Metal Coordination Dynamics Governs Selective Halogenation in α-KG/Fe-dependent SyrB2

α-ketoglutarate (α-KG)-dependent non-heme iron enzymes catalyze a diverse array of oxidative transformations essential for natural product biosynthesis. However, the mechanism by which the α-KG/Fe -dependent halogenase SyrB2 achieves selective halogenation while circumventing the thermodynamically favored hydroxylation pathway remains a subject of intense debate. In this study, we elucidate the halogenation mechanism catalyzed by SyrB2 through extensive computational and crystallographic investigations. Notably, we identify a previously unrecognized α-KG binding mode. Furthermore, our work reveals that the coordination dynamics of the iron center plays a pivotal role in controlling selective C−H bond activation and chlorination in SyrB2. The proposed mechanistic model is supported by multiple experimental observations, including Mössbauer, nuclear resonance vibrational, and 2 H-HYSCORE spectroscopies, alongside kinetic analysis of reactions with various substrates. These findings underscore the central role of metal coordination dynamics in enabling non-hydroxylative reactivity among α-KG/Fe-dependent enzymes, and provide valuable insights for engineering α-KG-dependent oxygenases for C−H halogenation.