Submission Date


Document Type

Paper- Restricted to Campus Access




Amanda J. Reig

Committee Member

Samantha Wilner

Committee Member

Ann Karreth

Department Chair

Amanda J. Reig

External Reviewer

Rachel N. Austin

Distinguished Honors

This paper has met the requirements for Distinguished Honors.

Project Description

Due Ferri single chain, a de novo protein initially designed to mimic the catalytic activity of diiron proteins, has been shown to bind a variety of metals, including copper and zinc. It has been observed that when zinc coordinates to the active site of this protein rather than iron, the protein cleaves phosphate bonds via hydrolysis. To better understand, and potentially enhance this reactivity, mutations have been made in or near the metal binding active site. We have developed an assay using phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) to monitor the catalytic activity of DFsc by observing the conversion of bis(4-nitrophenyl) phosphate (BNPP) to para-nitrophenolate (PNP) and para-nitro-phenylphosphate (pNPP). Using integral calculations, rates of reaction for DFsc and its mutants were determined. Additionally, the developed 31P-NMR protocol was replicated on a UV-Vis plate reader.

Type 3 copper proteins, or coupled binuclear copper proteins (CBC), are known for their oxidase chemistry, including their ability to transform catechols into quinones. High histidine variants of DFsc share structural similarities with coupled binuclear copper proteins and have been used to model the activity of these proteins without the structural complexity often found in the naturally occurring proteins. Herein, we characterized the copper-binding and oxidase activity of several DFsc variants (G4Y-G4DFsc, DFsc, and 3-His-G4DFsc) through 2-2’-biquinoline stoichiometry assays and the oxidation assays with trihydroxybenzene, pyrocatechol, and di-tert-butyl catechol as substrates to further understand the structure-function relationship between the DFsc protein family and CBC-like phenolase chemistry.