Redox-Polymer-Wired [NiFeSe] Hydrogenase Variants with Enhanced O₂ Stability for Triple-Protected...

Area of Science:

• Biocatalysis and Biofuel Cells
• Enzyme Engineering and Immobilization
• Renewable Energy Technologies

Background:

• Hydrogenases are crucial enzymes for hydrogen oxidation in biofuel cells.
• Oxygen sensitivity of wild-type hydrogenases limits their application in H2/O2 biofuel cells.
• Development of oxygen-tolerant hydrogenase variants is essential for efficient bioelectrocatalysis.

Purpose of the Study:

• To utilize engineered [NiFeSe] hydrogenase variants with enhanced oxygen tolerance as H2-oxidation catalysts.
• To evaluate the performance and stability of immobilized variants in H2/O2 biofuel cells.
• To establish a robust bioanode system for efficient hydrogen oxidation.

Main Methods:

• Electrochemical immobilization of two O2-tolerant [NiFeSe] hydrogenase variants (G491A, G941S) using viologen-modified redox polymers on glassy carbon electrodes.
• Evaluation of H2-oxidation activity and stability against O2 in the immobilized state.
• Construction and testing of gas-diffusion bioanodes and membrane-free biofuel cells incorporating these variants.

Main Results:

• The immobilized variants achieved high maximum current densities (450–476 μA cm−2) and exhibited superior O2 tolerance compared to the wild type.
• The redox polymer provided a triple protective layer for the enzyme against O2 damage and inactivation.
• Gas-diffusion bioanodes yielded H2-oxidation current densities up to 6.3 mA cm−2, leading to benchmark power densities of 4.4 mW cm−2 in a biofuel cell.

Conclusions:

• Engineered [NiFeSe] hydrogenase variants offer a promising solution for developing stable and efficient H2-oxidation bioanodes.
• The combination of enzyme engineering and protective immobilization strategies significantly enhances biofuel cell performance.
• This work establishes a new benchmark for power density in membrane-free H2/O2 biofuel cells using biocatalysts.