Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme.

KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H2O2-induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H2O2 to O2 and H2O only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of H2O2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting (i.e. ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with H2O2 Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates.

Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG.

Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation.

Stimulation of KatG catalase activity by peroxidatic electron donors.

Catalase-peroxidases (KatGs) use a peroxidase scaffold to support robust catalase activity, an ability no other member of its superfamily possesses. Because catalase turnover requires H(2)O(2) oxidation, whereas peroxidase turnover requires oxidation of an exogenous electron donor, it has been anticipated that the latter should inhibit catalase activity. To the contrary, we report peroxidatic electron donors stimulated catalase activity up to 14-fold, particularly under conditions favorable to peroxidase activity (i.e., acidic pH and low H(2)O(2) concentrations). We observed a "low-" and "high-K(M)" component for catalase activity at pH 5.0. Electron donors increased the apparent k(cat) for the "low-K(M)" component. During stimulated catalase activity, less than 0.008 equivalents of oxidized donor accumulated for every H(2)O(2) consumed. Several classical peroxidatic electron donors were effective stimulators of catalase activity, but pyrogallol and ascorbate showed little effect. Stopped-flow evaluation showed that a Fe(III)-O(2)(·-)-like intermediate dominated during donor-stimulated catalatic turnover, and this intermediate converted directly to the ferric state upon depletion of H(2)O(2). In this respect, the Fe(III)-O(2)(·-) -like species was more prominent and persistent than in the absence of the donor. These results point toward a much more central role for peroxidase substrates in the unusual catalase mechanism of KatG.