Catalase-peroxidase (KatG) in M. tuberculosis is a bifunctional heme protein that exhibits both high catalase activity (2H₂O ₂ → 2H₂O + O₂) and a broad-spectrum peroxidase activity (2AH + H₂O₂ → 2A^· + 2H₂O) and is responsible for activation of isoniazid (INH), a pro-drug used to treat TB infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. Nevertheless, there is a big gap in the M. tuberculosis literature with respect to the molecular origins of isoniazid resistance due to mutations in KatG. Here, we examined the origin of INH resistance caused by the KatG[S315G] mutant enzyme. Overexpressed KatG[S315G] was characterized by optical, EPR and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. INH resistance is suggested to arise from a redirection of catalytic heme intermediates into non-productive reactions that interfere with oxidation of INH.

Previous studies have shown the formation of amino acid based radicals in KatG upon reaction with alkyl peroxide. However, the location and the possible function of these radicals are far from being resolved. In this study we tried to gain insights into the loci of radical formation through the analysis of cross-linking during turnover of KatG in the presence and absence of reducing substrate. SDS and Native-PAGE of KatG treated with peracetic acid or hydrogen peroxide under a variety of conditions demonstrate oligomers of molecular weight greater than that of the native dimer. The results are consistent with the hypothesis that cross-linking of KatG can occur in the absence of peroxidase substrates and that under physiological conditions, the activation of INH as well as the stability of KatG may be altered by this process.

One of the most interesting structural features of Mtb KatG is the post-translational modification of residues Met 255, Tyr229 and Trp107, the side chains of which form an adduct on the distal side of the heme. Mutation of any of these three residues completely abolishes the catalase activity of KatG. A mechanism accounting for the robust catalase activity and the function of this adduct in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. Here, optical stopped-flow spectrophotometry, rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) spectroscopy both at X-band and at D-band, and mutagenesis were used to identify catalase reaction intermediates in Mtb KatG. Using rapid-freeze-quench EPR at X-band under catalase activity conditions (excess H₂O ₂), a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical persists in wild-type KatG only during the time course of turnover of excess H₂O₂ (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, abolishes this radical and the catalase activity. Therefore, a catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.