The engineering of single chain peptides, which can assemble into 4-alpha helix bundles with diiron centers (Due Ferri single chain, DFsc), has enabled the formation of models of binuclear non-heme iron enzymes that are both structural and functional. Here, we describe how perturbations on the substrate access cavity and modification of first sphere residues from a 2 His to a 3 His ligand environment at the diiron site successfully tune the functionality of these proteins to catalyze the oxygen-dependent oxidation of aminophenol and the selective N-hydroxylation of arylamines. Employing near-IR circular dichroism (CD), magnetic CD (MCD), and variable temperature variable field MCD (VTVH MCD) spectroscopies, we have studied the structure and reactivity of three systematic perturbations of DFsc: 1) a form with four A to G mutations that should allow for better substrate access to the diiron site (G4DFsc), 2) a perturbation of G4DFsc that includes second and third shell mutations for stabilization of a 3rd His (MutGDFsc), and 3) the incorporation of the 3rd His in MutGDFsc (3His-MutGDFsc). We investigate how the ligand environment of these forms affects their oxygen reactivity and substrate binding and how these properties tune the protein's catalytic ability.

Structure and function studies of systematic perturbations on <i>de novo</i> Due Ferri proteins: Insights into oxygen-dependent reactivity

248th National Meeting (2014)

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Structure and function studies of systematic perturbations on de novo Due Ferri proteins: Insights into oxygen-dependent reactivity