Voltammetry and Electrocatalysis of Achromobacter Xylosoxidans Copper Nitrite Reductase on Functionalized Au(111)-Electrode Surfaces

Abstract

A long-standing issue in protein film voltammetry (PFV), particularly electrocatalytic voltammetry of redox enzyme monolayers, is the variability of protein adsorption modes, reflected in distributions of catalytic activity of the adsorbed protein/enzyme molecules. Use of well-defined, atomically planar electrode surfaces is a step towards the resolution of this central issue. We report here the voltammetry of copper nitrite reductase (CNiR, Achromobacter xylosoxidans) on Au(111)-electrode surfaces modified by monolayers of a broad variety of thiol-based linker molecules. These represent positively charged and electrostatically neutral, hydrophobic and hydrophilic, aliphatic and aromatic, and variable-length micro-environments, as well as their combinations. Optimal conditions for enzyme function seems to be a combination of hydrophobic and hydrophilic surface linker properties, which can lead to close to complete non-catalytic monolayer interfacial electron transfer function and electrocatalysis with activity approaching enzyme activity in homogeneous solution. Thiophenol (combined hydrophobic stacking and interdispersed water molecules), 4-methyl-thiophenol (hydrophobic and water molecules), and 3- and 4-aminothiophenol (hydrophilic, hydrophobic) offer the overall most efficient micro-environments. Subtle differences with even small structural linker differences, however, lead to widely different electrocatalytic properties, strikingly illuminated by the ω-mercaptoamines. CuNiR thus shows highly efficient, close to ideal reversible electrocatalytic voltammetry on cysteamine-covered Au(111)-electrode surfaces, most likely due to two cysteamine orientations previously disclosed by in situ scanning tunnelling microscopy. Such a dual orientation exposes both a hydrophobic and a positively charged, hydrophilic surface feature. In contrast, the higher cysteamine homologues expose only the hydrophilic component with no electrocatalytic activity on these surfaces. These results offer a basis for rational surface design in forthcoming biological electrocatalysis useful both fundamentally and in novel biosensor technology.