New Insight into the Protein‐based Cofactor of Mycobacterium tuberculosis KatG: Toward Better Understanding an Unusual Catalase Mechanism

Abstract

Catalase-peroxidases (KatG) has been engineered by nature to exhibit dual functionalism of catalase and peroxidase mechanisms, serving to protect the organisms that carry it against peroxide-dependent oxidative damage. Despite bearing no resemblance to monofunctional (i.e., typical) catalases, KatGs have robust catalase activity due at least in part to a novel covalent linkage between three side chains (by Mycobacterium tuberculosis KatG [MtKatG] numbering, Met-255, Tyr-229, and Trp-107) (MYW). This MYW cofactor redox cycles between its radical (MYW•+) and fully covalent states, enabling KatG to leverage heme intermediates for catalatic O2 production. However, the molecular mechanism by which the adduct is formed and how this unique structure contributes to overall catalytic mechanism of KatG is yet to be explored. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy and mutagenesis have been used to investigate the mechanism of MYW adduct formation. We have expressed and purified MtKatG lacking heme and reconstituted with the cofactor after purification. This produces KatG lacking the MYW crosslink, allowing us to monitor its formation upon reaction with peroxides. Under multiple-turnover conditions using H2O2, optical stopped-flow experiments showed an initial appearance of a high-valent ferryl-like (FeIV=O) intermediate instead of the typical FeIII-O2•- -like steady-state intermediate of KatG's catalase activity. Nevertheless, in contrast to catalase-negative canonical heme peroxidases, full catalase H2O2 decomposition did emerge, returning the enzyme to its FeIII state. EPR experiments revealed that an admixture of radical species appeared before MYW cofactor radical intermediate, suggestive of the preferred site of crosslink initiation. In addition to that, KatG variants lacking the MYW adduct (M255I, Y229F and W107F) were also constructed, expressed, purified, and reconstituted with heme. Stopped flow and UV-vis analysis of these variants showed disrupted catalase activities and incomplete catalatic turnover. These observations strongly suggest that, these distal side residues actively participate in intramolecular electron transfer, thereby fulfilling a mechanistic role in KatG catalase mechanism. Our reconstituted KatG proteins may permit investigation of radical transfer reactions leading to formation of KatG's novel MYW cofactor as well as the influence of other protein radical transfer reactions on that process.