Figure 5. Proposed catalytic cycle of TglHI.

(A) Zoom-in of the structure of TglH with the C-terminal Cys of TglA modeled by comparison with the structures of VcMbnB and VcMbnABC (PDB accession No. 7DZ9). The AlphaFold2 model positions the side chain amide of Asn73 near the sulfur of Cys.

(B) In vitro activity of TglHI variants (TglH^N73AI, TglH^N73DI and TglH^N73LI) to modify the minimum substrate analog of TglA-Cys as measured by the formate production assay.

(C) The TglA-Cys modification process mediated by TglHI. Asn73 of TglH is suggested to orient the Cys ligated to Fe1 for hydrogen atom abstraction by the peroxo intermediate through formation of a hydrogen bond between the thiolate of Cys and the amide −NH₂ of Asn73. See Figure S6F for a detailed chemical description.