Molecular-Level Insight into the Differential Oxidase and Oxygenase Reactivities of de Novo Due Ferri Proteins
Rae Ana Snyder †, Susan E. Butch ‡, Amanda J. Reig *‡, William F. DeGrado *⊥, and Edward I. Solomon *†§
† Department
of Chemistry, Stanford University, Stanford, California 94305, United States,
‡ Department
of Chemistry, Ursinus College, Collegeville, Pennsylvania 19426, United States,
⊥ Department
of Pharmaceutical Chemistry, University
of California, San Francisco, California 94143, United States
§ Stanford
Synchrotron Radiation Laboratory, SLAC, Stanford University, Menlo Park, California 94025, United States
J. Am. Chem. Soc., Article ASAP
DOI: 10.1021/jacs.5b03524
Publication Date (Web): June 19, 2015
Copyright © 2015 American Chemical Society
http://pubs.acs.org/doi/pdf/10.1021/jacs.5b03524
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.
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