Reconstitution of [Fe]-Hydrogenase with Model Complexes Reveals Functional Roles of Methyl Groups in the...

Area of Science:

• Biocatalysis
• Bioinorganic Chemistry
• Metalloenzyme Engineering

Background:

• [Fe]-hydrogenase is a promising bio-alternative to noble-metal hydrogenation catalysts.
• Production and genetic manipulation challenges limit [Fe]-hydrogenase research and application.
• Semisynthetic approaches offer a solution but face low activity due to incomplete cofactor mimics.

Purpose of the Study:

• Investigate the impact of methyl substituents on the [Fe]-hydrogenase cofactor mimic's pyridinol ligand.
• Enhance the activity and stability of semisynthetic [Fe]-hydrogenases.
• Develop a tunable platform for efficient biohydrogenation catalysts.

Main Methods:

• Design and synthesis of model cofactor complexes with 3- and 5-methyl substituents.
• Reconstitution of apoenzymes with synthetic cofactor mimics.
• Characterization of catalytic activity and oxidative stability of semisynthetic enzymes.

Main Results:

• The 3-methyl substituent significantly increased catalytic activity (35-fold) and reconstitution kinetics.
• The 5-methyl substituent enhanced the oxidative stability of the semisynthetic enzyme.
• The optimized variant, jHmd-4, achieved 8% of native enzyme activity, the highest reported for semisynthetic systems.

Conclusions:

• Methyl group modifications on the pyridinol ligand are crucial for enhancing semisynthetic [Fe]-hydrogenase performance.
• This work provides a tunable platform for developing efficient biohydrogenation catalysts.
• Key insights into metalloenzyme engineering were gained, paving the way for future catalyst development.