Sequence-specific destabilization of azurin by tetramethylguanidinium-dipeptide ionic liquids

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing tetramethylguanidinium as the cation. The four dipeptides have different sequences including the amino acids Ser and Asp: TMG-AspAsp, TMG-SerSer, TMG-SerAsp, and TMG-AspSer. Thermal unfolding curves generated from temperature-dependent fluorescence spectroscopy experiments showed that TMG-AspAsp and TMG-SerSer have minor destabilizing effects on the protein while TMG-AspSer and TMG-SerAsp strongly destabilize azurin. Red-shifted fluorescence signatures in the 25 °C correlate with the observed protein destabilization in the solutions with TMG-AspSer and TMG-SerAsp. These signals could correspond to interactions between the Asp residue in the dipeptide and the azurin Trp residue in the unfolded state. These results, supported by appropriate control experiments, suggest that dipeptide sequence-specific interactions lead to selective protein destabilization and motivate further studies of TMG-dipeptide ILs.     

Silver binding toPseudomonas aeruginosa azurin

Summary The interaction between azurin and silver ions was investigated, by means of ultraviolet, fluorescence and atomic absorption spectroscopies, as a function of the redox state of the protein. The Ag(I) ion has a very low affinity for oxidized azurin. Interestingly, the affinity is much higher for reduced azurin; in this case Ag(I) completely displaces the Cu(I) ion from the native binding site. The effect is very specific for silver ions since other ions, such as Hg(II), Ni(II) and Cd(II), do not produce the same effect. Treatment of reduced and oxidized azurin with excess Ag(I) (2-8-fold stoichiometric) shows that there is a second binding site for silver ions on the protein which can also bind Cu(II) and Hg(II) with comparable affinities.     

Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5.

A blue copper protein was purified together with a type II quinohemoprotein alcohol dehydrogenase (ADH IIB) from the soluble fraction of Pseudomonas putida HK5 grown on n-butanol. The purified blue copper protein was shown to be azurin, on the basis of several properties such as its absorption maximum (623 nm), its low molecular mass (17 500 Da), its acidic nature (pI of 4.1), its relatively high redox potential (306 mV), the presence of an intramolecular disulfide bond, and N-terminal amino acid sequence homology with respect to azurins from other sources, especially from P. putida NCIB 9869 and Pseudomonas fluorescens. Direct electron transfer from ADH IIB to azurin was shown to occur at a rate of 48-70 s-1. The apparent Km value of ADH IIB for azurin, determined by steady-state kinetics, was decreased several-fold by increasing the ionic strength. Furthermore, the extent of fluorescence quenching of ADH IIB due to the interaction with azurin was increased by increasing the ionic strength, but the binding constant for binding between ADH IIB and azurin was unchanged. The redox potential of azurin was increased 12 mV by incubation with ADH but not vice versa. Furthermore, the redox potential gap between ADH and azurin was increased from 102 to 126 mV by increasing the ionic strength. It is conceivable that a hydrophobic interaction is involved in the electron transfer between both proteins, and it is also suggested that the electron transfer may occur by a freely reversible on and off binding process but may not be related to the global binding process of both proteins. Thus, the results presented here strongly suggest that azurin works as an electron-transfer mediator in a PQQ-dependent alcohol oxidase respiratory chain in P. putida HK5.     

Metal binding to Pseudomonas aeruginosa azurin: a kinetic investigation

The interaction between azurin from Pseudomonas aeruginosa and Ag(I), Cu(II), Hg(II), was investigated as a function of protein state, i.e. apo-, reduced and oxidised azurin. Two different metal binding sites, characterized by two different spectroscopic absorbancies, were detected: one is accessible to Ag(I) and Cu(II) but not to Hg(II); the other one binds Ag(I) and Hg(II) but not copper. When added in stoichiometric amount, Ag(I) shows high affinity for the redox center of apo-azurin, to which it probably binds by the -SH group of Cys112; it can displace Cu(I) from reducedazurin, while it does not bind to the redox center of oxidizedazurin. Kinetic experiments show that Ag(I) binding to the reduced form is four times faster than binding to the apo-form. This result suggests that metal binding requires a conformational rearrangement of the active site of the azurin. Interaction of Ag(I) or Hg(II) ions to the second metal binding site, induces typical changes of UV spectrum and quenching of fluorescence emission.     

A time-resolved fluorescence study of azurin and metalloazurin derivatives

Nickel and cobalt derivatives of Pseudomonas fluorescens (ATCC 13525) azurin were prepared and their steady-state fluorescence and time-resolved fluorescence monitored. Like the copper-containing native protein, the fluorescence decay of both metallo derivatives was best fit to a sum of three exponentials, whereas the apoazurin from which they were prepared obeyed single-exponential decay kinetics. However, comparison of the lifetimes and fractional of each of the components in these derivatives to those in the oxidized and reduced native proteins revealed significant differences. These results suggest that the presence of a metal center in azurin imparts a conformational heterogeneity which is strongly dependent on the nature of the metal center. Further, the results are used to comment on current ideas concerning the geometry of the active site in this redox protein.     

pH dependence of the reduction-oxidation reaction of azurin with cytochrome c-551: role of histidine-35 of azurin in electron transfer

A fluorescence quenching experiment confirms that in the redox reaction between cytochrome c-551 and azurin, protein complexing is negligible. Azurin-pH indicator T-jump experiments show that Pseudomonas aeruginosa (Ps.) azurin exhibits a slow time constant, tau, in its return to pH equilibrium but Alcaligenes faecalis (Alc.) azurin does not. The decrease of l/tau with increasing pH shows that the rate-determining process is a slow transformation of the imidazolium form of histidine-35 from a conformation where it cannot ionize to one in which it can. The fast relaxation time constant of the redox reaction varies little with pH, but the slow time constant increased by a factor of approximately 2.5 increasing pH between pH 5 and pH 8. The corresponding amplitudes, especially the slow one, vary with pH. On the basis of all the present evidence it is concluded that, while some differences of redox reactivity do occur on protonation, these differences are not major. In general, the two proteins cyt c-551 and azurin react with each other with rates only weakly dependent upon pH. A classical pH titration was carried out on the reduced and oxidized form of Ps. and Alc. azurin with the result that two protons were released between pH 6 and pH 8, in the former from His-35 and -83 and in the latter from His-83 and Ala-1.     

A Förster-resonance-energy transfer-based method for fluorescence detection of the protein redox state

A method for fluorescence detection of a protein's redox state based on resonance energy transfer from an attached fluorescence label to the prosthetic group of the redox protein is described and tested for proteins containing three types of prosthetic groups: a type-1 copper site (azurin, amicyanin, plastocyanin, and pseudoazurin), a heme group (cytochrome c550), and a flavin mononucleotide (flavodoxin). This method permits one to reliably distinguish between reduced and oxidized proteins and to perform potentiometric titrations at submicromolar concentrations.     

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.     

Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species,but not redox activity,as critical elements in cytotoxicity

Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054-7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.     

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.     

Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.     

The effect of iron-hexacyanide binding on the determination of redox potentials of cytochromes and copper proteins

The midpoint redox potentials of Pseudomonas aeruginosa cytochrome c-551 and Rhodopseudomonas viridis cytochrome c₂ were measured as a function of pH in the presence of Euglena cytochrome c-558 and the results compared with those obtained in the presence of ferro-ferricyanide. The pattern of pH dependence observed for the two bacterial cytochromes was the same whether it was measured by equilibrium with another redox protein or with the inorganic redox couple. Thus, the pH dependence of redox potential is not a consequence of pH-dependent ligand binding. The midpoint potential of Ps. aeruginosa azurin was measured as a function of pH using both ferro-ferricyanide mixtures and redox equilibrium with horse cytochrome c or Rhodopseudomonas capsulata cytochrome c₂. In this case also the pattern of pH dependence obtained did not vary with the redox system used and it closely resembled that of Ps. aeruginosa cytochrome c-551. This is consistent with the observation that the equilibrium between cytochrome c-551 and azurin is relatively independent of pH. An equation was derived which described pH-dependent ligand binding and which can produce theoretical curves to fit the experimental pH dependence of redox potential for both cytochrome and azurin. However, the pronounced effect on such curves produced by varying the ligand association constants, and the insensitivity of the experimental data to changes in ionic strength, suggest that ligand binding effects do not account for the pH dependence of redox potential.     

Importance of polarization effect in the study of metalloproteins: Application of polarized protein specific charge scheme in predicting the reduction potential of azurin

Molecular dynamics (MD) simulation is commonly used in the study of protein dynamics, and in recent years, the extension of MD simulation to the study of metalloproteins is gaining much interest. Choice of force field is crucial in MD studies, and the inclusion of metal centers complicates the process of accurately describing the electrostatic environment that surrounds the redox centre. Herein, we would like to explore the importance of including electrostatic contribution from both protein and solvent in the study of metalloproteins. MD simulations with the implementation of thermodynamic integration will be conducted to model the reduction process of azurin from Pseudomonas aeruginosa. Three charge schemes will be used to derive the partial charges of azurin. These charge schemes differ in terms of the amount of immediate environment, respective to copper, considered during charge fitting, which ranges from the inclusion of copper and residues in the first coordination sphere during density functional theory charge fitting to the comprehensive inclusion of protein and solvent effect surrounding the metal centre using polarized protein‐specific charge scheme. From the simulations conducted, the relative reduction potential of the mutated azurins respective to that of wild‐type azurin (ΔE_cal) were calculated and compared with experimental values. The ΔE_cal approached experimental value with increasing consideration of environmental effect hence substantiating the importance of polarization effect in the study of metalloproteins. This study also attests the practicality of polarized protein‐specific charge as a computational tool capable of incorporating both protein environment and solvent effect into MD simulations. Proteins 2014; 82:2209–2219. © 2014 Wiley Periodicals, Inc.     

Optical investigation of the electron transfer protein azurin-gold nanoparticle system

The hybrid system obtained by conjugating the protein azurin, which is a very stable and well-described protein showing a unique interplay among its electron transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy transfer from protein to metal surface and it is discussed in terms of the involvement of the Az electron transfer route in the interaction of the protein with the nanoparticle.     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Structure and reactivity of type-I copper sites

Summary Biological electron transfer is not well understood. The question is addressed in this contribution with reference to the so-called blue copper proteins, each of which has a single copper atom at its active centre. The redox activity (as probed by the electron self exchange reaction) of the Cu centre seems not to be affected. The electron self exchange reaction is known to proceed through His-117, and the hydrophobic patch is most important in the formation of the azurin/azurin encounter complex. Ph effects have not been observed on the three-dimensional structure ofA. denitrificans azurin, which may indicate that if present at all these have no direct physiological implications. Mutants are in process of construction.     

Optical and magnetic properties of azurin from Pseudomanas aeruginosa]

Optical, fluorescence and EPR spectra of azurin from Pseudomonas aeruginosa are described. Some properties of this protein are found to be similar to those of copper-containing proteins from plants (plastocyanin and plantacyanin). The interaction of ferricyanide with azurin bleached in alkaline media results in the formation of free radicals and an alteration in the shape of the EPR signal of azurin.     

Photophysics of metalloazurins

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.     

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.