Molecular recognition: design of a biosensor with genetically engineered azurin as redox mediator

Abstract

The development of a second generation biosensor that uses azurin as mediator for electron transport at the electrode surface is reported. Azurin is a small (14.6 kDa) bacterial protein that is relatively stable and that can be expressed in high yield in a heterologous system (E. coli) making it an ideal candidate for protein engineering studies and biosensor development. This protein has a role as a natural electron transfer carrier in the bacterial anaerobic respiratory chain and is able to transfer electrons from cytochrome C-551 to nitrate reductase. The active centre of azurin is a type-1 copper centre. The copper ligand His(117) protrudes through the protein surface, and it has been mutated by site-directed mutagenesis into a glycine (His(117)Gly). The absence of the side-chain of the glycine residue creates an aperture on the protein surface that makes the copper centre accessible to external ligands. In this way the azurin His(117)Gly mutant can accept external ligands, such as 4-methylimidazole, that can be bound to the electrode surface. Attempts to establish fully reversible redox chemistry with the mutated azurin are underway. In principle, this allows the immobilization of azurin on an electrode surface for the construction of a biosensor. In this way azurin can be used as mediator in transferring electrons between the enzymatic system present in solution and the electrode. The combination of protein engineering with electrode surface modification allows further advances in biosensor technology.