Abstract
Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme.
Highlights
We have combined surface enhanced infrared absorption (SEIRA) spectroscopy, atomic force microscopy (AFM), and protein film voltammetry (PFV) with molecular dynamics (MD) simulations to elucidate enzyme-surface interactions, thereby providing comprehensive insights into a bioelectronic hybrid system consisting of the oxygen-tolerant Ralstonia eutropha (Re) membrane-bound [NiFe]-hydrogenases (MBHs) immobilized on Au electrodes coated with a selfassembled monolayers (SAMs) consisting of 6-amino-1-hexanethiol
We applied an integral approach of SEIRA spectroscopy, AFM, and PFV in combination with MD simulations to study immobilization, structural integrity, and catalytic activity of an oxygen-tolerant [NiFe] hydrogenase on SAM-coated Au electrodes
We identified at least two enzyme populations that operate with different types of electronic communication with the electrode, i.e. direct and mediated electron transfer
Summary
[NiFe]-hydrogenases catalyze the reversible cleavage of molecular hydrogen into electrons and protons. A systematic study using atomic force microscopy (AFM), PFV, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS) revealed that the H2-driven catalytic currents of the oxygen-tolerant Aa MBH correlate with the electrode surface functionalities used for enzyme immobilization on gold. The latter influence interactions of the protein with the SAM, which were suggested to be governed by the C-terminal hydrophobic tail of the Aa MBH [12]. We have combined SEIRA spectroscopy, AFM, and PFV with MD simulations to elucidate enzyme-surface interactions, thereby providing comprehensive insights into a bioelectronic hybrid system consisting of the oxygen-tolerant Re MBH immobilized on Au electrodes coated with a SAM consisting of 6-amino-1-hexanethiol. The present integral approach is shown to successfully identify those parameters that control enzyme adsorption and orientation on electrodes, thereby ensuring structural integrity and highest catalytic efficiency
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