We have characterized the intermediates formed in the peroxidase cycle of the multifunctional heme-containing enzyme KatG of M. tuberculosis. Selected Trp variants from the heme proximal (W321F) and distal (W107F and W91F) sides were analyzed together with the wild-type enzyme with regard to the reaction with peroxyacetic acid and hydrogen peroxide (in the catalase-inactive W107F). The 9 GHz EPR spectrum of the enzyme upon reaction with peroxyacetic acid showed the contribution of three protein-based radical species, two Trp(center dot) and a Tyr(center dot), which could be discerned using a combined approach of multifirequency Electron Paramagnetic Resonance (EPR) spectroscopy with selective deuterium labeling of tryptophan and tyrosine residues and site-directed mutagenesis. Trp321, a residue in H-bonding interactions with the iron through Asp381 and the heme axial ligand His270, was identified as one of the radical sites. The 9 GHz EPR signal of the Trp321 radical species was consistent with an exchange-coupled species similar to the oxoferryl-Trp radical intermediate in cytochrome c peroxidase. On the basis of the possibility of distinguishing among the different radical intermediates of the peroxidase cycle in M. tuberculosis KatG (MtKatG), we used EPR spectroscopy to monitor the reactivity of the enzyme and its W321F variant with isoniazid, the front-line drug used in the treatment of tuberculosis. The EPR experiments on the W321F variant preincubated with isoniazid allowed us to detect the short-lived [Fe(IV)=O Por(center dot+)] intermediate. Our results showed that neither the [Fe(IV)=O Por(center dot+)] nor the [Fe(IV)=O TrP321 center dot+] intermediates were the reactive species with isoniazid. Accordingly, the subsequent intermediate (most probably the other Trp(center dot)) is proposed to be the oxidizing species. Our findings demonstrate that the protein-based,radicals formed as alternative intermediates to the [Fe(IV)=O Por(center dot+)] can play the role of cofactors for substrate oxidation in the peroxiclase cyle of KatGs.