Affinity binding via Zinc(II) for controlled orientation and electrochemistry of Histidine-tagged nitrate reductase in self-assembled monolayers

Monolayers of CuII-complexes on electrode surfaces are frequently applied for the immobilization and controlled orientation of His-tagged redox proteins. However, affinity binding is limited to applications that require potentials less negative than the reduction potential of the metal complexes. In order to overcome this limitation, we used Zn2 + cations on nitrilotriacetic acid (NTA) modified carbon electrodes for the coordination of His-tagged nitrate reductase (NaR). The NTA modified electrodes were prepared upon diazotation and electrochemical reduction of an aniline functionalized NTA ligand. After coordination of Zn2 + to the bound NTA ligand, self-assembly of NaR is achieved via coordination of the imidazole groups from the His-tag to the NTA–ZnII complex. The electrochemical investigations of the NaR monolayer on NTA–ZnII films demonstrate the catalytic activity for reduction of nitrate to nitrite in the presence of methyl viologen. The catalytic current density correlates with the one expected for a fully active enzyme monolayer. Moreover, the reduction of Zn2 + is not observed at the potential necessary for the reduction of methyl viologen. Therefore, affinity binding based on Zn2 + may be used for the immobilization and electrochemical applications of His-tagged NaR.

► NTA–ZnII complexes for affinity binding on electrode surfaces
► Self-assembled monolayers of fully active His-tagged nitrate reductase
► Attachment unaffected by potentials necessary for nitrate reduction