Improving enzyme-electrode contacts by redox modification of cofactors

Efficient electron transfer of redox proteins to and from theirenvironment is essential for the use of such proteins in biotechno-logical applications such as amperometric biosensors and photo-synthetic biocatalysts^1-3. But most redox enzymes lack pathwaysthat can transport an electron from their embedded redox siteto an electrode^4,5 or a diffusing photoexcited species6. Electricalcommunication between redox proteins and electrode surfaces hasbeen improved by aligning proteins on chemically modifiedelectrodes^7-9, by attaching electron-transporting groups^10,11 andby immobilizing proteins in polymer matrices tethered by redoxgroups^2-14. Generally these methods involve contacting theenzymes at random with electron relay units. Here we report anapproach that allows site-specific positioning of electron-mediatingunits in redox proteins. We strip glucose oxidase of its flavinadenine dinucleotide (FAD) cofactors, modify the latter withredox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solutionis greatly enhanced in a controlled and reproducible way.