Mononuclear non-heme Fe(II)- and α-ketoglutarate-dependent
oxygenases (FeDOs) catalyze a site-selective C–H hydroxylation.
Variants of these enzymes in which a conserved Asp/Glu residue in
the Fe(II)-binding facial triad is replaced by Ala/Gly can, in some
cases, bind various anionic ligands and catalyze non-native chlorination
and bromination reactions. In this study, we explore the binding of
different anions to an FeDO facial triad variant, SadX, and the effects
of that binding on HO• vs X• rebound.
We establish not only that chloride and bromide enable non-native
halogenation reactions but also that all anions investigated, including
azide, cyanate, formate, and fluoride, significantly accelerate and
influence the site selectivity of SadX hydroxylation catalysis. Azide
and cyanate also lead to the formation of products resulting from
N3
•, NCO•, and OCN• rebound. While fluoride rebound is not observed, the
rate acceleration provided by this ligand leads us to calculate barriers
for HO• and F• rebound from a
putative Fe(III)(OH)(F) intermediate. These calculations suggest that
the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states
rather than an inaccessible barrier for F• rebound.
Together, these results improve our understanding of the FeDO facial
triad variant tolerance of different anionic ligands, their ability
to promote rebound involving these ligands, and inherent rebound preferences
relative to HO• that will aid efforts to develop
non-native catalysis using these enzymes.