Studying direct electron transfer by site-directed immobilization of cellobiose dehydrogenase.

Covalent coupling between a surface exposed cysteine residue and maleimide groups was used to immobilize variants of cellobiose dehydrogenase (CDH) at multiwall carbon nanotube electrodes. By introducing individual cysteine residues at particular places on the surface of the flavodehydrogenase domain of the flavocytochrome we are able to immobilize the different variants in different orientations. Our results show that direct electron transfer (DET) occurs exclusively through the haem cofactor and that the redox potential of the haem is unaffected by the orientation of the enzyme. Electron transfer between the haem and the electrode is fast in all cases and at high glucose concentrations the catalytic currents are limited by the rate of inter-domain electron transfer (IET) between the FAD and the haem. Using ferrocene carboxylic acid as a mediator we find that the total amount of immobilized enzyme is 4 to 5 times greater than the amount of enzyme that participates in DET. The role of IET in the overall DET catalysed oxidation was also demonstrated by the effects of changing Ca concentration and by proteolytic cleavage of the cytochrome domain on the DET and MET currents.