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Title: Systematic Perturbations of Binuclear Non-heme Iron Sites: Structure and Dioxygen Reactivity of de Novo Due Ferri Proteins

We report that 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 O 2 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 O 2 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 O 2 reactivity correlate with exchange coupling between the Fe(II) sites and suggest that the two-electron reduction of Omore » 2 occurs through end-on binding at one Fe(II) rather than through a peroxy-bridged intermediate. Finally, 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).« less
 [1] ;  [2] ;  [2] ;  [2] ;  [3] ;  [4]
  1. Stanford Univ., CA (United States). Dept. of Chemistry
  2. Ursinus College, Collegeville, PA (United States). Dept. of Chemistry
  3. Univ. of California, San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry
  4. Stanford Univ., CA (United States). Dept. of Chemistry; SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lab.
Publication Date:
OSTI Identifier:
Grant/Contract Number:
MCB-1404866; F32-GM808852; GM54616; GM71628; R15-GM110657; CHE-1413295; AC02-76SF0051
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 54; Journal Issue: 30; Journal ID: ISSN 0006-2960
American Chemical Society (ACS)
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
National Science Foundation (NSF); National Institutes of Health (NIH); USDOE
Country of Publication:
United States