Esben P. Friis

In situ STM and AFM of the copper protein Pseudomonas aeruginosa azurin

Abstract Scanning tunnel (STM) and atomic force microscopy (AFM) in the in situ mode under potentiostatic control have opened new perspectives for mapping the two-dimensional organization of surface adsorbates in aqueous solution. In situ STM and AFM, however, also raise recognized problems. In the context of biological macromolecules, sample immobilization and the mechanism of the imaging process are, for example, outstanding issues. We have shown that the blue single-copper redox protein azurin is well suited for gentle surface immobilization and mapping. Azurin has a surface disulphide group which adsorbs to gold and facile electron tunnel routes between this group and the copper atom. Azurin adsorbed on Au(111) can be imaged to molecular resolution by in situ STM and shows regular arrays of individual structures corresponding well to the known molecular size of azurin. The current falls off approximately exponentially with increasing distance with a decay constant of 0.4–0.5 A −1 . In comparison in situ AFM shows structures laterally convoluted with the tip while the vertical extension is in the same range as the structural size of azurin. The results are of interest in relation to electron tunnel mechanisms of redox metalloproteins and in technological contexts such as electrochemical biosensors, microbial corrosion and broadly for protein adsorption from biological liquids.

Electrochemistry of Self-Assembled Monolayers of the Blue Copper Protein Pseudomonas Aeruginosa Azurin on Au(111)

Abstract We report the self-assembly and electrochemical behaviour of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) electrodes in aqueous acetate buffer (pH=4.6). The formation of monolayers of this protein is substantiated by electrochemical measurements. Capacitance results indicate qualitatively that the protein is strongly adsorbed at sub-μM 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 (pH=13), 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.

Metalloprotein adsorption on Au(111) and polycrystalline platinum investigated by in situ scanning tunneling microscopy with molecular and submolecular resolution

Redox metalloproteins exhibit interesting features such as long-range electron transfer (ET), cooperative effects etc. of importance in relation to fundamental ET theory, and mapped in considerable detail. Adsorption and interfacial electrochemical ET of metalloproteins at metallic surfaces is also broadly important in a range of contexts, and has been addressed by spectroscopic, voltammetric, and thermodynamic methods. In situ scanning tunneling (STM) and atomic force microscopy (AFM) have opened new perspectives for addressing adsorbed metalloproteins in their natural functional aqueous medium at the molecular level. In addition to broadly recognized problems of in situ STM AFM imaging, sample preparation, mobility, and adsorbate stability are, however, particular problems. We illustrate here the perspectives by recent in situ STM imaging of covalently bound horse heart cytochrome c on polycrystalline platinum, and of chemisorbed Pseudomonas aeruginosa azurin on Au(111). Molecular resolution is achieved, but azurin gives by far the best images which show, moreover, an interesting submolecular feature. This is likely to be associated with the disulphide group as a natural unit for gentle linking, facile ET routes through the protein, and tunnel enhancement by the low-lying redox level of the copper atom. The particular electronic-vibrational three-level configuration in in situ STM of metalloproteins. finally, offers a new way of distinction between superexchange, coherent, and sequential ET modes in the long-range ET patterns of metalloproteins.

Creating nanoscale pits on solid surfaces in aqueous environment with scanning tunnelling microscopy

A novel method has been developed to fabricate nanoscale pits on Au(111) in aqueous environments by in situ scanning tunnelling microscopy (STM), based on critical interactions between tip and substrate. The most striking advantages of the present method are that the dimension and position of the pits can be controlled well in aqueous environments, and the operations are simple. Parameters affecting the pit formation and size have been systematically characterized to show that pit formation is dominated by bias voltage. A mechanism is proposed based on local surface reconstruction induced by electronic contact between tip and substrate.

Dynamics of Pseudomonas aeruginosa azurin and its Cys3Ser mutant at single-crystal gold surfaces investigated by cyclic voltammetry and atomic force microscopy

Abstract Cyclic voltammetry of Pseudomonas aeruginosa azurin on polycrystalline gold is reversible ( E 0 = 360 mV vs she; 50 mM ammonium acetate) but the voltammetric signals decay with time constants of about 3 × 10 −3 s −1 . No signal is observed for monocrystalline Au(111). Cys3Ser azurin is electrochemically inactive on either type of gold electrode but shows a reversible although decaying peak (362 mV, 50 mM ammonium acetate; decay time constant ≈2 × 10 −3 s −1 ) on edge-plane pyrolytic graphite. Ex situ and in situ atomic force microscopy (AFM) of the azurins on Au(111) show initially arrays of protein structures of lateral 100–200 A and vertical ≈50 A extension. These could be individual molecular images convoluted with the tip curvature. As scanning proceeds the structures in the ex situ mode collect into large two-dimensional assemblies while the adsorbed protein in the in situ mode is largely swept into the solution, recovering the free Au(111) surface. The cyclic voltammetry and AFM data are consistent with time dependent adsorption of the azurins on gold via the disulphide bridge (wild-type) or free thiol group (Cys3Ser mutant).

Sequential and coherent long-range electron transfer close to resonance with intermediate bridge groups, and new perspectives for in situ scanning tunnelling microscopy of adsorbed metalloproteins

Abstract Three-level electron transfer follows superexchange patterns when the intermediate electronic level is off-resonance with the donor and acceptor levels. Close to resonance, new patterns emerge where the intermediate level is temporarily populated in vibrationally coherent or incoherent modes. We discuss energy and distance relations associated with such electron transfer modes. These appear to accord with fast electron transfer in several chemical and biological systems. We also discuss some recent observations on in situ scanning tunnelling microscopy of metalloproteins and large transition metal complexes which enable, in principle, a distinction between superexchange, coherent, and sequential three-level electron transfer.

In situ scanning probe microscopy and new perspectives in analytical chemistry

Abstract The resolution of scanning tunnelling microscopy (STM) and other scanning probe microscopies is unprecedented but the techniques are fraught with limitations as analytical tools. These limitations and their relationship to the physical mechanisms of image contrast are first discussed. Some new options based on in situ STM, which hold prospects for molecular- and mesoscopic-scale analytical chemistry, are then reviewed. They are illustrated by metallic electro-crystallisation and -dissolution, and in situ STM spectroscopy of large redox molecules. The biophysically oriented analytical options of in situ atomic force microscopy, and analytical chemical perspectives for the new microcantilever sensor techniques are also discussed.