III) electron transfer falls in the range (0.6-0.8 eV) determined experimentally for the reaction in solution. Our work suggests that outer-sphere protein reorganization is the dominant activation component required for electron tunneling.

Azurin↗

PubMed2002

An engineered azurin variant containing a selenocysteine copper ligand.

Modulating the properties of proteins through de novo design or redesign of existing proteins has been a longstanding goal in protein chemistry. Over the past two decades, site-directed mutagenesis has been a powerful tool to probe the role of certain residues and to fine-tune the activity of proteins. A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. The more recent technique of expressed protein ligation (EPL) provides an alternative route that allows efficient incorporation of nonnatural residues into proteins. We report here the preparation and spectroscopic characterization of an azurin variant in which a cysteine ligand to the blue copper center has been replaced by EPL with selenocysteine (Sec). This reports marks the first time that selenocysteine is artificially incorporated into the active site of a metalloprotein. The variant displays a significantly increased A(parallel) (from 56 to 104 G) and red-shifted CT band (from 625 to 677 nm), while maintaining the general type 1 copper characteristics, including similarity in reduction potentials. This study illustrates that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of most of its characteristics. Further spectroscopic and X-ray crystallographic studies of this and other EPL variants will provide new insights into the fine-control of the structure and function of metalloproteins.

Amino Acid Sequence↗

PubMed2004

X-ray magnetic circular dichroism of Pseudomonas aeruginosa nickel(II) azurin.

We show that X-ray magnetic circular dichroism (XMCD) can be employed to probe the oxidation states and other electronic structural features of nickel active sites in proteins. As a calibration standard, we have measured XMCD and X-ray absorption (XAS) spectra for the nickel(II) derivative of Pseudomonas aeruginosa azurin (NiAz). Our analysis of these spectra confirms that the electronic ground state of NiAz is high-spin (S = 1); we also find that the L(3)-centroid energy is 853.1(1) eV, the branching ratio is 0.722(4), and the magnetic moment is 1.9(4) mu(B). Density functional theory (DFT) calculations on model NiAz structures establish that orbitals 3d(x2-y2) and 3d(z2) are the two valence holes in the high-spin Ni(II) ground state, and in accord with the experimentally determined orbital magnetic moment, the DFT results also demonstrate that both holes are highly delocalized, with 3d(x2-y2) having much greater ligand character.

Azurin↗

PubMed2006

Reduction potential tuning of the blue copper center in Pseudomonas aeruginosa azurin by the axial methionine as probed by unnatural amino acids.

The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in < 6 nm shifts in the S(Cys)-Cu charge transfer (CT) band in the electronic absorption spectra and < 8 gauss changes in the copper hyperfine coupling constants (AII) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of AII differs, suggesting additional factors influencing AII. In contrast to the small changes in the UV-vis and EPR spectra, a large variation of > 227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.

Amino Acids↗

PubMed2005

The reorganization energy of azurin in bulk solution and in the electrochemical scanning tunneling microscopy setup.

The total reorganization energy lambda of azurin is theoretically studied both for the electron self-exchange reaction and for the protein in the electrochemical scanning tunneling microscopy (ECSTM) setup. The results demonstrate the importance of the proximity between the active sites in the encounter complex to reduce lambda for the electron self-exchange reaction and quantifies the effects of the presence of an STM environment (tip and substrate) on lambda. A comparison of the calculated results with experimental data is performed, and the relative magnitudes of the inner and outer contributions to lambda are discussed.

Azurin↗

PubMed2006

Comparison of intra- vs intermolecular long-range electron transfer in crystals of ruthenium-modified azurin.

Selective metal-ion incorporation and ligand substitution are employed to control whether electrons tunnel over intra- or intermolecular separations in crystals of P. aeruginosa azurin modified with Ru-polypyridine complexes. Cu(1+)-to-Ru3+ electron transfer (ET) across a specific protein-protein interface in the crystal lattice has a time constant 5-10 times longer than ET between the same donor and acceptor within a single protein (tauET = 5 vs 0.5-1.0 micros). Slower intermolecular ET agrees well with a longer distance between redox centers across the inter-protein (18.9 A) compared to the intra-protein separation (17.0 A) and indicates that the closest donor/acceptor pair dominates crystal ET. Lowering the crystal pH accelerates inter-protein ET (tauET = 1.0 micros) but not intra-protein ET. Faster inter-protein ET likely results from a pH-induced peptide bond flip that perturbs hydrogen bonding in the path between Ru and Cu centers on adjacent molecules.

Azurin↗

PubMed2006

Water effects on electron transfer in azurin dimers.

Recent experimental and theoretical analyses indicate that water molecules between or near redox partners can significantly affect their electron-transfer (ET) properties. Here, we study the effects of intervening water molecules on the electron self-exchange reaction of azurin (Az) by using a newly developed ab-initio method to calculate transfer integrals between molecular sites. We show that the insertion of water molecules in the gap between the copper active sites of Az dimers slows down the exponential decay of the ET rates with the copper-to-copper distance. Depending on the distance between the redox sites, water can enhance or suppress the electron-transfer kinetics. We show that this behavior can be ascribed to the simultaneous action of two competing effects: the electrostatic interaction of water with the protein subsystem and its ability to mediate ET coupling pathways.

Azurin↗

PubMed2006

First-principles density-functional theory calculations of electron-transfer rates in azurin dimers.

We have conceived and implemented a new method to calculate transfer integrals between molecular sites, which exploits few quantities derived from density-functional theory electronic structure computations and does not require the knowledge of the exact transition state coordinate. The method uses a complete multielectron scheme, thus including electronic relaxation effects. Moreover, it makes no use of empirical parameters. The computed electronic couplings can then be combined with estimates of the reorganization energy to evaluate electron-transfer rates that are measured in kinetic experiments: the latter are the basis to interpret electron-transfer mechanisms. We have applied our approach to the study of the electron self-exchange reaction of azurin, an electron-transfer protein belonging to the family of cupredoxins. The transfer integral estimates provided by the proposed method have been compared with those resulting from other computational techniques, from empirical models, and with available experimental data.

Algorithms↗

PubMed2005

Role of structural determinants in folding of the sandwich-like protein Pseudomonas aeruginosa azurin.

An invariant substructure that forms two interlocked pairs of neighboring beta-strands occurs in essentially all known sandwich-like proteins. Eight conserved positions in these strands were recently shown to act as structural determinants. To test whether the residues at these invariant positions are conserved for mechanistic (i.e., part of folding nucleus) or energetic (i.e., governing native-state stability) reasons, we characterized the folding behavior of eight point-mutated variants of the sandwich-like protein Pseudomonas aeruginosa apo-azurin. We find a simple relationship among the conserved positions: half of the residues form native-like interactions in the folding transition state, whereas the others do not participate in the folding nucleus but govern high native-state stability. Thus, evolutionary preservation of these specific positions gives both mechanistic and energetic advantages to members of the sandwich-like protein family.

Azurin↗

PubMed2006

Role of protein frame and solvent for the redox properties of azurin from Pseudomonas aeruginosa.

We have coupled hybrid quantum mechanics (density functional theory; Car-Parrinello)/molecular mechanics molecular dynamics simulations to a grand-canonical scheme, to calculate the in situ redox potential of the Cu(2+) + e(-) --> Cu(+) half reaction in azurin from Pseudomonas aeruginosa. An accurate description at atomistic level of the environment surrounding the metal-binding site and finite-temperature fluctuations of the protein structure are both essential for a correct quantitative description of the electronic properties of this system. We report a redox potential shift with respect to copper in water of 0.2 eV (experimental 0.16 eV) and a reorganization free energy lambda = 0.76 eV (experimental 0.6-0.8 eV). The electrostatic field of the protein plays a crucial role in fine tuning the redox potential and determining the structure of the solvent. The inner-sphere contribution to the reorganization energy is negligible. The overall small value is mainly due to solvent rearrangement at the protein surface.

Azurin↗

PubMed2001

Deuterium isotope effect on the intramolecular electron transfer in Pseudomonas aeruginosa azurin.

Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k(H)/k(D), is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (-56.5 J K(-1) mol(-1)) and in deuterium oxide (-35.7 J K(-1) mol(-1)). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential (E(0')) of Cu(2+/+) at 298 K is 10 mV more positive in D(2)O than in H(2)O. The temperature dependence of E(0') is also different, yielding entropy changes of -57 J K(-1) mol(-1) in water and -84 J K(-1) mol(-1) in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to DeltaS from the temperature dependence of E(0') is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H(2)O than in D(2)O (0.001 nm K(-1)) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E(0'). Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.

Azurin↗

PubMed2002

A new type 2 copper cysteinate azurin. Involvement of an engineered exposed cysteine in copper binding through internal rearrangement.

The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell.

Azurin↗

PubMed1991

Cassette mutagenesis of Met121 in azurin from Pseudomonas aeruginosa.

Cassette mutagenesis was used to exchange the suggested copper ligand Met121 in azurin to all other amino acids, and a stop codon. The mutant proteins were characterized by optical absorption spectroscopy and EPR. At low pH, all mutants exhibit the characteristics of a blue type 1 copper protein, indicating that methionine is not needed to create a blue copper site. At high pH, the Glu121 and the Lys121 mutants constitute a new form of protein-bound copper that is outside the range of type 1 copper.

Azurin↗