**3. Singular aspects of the antioxidant defense systems of** *M. tuberculosis*

The antioxidant defenses of *Mycobacterium tuberculosis* are unusual in many aspects. As most other Actinobacteria, it lacks glutathione, and contains millimolar concentration of 1-D-myo-inosityl-2-deoxy-2-(N-acetyl-L-cysteinyl)amino-D-glucopyranoside, or mycothiol (MSH), as main low molecular weight thiol (Newton & Fahey, 2002). MSH is maintained in the reduced form by mycothione reductase using NADPH as electron donor (Patel & Blanchard, 2001). It participates in drug detoxification pathways by forming adducts with alkylating agents and antibiotics that are subsequently cleaved by MSH S-conjugate amidase to generate a mercapturic acid (excreted outside the cell) and glucosamine inositol (used to regenerate MSH) (Newton *et al.*, 2000). MSH can function as a resource for metabolic precursors and for energy production (Bzymek *et al.*, 2007). Mycothiol-deficient *M. smegmatis* strains are more sensitive to •NO- and H2O2-mediated toxicity than wild type strains (Rawat *et al.*, 2002; Miller *et al.*, 2007). However, there is currently no evidence for MSH acting as a reducing substrate for any peroxidase. Mycobacteria, among other organisms, also synthesize ergothioneine, which is a thiourea derivative of histidine containing a sulfur atom in the imidazole ring. Its synthesis is increased in *M. smegmatis* mutants in MSH synthesis suggesting a compensation mechanism (Ta *et al.*, 2011), although the actual function of this unusual thiol remains to

Thiol-Dependent Peroxidases in *Mycobacterium tuberculosis* Antioxidant Defense 297

In addition to *Mt*KatG, the genome of *M. tuberculosis* codifies for a putative lignin peroxidase (Rv1900c) and other putative non-heme non-thiol -dependent peroxidases whose

Peroxidases with catalytic activities dependent on critical cysteine residues are called thioldependent peroxidases. These enzymes catalyze the reduction of H2O2, organic hydroperoxides and/or peroxynitrous acid (ONOOH) to water, organic alcohols and nitrite, respectively, at the expense of a reducing substrate, usually thioredoxin (Trx) or a Trxrelated protein, via a double-displacement or ping-pong kinetic mechanism (Flohe *et al.*,

(1)

(2)


functional characterization is lacking (Cole *et al.*, 1998)(http://www.webtb.org/).

where ROOH is organic peroxide; ONOOH is peroxynitrous acid; NO2

organic alcohol; Trx(SH)2 is reduced thioredoxin and TrxS2 is oxidized thioredoxin.

The oxidizing part of the catalytic cycle involves a SN2 reaction occurring through a nucleophilic attack of the deprotonated thiol at the so called peroxidatic cysteine residue (CP) on one of the peroxide oxygens. In the transition state, the negative charge is distributed among the two oxygen and the sulfur atoms, and the reaction is completed by the break of the peroxide bond forming an alcoxide as leaving group, which may protonate depending on its basicity. Thus, the thiolate in CP suffers a two-electron oxidation to sulfenic

The rest of the catalytic cycle differs depending on the kind of thiol-dependent peroxidase. In most cases, it consists on the formation of a disulfide bridge through the reaction between the sulfenic acid intermediate in CP and another cysteine residue, which is called the resolving cysteine residue (CR), which is then reduced by thioredoxin (Trx) (or another thioldisulfide oxidoreductase protein) that is maintained at reduced state by thioredoxin reductase and NADPH (Poole, 2007). For all thiol-dependent peroxidases tested so far, the acidity constants of the peroxidatic thiols are quite high (p*K*a ~ ‹5 - 6.3, (Bryk *et al.*, 2000; Ogusucu *et al.*, 2007; Trujillo *et al.*, 2007; Nelson *et al.*, 2008; Hugo *et al.*, 2009)). Thus, under physiological conditions they are expected to be mostly under thiolate form, the reactive species. However, the rate constants of reactions of CP in thiol-dependent peroxidases with peroxide substrates are several orders of magnitude faster than the corresponding reactions of low molecular weight or most protein thiolates, indicating the existence of protein factors involved in specific peroxide reduction by these enzymes that are only starting to be unraveled (Trujillo *et al.*, 2007; Flohe *et al.*, 2010; Hall *et al.*, 2010; Ferrer-Sueta *et al.*, 2011).

**5. Thiol-dependent peroxidases of** *M. tuberculosis*

**5.1 Thiol-dependent peroxidases** 

2003; Wood *et al.*, 2003; Trujillo *et al.*, 2007).

acid (E-SOH).

be investigated (Seebeck, 2010). Related to enzymatic mechanisms of reactive oxygen species detoxification, *M. tuberculosis* expresses a Fe-dependent superoxide dismutase, SODA (Rv3846), which is released to the extracellular medium and is considered to be important for pathogenesis (Edwards *et al.*, 2001); it also express a Cu-dependent SODC (Rv0432) that is not essential for intracellular growth within macrophages and seems to play a minor role in pathogenicity (Dussurget *et al.*, 2001). *M. tuberculosis* contains different thioredoxin-related enzymes which are maintained at reduced state by thioredoxin reductase and NADPH (Jaeger *et al.*, 2004). In spite of the absence of glutathione, *M. tuberculosis* genome codifies for different glutaredoxin-like proteins whose functional role awaits further investigation (Cole *et al.*, 1998). The bacterium expresses a heme-dependent peroxidase (catalase peroxidase, KatG) and several thiol-dependent peroxidases of the peroxiredoxin (Prx) type (see below). Moreover, *M. tuberculosis* lacks a functional OxyR, that in *E. coli* controls the transcription of a regulon of ~ 20 antioxidant genes (Zahrt & Deretic, 2002). The regulation of oxidative stress responses in *M. tuberculosis* is at least partially dependent on the alternative sigma factor H/antisigma factor H, a zinc-thiolate redox sensor (Raman *et al.*, 2001).
