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.