De novo proteins provide a unique opportunity for investigating the structure-function relationships that govern reactivity of binuclear metalloproteins. The Due Ferri single chain protein is a computationally designed de novo four-helix bundle protein originally created to mimic the structure, spectroscopic features, and reactivity of diiron carboxylate enzymes. The active site, which contains two histidine and four carboxylate amino acid residues, has been shown to bind a wide variety of first-row transition metals, including copper, suggesting that the scaffold could also be used to mimic the structure and reactivity of dicopper enzymes. We have systematically inserted four Glu to His mutations in the DFsc active site to model the 6-His configuration found in natural coupled binuclear copper enzymes. The protein variants were overexpressed in E. coli and purified by high performance liquid chromatography. Biophysical characterization indicates that the proteins are well-folded and most bind copper ions in the expected 2:1 ratio. Reactivity studies indicate that the proteins can activate molecular oxygen and are capable of performing simple 2-electron oxidation reactions. This library of proteins allow us to explore how the active site composition affects the metal binding and catalytic properties of binuclear metalloenzymes.

Altering the de novo Due Ferri single chain protein to mimic the structure and reactivity of coupled binuclear copper enzymes

255th National Meeting (2018)

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