Systematic Perturbations of Binuclear Non-heme Iron Sites: Structure and Dioxygen Reactivity of de Novo Due Ferri Proteins
Rae Ana Snyder †, Justine Betzu ‡, 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, San
Francisco, California 94143, United States
∥ Stanford
Synchrotron Radiation Laboratory, Stanford
University, SLAC, Menlo Park, California 94025, United States
Biochemistry, Article ASAP
DOI: 10.1021/acs.biochem.5b00324
Publication Date (Web): July 8, 2015
Copyright © 2015 American Chemical Society
*E-mail: areig@ursinus.edu. Phone: (610) 409-3383., *E-mail: Bill.DeGrado@ucsf.edu. Phone: (415) 476-9679., *E-mail: edward.solomon@stanford.edu. Phone: (650) 723-9104.
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).
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