Rubrerythrins (Rbr) and symerythrins (Sym) are diiron carboxylate enzymes that exhibit ferroxidase and uniquely 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 carboxylate active site amino acids, compared to the FLSF archetype. However, the relationship between these structural features and the altered function is currently not well understood. Model proteins constructed from the <i>de novo-</i>designed G4DFsc system have been created to investigate this relationship. G4DFsc demonstrates the canonical 4-helix bundle and 2His/4-carboxylate active site structure of FLSF enzymes to mimic their reactivities. Aspartate (D) or glutamate (E) residues were introduced at positions 14, 43, 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 and hydrogen peroxide.

Biophysical characterization and catalytic reactivity of rubrerythrin and symerythrin model proteins

253rd National Meeting (2017)

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