Engineering the Electron Relay in [FeFe]-Hydrogenase Enhances Electrocatalytic H₂ Evolution

Area of Science:

• Biotechnology
• Bioinorganic Chemistry
• Enzyme Engineering

Background:

• Hydrogen (H2) is a clean energy vector, but efficient catalysts for its production from water are lacking.
• [FeFe]-hydrogenases are nature's most active H2-converting enzymes, featuring a unique organometallic active site.
• M3 type [FeFe]-hydrogenases are highly active and oxygen-tolerant but challenging due to size and complexity.

Purpose of the Study:

• To engineer the [FeFe]-hydrogenase from *C. acetobutylicum* for improved biotechnological applications.
• To create smaller, more manageable catalytic systems while preserving enzymatic function.
• To investigate the impact of truncating iron-sulfur clusters on enzyme activity and stability.

Main Methods:

• Systematic engineering of the [FeFe]-hydrogenase by truncating iron-sulfur-containing regions of the F-domain.
• Detailed biochemical and biophysical characterization of the engineered variants.
• Electrocatalytic performance assessment of the truncated enzymes.

Main Results:

• Successfully generated smaller, truncated variants of the [FeFe]-hydrogenase.
• The engineered variants retained high electrocatalytic performance for H2 production.
• Essential properties of the natural enzyme were preserved in the truncated forms.

Conclusions:

• Truncation of iron-sulfur regions yields smaller, easier-to-produce [FeFe]-hydrogenase systems.
• These engineered variants maintain high catalytic efficiency, making them promising for H2 biotechnology.
• The study demonstrates a viable strategy for simplifying complex metalloenzymes for practical applications.