Redox Potential Measurements of the Mycobacterium tuberculosis Heme Protein KatG and the Isoniazid-Resistant Enzyme KatG(S315T):  Insights into Isoniazid Activation

Abstract

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe(3+)/Fe(2+) couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and II or oxyferrous KatG.

The Met-Tyr-Trp Cross-link in Mycobacterium tuberculosis Catalase-peroxidase (KatG)

“…2, and relevant spectral features and analysis are presented in Table I. The optical purity ratio (Reinheitzahl or R z , defined as A Soret /A 280 ) for WT KatG was found to be 0.62, whereas that for KatG(Y229F) was 0.59, both being consistent with literature values (46,47). Pyridine hemochrome assays yielded 0.97-1.04 heme/monomer, indicating complete holoenzyme formation for both KatGs.…”

Crystal Structure of Mycobacterium tuberculosis Catalase-Peroxidase

“…Ser-315 has been reported to be mutated to asparagine, isoleucine, arginine, and glycine, although the most frequently occurring mutation is to threonine. As a result, a number of in vitro studies have been undertaken to understand the origins of resistance using the S315T mutant as a model (50,(77)(78)(79)(80)(81)(82)(83)(84)(85)(86). In the absence of a structure for mtCP, it was recently postulated that Ser-315 forms hydrogen bonds to one of the heme propionate groups and that mutation to threonine would, therefore, modify the heme pocket, altering INH binding (16,50).…”

Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

“…There is a correlation between the standard reduction potential of the ferric/ferrous couple and the stability of high valent porphyrin species (25). In the case of catalase-peroxidases the standard reduction potential of the ferric/ferrous couple of M. tuberculosis KatG was determined to be Ϫ50 mV (26), which is much more positive than that of other plant-type peroxidases (27). This fits with the observation that Synechocystis KatG is able to oxidize chloride (27) a two-electron oxidation reaction, which needs a redox potential of the KatG couple compound I/native enzyme to exceed 1.1 V. But regarding the catalatic reactivity (i.e.…”