A structural view of synthetic cofactor integration into [FeFe]-hydrogenases

Area of Science:

• Biochemistry
• Structural Biology
• Bioinorganic Chemistry

Background:

• [FeFe]-hydrogenases are highly efficient biological catalysts for hydrogen metabolism.
• Synthetic models of the [2Fe] subcluster have not replicated enzyme activity.
• A specific synthetic complex (Fe2[μ-(SCH2)2NH](CN)2(CO)42-) can integrate into hydrogenase apo-enzymes.

Purpose of the Study:

• To elucidate the structural basis for the activity of semisynthetic [FeFe]-hydrogenases.
• To investigate the role of the dithiolate bridge in the [2Fe] subcluster of hydrogenases.
• To compare the structures of active and inactive semisynthetic hydrogenases.

Main Methods:

• X-ray crystallography of apo- and semisynthetic [FeFe]-hydrogenases.
• High-resolution structural analysis at atomic detail.
• Comparison of structural features between active and inactive enzyme variants.

Main Results:

• Crystal structures of apo- and active semisynthetic [FeFe]-hydrogenase (CpI^ADT) were determined.
• Significant ligand coordination changes occur upon integration and activation of the [2Fe] complex.
• Structures of inactive semisynthetic variants (CpI^PDT, CpI^ODT, CpI^SDT) show minimal changes in the [2Fe] subcluster and protein environment.
• An open coordination site is present in all structures, suggesting steric hindrance is not the cause of inactivity.

Conclusions:

• The protein cavity for the [2Fe] subcluster is rigid and dictates structural changes upon cofactor integration.
• The chemical properties of the dithiolate bridge, not steric factors, are critical for [FeFe]-hydrogenase catalytic activity.
• Semisynthetic approaches combined with structural biology provide insights into enzyme mechanisms.