Fundamental signatures of short- and long-range electron transfer for the blue copper protein azurin at Au/SAM junctions (original) (raw)
Related papers
The Journal of Physical Chemistry B, 2001
We have shown that Pseudomonas aeruginosa azurin can be immobilized on alkanethiol monolayers selfassembled on Au(111). Immobilization is achieved through hydrophobic interactions between the hydrophobic area around the copper atom in azurin and methyl heads of alkanethiol to form submonolayers or monolayers. In this orientation mode azurin molecules on Au(111) are oriented with the redox center (copper atom) facing the electrode surface. This is opposite to the orientation of azurin on bare gold which is via a surface disulfide group such as recently reported. Scanning tunneling microscopy (STM) with molecular resolution reveals that both well-ordered alkanethiol and protein adlayers are present. Adsorbed azurin molecules exhibit high stability and retain electron transfer (ET) function. Long-range interfacial ET between azurin and Au(111) across variable-length alkanethiol bridges was systematically investigated by different electrochemical techniques. Distance-dependent ET can be controlled by adjusting the length of the alkanethiol chain. The electrochemical ET rate constant is almost independent of the chain length up to ca. 9 methylene units but follows exponential distance decay with a decay factor ( ) of 1.03 ( 0.02 per CH 2 unit at longer chain lengths. Overvoltage-dependent ET was also examined. The results provide a strategy to ordered molecular assemblies, and controlled orientation and ET of azurin at atomically planar metallic surfaces. This approach can in principle be extended to other redox metalloproteins. reorganization free energy of electrochemical ET of azurin in this adsorption mode.
Molecular Monolayers and Interfacial Electron Transfer of Pseudomonas aeruginosa Azurin on Au(111)
Journal of the American Chemical Society, 2000
We provide a comprehensive approach to the formation and characterization of molecular monolayers of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) in aqueous ammonium acetate solution. Main issues are adsorption patterns, reductive desorption, properties of the double layer, and long-range electrochemical electron transfer between the electrode and the copper center. Voltammetry, electrochemical impedance spectroscopy (EIS), in situ scanning tunneling microscopy (STM), and X-ray photoelectron spectroscopy (XPS) have been employed to disclose features of these issues. Zn-substituted azurin, cystine, and 1-butanethiol are investigated for comparison. Cyclic voltammetric and capacitance measurements show qualitatiVely that azurin is adsorbed at submicromolar concentrations over a broad potential range. The characteristics of reductive desorption suggest that azurin is adsorbed via its disulfide group to form a monolayer. The adsorption of this protein on Au(111) via a gold-sulfur binding mode is further supported by XPS measurements. In situ STM images with molecular resolution have been recorded and show a dense monolayer organization of adsorbed azurin molecules. Direct electron transfer (ET) between the copper atom of adsorbed azurin and the electrode has been revealed by differential pulse voltammetry. The rate constant is estimated from electrochemical impedance spectroscopy and shows that ET is compatible with a long-range ET mode such as that anticipated by theoretical frames. The results constitute the first case of an electrochemically functional redox protein monolayer at single-crystal metal electrodes.
Electrochemistry Communications, 1999
We report the self-assembly and electrochemical behaviour of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) electrodes in aqueous acetate buffer (pHs4.6). The formation of monolayers of this protein is substantiated by electrochemical measurements. Capacitance results indicate qualitatively that the protein is strongly adsorbed at sub-mM concentrations in a broad potential range (about 700 mV). This is further supported by the attenuation of a characteristic cyclic voltammetric peak of Au(111) in acetate solution with increasing azurin concentration. Reductive desorption is clearly disclosed in NaOH solution (pHs13), strongly suggesting that azurin is adsorbed via its disulphide group. An anodic peak and a cathodic peak associated with the copper centre of azurin are finally observed in the differential pulse voltammograms. These peaks are, interestingly, indicative of long-range electrochemical electron transfer such as paralleled by intramolecular electron transfer between the disulphide anion radical and the copper atom in homogeneous solution, and anticipated by theoretical frames. Together with reported in situ scanning tunnelling microscopy (STM) results they constitute the first case for electrochemistry of self-assembled monolayers of azurin, even redox proteins. This integrated investigation provides a new approach to both structure and function of adsorbed redox metalloproteins at the molecular level.
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.
Electron tunneling through Pseudomonas aeruginosa azurins on SAM gold electrodes
Inorganica Chimica Acta, 2008
Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH 3 (CH 2) n SH:HO(CH 2) m SH (n=m=4,6,8,11; n=13,15 m=11) self-assembled monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6 to 11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn 47 caused by deprotonation of nearby surface residues.
Long-range interfacial electron transfer of metalloproteins based on molecular wiring assemblies
Faraday Discussions, 2006
We address some physical features associated with long-range interfacial electron transfer (ET) of metalloproteins in both electrochemical and electrochemical scanning tunneling microscopy (ECSTM) configurations, which offer a brief foundation for understanding of the ET mechanisms. These features are illustrated experimentally by new developments of two systems with the blue copper protein azurin and enzyme nitrite reductase as model metalloproteins. Azurin and nitrite reductase were assembled on Au(111) surfaces by molecular wiring to establish effective electronic coupling between the redox centers in the proteins and the electrode surface for ET and biological electrocatalysis. With such assemblies, interfacial ET proceeds through chemically defined and well oriented sites and parallels biological ET. In the case of azurin, the ET properties can be characterized comprehensively and even down to the single-molecule level with direct observation of redox-gated electron tunnelling resonance. Molecular wiring using a p-conjugated thiol is suitable for assembling monolayers of the enzyme with catalytic activity well-retained. The catalytic mechanism involves multiple-ET steps including both intramolecular and interfacial processes. Interestingly, ET appears to exhibit a substrate-gated pattern observed preliminarily in both voltammetry and ECSTM.
The electrochemical characteristics of blue copper protein monolayers on gold
Journal of Electroanalytical Chemistry, 2004
Site-specifically engineered disulphide or surface cysteine residues have been introduced into two blue copper proteins, Pseudomonas aeruginosa azurin and Populus nigra plastocyanin, in order to facilitate protein chemisorption on gold electrodes. The subsequently formed well-defined protein monolayers gave rise to robust electrochemical responses and electron transfer rates comparable to those observed at modified electrode surfaces. Proximal probe characterisation confirms the presence, at high coverage, of well-ordered protein adlayers. Additionally, gold-metalloprotein affinity is such that molecular-level tunnelling and topographic analyses can be carried out under aqueous solution. The approaches outlined in this work can, in principal, be extended to the generation of arrays of any redox-active biomolecule.
A model system for the electrochemical investigation of vectorial electron transfer in biological systems was designed, assembled and characterized. Gold electrodes, functionalized with a -OCH3 terminated, aromatic self-assembled monolayer, were used as a substrate for the adsorption of variants of copper- containing, redox metalloprotein azurin. The engineered azurin bears a polyhistidine tag at its C-terminus. Thanks to the presence of the solvent exposed tag, which chelates Cu2+ ions in solution, we introduced an exogenous redox centre. The different reduction potentials of the two redox centres and their positioning with respect to the surface are such that electron transfer from the exogenous copper centre and the electrode is mediated by the native azurin active site, closely paralleling electron transfer processes in naturally occurring multicentre metalloproteins.
The pH dependence of intramolecular electron transfer in azurins
Inorganica Chimica Acta, 1996
Long range electron transfer (LRET) can be induced between the disulfide radical anion, produced pulse radiolytically, and the copper(II) center in the single blue copper protein, azurin. The rate constant of this intramolecular process increases by one order of magnitude upon decreasing the pH from 8 to 4 in all azurins (wild types as well as single site mutants of Pseudomonas aeruginosa azurin) studied so far. In order to pursue the structural basis for the observed pH dependence we have extended our studies to a new mutant, Asp23Ala. In this derivative the aspartate residue is proximal to the electron donating cystine disulfide bridge. However, LRET in this azurin was found to exhibit similar pH dependence as all other wild type and single-site mutants with residues potentially being able to affect the electron transfer process, A detailed consideration of the parameters that determine the efficiency of this process leads to the suggestion that a pH induced change either in the electron transfer distance or in the electronic coupling may be the cause of this behavior.