Redox‐Polymer‐Wired [NiFeSe] Hydrogenase Variants with Enhanced O 2 Stability for Triple‐Protected High‐Current‐Density H 2 ‐Oxidation Bioanodes

Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O 2 tolerance were used as H 2 ‐oxidation catalysts in H 2 /O 2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low‐potential viologen‐modified redox polymers and evaluated with respect to H 2 ‐oxidation and stability against O 2 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 O 2 tolerance than the wild type. In addition, the polymer protected the enzyme from O 2 damage and high‐potential inactivation, establishing a triple protection for the bioanode. The use of gas‐diffusion bioanodes provided current densities for H 2 ‐oxidation of up to 6.3 mA cm −2 . Combination of the gas‐diffusion bioanode with a bilirubin oxidase‐based gas‐diffusion O 2 ‐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.