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.

Microscopic structures for molecular monolayers of L-cysteine and L-cystine assembled on Au(111) have been disclosed by employing electrochemistry and in situ scanning tunneling microscopy (STM). Highresolution STM images show that the adlayers of both cyteine and cystine exhibit highly-ordered networklike clusters with (3 3 × 6)R30°structure. By combining the surface coverage estimated from voltammetric data, each cluster is demonstrated to include six individual cysteine molecules or three cystine molecules. As a comparison, no cluster structure is observed for the 1-butanethiol adlayer prepared and examined under the same conditions as those for cysteine and cystine. This suggests that intermolecular and intramolecular hydrogen bonds among adsorbed cysteine or cystine molecules could be responsible for the origin of the cluster-network structures for the adlayers. Several models are proposed and used to explain the in situ STM observations in detail.

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In situ scanning tunneling microscopy (STM) of redox molecules, in aqueous solution, shows interesting analogies and differences compared with interfacial electrochemical electron transfer ( Molecular long-range electron transfer (ET) in solid phases or liquid solution, in which the ET distance exceeds the structural extension of the donor and acceptor, has been in focus over the last decade and a half (1-5). Progress has rested on synthetic donor-acceptor molecules (1-3), metalloproteins (2, 4, 6, 7), and on new electrochemical systems in which electrons are brought to tunnel across well characterized, self-assembled films (8, 9). These achievements have prompted new theoretical efforts with notions such as directional tunneling along chemical bond networks (10), fluctuating tunnel barriers (11, 12), coherent and resonance ET (13), and self-consistent electronic-vibrational interaction (11).In a parallel development, scanning tunneling and atomic force microscopy (STM and AFM, respectively) have opened exciting new perspectives for mapping molecular adsorbate patterns (14, 15). The primary basis for adsorption patterns in vacuum or air is imaging of small and intermediate-size adsorbate molecules with molecular and submolecular resolution (14-17), combined with theoretical frames for tunneling through adsorbate molecules based on different methodologies (17-21). There are also reported experimental approaches to functional mapping of intermediate-size molecules, most prominently in the form of correlations between the tunnel current and the bias voltage. Target adsorbates for ex situ STM imaging have been, for example, benzene (22, 23), methylazulenes (17, 24), C 60 (25), alkyl and aryl thiolates (17,21,(26)(27)(28), and transition metal phthalocyanins (29) on highly oriented pyrolytic graphite or crystalline surfaces of electronically ''soft'' metals compatible with the adsorbates. STM imaging at the solid͞air interface to molecular and occasionally submolecular resolution also has been extended to biological macromolecules including DNA (30) and a number of redox and nonredox proteins (for an overview, see ref. 31). Functional properties addressed particularly have been the electrical potential distribution and conductivity patterns of the tunnel gap (21, 27-29), resonance tunneling via suitable highest occupied molecular orbitals, lowest unoccupied molecular orbitals, or (transition metal) redox levels (17-21), and the notion of orbital-specific mediation of the tunnel current (29).Combination of STM with concepts of long-range ET in chemical and, particularly, metalloprotein systems must, however, give explicit attention to the fact that the natural medium for most chemical and biological reactivity is aqueous solution. Extension of STM͞AFM to aqueous solution (in situ STM͞ AFM) is established (32, 33) but has raised issues related to adsorbate immobilization, ultrapure solutions, tip coating, independent tip and substrate potential control (32-34), and the fundamental STM and AFM phenomena. In situ...

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We provide a theoretical frame for in situ scanning tunneling microscopy (STM) of adsorbate molecules with low-lying redox levels strongly coupled to the environmental nuclear motion. The STM process is viewed as a coherent two-step electron transfer (ET) involving electron exchange between the local redox level and the manifolds of electronic levels in the substrate and tip. The notion coherence is here taken to imply that the intermediate electron or hole state after the first ET step does not fully relax vibrationally before the second ET step. These views and the theoretical formalism are appropriate to in situ STM of large transition metal complexes and redox metalloproteins. The formalism offers two kinds of spectroscopic features. One is the relation between the tunnel current and the bias voltage at fixed overvoltage of either the tip or the substrate relative to a reference electrode. The other one is the tunnel current dependence on the overvoltage, at fixed bias voltage. Recent data on tunneling current patterns for adsorbed or covalently tethered metalloporphyrins and the blue single-copper protein azurin are discussed in terms of the formalism.

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