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Biophysical Characterization and Catalytic Reactivity of Rubrerythrin and Symerythrin Model Proteins
Author(s) -
Pellegrino Jenna,
Bell Katherine A.,
Polinski Rachel Z.,
Cimerol Sabri.,
Jacobs Ari,
Solomon Edward I.,
Reig Amanda
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.922.11
Subject(s) - active site , chemistry , carboxylate , reactivity (psychology) , stereochemistry , enzyme , amino acid , catalysis , protein structure , biochemistry , medicine , alternative medicine , pathology
Rubrerythrins (Rbr) and symerythrins (Sym) are diiron carboxylate enzymes that function as both ferroxidases and peroxidases in two‐electron reduction reactions. This peroxidase activity, uniquely preferential in Rbr and Sym as compared to other members of the ferritin‐like superfamily (FLSF), is thought to result from the one or two additional carboxylate active site amino acids, compared to the FLSF archetype. The relationship between these structural features and the altered function is currently not well understood. To investigate these phenomena, a de novo‐ designed protein model system was computationally designed to demonstrate the canonical 4‐helix bundle and 2His/4‐carboxylate active site structure of FLSF enzymes and to mimic their reactivities. Modifications to this model resulted in G4DFsc, which exhibits higher solubility and stability as well as increased active site accessibility. Using the G4DFsc system, Rbr‐ and Sym‐like proteins were achieved by introducing either aspartate (D) or glutamate (E) residues at active site positions 14, 43, and/or 47. 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. Preliminary cobalt titration curves show the tightest binding between the metal and G4DFsc. Interestingly, the dissociation constant of G47D is 4 times greater. Support or Funding Information Howard Hughes Medical Institute (HHMI), National Institutes of Health (NIH) R15GM110657