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.

FeII‐ and α‐ketoglutarate‐dependent halogenases and oxygenases can catalyze site‐selective functionalization of C−H bonds via a variety of C−X bond forming reactions, but achieving high chemoselectivity for functionalization using non‐native functional groups remains rare. The current study shows that directed evolution can be used to engineer variants of the dioxygenase SadX that address this challenge. Site‐selective azidation of succinylated amino acids and a succinylated amine was achieved as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C−H functionalization with other non‐native functional groups.

Fe II -and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of CÀ H bonds via a variety of CÀ X bond forming reactions, but achieving high chemoselectivity for functionalization using non-native functional groups remains rare. The current study shows that directed evolution can be used to engineer variants of the dioxygenase SadX that address this challenge. Siteselective azidation of succinylated amino acids and a succinylated amine was achieved as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic CÀ H functionalization with other non-native functional groups.

Mononuclear non-heme Fe(II)- and -ketoglutarate dependent oxygenases (FeDOs) catalyze 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 a FeDO facial triad variant, SadX, and the effects of that binding on HO• vs. X• rebound. We establish that chloride and bromide not only enable non-native halogenation reactions but 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 led 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 FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving those ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

Fe(II)- and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of C-H bonds via a variety of C-X bond forming reactions. Achieving high chemoselectivity for functionalization using non-native functional groups remains rare, however, particularly for non-native substrates. The current study shows that directed evolution can be used to engineer variants of an engineered dioxygenase, SadX, that address this challenge. Site-selective azidation of succinylated amino acids and a succinylated amine was achieved using variants with improved azidation yield and selectivity on a probe substrate as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C-H functionalization with other non-native functional groups.