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

Abstract

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.