Binuclear copper enzymes, such as tyrosinase and catechol oxidase, are involved in the melaninization and sclerotization processes. Understanding the mechanism of the diphenol oxidation activity in these enzymes is crucial to design efficient target drugs and inhibitors. One way to gain mechanistic insight into these processes is through the design and characterization of protein-based model systems. The de novo designed Due Ferri Single Chain (DFsc) proteins are water-soluble monomeric four-helix bundles that can readily bind metal cofactors. Several variants on this scaffold, namely mutants of G4DFsc and 3HisG4DFsc, have been shown to mimic natural diiron oxidases. Interestingly, copper titrations have shown that G4DFsc, E11H/E44H-G4DFsc, and E11H-3HisG4DFsc bind two copper ions. Catalytic oxidase activity for these DFsc variants was determined using UV-Visible spectrophotometric assays. Specifically, the formation of an o-quinone coupling product with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of the copper-protein complex and trihydroxybenzene (THB) is indicative of catechol oxidase–like behavior. These results are supported by Michealis-Menten kinetics studies. Catalytic oxidation was further supported by changes in activity when the assays were conducted under anaerobic and reductive conditions, and in response to active site variations.

Copper binding and reactivity of de novo designed Due Ferri single chain (DFsc) proteins

253rd National Meeting (2017)

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