An S=1 Iron(IV) Intermediate Revealed in a Non‐Heme Iron Enzyme‐Catalyzed Oxidative C−S Bond Formation

Abstract

Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol‐histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2‐dependent C−S bond formation catalyzed by non‐heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four‐histidine ligand environment (three from the protein residues and one from the substrate) in enabling C−S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non‐heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C−S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure‐function relationship of high‐valent iron intermediates supported by a histidine rich ligand environment.

Conformational Isomerization of the Fe(III)–OH Species Enables Selective Halogenation in Carrier-Protein-Independent Halogenase BesD and Hydroxylase-Evolved Halogenase

“…This mechanistic interpretation is further supported by the QM/MM study of the N219A mutant of BesD, in which the removal of the hydrogen-bonding interaction between Asn219 and succinate leads to the bidentate coordinate of succinate, inhibiting the conformational flip of Fe­(III)–OH. These findings also have implications on the other nonheme Fe/2OG-dependent oxygenases-catalyzed C–H functionalization beyond the hydroxylation, such as the C–N bond formation, − C–S bond formation, , C–C bond formation, − and C–O bond formation, − in which the thermodynamically favored hydroxylation reactions have been inhibited.…”

Conformational Isomerization of the Fe(III)-OH Species Enables Selective Halogenation in Non-Heme Iron Halogenase BesD and Hydroxylase-Evolved Halogenase

“…This mechanistic interpretation is further supported by the QM/MM study of the N219A mutant of BesD, in which the removal of the hydrogen bonding interaction between N219 and succinate leads to the bidentate coordinate of succinate, inhibiting the conformational flip of Fe(III)-OH. These findings also have implications on the other non-heme Fe/2OG catalyzed C-H functionalization beyond the hydroxylation, such as the C-N bond formation, [103][104][105] C-S bond formation, 106,107 C-C bond formation, [108][109][110][111][112] as well as the C-O bond formation, [113][114][115][116][117][118][119][120][121] in which the thermodynamically favored hydroxylation reactions have been inhibited.…”