Surface characterization and direct electrochemistry of redox copper centers of bilirubin oxidase from fungi Myrothecium verrucaria.

The key characteristics of multicopper oxidases are redox potentials of Type 1, Type 2 and Type 3 copper centers of enzymes. However, there is still a challenge to obtain a value of the redox "signature" of the enzymes. In this study, the electrochemical behavior of T1 and T2/T3 redox copper centers of bilirubin oxidase (BOD) from the fungi Myrothecium verrucaria was studied based on direct bioelectrocatalysis. Two distinct redox peaks corresponding to reduction and oxidation of T1 and T2/T3 redox centers of enzymes have been clearly detected in anaerobic conditions. The bioelectrocatalytic activity of the enzyme was studied in the presence of oxygen and redox mediators. The electron-transfer rate constant for BOD immobilized on carbon electrode (CE) is 1.5 s(-1). The mechanism of enzyme inactivation by ABTS has been proposed. The physical architecture of BOD layers immobilized on the electrode surface, including elemental and chemical composition, relative thickness and assembly of layers was investigated by Angle Resolved X-ray photoelectron spectroscopy. Unique peaks of BOD at 288.5 eV and of CE at 284.6 eV were used in a substrate over layer model for estimation of the thickness of the of BOD film on the carbon electrode surface.