Probing Ferryl Reactivity in a Nonheme Iron Oxygenase Using an Expanded Genetic Code

Area of Science:

• Biochemistry
• Enzyme kinetics
• Bioinorganic chemistry

Background:

• Nonheme iron enzymes, including 2-oxoglutarate (2OG) dependent oxygenases, utilize reactive ferryl intermediates for C-H functionalization.
• Heme enzymes have been successfully studied using noncanonical amino acids as axial ligands to probe ferryl intermediates.

Purpose of the Study:

• To investigate the feasibility of perturbing the iron coordination environment in 2OG dependent oxygenases using modified ligands.
• To understand the role of axial ligands in the reactivity of nonheme iron-containing enzymes.

Main Methods:

• Site-directed mutagenesis was used to replace a histidine ligand in the VioC oxygenase with Nδ-methyl-histidine (MeHis).
• Catalytic activity and reaction selectivity of the wildtype and mutant VioC were assessed.
• Accumulation and characterization of the ferryl intermediate were performed.

Main Results:

• Replacement of histidine with MeHis in VioC preserved catalytic function and reaction selectivity.
• The key ferryl intermediate involved in C-H activation could be accumulated in both wildtype and modified VioC.
• Axial ligand modification minimally affected the rates of C-H activation and kinetic isotope effects, unlike in heme enzymes.

Conclusions:

• Modulating the coordination sphere of nonheme iron enzymes is achievable through ligand alteration.
• Axial ligand identity has a less pronounced effect on the C-H activation mechanism in VioC compared to heme enzymes.
• This approach provides a new tool for studying nonheme iron enzyme mechanisms and factors influencing their activity.