Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis

Area of Science:

• Biochemistry and Molecular Biology
• Bioinorganic Chemistry
• Protein Engineering

Background:

• Copper sites in proteins are crucial for electron transfer and redox catalysis.
• Type 1 and Cu_A sites function as electron transfer hubs within rigid protein structures, limiting exogenous ligand binding and side reactions.

Purpose of the Study:

• To engineer novel Type 1 copper sites with unique electronic structures and functional properties.
• To investigate the impact of loop-directed mutagenesis within a Cu_A scaffold on copper site characteristics.

Main Methods:

• Loop-directed mutagenesis was employed to engineer Type 1 copper sites within a Cu_A protein scaffold.
• Characterization of the engineered sites focused on electronic structure, reduction potential, and ligand-binding capabilities.

Main Results:

• Two engineered Type 1 sites exhibited unique electronic structures, attributed to a shorter copper-thioether axial bond compared to the copper-thiolate bond.
• These sites displayed a high reduction potential, despite strong axial ligand interaction, linked to alterations in the hydrogen bond network.
• The engineered sites demonstrated the ability to bind exogenous ligands, including imidazole and azide.

Conclusions:

• Protein engineering strategies can create novel functional features not found in natural copper sites.
• The study demonstrates the successful modification of natural protein scaffolds to achieve unique electronic and functional properties.
• This approach broadens the scope for utilizing protein scaffolds in bioinorganic chemistry and enzyme design.