Non-Native Anionic Ligand Binding and Reactivity in Engineered Variants of the Fe(II)- and -Ketoglutarate-Dependent Oxygenase, SadA

Mononuclear non-heme Fe(II)- and alpha-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 center dot vs X-center dot 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 N-3(center dot), NCO center dot, and OCN center dot rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand leads us to calculate barriers for HO center dot and F-center dot 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 center dot and F-center dot rebound transition states rather than an inaccessible barrier for F-center dot 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 center dot that will aid efforts to develop non-native catalysis using these enzymes.