Indole N‐Linked Hydroperoxyl Adduct of Protein‐Derived Cofactor Modulating Catalase‐Peroxidase Functions

Bifunctional catalase‐peroxidase (KatG) features a posttranslational methionine‐tyrosine‐tryptophan (MYW) crosslinked cofactor crucial for its catalase function, enabling pathogens to neutralize hydrogen peroxide during infection. We discovered the presence of indole nitrogen‐linked hydroperoxyl adduct (MYW‐OOH) in Mycobacterium tuberculosis KatG in the solution state under ambient conditions, suggesting its natural occurrence. By isolating predominantly MYW‐OOH‐containing KatG protein, we investigated the chemical stability and functional impact of MYW‐OOH. We discovered that MYW‐OOH inhibits catalase activity, presenting a unique temporary lock. Exposure to peroxide or increased temperature removes the hydroperoxyl adduct from the protein cofactor, converting MYW‐OOH to MYW and restoring the detoxifying ability of the enzyme against hydrogen peroxide. Thus, the N‐linked hydroperoxyl group is releasable. KatG with MYW‐OOH represents a catalase dormant, but primed, state of the enzyme. These findings provide insight into chemical strategies targeting the bifunctional enzyme KatG in pathogens, highlighting the role of N‐linked hydroperoxyl modifications in enzymatic function. Catalase‐peroxidase (KatG) neutralizes hydrogen peroxide during infection. Mycobacterium tuberculosis KatG from cells grown at ambient temperature contains an indole nitrogen‐linked hydroperoxyl adduct, MYW‐OOH (golden color). MYW‐OOH transiently inhibits catalase activity, presenting a unique off switch. Exposure to peroxide or increased temperature converts MYW‐OOH to MYW (blue color) and restores KatG's detoxifying ability.