Potential-induced resonant tunneling through a redox metalloprotein investigated by electrochemical scanning probe microscopy (original) (raw)
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Ultramicroscopy, 2002
We have applied scanning tunnelling microscopy (STM) to the study of two proteins: pseudoazurin and apo-pseudoazurin. Both proteins adsorbed onto a Au (1 1 1) surface are visible to STM individually, forming into layers and multilayers, with currents from about 55 to 600 pA. The images reproduce well the expected dimensions laterally but not in the z direction. The apparent height of the proteins varies with the voltage polarity, being higher at negative sample voltages. The bias also affects their shape. Negative sample voltages of more than 1.5 V orient the proteins present on a gold terrace in parallel rows. The layer of water adsorbed on surfaces in ambient conditions can be related to our results to explain the reduced z dimensions, the asymmetry with the voltage polarity and the alignment of proteins at voltages more negative than −1.5 V.
Proceedings of the National Academy of Sciences, 2005
A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi-biological environment, suitable for detailed observations of long-range protein interfacial ET at the nanoscale and single-molecule levels. Because azurin is located at clearly identifiable fixed sites in well controlled orientation, the ET configuration parallels biological ET. The ET is nonadiabatic, and the rate constants display tunneling features with distance-decay factors of 0.83 and 0.91 Å ؊1 in H2O and D2O, respectively. Redoxgated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on͞off current ratio of Ϸ9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative information for rational understanding of the ET mechanism.
Electron tunnelling through azurin is mediated by the active site Cu ion
Chemical Physics Letters, 2003
Cu-and Zn-azurin chemisorbed on Au(1 1 1) have been comparatively investigated by electrochemical scanning tunnelling microscopy in buffer solution. Cu-azurin shows a marked tunnelling current resonance upon substrate potential at)0.21 V (vs SCE), whereas Zn counterparts do not. These data, discussed in terms of current theories on electron tunnelling through redox adsorbates, demonstrate the role of the electroactive metal ion present in the active site in assisting electron transfer via this metalloprotein.
Electronic Properties of Functional Biomolecules at Metal/Aqueous Solution Interfaces
The Journal of Physical Chemistry B, 2002
Monolayers of molecules, which retain their function in the adsorbed state on solid surfaces, are important in materials science, analytical detection, and other technology approaching the nanoscale. Molecular monolayers, including layers of functional biological macromolecules, offer new insight in electronic properties and stochastic single-molecule features and can be probed by new methods which approach the single-molecule level. One of these is in situ scanning tunneling microscopy (STM) in which single-molecule electronic properties directly in aqueous solution are probed. In situ STM combined with physical electrochemistry, single-crystal electrodes, and spectroscopic methods is now a new dimension in interfacial bioelectrochemistry. We overview first some approaches to spectroscopic single-molecule imaging, including fluorescence spectroscopy, chemical reaction dynamics, atomic force microscopy, and electrochemical single-electron transfer. We then focus on in situ STM. In addition to high structural resolution, in situ STM offers a singlemolecule spectroscopic perspective. This emerges most clearly when adsorbate molecules contain accessible redox levels, and the tunneling current decomposes into successive single-molecule interfacial electron transfer (ET) steps. Theories of electrochemical ET and in situ STM of redox molecules as well as specific cases are addressed. Two-step in situ STM represents different molecular mechanisms and even new ET phenomena, related to coherent many-electron transfer. A number of systems are noted to accord with these views. The discussion is concluded by attention to one of the still very few redox proteins addressed by in situ STM, the blue copper protein Pseudomonas aeruginosa azurin. Use of comprehensive electrochemical techniques has ascertained that well-defined protein monolayers in two opposite orientations can be formed and interfacial tunneling patterns disclosed. P. aeruginosa azurin emerges as by far the most convincing case where in situ STM of functional metalloproteins to single-molecule resolution has been achieved. This comprehensive approach holds promise for broader use of in situ STM as a single-molecule spectroscopy of metalloproteins and illuminates prerequisites and limitations of in situ STM of biological macromolecules. E-FAD + S a E-FAD-S f E-FADH 2 + P E-FADH 2 + O 2 a E-FADH 2 -O 2 f E-FAD + H 2 O 2 (1) Feature Article
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.
ACS Nano, 2015
Surprisingly efficient solid-state Electron Transport has recently been demonstrated through "dry" proteins (with only structural, tightly bound H 2 O left), suggesting proteins as promising candidates for molecular (bio)electronics. Using inelastic electron tunneling spectroscopy (IETS), we explored electron-phonon interaction in metal/protein/metal junctions, to help understanding solid-state electronic transport across the redox protein Azurin. To that end an oriented Azurin monolayer on Au is contacted by soft Au electrodes. Characteristic vibrational modes of amide and amino-acid side groups as well as of Az-electrode contact were observed, revealing the Az native conformation in the junction and the critical role of side groups in the charge transport. The lack of abrupt changes in the conductance and the line shape of IETS point to far off-resonance tunneling as the dominant transport mechanism across Azurin, in line with previously reported (and herein confirmed) Azurin junctions. The inelastic current and hence electron-phonon interaction appears to be rather weak and comparable in magnitude with the inelastic fraction of tunneling current via alkyl chains, which may reflect the known structural rigidity of Azurin.
We observe reversible, bias-induced switching of conductance via a blue copper protein azurin mutant, N42C Az, with a nearly 10-fold increase at |V| > 0.8 V than at lower bias. No such switching is found for wild-type azurin, WT Az, up to |1.2 V|, beyond which irreversible changes occur. The N42C Az mutant will, when positioned between electrodes in a solid-state Au− protein−Au junction, have an orientation opposite that of WT Az with respect to the electrodes. Current(s) via both proteins are temperature-independent, consistent with quantum mechanical tunneling as dominant transport mechanism. No noticeable difference is resolved between the two proteins in conductance and inelastic electron tunneling spectra at <|0.5 V| bias voltages. Switching behavior persists from 15 K up to room temperature. The conductance peak is consistent with the system switching in and out of resonance with the changing bias. With further input from UV photoemission measurements on Au−protein systems, these striking differences in conductance are rationalized by having the location of the Cu(II) coordination sphere in the N42C Az mutant, proximal to the (larger) substrate-electrode, to which the protein is chemically bound, while for the WT Az that coordination sphere is closest to the other Au electrode, with which only physical contact is made. Our results establish the key roles that a protein's orientation and binding nature to the electrodes play in determining the electron transport tunnel barrier.
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.