Diffusion network of CO in FeFe-Hydrogenase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Computational biophysics

Background:

• Iron-iron hydrogenase (FeFe-hydrogenase) efficiently produces hydrogen but is inhibited by carbon monoxide (CO) and oxygen (O2).
• Understanding gas diffusion pathways is crucial for developing robust enzyme variants resistant to inhibitory gases.
• The active site of FeFe-hydrogenase is susceptible to irreversible damage from CO and O2.

Purpose of the Study:

• To investigate the diffusion pathways of carbon monoxide (CO) within the CpI FeFe-hydrogenase from Clostridium pasteurianum.
• To identify thermodynamically favorable pathways for CO entry and proximity to the active site.
• To propose candidate residues for mutagenesis to enhance enzyme tolerance to inhibitory gases.

Main Methods:

• Utilized enhanced sampling and free-energy simulation techniques.
• Reconstructed a 3D free-energy surface to map CO diffusion.
• Analyzed free-energy minima to identify interconnected pathways.

Main Results:

• Discovered a network of 45 free-energy minima representing CO diffusion pathways.
• Identified multiple minimal free-energy pathways, challenging previous assumptions about hydrophobic routes.
• Located the global free-energy minimum near the active site's H-cluster, indicating high CO affinity.

Conclusions:

• The study elucidates specific, thermodynamically favorable CO diffusion pathways in FeFe-hydrogenase.
• Identified 19 candidate residues for mutagenesis, with 11 overlapping with those known to confer O2 tolerance.
• Hypothesizes that these shared residues could be key for engineering dual CO and O2 tolerance in FeFe-hydrogenase variants.