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 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) mu A cm(-2) for G491A and (476 +/- 172) mu A 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.