Spectroelectrochemical study of cellobiose dehydrogenase and diaphorase in a thiol-modified gold capillary in the absence of mediators

Abstract

A spectroelectrochemical cell was constructed from a gold capillary with 200 μm inner diameter as a working electrode. This allowed spectroelectrochemical study of liquid samples with available volumes less than 5 μl. The optical measurements were accomplished with an optical fibre spectrometer. The optical path of the cell was about 1 cm. To facilitate electrochemistry of biomolecules, the surface of the gold capillary was modified with thiols. The formal potential, E°′, of the heme in cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium was determined by spectroelectrochemistry in the absence of redox mediators. The number of electrons per redox conversion of heme in CDH was found to be equal to 0.98±0.04 corresponding well to a theoretical value representing the redox reaction Fe 3++ e −=Fe 2+. Similar spectroelectrochemical experiments with diaphorase from Bacillus stearothermophilus showed the redox conversion of the flavin mononucleotide in diaphorase in the absence of external redox mediators.