Redox‐Polymer‐Wired [NiFeSe] Hydrogenase Variants with Enhanced O2 Stability for Triple‐Protected High‐Current‐Density H2‐Oxidation Bioanodes

Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O2 tolerance were used as H2‐oxidation catalysts in H2/O2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low‐potential viologen‐modified redox polymers and evaluated with respect to H2‐oxidation and stability against O2 in the immobilized state. The two variants showed maximum current densities of (450±84) μA cm−2 for G491A and (476±172) μA cm−2 for variant G941S on glassy carbon electrodes and a higher O2 tolerance than the wild type. In addition, the polymer protected the enzyme from O2 damage and high‐potential inactivation, establishing a triple protection for the bioanode. The use of gas‐diffusion bioanodes provided current densities for H2‐oxidation of up to 6.3 mA cm−2. Combination of the gas‐diffusion bioanode with a bilirubin oxidase‐based gas‐diffusion O2‐reducing biocathode in a membrane‐free biofuel cell under anode‐limiting conditions showed unprecedented benchmark power densities of 4.4 mW cm−2 at 0.7 V and an open‐circuit voltage of 1.14 V even at moderate catalyst loadings, outperforming the previously reported system obtained with the [NiFeSe] wild type and the [NiFe] hydrogenase from D. vulgaris Miyazaki F. Triple protection: A stable, high‐current‐density‐based H2‐oxidation bioanode is presented. It is equipped with [NiFeSe] variants that show enhanced O2 tolerance, which are immobilized and wired to electrode surfaces with a low‐potential viologen‐modified polymer. The polymer acts simultaneously as a high‐potential and O2 shield. The triply protected bioanodes are incorporated in membrane‐free biofuel cells, which reveal benchmark performances at moderate catalyst loading.