Dramatic Electronic Perturbations of Cu_A Centers via Subtle Geometric Changes.

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Protein Engineering

Background:

• The copper-A (Cu_A) site is a binuclear copper center crucial for electron entry in terminal heme-copper oxidases.
• In its oxidized state, Cu_A exhibits a mixed-valence character, described by a potential energy surface with two minima (σ_u* and π_u) populated at room temperature.

Purpose of the Study:

• To investigate the impact of mutations in the coordination spheres of the binuclear metallocofactor on the Cu_A electronic structure.
• To tune the energy gap between the σ_u* and π_u states and their relative populations through protein engineering.

Main Methods:

• Site-directed mutagenesis was employed to modify the first and second coordination spheres of the Cu_A site.
• Spectroscopic and computational methods were used to characterize the electronic structure and energy gaps of engineered mutants.

Main Results:

• Mutations allowed additive tuning of the σ_u*/π_u energy gap, ranging from 900 to 13 cm^-1.
• A minimal Cu-Cu distance increase of <0.06 Å significantly altered the energy gap, favoring the π_u state population.
• Engineered sites maintained mixed-valence character and electron transfer functionality.

Conclusions:

• Protein environment engineering offers precise control over the electronic structure of the Cu_A site, contrasting with synthetic models.
• Minor geometric modifications within the protein scaffold can induce substantial changes in the electronic properties of the Cu_A center.
• This study highlights the power of protein engineering to modulate metalloenzyme function.