Ordered Assembly and Controlled Electron Transfer of the Blue Copper Protein Azurin at Gold (111) Single-Crystal Substrates Qijin Chi, Jingdong Zhang, Jens E. T. Andersen, and Jens Ulstrup* Department of Chemistry, Building 207, Technical UniVersity of Denmark, DK-2800 Kgs. Lyngby, Denmark ReceiVed: February 14, 2001 We have shown that Pseudomonas aeruginosa azurin can be immobilized on alkanethiol monolayers self- assembled 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. Introduction Important features of pure and applied nanoscale biochemistry involve two-dimensional organization of biomolecules on solid surfaces or other interfaces. The molecules range from inter- mediate-size organic molecules such as nucleotide bases and amino acids to biological macromolecules such as DNA and proteins. 1-11 Langmuir-Blodgett techniques and molecular self- assembly have been used to prepare the two-dimensional layers. Self-assembly has proved efficient for small organic molecules and some biological macromolecules, particularly molecules with thiol groups. Monolayers of DNA and proteins can be prepared either by using thiolate-containing DNA 12-17 or proteins, 18-20 or by immobilizing proteins on self-assembled monolayers (SAMs) of pure or functionalized thiolates. 21-30 Structural and functional characterization has been explored using various spectroscopies, scanning probe microscopies, and electrochemistry. Such studies are important both for funda- mental understanding and technological utility. The systems provide attractive models to disclose mechanisms of biological macromolecule adsorption and function at surfaces, while new generation nanoscale devices based on such studies could hold technological perspectives. Self-assembly of redox metalloproteins offers not only an immobilization method but also control of electron (ET) routes. Tarlov and Bowden first used carboxylic acid terminated alkanethiol monolayers to immobilize cytochrome c. 22 Direct electronic communication between adsorbed cyt c and the Au electrode could be detected by cyclic voltammetry. 29 The attachment of protein molecules is most likely by electrostatic interactions between deprotonated carboxylic groups in the thiol layer and positively charged lysine residues in cyt c. 30 This system has subsequently been characterized broadly by elec- trochemistry, electroreflectance spectroscopy, and resonance Raman spectroscopy. 31-39 A recent report by Gaigalas et al. 40 showed that the blue copper protein, azurin, can be adsorbed on methyl-terminated hexanethiol SAMs at polycrystalline Au electrodes, and absorbed protein molecules exhibit quasi- reversible electron exchange with the underlying Au substrate. In previous investigations, we have shown that Pseudomonas aeruginosa azurin can assemble directly on Au(111) by adsorp- tion via the surface disulfide group. Structures and ET function of molecular monolayers were characterized by electrochemistry, in situ STM, and X-ray photoelectron spectroscopy (XPS). 41-43 The molecular orientation is shown in Figure 1A. The copper redox center is opposite to the electrode surface in this orientation, and the distance between the copper center and the electrode surface about 25 Å. Electrochemical impedance spectra gave an ET rate constant of 30 s -1 , but no signal could be detected from cyclic voltammetry. 42,43 This is most likely due to the dispersion of ET rate constants and the capacitive background from the azurin film. In the present work, we have provided conditions for an alternative molecular orientation with the copper center facing the single-crystal electrode surface, such as shown in Figure 1B. This orientation has been achieved by using methyl-terminated alkanethiol SAM on which azurin molecules are immobilized through hydrophobic interactions between the hydrophobic area around the copper center in azurin and methyl heads in the alkanethiol. These systems are stable and possess high signal-to-noise ratio. The interfacial ET rate can, moreover, be controlled by varying the alkyl chain length. This study has disclosed a new case for tunneling between the electrode and a protein and provided a value for the nuclear * Author to whom correspondence should be addressed. Phone: +45 45252359. Fax: +45 45883136. E-mail: ju@kemi.dtu.dk. 4669 J. Phys. Chem. B 2001, 105, 4669-4679 10.1021/jp0105589 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/01/2001