Bioelectrocatalytic activity of reconstituted peroxidase on hemin-terminated PQQ-modified electrodes

Abstract

Efficient direct electrical communication between an enzyme redox center and an electrode is of great interest from the viewpoints of application to reagentless biosensors and biofuel cells. To immobilize redox enzymes in a favorable orientation for electron transfer at the electrode surface, we reconstituted apo-horseradish peroxidase (apo-HRP) onto a hemin cofactor linked to pyrroloquinoline quinone (PQQ) spacer at an indium-tin-oxide (ITO) electrode surface. The bioelectrocatalytic activity of reconstituted HRP for H2O2 reduction was evaluated by means of amperometry at +0.150 V (vs. Ag|AgCl), which value is larger than that for the redox potential of PQQ (ca. –0.140 V). The complex-formation reaction rate of reconstituted HRP with H2O2 enhanced in comparison with that for the hemin linked to PQQ electrode without apo-HRP by a factor of 200. PQQ served as the part of spacer for the insertion of hemin into a certain desired position in apo-HRP. Although native HRP cannot electrochemically communicate with the ITO electrode in general, the quasi-direct electron transfer from the ITO electrode to reconstituted HRP oxidized by H2O2 was also achieved most likely by electron hopping and/or resonant transport without the redox mediation thanks to the π-conjugated PQQ molecule in the spacer.

Keywords

• Enzyme orientation
• π-Conjugated molecules
• Enzyme activity regulation
• Kinetic analysis
• Heme proteins