Stabilization role of a phenothiazine derivative on the electrocatalytic oxidation of hydrogen via Aquifex aeolicus hydrogenase at graphite membrane electrodes

The [NiFe] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic Aquifex aeolicus bacterium (Aa Hase) presents oxygen, carbon monoxide, and temperature resistances. Since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. Efficient immobilization of the enzyme onto electrodes is however a mandatory step. To gain further insight into the parameters governing the interfacial electron process, cyclic voltammetry was performed combining the use of a phenothiazine dye with a membrane electrode design where the enzyme is entrapped in a thin layer. In the absence of the phenothiazine dye, direct electron transfer (DET) for H(2) oxidation is observed due to Aa Hase adsorbed onto the PG electrode. An unexpected loss of the catalytic current with time is however observed. The effect of toluidine blue O (TBO) on the catalytic process is first studied with TBO in solution. In addition to the expected mediated electron transfer process (MET), TBO is demonstrated to reconnect directly some Aa Hase molecules possibly released from the electrode but still entrapped in the thin layer. On adsorbed TBO the two same processes occur demonstrating the ability of the TBO film to connect Aa Hase via a DET process. Loss of activity is however observed due to the poor stability of adsorbed TBO at high temperatures. Aa Hase immobilization is then studied on electropolymerized TBO (pTBO). The effect of film thickness, temperature, presence of inhibitors and pH is evaluated. Given a film thickness less than 20 nm, H(2) oxidation proceeds via a mixed DET/MET process through the pTBO film. A high and very stable H(2) oxidation activity is reached, showing the potential applicability of the bioelectrode for biotechnologies. Finally, the multifunctional roles of TBO-based matrix are underlined, including redox mediator, Aa Hase anchor, but also buffering and ROS scavenger capabilities to drive pH local changes and avoid oxidative damage.