G4DFsc is a de novo designed single-chain four-helix bundle protein generated to mimic the properties of di-iron carboxylate enzymes. The active site of G4DFsc is made up of two iron atoms ligated by two histidine and four carboxylate residues. The di-iron protein, flavin-dependent nitric oxide reductase (FprA), contains a 4-His/3-carboyxlate iron-binding motif and initiates the reduction of NO to N2O and H2O. Two G4DFsc variants with 4-His/3-carboxylate active sites (E11H-3HisG4DFsc and E44H-G4DFsc) have been created to explore how enzymes like FprA are optimized for NO reduction relative to O2 dependent oxidation. NMR structural studies coupled with UV-CD and metal binding titrations indicate that E11H-3HisG4DFsc is well structured, helical, and binds two metal ions. E44H-3HisG4DFsc is also well folded but only binds one metal ion. The ability of these proteins to catalyze substrate oxidation and oxygenation, along with NO reduction, was explored through spectroscopic studies.

Structure-function relationships in G4DFsc variants containing a 4-His/3-carboxylate active site

253rd National Meeting (2017)

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