Abstract
One of the processes most studied in bioenergetic systems in recent years is the oxygen reduction reaction (ORR). An important challenge in bioelectrochemistry is to achieve this reaction under physiological conditions. In this study, we used bilirubin oxidase (BOD) from Myrothecium verrucaria, a subclass of multicopper oxidases (MCOs), to catalyse the ORR to water via four electrons in physiological conditions. The active site of BOD, the T2/T3 cluster, contains three Cu atoms classified as T2, T3α, and T3β depending on their spectroscopic characteristics. A fourth Cu atom; the T1 cluster acts as a relay of electrons to the T2/T3 cluster. Graphite electrodes were modified with BOD and the direct electron transfer (DET) to the enzyme, and the mediated electron transfer (MET) using an osmium polymer (OsP) as a redox mediator, were compared. As a result, an alternative resting (AR) form was observed in the catalytic cycle of BOD. In the absence and presence of the redox mediator, the AR direct reduction occurs through the trinuclear site (TNC) via T1, specifically activated at low potentials in which T2 and T3α of the TNC are reduced and T3β is oxidized. A comparative study between the DET and MET was conducted at various pH and temperatures, considering the influence of inhibitors like H2O2, F−, and Cl−. In the presence of H2O2 and F−, these bind to the TNC in a non-competitive reversible inhibition of O2. Instead; Cl− acts as a competitive inhibitor for the electron donor substrate and binds to the T1 site.
Highlights
One of the most interesting phenomena to have been intensively studied over the last 25 years is the electronic coupling between the redox cofactor of proteins and electrodes by direct (DET) or mediated electron transfer (MET) reactions [1,2]
It is important to note that electrocatalysis by adsorbed enzymes presents a residual slope and voltammetry curves show a linear response, suggesting that the electrode follows Ohm’s law
Spectroscopies and electrochemistry are used to identify different resting forms of multicopper oxidases (MCOs). One of these forms corresponds to a fully oxidized resting form (RO), which is derived from the decay of the native intermediate (NI) [27]
Summary
One of the most interesting phenomena to have been intensively studied over the last 25 years is the electronic coupling between the redox cofactor of proteins and electrodes by direct (DET) or mediated electron transfer (MET) reactions [1,2]. BODs, a sub-class of MCOs containing four Cu ions per one enzyme molecule, were discovered in 1981 by Tanaka and Murao [18], and first used for the detection of bilirubin [19] and later for the reduction of O2 [20,21] According to their magnetic and optical properties, the Cu atoms are classified into three types T1, T2 and a binuclear T3 (T2 and T3 sites are combined in a tri-nuclear cluster or TNC) [16,22,23,24]. The electron transfer processes for the different states of BOD in the presence of an osmium polymer were studied and compared to DET. F− anions were analysed in the activation of the AR form in the DET and MET of bilirubin oxidase on graphite electrodes
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