Mechanistic insight into the initiation step of Methionine‐Tyrosine‐Tryptophan (MYW) adduct in <i>Mycobacterium tuberculosis</i> KatG

KatG is produced by a broad range of pathogenic fungi and bacteria, including Mycobacterium tuberculosis . Rapid disproportionation of H 2 O 2 prevents its use by host defensive responses for production of far more bactericidal/fungicidal compounds (e.g, HOCl). KatG’s robust catalase activity depends on a unique post‐translationally generated protein‐based cofactor that arises from covalent links between the side chains of Met255, Tyr229, and Trp107 (MYW). In this study, we addressed the early stages of formation of this unique adduct and how its absence impacts the structure and function of KatG. We have capitalized on our protocols for first expressing/purifying KatG lacking heme (i.e., apoKatG or a KatG) and reconstituting it with heme during purification (i.e., r KatG). We have shown that our r WT KatG contains heme but has yet to form the MYW cofactor; the MYW structure is formed upon reaction with peroxide to generate the mature form of the enzyme ( m WT KatG). We have also generated MYW‐disruptive variants in their reconstituted forms (i.e., r M255I, r Y229F, r W107F). All r KatGs showed spectral features as well as catalase and/or peroxidase activities consistent the forms expressed and purified already containing the heme cofactor. Analysis of tryptic peptides of r KatG and m KatG in LC/MS/MS revealed the presence of MYW‐related ions, though to a much lesser extent in the r KatG as compared to the m KatG, suggesting that the MYW adduct has yet to be generated in r KatG upon reacting with peroxides. In our observation, MYW lacking variants are partially or completely devoid of MYW‐related ions regardless of their treatment with peroxides indicating that the r M255I variant was only able to form the partial YW adduct whereas adduct formation was completely abrogated in the r Y229F and r W107F KatG variants. The resulting mechanistic changes in these mutants are probed in our study using stopped‐flow and rapid freeze‐quench experiments. Stopped‐flow spectroscopy captured [Fe IV =O]‐like intermediate (compound II), followed by Fe III ‐O 2 •– species (compound III) in r M255I and r Y229F variants, consistent with the observation of r WT, although the ferric state has never emerged with the variants. In contrast, compound II has not been detected, but compound III has been the very first intermediate detected in the matured enzymes even when they reacted with the very low equivalences of H 2 O 2 . These early intermediates, observed only with the reconstituted enzymes, demonstrate the feasibility of KatG’s novel MYW cofactor elucidation and characterization, the step essential for exhibiting catalase activity. However, the variant W107F showed identical absorption features of heme‐based intermediates in reconstituted and matured form. This mutation leads to a damage in the hydrogen bonding network, therefore inhibiting the formation of early intermediates. Further investigation of the radical intermediates by EPR will provide valuable insight on the preferred site of crosslink initiation.