title: Molecular-Level Insight into the Differential Oxidase and Oxygenase Reactivities of de Novo Due Ferri Proteins.
abstract: Using the single-chain due ferri (DFsc) peptide scaffold, the differential oxidase and oxygenase reactivities of two 4A→4G variants, one with two histidines at the diiron center (G4DFsc) and the other with three histidines (3His-G4DFsc(Mut3)), are explored. By controlling the reaction conditions, the active form responsible for 4-aminophenol (4-AP) oxidase activity in both G4DFsc and 3His-G4DFsc(Mut3) is determined to be the substrate-bound biferrous site. Using circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies, 4-AP is found to bind directly to the biferrous sites of the DF proteins. In G4DFsc, 4-AP increases the coordination of the biferrous site, while in 3His-G4DFsc(Mut3), the coordination number remains the same and the substrate likely replaces the additional bound histidine. This substrate binding enables a two-electron process where 4-AP is oxidized to benzoquinone imine and O2 is reduced to H2O2. In contrast, only the biferrous 3His variant is found to be active in the oxygenation of p-anisidine to 4-nitroso-methoxybenzene. From CD, MCD, and VTVH MCD, p-anisidine addition is found to minimally perturb the biferrous centers of both G4DFsc and 3His-G4DFsc(Mut3), indicating that this substrate binds near the biferrous site. In 3His-G4DFsc(Mut3), the coordinative saturation of one iron leads to the two-electron reduction of O2 at the second iron to generate an end-on hydroperoxo-Fe(III) active oxygenating species.
authors.affiliation: †Department of Chemistry, Stanford University, Stanford, California 94305, United States.
authors.forename: Rae Ana
authors.initials: RA
authors.lastname: Snyder
authors.affiliation: ‡Department of Chemistry, Ursinus College, Collegeville, Pennsylvania 19426, United States.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Butch
authors.affiliation: ‡Department of Chemistry, Ursinus College, Collegeville, Pennsylvania 19426, United States.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Reig
authors.affiliation: ⊥Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States.
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.affiliation: †Department of Chemistry, Stanford University, Stanford, California 94305, United States.|§Stanford Synchrotron Radiation Laboratory, SLAC, Stanford University, Menlo Park, California 94025, United States.
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D006639:Histidine / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D010455:Peptides; D006639:Histidine; D007501:Iron; D010088:Oxidoreductases; D010105:Oxygenases