Crystal Structure, Exogenous Ligand Binding, and Redox Properties of an Engineered Diiron Active Site in a Bacterial Hemerythrin

A nonheme
diiron active site in a 13 kDa hemerythrin-like domain of the bacterial
chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate
groups from Glu and Asp residues, and five terminally ligated His
residues. We created a unique diiron coordination sphere containing
five His and three Glu/Asp residues by replacing an Ile residue with
Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119
to Fe2 of the diiron site in the I119E variant was confirmed by X-ray
crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible
tunnel. UV–vis absorption spectra indicate that the additional
coordination of E119 inhibits the binding of the exogenous ligands
azide and phenol to the diiron site. The extent of azide binding to
the diiron site increases at pH ≤ 6, which is ascribed to protonation
of the carboxylate ligand of E119. The diferrous state (deoxy form)
of the engineered diiron site with the extra Glu residue is found
to react more slowly than wild type with O2 to yield the
diferric state (met form). The additional coordination of E119 to
the diiron site also slows the rate of reduction from the met form.
All these processes were found to be pH-dependent, which can be attributed
to protonation state and coordination status of the E119 carboxylate.
These results demonstrate that modifications of the endogenous coordination
sphere can produce significant changes in the ligand binding and redox
properties in a prototypical nonheme diiron-carboxylate protein active
site.