A friendly detergent for H2 oxidation by Aquifex aeolicus membrane-bound hydrogenase immobilized on graphite and Self-Assembled-Monolayer-modified gold electrodes

Immobilization of membrane bound enzymes onto electrodes is of great interest for studying physiological electron transfer processes and for biotechnological devices. Hydrogenases are the key enzymes for hydrogen metabolism in many microorganisms. Due to the high efficiency and specificity they develop for H2 oxidation, research in the last 5 years has aimed towards their use as biocatalysts for H2/O2 biofuel cells to replace platinum-based bioanodes. We demonstrate in this work that the O2-, CO- and T°-resistant membrane-bound [NiFe] hydrogenase purified from the hyperthermophilic bacterium Aquifex aeolicus can be efficiently immobilized onto electrodes. Both pyrolytic graphite (PG) and hydrophobic Self-Assembled-Monolayers (SAMs) on gold electrodes are used for hydrogenase immobilization. According to the chemistry and structure of the electrochemical interface, a different process for H2 oxidation is observed, from direct to mediated electron transfer process. To gain new insight in the catalytic process, a quantification of the remaining detergent surrounding the membrane protein is performed. Adsorption isotherms of hydrogenase are determined as a function of the electrode material and the amount of detergent. Competitive adsorption of free detergent and hydrogenase is demonstrated coupling electrochemistry and Polarization Modulation Infrared Reflexion Adsorption Spectroscopy (PM-IRRAS). The efficiency of the enzymatic process is then analyzed according to the tiny interaction between the lipophilic redox mediator (methylene blue), the detergent, the enzyme and the electrochemical interface.