Submission Date


Document Type

Paper- Restricted to Campus Access




Amanda Reig

Committee Member

Ryan Walvoord

Committee Member

Rebecca Roberts

Department Chair

Amanda Reig

External Reviewer

Kenneth Karlin

Distinguished Honors

This paper has met the requirements for Distinguished Honors.

Project Description

Coupled binuclear copper proteins (CBCs) such as hemocyanins, catechol oxidases, and tyrosinases utilize dioxygen for storage, oxidase, and monoxygenase behavior, respectively. However, all three protein families have a common 6-His dicopper coordination environment. It is believed that nuanced structure-function relationships are the cause of this role diversity. The development and characterization of protein-based model systems for CBCs allows for in-depth investigation of these relationships. One such model system is the due ferri single chain (DFsc) protein – a computationally designed de novo four-helix bundle containing an active site that mimics the 2-His/4-carboxylate configuration of naturally-occurring binuclear non-heme iron enzymes. To investigate the potential of this scaffold as a dicopper model system, we created a library of eight G4DFsc variants with between two and six His residues by performing iterative site-selective mutagenesis on each Glu residue. Circular dichroism spectroscopy indicates that the apo-variants had either similar or increased alpha-helical character compared to the original DFsc protein upon incorporation of the additional His residues. The addition of copper (II) ions increased the thermodynamic stability of the variants and metal binding assays are mostly consistent with 2:1 metal-to-protein ratios. Aerobic copper(II) addition leads to the formation of a putative Cu(II)2(bis-μ-hydroxo) species with an absorption feature between 615-675 nm and a shoulder between 310-400 nm. Anaerobic reduction of the protein-Cu(II) complexes followed by exposure to O2-saturated buffer resulted in a yellow solution with absorption features consistent with a Cu(II)2(μ-η2:η2-peroxo) species. The Cu(II)-bound protein variants modestly catalyze oxygen-dependent 2-electron oxidation of catechols and 3-amino-4-hydroxybenzoic acid, but lack phenol monoxygenation activity. These results demonstrate that it is feasible to create an artificial coupled binuclear copper enzyme within a de novo protein scaffold. To further examine the subtle catalytic differences between the variants, we have also begun to develop a turn-on fluorescent probe for catechol oxidase activity. Current work is focused on linking a coumarin fluorophore with 3-amino-4-hydroxybenzoic acid to create an ester susceptible to hydrolysis after oxidation of the o-aminophenol trigger to the electron-withdrawing iminoquinone. This modular approach can allow for the synthesis of a library of ester linked fluorophore turn-on probes for catechol oxidase behavior.