Hydrogen bioelectrooxidation in ionic liquids: From cytochrome c3 redox behavior to hydrogenase activity

Electrochemistry of polyheme bacterial cytochrome c₃ and catalytic oxidation of hydrogen by two different bacterial [NiFe] hydrogenases were investigated for the first time in pure room-temperature ionic liquids (RTILs) as electrolyte. Direct electrochemical response of Desulfovibrio vulgaris Hildenborough cytochrome c₃ (DvH cytc₃) adsorbed at a pyrolytic graphite (PG) electrode was observed in the RTILs used in this work: 1-butyl-3-methylimidazolium tetrafluoroborate (BmimBF4), 1-ethyl-3-methylimidazolium tetrafluoroborate (EmimBF4) and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (EmimNTf2). The electrochemical signal differed however from that obtained in aqueous buffer, and depended on the type of RTIL. UV–vis measurements as well as transfer experiments from aqueous buffer to RTILs or RTILs to aqueous buffer strongly suggested that the protein was not denatured in the presence of RTILs. EmimNTf2, as a hydrophobic non-water-miscible RTIL, was demonstrated to stabilize the native form of DvH cytc₃. Moreover it allowed an amount of electroactive DvH cytc₃ 30-fold higher than observed in aqueous buffer. Catalytic oxidation of H₂ via Desulfovibrio fructosovorans [NiFe] hydrogenase (Df Hase) mediated by DvH cytc₃ failed however. Further investigation suggested that Df Hase could be inhibited in the presence of RTILs. Reasons for such an inhibition were explored, including the blocking up of the substrate channels. By using hyperthermophilic [NiFe] membrane-bound hydrogenase from Aquifex aeolicus (Aa Hase) an efficient direct catalytic oxidation process was obtained in mixed aqueous buffer/RTILs electrolytes, although direct H₂ oxidation was not observed in pure RTIL. Chronoamperometric experiments showed that Aa Hase could afford 80% RTILs in aqueous buffer, thus giving the opportunity of future electrolytes with uncommon and variable properties for biofuel cell design.