title: Systematic Perturbations of Binuclear Non-heme Iron Sites: Structure and Dioxygen Reactivity of de Novo Due Ferri Proteins.
abstract: DFsc (single-chain due ferri) proteins allow for modeling binuclear non-heme iron enzymes with a similar fold. Three 4A → 4G variants of DFsc were studied to investigate the effects of (1) increasing the size of the substrate/solvent access channel (G4DFsc), (2) including an additional His residue in the first coordination sphere along with three additional helix-stabilizing mutations [3His-G4DFsc(Mut3)], and (3) the three helix-stabilizing mutations alone [G4DFsc(Mut3)] on the biferrous structures and their O2 reactivities. Near-infrared circular dichroism and magnetic circular dichroism (MCD) spectroscopy show that the 4A → 4G mutations increase coordination of the diiron site from 4-coordinate/5-coordinate to 5-coordinate/5-coordinate, likely reflecting increased solvent accessibility. While the three helix-stabilizing mutations [G4DFsc(Mut3)] do not affect the coordination number, addition of the third active site His residue [3His-G4DFsc(Mut3)] results in a 5-coordinate/6-coordinate site. Although all 4A→ 4G variants have significantly slower pseudo-first-order rates when reacting with excess O2 than DFsc (∼2 s(-1)), G4DFsc and 3His-G4DFsc(Mut3) have rates (∼0.02 and ∼0.04 s(-1)) faster than that of G4DFsc(Mut3) (∼0.002 s(-1)). These trends in the rate of O2 reactivity correlate with exchange coupling between the Fe(II) sites and suggest that the two-electron reduction of O2 occurs through end-on binding at one Fe(II) rather than through a peroxy-bridged intermediate. UV-vis absorption and MCD spectroscopies indicate that an Fe(III)Fe(III)-OH species first forms in all three variants but converts into an Fe(III)-μ-OH-Fe(III) species only in the 2-His forms, a process inhibited by the additional active site His ligand that coordinatively saturates one of the iron centers in 3His-G4DFsc(Mut3).
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: Justine
authors.initials: J
authors.lastname: Betzu
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, 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, Stanford University, SLAC, Menlo Park, California 94025, United States.
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D001665:Binding Sites; D007501:Iron / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry*; D017354:Point Mutation; D017433:Protein Structure, Secondary
chemical_list: D008667:Metalloproteins; D007501:Iron; D010100:Oxygen