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Title: Molecular-Level Insight into the Differential Oxidase and Oxygenase Reactivities of de Novo Due Ferri Proteins

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. Lastly, in 3His-G4DFsc(Mut3), the coordinative saturation of one iron leads tomore » the two-electron reduction of O2 at the second iron to generate an end-on hydroperoxo-Fe(III) active oxygenating species.« less
Authors:
 [1] ;  [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 Lightsource (SSRL)
Publication Date:
OSTI Identifier:
1260959
Grant/Contract Number:
MCB-0919027; R15-GM110657; CHE-1413295; AC02-76SF0051; GM71628
Type:
Accepted Manuscript
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 137; Journal Issue: 29; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society (ACS)
Research Org:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org:
National Science Foundation (NSF); USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY