Site-Directed Immobilization of Bilirubin Oxidase for Electrocatalytic Oxygen Reduction

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In this work, we extended the generic approach for the site-directed immobilization of enzymes based on maleimide coupling of engineered enzymes to the oriented immobilization of variants of bilirubin oxidase from Magnaporthe oryzae ( Mo BOD) to electrodes. We show that this approach leads to the stable attachment of the enzyme to the electrode surface and that the immobilized Mo BOD 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 Mo BOD 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 – .