Comparison of Direct and Mediated Electron Transfer for Bilirubin Oxidase from Myrothecium Verrucaria. Effects of Inhibitors and Temperature on the Oxygen Reduction Reaction

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.

Use of a Thermophile Desiccation-Tolerant Cyanobacterial Culture and Os Redox Polymer for the Preparation of Photocurrent Producing Anodes

“…At 0.26 V, it is possible to observe the reversible peak for the OsP, corresponding to the Os(III)/Os(II) redox couple. Similar results were obtained in previous works ( Hasan et al, 2014 ; Hamidi et al, 2015 ; Antiochia et al, 2013 , 2019 ). Interestingly, at higher potential (0.65 V), a second oxidation peak is obtained.…”

“…In an attempt to improve electrical wiring of electroactive bacteria, GE were modified with an OsP, an electrode wiring mediator previously used for wiring both enzymes and microorganisms to electrodes ( Ohara et al, 1993 ; Vostiar et al, 2004 ; Timur et al, 2007a , b ; Coman et al, 2009 ). Increased photocurrent outputs are usually obtained when in the presence of OsP because a higher number of molecules or living cells can be wired to the electrode because the OsP is usually present in excess and can penetrate the protein and membrane structures ( Scheme 1B ) ( Kurbanoglu et al, 2018 ; Antiochia et al, 2019 ). In Figure 5 , a typical CV obtained with electrodes modified with both OsP and Gl is presented.…”

Inhibition in multicopper oxidases: a critical review

“…175,250 The CV trace of the AR state is characterized by cross-over features as well as a shift in the E cat from the first to the second scan, and has been observed by others reporting Cl − inhibition of BODs. 176,184,251 The forward and reverse traces can loop or cross over each other, as they appear on the 12 mM Cl − trace in Fig. 6B at around 0.5 V. This hysteresis between the reductive and oxidative scans has been proposed to be the result of an inhibitor binding near the T1 site, which creates an electrostatic barrier to interfacial electron transfer to the T1 Cu.…”

Recent Advances in the Direct Electron Transfer-Enabled Enzymatic Fuel Cells

“…Carbon-based nanomaterials exhibit high electrical conductivity and good mechanical properties, as well as various physical and electrical properties depending on the control of their morphologies. Various carbonaceous materials such as carbon fibers or papers (Xu and Minteer, 2012 ; Kuo et al, 2013 ), carbon black (Kamitaka et al, 2007 ; Gupta et al, 2011 ; Haneda et al, 2012 ; Xia et al, 2016a ), carbon nanoparticles (Selloum et al, 2014 ), graphene (Chen et al, 2012 ; Campbell et al, 2015 ; Song et al, 2015 ), graphite (Tasca et al, 2015 ; Antiochia et al, 2019 ), and carbon nanotubes (Gao et al, 2010 ; Ciaccafava et al, 2012 ; Agnès et al, 2013 ) have been employed as EFC electrode materials. For example, buckypaper form of multiwalled carbon nanotubes (MWNTs) demonstrated excellent potential as enzymatic electrode material by 68-fold increase in current density from oxygen reduction reaction catalyzed by laccase compared to as-prepared agglomerates of MWNTs (Hussein et al, 2011 ).…”