Bilirubin oxidase bioelectrocatalytic cathodes: the impact of hydrogen peroxide

Abstract

Mediator-less, direct electro-catalytic reduction of oxygen to water by bilirubin oxidase (Myrothecium sp.) was obtained on anthracene-modified, multi-walled carbon nanotubes. H₂O₂ was found to significantly and irreversibly affect the electro-catalytic activity of bilirubin oxidase, whereas similar electrodes comprised of laccase (Trametes versicolor) were reversibly inhibited.

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Multi-copper oxidases (MCOs), such as bilirubin oxidase (BOd) and laccase, are commonly incorporated within enzymatic biological fuel cells (EBFCs) as cathodic biocatalysts responsible for the 4-electron reduction of dissolved O₂ to H₂O.^1–6 BOd and laccase both contain 3 different copper centres that are responsible for the catalysis of substrates, and they are classified as type 1 (T1), type 2 (T2) and a binuclear type 3 (T3). The T2 and T3 sites are combined in a tri-nuclear cluster (TNC).⁷ The single-electron oxidation of substrates takes place at the proximal T1 site, with the 4-electron reduction of O₂ taking place at the TNC. Following the oxidation of substrates, 4 electrons are quickly transferred between the T1 site and the TNC (in 4 × 1-electron transfers) of MCOs via a His-Cys-His chain.^7,8 It has also been reported that laccase and BOd can promote the reduction of O₂ to H₂O via a (albeit unfavourable) 2-electron peroxide intermediate step.^9,10

In contrast to laccase (Trametes versicolor), BOd (Myrothecium sp.) is capable of reducing O₂ in neutral or near-neutral pH conditions.¹¹ The operation of a cathode in neutral pH conditions is favourable for enzymatic devices incorporating glucose-oxidising enzymes such as glucose oxidase (GOx) or glucose dehydrogenase (GDH) that commonly have neutral optimum pH's. This has implications for devices in physiological conditions, pH 7.3–7.4 is being considered to be physiological pH.^3,12

The electro-catalytic reduction of O₂ by MCOs can be facilitated by an electron-mediated route (mediated electron transfer, MET) or by direct electron transfer between the enzyme and electrode (DET). Common electron mediators for the oxygen reduction reaction (ORR) with BOd or laccase include ABTS‡^13,14 and osmium-based redox polymers.^15,16 DET of laccase has been reported at gold, graphite and carbon nanotube surfaces, where anthracene moieties induce and enhance communication between the T1 site of the MCO and the supporting electrode.^2,17–21 The reduction of O₂ by DET can even be observed for laccase on unmodified gold electrodes.²² Furthermore, DET by BOd has also been demonstrated on many electrode architectures,^4,5,23 including by the attachment of poly-cyclic moieties to graphite electrodes.²⁴

It is widely reported that the activity of laccase and BOd can be inhibited by halide anions, but BOd is reported to be less-sensitive to Cl⁻ ions.^11,12,25 It has been shown that the mechanisms of inhibition of MCOs by F⁻ and Cl⁻ differ; F⁻ has been demonstrated to act as a non-competitive inhibitor that directly disrupts electron transfer between the T1 and TNC of MCOs whereas Cl⁻ has been shown to behave as a competitive inhibitor (with a physiological concentration of around 150 mM), that suppresses the oxidation of substrates (and communication with mediators) at the T1 site.^11,22,26,27 Thus, it has been shown that DET with the T1 site can introduce varying degrees of Cl⁻ resistance to MCOs. F⁻, however, still remains a problem for DET-based cathodes.²²

Work by Calvo et al. in 2010 reported the inhibition of laccase (Trametes trogii) by H₂O₂ during O₂ reduction by MET via an osmium redox polymer.¹⁵ We have previously demonstrated the inhibition of laccase (Trametes versicolor) by H₂O₂ on DET-based cathodes; it was shown that GOx anodes produced significant quantities of H₂O₂ to effectively inhibit laccase in membrane-less glucose/O₂ EBFCs.²⁸ This could suggest that H₂O₂ inhibits MCOs in a similar fashion to F⁻ (non-competitive); this may differ between MCOs.

Fig. 1 presents cyclic voltammograms of BOd cathodes immobilised on a Toray carbon paper electrodes comprised of anthracene-modified multi-walled carbon nanotubes (Ac-MWCNTs). Complete electrode preparation procedures are detailed within the ESI.† Comparison of cyclic voltammograms recorded under argon and under flowing air indicates the DET reduction of O₂ by BOd, at an onset potential of approximately 0.57 V (vs. Ag/AgCl) at pH 6.5 and reaching a current density of approximately 0.28 mA cm⁻² (at a scan rate of 1 mV s⁻¹). Further potentiostatic experiments (Fig. 2 and in the ESI† in Fig. S3) under air-purging conditions provide catalytic current densities of approximately 0.36 mA cm⁻² at 0.2 V (vs. Ag/AgCl). The reduction of O₂via DET by these cathodes was also evaluated between pH 4.5 and 7.5 (Fig. S1 and S2A and B, ESI†), with the optimum pH found to be 6.5. The reduction of O₂via DET by BOd was also investigated on hydroxylated MWCNTs (OH-MWCNTs) that serve as a precursor to the Ac-MWCNTs. Ac-MWCNTs showed improved cyclic voltammograms, suggesting that the presence of anthracene moieties can favourably orientate BOd to facilitate DET with a lower over-potential for O₂ reduction. Furthermore, the reduction of O₂via DET by BOd on Ac-MWCNTs gave comparable performance to laccase/Ac-MWCNTs cathodes under similar conditions and optimal pH (ESI,† Fig. S4).

Fig. 1 Cyclic voltammograms of BOd/Ac-MWCNTs Toray-paper electrodes under argon purged (dotted line) and air-purged (solid line) hydrodynamic conditions, at a scan rate of 1 mV s⁻¹ (0.2 M citrate/phosphate buffer, pH = 6.5). The dashed line presents a BOd/OH-MWCNTs Toray-paper electrode (same conditions), where the OH-MWCNTs is the precursor to the Ac-MWCNTs.

Fig. 2 Chronoamperometric trace for a (A) BOd/Ac-MWCNTs (pH 6.5) and (B) laccase/Ac-MWCNTs (pH 4.5) Toray-paper electrode in a hydrodynamic citrate/phosphate buffer solution (0.2 M), poised at 0.2 V (vs. Ag/AgCl). n.b. air and argon gasses were purged individually – air purging was terminated at t = 3600 seconds.

Fig. 2A presents the chronoamperometric trace of a BOd/Ac-MWCNTs electrode with additions of air, H₂O₂ and catalase at differing times during the experiment. Upon the initiation of air-purging, a current response of approximately 0.36 mA cm⁻² is observed, for the reduction of O₂ by BOd via DET. The addition of ABTS to the electrolyte (final concentration of 0.2 mM) results in an approximate improvement of 11% of the current density, suggesting that most of the immobilised BOd undergoes DET. H₂O₂ was added to the electrolyte, and after 2000 seconds of exposure (t = 3000 seconds) an addition of catalase was made (final concentration of 20 μg ml⁻¹). After subsequent intermittent periods of argon-purging (with a further addition of catalase), no significant recovery of the catalytic current for the reduction of O₂ was observed for both MET and DET. This could suggest that exposure to H₂O₂ had denatured or strongly (perhaps irreversibly) inhibited the catalytic activity of BOd (measured up to 3000 seconds). Electrode rinsing and electrolyte replacement did not recover any lost catalytic current.

The effect of H₂O₂ on the laccase/Ac-MWCNTs cathodes was also investigated under similar conditions (Fig. 2B). Although a current density of approximately 0.29 mA cm⁻² was observed, the addition of ABTS (0.2 mM) results in an approximate improvement of 59% of the DET current (implying not all immobilised laccase undergoes DET). A concentration of 20 mM H₂O₂ was found to sharply remove approximately 75% of the total catalytic current, which was recovered to 98% after the first addition of catalase (20 μg ml⁻¹). Argon-purging confirmed enzymatic activity (and no interference from catalase). During argon cycling, the total catalytic current did not return to 98%; no air-purging was performed and therefore the dissolved O₂ concentration was recovered via solution perturbation (hydrodynamic) and the decomposition of any remaining H₂O₂ by catalase.

The catalytic activities of these BOd/Ac-MWCNTs and laccase/Ac-MWCNTs electrodes were also tested in the presence of 150 mM Cl⁻ and 15 mM F⁻ (Fig. S3A and B, respectively (ESI†)). For the BOd/Ac-MWCNTs electrode, the addition of ABTS (final concentration of 0.2 mM) saw an approximate increase in 3% of the catalytic current; the addition of Cl⁻ and F⁻ gave decreases in total catalytic current of approximately 15% and 11%, respectively. Furthermore, argon-purging the solution removed catalytic activity. For the laccase/Ac-MWCNTs electrode, the addition of ABTS (final concentration of 0.2 mM) gave an increase of approximately 70% of the catalytic current from DET. The addition of Cl⁻ removed the current resulting from mediated electro-catalysis, with further removal of approximately 19% of the DET current. The addition of F⁻ completely inhibited catalytic activity of laccase. Control experiments with bovine serum albumin as a protein showed no significant response upon the addition of any of ABTS, Cl⁻, F⁻ and H₂O₂ (data not shown). The catalytic activity of laccase (determined by chronoamperometry) in the presence of H₂O₂ was also studied (Fig. S5, ESI†).

Specific enzymatic activities were spectrophotometrically determined to be 12.1 ± 0.2 U mg⁻¹ and 10.3 ± 0.2 U mg⁻¹ for BOd (Myrothecium sp., pH 6.5) and laccase (Trametes versicolor, pH 4.5) respectively, using ABTS as the substrate. For BOd, this decreased by approximately 65% after 5 min of exposure to a 25 mM H₂O₂ solution. The rapid removal of H₂O₂ by catalase did not recover specific enzymatic activity (ESI†).

Although the laccase/Ac-MWCNTs electrodes presented within this study are sharply and significantly inhibited by H₂O₂, the addition of catalase can effectively restore enzymatic activity. Thus, the co-immobilisation of catalase (or other H₂O₂-decomposing moiety) within EBFC electrodes (albeit effective) could be replaced with “on the spot” treatments to remove H₂O₂. For BOd, however, it is suggested that catalase (or another H₂O₂-decomposing moiety) is permanently required within EBFCs that are likely to produce H₂O₂; this study suggests that BOd is less resistant to H₂O₂ than laccase and that enzymatic activity cannot be easily restored by the removal of H₂O₂ (albeit at relatively high concentrations). Further studies are required to determine whether the concentrations of H₂O₂ produced as a function of anodic side reactions (for example, by GOx) in common EBFCs, are sufficient to be detrimental to the performance of BOd-containing devices.

The authors thank Sekisui Diagnostics (UK) for their kind donation of BOd. This research was financially supported by the U.K. Engineering and Physical Sciences Research Council's SuperGen Biological Fuel Cells Consortium (EPSRC contract EP/H019480/1). The authors thank National Science Foundation Grant #105797 and the Air Force Office of Scientific Research for funding.

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Footnotes

† Electronic supplementary information (ESI) available: Fig. S1–S5, experimental details and procedures. See DOI: 10.1039/c3cc47689h

‡ ABTS = 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid).

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