Cytochromes as electron shuttles from FAD-dependent glucose dehydrogenase to electrodes

Abstract

Cytochromes are a large and well-known group of electron transfer proteins, but only very few are used in bioelectrochemistry, e.g. to serve as electron shuttles between biocatalysts and electrodes. There, cytochromes generate an artificial electron transfer pathway if their structural and electrochemical properties are fitting. Cytochromes considered suitable are small and feature an exposed heme cofactor, a suitable redox potential, an electrochemically reversible redox process, sufficient protein production, and a fast electron transfer between other enzymes and hemoproteins. Out of 850 screened hemoproteins, 58 cytochromes met the requirements to function as an electron shuttle. They can be classified into five distinguished protein folds. Four representative b-type and three c-type cytochromes were selected for further analyses. The proteins were obtained in a yield of 0.075 to 375 mg L-1 in Escherichia coli or Komagataella phaffii. The electrochemical reversibility on 1-thioglycerol functionalized gold electrodes of all cytochromes was confirmed. The pH-dependent redox potentials range from -50 mV to +298 mV vs. SHE at pH 7.0. The electronic wiring ability was tested using the biocatalyst FAD-dependent glucose dehydrogenase (GDH). Cytochrome reduction by the GDH was monitored by spectrophotometry and amperometry. c-Type cytochromes showed a faster electron uptake from GDH than b-type cytochromes. Complementary surface charges at the cytochrome/GDH interface were not a predictor for a fast electron transfer but a higher difference in the redox potential of the cofactors was. This study supports the design of bioelectrocatalysts with high electron transfer rates, which are of great interest in 3rd generation biosensors and for direct bioelectrocatalysis.