Ordered assembly and controlled electron transfer of the blue copper protein azurin at gold (111) single-crystal substrates

We have shown that Pseudomonas aeruginosa azurin can be immobilized on alka nethiol monolayers self-assembled on Au(111). Immobilization is achieved th rough hydrophobic interactions between the hydrophobic area around the copp er atom in azurin and methyl heads of alkanethiol to form submonolayers or monolayers. In this orientation mode azurin molecules on Au(111) are orient ed with the redox center (copper atom) facing the electrode surface. This i s 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 a nd protein adlayers are present. Adsorbed azurin molecules exhibit high sta bility and retain electron transfer (ET) function. Long-range interfacial E T between azurin and Au(111) across variable-length alkanethiol bridges was systematically investigated by different electrochemical techniques. Dista nce-dependent ET can be controlled by adjusting the length of the alkanethi ol 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 (beta) of 1.03 +/- 0.02 per CH2 unit at longer ch ain lengths. Overvoltage-dependent ET was also examined. The results provid e a strategy to ordered molecular assemblies, and controlled orientation an d ET of azurin at atomically planar metallic surfaces. This approach can in principle be extended to other redox metalloproteins.