Fig. 6. Implications of the GriE crystal structures on the reaction mechanism. (A) Active site as observed in the crystal structure of the substrate complex. O₂ would bind to the vacant coordination site (purple arrow). After the decarboxylation of α-ketoglutarate, the reactive oxygen intermediate (O) would abstract a hydrogen atom from the nearest methyl group of l-leucine (red arrow), leading to (2S,4R)-5-hydroxyleucine (see also Fig. S9 and S10†). (B) Reaction scheme derived from the crystal structures of the GriE ligand complexes. Starting with the state corresponding to the Co²⁺/substrate-containing crystal structure (highlighted in blue), dioxygen can bind to the vacant coordination site trans to His110 and perform a nucleophilic attack on the carbonyl group of α-ketoglutarate, forming a Fe(iv)-peroxy-hemiketal transition state. In order to react with the substrate, the reactive oxygen species has to swap its position with CO₂ during the subsequent decarboxylation step. Alternatively, CO₂ might already have left the active site and been replaced by a water molecule (not shown). The oxo-ferryl intermediate abstracts a hydrogen from the closest C_δ methyl group of l-leucine, leaving a radical at the substrate which then reacts with the hydroxyl group of the Fe(iii)-hydroxo species. The state with the reaction products succinate and (2S,4R)-5-hydroxyleucine still bound to the metal, but CO₂ being replaced by a water molecule, corresponds to the crystal structure of the Mn²⁺/product complex (highlighted in red).