The pH dependence of intramolecular electron transfer in azurins (original) (raw)
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European Journal of Biochemistry, 1992
An intramolecular electron-transfer process has previously been shown to take place between the Cys3 -Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(I1) ion in the blue singlecopper protein, azurin [Farver, 0. & Pecht, I. (1 989) Proc. Nut1 Acad. Sci. USA 86, further investigate the nature of this long-range electron transfcr (LRET) proceeding within the protein matnx, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of thc copper sitc and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site.
Chemical Physics, 1996
Intramolecular electron transfer (ET) between the Cys3-Cys26 radical ion (RSSR-) produced pulse radiolytically and the C&I) ion has been studied in four wild type and nine different single site mutants of the blue single-copper protein, azurin. This enabled examination of the rate of this intramolecular ET as a function of driving force and the nature of the medium separating the electron donor and acceptor. Using a tunneling pathway model for ET from donor (RSSR-) to acceptor (Cu[II]) through a combination of covalent bonds, hydrogen bonds, and space (van der Waals contact) jumps, the electronic coupling decays for protein mediated ET were calculated and potential pathways operating within the different azurins could be predicted. The rates of intramolecular ET and activation parameters for the above azurins correlate well with pathway distance and driving force as predicted by the Marcus theory, using a through-bond ET mechanism. ' Corresponding author. Institute of Analytical and Pharmaceutical Chemistty, Royal 0301-0104/96/$15OO 0 1996 Elsevier Science B.V. All rights reserved SSDI 0301.0104(95)00294-4 0. Faruer et al./Chemicd Physics 204 (1996) 271-277 273
Electron-Transfer Properties of Pseudomonas Aeruginosa [Lys44, Glu64]azurin
European Journal of Biochemistry, 1997
In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.
European Journal of Biochemistry, 1993
The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5 -8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-+Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK, 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR gll value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation. Fax: +31 71 274537. Abbreviations. Ches, 2-(N-cyclohexylamino)ethmesulfonic acid; Cu(1) azurin, reduced azurin; Cu(I1) azurin, oxidized azurin; Em, midpoint potential ; ESE, electron self-exchange ; ESEEM, electron spin-echo envelope modulation; M44K, Met44+Lys ; RR, resonance Raman; wt, wild type; FT, Fourier transform. due His117 is located in the centre of this patch. The replacement of Met44 by the protonated lysine residue hardly affects the spectroscopic properties of the Cu site, but causes a considerable decrease of the k,,, value of azurin. At pH >8, deprotonation of Lys44 produces a new type-] Cu site, whereas the magnitude of the k,,, value of the protein is largely restored. At pH 5 and pH 8, the midpoint potential, Em, of Met44+Lys (M44K) azurin is higher than the wildtype (wt) azurin Em by 60 mV. At pH > 8, deprotonation of Lys44 in M44K azurin reduces the En, difference from 60 mV to 40 mV. The pH dependence of the wt and M44K azurin En, values is analyzed in terms of the titration of His35, His83 and Lys44. In addition, the effects of the M44K mutation and the Cu-ion oxidation state (I or TI) on the pK, values of the titratable His35 and His83 of azurin are reported and analyzed. The structural and functional implications of the findings are discussed.
Advanced Science, 2015
Electron transfer (ET) reactions are central to a wide range of biological processes, notably those of energy conversion such as the respiratory and photosynthetic chains. These employ mostly proteins, between which electrons are shuttled from one electron mediator to another by a redox process, driven by a free energy difference (driving force) between or within the mediators. The separation distance over which an electron is transferred within proteins can reach in some cases around 2.5 nm. One of the most intensively studied group of ET proteins is the blue copper proteins (e.g., plastocyanin, azurin, and the family of multi-copper oxidases). [ 1 ] The copper binding sites were shown to confer unique spectroscopic and thermodynamic properties on the copper ions. [ 2 ] These properties attracted studies aiming at understanding their role in the ET via proteins. Here we focus on the bacterial blue copper protein azurin (Az), functioning as an electron mediator in certain bacterial respiratory chains. [ 3 ] Most of the studies of the ET process in Az were done in solution, using spectroscopy, mainly fl ash-quench [ 4 ] or pulse-radiolysis [ 5 ] techniques. A common denominator of these studies is monitoring the ET process between the Cu ion of Az and an intramolecular donor/acceptor. In another experimental approach for measuring intramolecular ET process in Az, the protein is bound to a conductive electrode and ET can be measured between the Cu ion and the electrode as a rate-determining step of an electrochemical redox process in an electrochemical cell. [ 6 ] In contrast, electronic transport (ETp) via the protein has more recently been studied by measuring conductance across Az, trapped between two electrodes in a solid-state confi guration. [ 7 ] One pronounced difference between measuring ET in solution by spectroscopy or electrochemistry and measuring the ETp in a solid-state confi guration is that while the former requires a redox process to occur and, thus, the presence of a redox-active center (such as the Cu ion, a disulphide bond or a bound external Ru complex), the latter does not. Electron transfer (ET) proteins are biomolecules with specifi c functions, selected by evolution. As such they are attractive candidates for use in potential bioelectronic devices. The blue copper protein azurin (Az) is one of the most-studied ET proteins. Traditional spectroscopic, electrochemical, and kinetic methods employed for studying ET to/from the protein's Cu ion have been complemented more recently by studies of electrical conduction through a monolayer of Az in the solid-state, sandwiched between electrodes. As the latter type of measurement does not require involvement of a redox process, it also allows monitoring electronic transport (ETp) via redox-inactive Az-derivatives. Here, results of macroscopic ETp via redox-active and-inactive Az derivatives, i.e., Cu(II) and Cu(I)-Az, apo-Az, Co(II)-Az, Ni(II)-Az, and Zn(II)-Az are reported and compared. It is found that earlier reported temperature independence of ETp via Cu(II)-Az (from 20 K until denaturation) is unique, as ETp via all other derivatives is thermally activated at temperatures >≈200 K. Conduction via Cu(I)-Az shows unexpected temperature dependence >≈200 K, with currents decreasing at positive and increasing at negative bias. Taking all the data together we fi nd a clear compensation effect of Az conduction around the Az denaturation temperature. This compensation can be understood by viewing the Az binding site as an electron trap, unless occupied by Cu(II), as in the native protein, with conduction of the native protein setting the upper transport effi ciency limit. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Role of the electronic properties of azurin active site in the electron-transfer process
International Journal of Quantum Chemistry, 2005
Electron transfer proteins, such as azurin (a blue copper protein), are promising candidates for the implementation of biomolecular nanoelectronic devices. To understand the details of electron transfer in redox active azurin molecules, we performed plane-wave pseudo-potential density functional theory (DFT) calculations of the protein active site in the two possible oxidation states Cu(I) and Cu(II). The ab initio results are used to discuss how the electronic spectrum and wavefunctions may mediate the shuttling of electrons through the copper ion. We find that the Cu-ligand hybridization is very similar in the two charge states of the metal center, but the energy spectrum changes substantially. This result might indicate important effects of electronic correlations in the redox activity and consequent electron transfer through the Cu site.
Resolution of two distinct electron transfer sites on azurin
Biochemistry, 1982
Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II), ions, yielding a substitution-inert Cr(II1) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(II1)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(II1)-labeled azurin and cytochrome c551 were studied by the temperature-jump
Water Effects on Electron Transfer in Azurin Dimers
The Journal of Physical Chemistry B, 2006
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.