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Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) enzymes have garnered strong research interest in past decades due to their ability to catalyze regio- and stereoselective C-H functionalization via a single reactive intermediate, the oxyferryl species. In addition to the hydroxylation reaction that is commonly observed, other reaction outcomes have also been discovered in Fe/2OG enzymes. Among them, halogenation has attracted much research effort with the goal of revealing the molecular determinants to favor halogenation over hydroxylation; however, a full mechanistic picture is still missing. In this study, by investigating a recently identified Fe/2OG halogenase, AdeV, from the biosynthetic pathway of Adechlorin, we show, via biochemical, kinetics, and spectroscopic characterizations, that two oxyferryl intermediates are formed during the AdeV reaction in a sequential manner, which interconvert but only one shows kinetic competency to enable C-H activation and leads to the conversion of 2'-deoxyadenosine monophosphate (2'-dAMP) and 2',3'-dideoxyadenosine monophosphate (ddAMP) to 2'-Cl-dAMP and 2'-Cl-ddAMP, respectively. By applying chemical synthesis and product characterization by detailed NMR analysis, the stereochemical assignment of the AdeV-catalyzed reaction is resolved, whereof the C-H bond cleavage and the C-Cl bond formation occur in a suprafacial manner. Using the experimental observations as a guide, the computational studies reveal that the kinetically competent oxyferryl intermediate structurally exhibits an offline configuration. However, this offline oxyferryl intermediate requires a structural conversion to a metastable inline configuration to perform a regio- and stereospecific C-H activation via a σ reaction channel. The subsequent conversion back to the offline configuration in the hydroxy-ferric state facilitates the final C-Cl bond formation.
Palacios et al. (Tue,) studied this question.