Site-Directed Immobilization of Bilirubin Oxidase for Electrocatalytic Oxygen Reduction

Abstract

In this work, we extended the generic approach for the sitedirected immobilization of enzymes based on maleimide\thiol coupling of engineered enzymes to the oriented immobilization of variants of bilirubin oxidase from Magnaporthe oryzae (MoBOD) to electrodes. We show that this approach leads to the stable attachment of the enzyme to the electrode surface and that the immobilized MoBOD variants are active for bioelectrocatalytic reduction of dioxygen through direct (unmediated) electron transfer (DET) from the electrode. For the three MoBOD variants studied, significant differences are observed in the kinetics of DET that relate to the orientation of the enzyme and the distance of the T1 site from the electrode surface. The stability of the immobilized enzymes allows us to compare the DET and mediated electron-transfer (MET) pathways and to investigate the effects of pH and Cl − . Our studies show a change in the slope of pH dependence at pH 6.0 and highlight the effect of Cl − on the direct oxygen reduction by MoBOD as a function of pH for the immobilized enzyme and the interconversion of the resting oxidized (RO) form of the immobilized enzyme and the alternative resting (AR) state formed in the presence of Cl − .

Rational Design of Enzyme‐Modified Electrodes for Optimized Bioelectrocatalytic Activity

“…We reported earlier the use of a mutant BOD for the site directed immobilization of the enzyme through a 4‐step coupling on gold electrodes . In the present work, the same mutant, (cys‐BOD), with a specifically located cysteine residue on the outer surface of the enzyme close to the T1 active center (Figure S2), was immobilized on a gold surface in only one step, allowing the formation of a layer of BOD and efficient DET.…”

Efficiency of Site‐Specific Clicked Laccase–Carbon Nanotubes Biocathodes towards O2 Reduction

“…Comparatively, reports targeting specific modifications of the surface of MCOs—that is, terminal tags, single surface exposed reactive variant or unnatural amino acid—are still few . Probably seen as complex because a modification of the DNA to encode a site‐selective anchor point is a prerequisite, this strategy has yet no equivalent to generate uniform enzyme–electrode interfaces where almost each enzyme molecule is bound through the same single point.…”

“…[13,[27][28][29] Comparatively,r eports targetings pecific modificationso f the surface of MCOs-that is, terminal tags, single surface exposed reactive variant or unnatural amino acid-are still few. [30][31][32] Probably seen as complex because am odification of the DNA to encodeasite-selective anchorp oint is ap rerequisite, this strategy has yet no equivalent to generate uniform enzyme-electrode interfaces where almost each enzymem olecule is bound through the same single point. Moreover, in principle, for any given enzyme-electrode couple the interface can be as finely adjusted as movingt he anchoring point( i.e., the reactive function) from one position to an adjacent surface exposed position.…”

Bismuth Vanadate/Bilirubin Oxidase Photo(bio)electrochemical Cells for Unbiased, Light‐Triggered Electrical Power Generation

“…The hydrophobic pocket enables a short electron transfer distance, which, in turn, leads to an efficient reduction of the T1 Cu ion. Subsequently, the process activates the oxygen reduction reaction at the T2/T3‐type Cu ion active site . The use of negative and positive charges on hydrophobic surfaces at different pH values was examined comprehensively by Lojou and co‐workers .…”

“…An elegant approach was developed recently in which BOD or copper oxidase was modified genetically in close proximity to the T1 Cu ion site. By the addition of a “click chemistry” step to introduce noncanonical amino acid or surface cysteine, short electron transfer distances between the electrodes and the T1 sites and improved enzyme orientation were achieved.…”