Submission Date


Document Type

Paper- Restricted to Campus Access


Biochemistry & Molecular Biology


Amanda Reig

Committee Member

Julin Everett

Committee Member

Rebecca Roberts

Committee Member

Amanda Reig

Department Chair

Eric Williamsen

Department Chair

Anthony Lobo

External Reviewer

Kate Buettner

Distinguished Honors

This paper has met the requirements for Distinguished Honors.

Project Description

Rubrerythrins (Rbr) and symerythrins (Sym) are diiron carboxylate enzymes that exhibit ferroxidase activity and enhanced preferential peroxidase activity compared to other members of the ferritin-like superfamily (FLSF). This peroxidase activity is thought to result from the one or two additional carboxylates in the active sites of Rbr and Sym, compared to the FLSF archetype. However, the relationship between these structural features and the altered function is currently not well understood. Model proteins based on the de novo-designed G4DFsc system have been created to investigate this relationship. G4DFsc structurally replicates the canonical 4-helix bundle and 2-histidine/4-carboxylate active site structure of FLSF enzymes as well as mimics their reactivities. Aspartate (Asp, D) or glutamate (Glu, E) residues were introduced at positions 14 and/or 47 to generate Rbr- and Sym-like active sites within the G4DFsc bundle. The structural and catalytic properties of these systems were investigated using metal-binding, protein-folding, and reactivity assays at pH 7 and pH 7.5. Data show that the double mutants exhibit the weakest metal-binding capacity at pH 7. These proteins also show slower rates of 4-aminophenol oxidation than the original G4DFsc protein. The G47D variant shows the greatest catalytic capacity, with 4-aminophenol oxidation rates increased 11-fold over control reactions and 2-fold compared to G4DFsc. These results provide insight into how particular carboxylate residues in the G4DFsc active site affect its ability to react with dioxygen.