Implications of active site orientation in myoglobin for direct electron transfer and electrocatalysis based on monolayer and multilayer covalent immobilization on gold electrodes

Cyclic voltammetry (CV) and atomic force microscopy (AFM) were used to investigate the importance of active site orientation of an immobilized protein for direct electron transfer (DET) and electrocatalysis. While the bioconjugated wild-type myoglobin (WT Mb) was immobilized on the modified gold electrode surface in a random multilayered fashion, the Ser3 replaced with unnatural amino acid, 3-amino-l-tyrosine, (NH2Tyr) mutant Mb was immobilized via a Diels–Alder reaction specific to NH2Tyr residue to form a homogeneous monolayer. Electrochemical calculations for the number of surface exposed redox-active molecules on the electrode surface (Γ) and heterogeneous rate constant for DET were 1.29 × 10−10 mol cm−2; 2.3 s−1 for the WT Mb and 1.54 × 10−10 mol cm−2; 1.3 s−1 for the S3NH2Tyr Mb mutant, respectively. Electro-catalytic conversion of thioanisole to sulfoxide products showed similar turnover frequencies (TOF) around 1.9 × 103 s−1 (with 87% conversion) for the WT Mb, and 1.5 × 103 s−1 for the mutant S3NH2Tyr Mb (with 81% conversion). These results indicate that site-directed single monolayer immobilization affords almost the same number of surface exposed Mb active sites as the random multilayer immobilization strategy, though the latter contains a greater number of protein molecules on the electrode surface, as observed from the AFM data.

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