16 Thiol-Dependent Peroxidases in Mycobacterium tuberculosis Antioxidant Defense

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis disease. According to the Global Tuberculosis Control 2010 report of the World Health Organization http://www.who.int/tb/publications/global_report/en/, approximately one-third of the world’s population is latently infected with M. tuberculosis and about two million people die of this disease every year. The emergence of multiand extensivelydrug resistant strains to the currently available drugs makes the development of new therapeutic strategies a priority. However, the mechanisms underlying pathogenesis, virulence and persistence of infections caused by M. tuberculosis are not completely understood (Nathan, 2009; Lawn & Zumla, 2011).


Introduction
Mycobacterium tuberculosis (M.tuberculosis) is the causative agent of tuberculosis disease.According to the Global Tuberculosis Control 2010 report of the World Health Organization http://www.who.int/tb/publications/global_report/en/,approximately one-third of the world's population is latently infected with M. tuberculosis and about two million people die of this disease every year.The emergence of multi-and extensively-drug resistant strains to the currently available drugs makes the development of new therapeutic strategies a priority.However, the mechanisms underlying pathogenesis, virulence and persistence of infections caused by M. tuberculosis are not completely understood (Nathan, 2009;Lawn & Zumla, 2011).
M. tuberculosis is one of the most successful human pathogens.It has evolved diverse strategies to ensure growth and survival inside the hostile environment of macrophages, its primary host cells (Ehrt & Schnappinger, 2009;Meena & Rajni, 2010).The molecular mechanisms of M. tuberculosis pathogenesis are under active investigation, since they could provide the basis for a rationalized drug design.These include inhibition of phagosome maturation into phagolysosomes (Armstrong & Hart, 1971;MacMicking et al., 2003), inhibition of the acidification of Mycobacterium-harboring phagosomes (Sturgill-Koszycki et al., 1994), DNA repair and protein repair or degradation (Boshoff et al., 2003;Gandotra et al., 2007;Lee et al., 2009), as well as decomposition of cytotoxic reactive nitrogen and oxygen species formed upon phagocytosis (Nathan & Shiloh, 2000;Shiloh & Nathan, 2000;Bedard & Krause, 2007).These should be considered as complementary survival mechanisms.Herein, we will focus in the antioxidant systems of M. tuberculosis, and particularly, in thioldependent peroxidases.oxidizing and nitrating moiety (Ferrer-Sueta & Radi, 2009;Alvarez et al., 2011).In the absence of direct targets, peroxynitrous acid (pK a = 6.5-6.8,(Goldstein & Czapski, 1995;Pryor & Squadrito, 1995;Kissner et al., 1997)) homolyses into nitrogen dioxide ( • NO 2 ) and • OH in 30% yields (k = 0.9 s -1 at pH 7.4 and 37 ºC) (Goldstein & Czapski, 1995;Gerasimov & Lymar, 1999).However, the importance of this reaction in vivo is probably limited, since in cells, most peroxynitrite is expected to be involved in direct reactions.For instance, peroxynitrite can react with carbon dioxide (CO 2 ) present in mM concentrations in biological systems (k = 4.6 x 10 4 M -1 s -1 at pH 7.4 and 37 ºC), leading to the formation of up to 35% carbonate (CO 3 •-) and • NO 2 radicals, which are also oxidizing species (Lymar & Hurst, 1995;Denicola et al., 1996;Bonini et al., 1999;Augusto et al., 2002).• OH and • NO 2 can participate in lipid peroxidation reactions, resulting in fatty acid hydroperoxide formation (Barber & Thomas, 1978;Prutz et al., 1985).These can also be synthesized by enzymatic mechanisms through lipoxygenase (LOX)-catalyzed reactions (Sevanian et al., 1983).Fatty acid hydroperoxides can be released from membranes by the action of phospholipase A 2 (PLA 2 ) (Bonney et al., 1985).Free arachidonic acid is toxic for M. tuberculosis acting in a synergistic way with reactive nitrogen species (Akaki et al., 2000).Although the mechanism of synergism has not been resolved, the fact that free fatty acid-dependent toxicity to Helicobacter pylori increases in peroxidase-deficient strains indicates that fatty acid hydroperoxides could participate in cytotoxicity (Wang et al., 2006).In summary, inside the phagosomes of activated macrophages, and among other reactive species, different peroxides can be formed, including H 2 O 2 , peroxynitrite and fatty acid hydroperoxides (Figure 1).All of these species have been reported to be cytotoxic against microorganisms including bacteria (Clifford & Repine, 1982;Denicola et al., 1993;Hurst & Lymar, 1997;Evans et al., 1998;Wang et al., 2006).The enzymatic mechanisms that allow reactive nitrogen and oxygen species detoxification, in general, and peroxide reduction, in particular, thus enabling the bacterium to infect and persist inside the phagosome of activated macrophages, is a field of active investigation.

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 H 2 O 2 -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 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).

Catalase peroxidase, the heme-dependent peroxidase of M. tuberculosis
M. tuberculosis constitutively expresses a catalase peroxidase (EC 1.11.1.6)(MtKatG, Rv1908) (Diaz & Wayne, 1974).The enzyme has attracted considerable attention due to its role in the activation of the first line antituberculosis prodrug isonicotinic acid hydrazide (isoniazid, INH) and the fact that loss-of-function mutations are a major mechanism of resistance to INH (Zhang et al., 1992).In vitro generated MtKatG negative strains were non pathogenic.Virulent catalase-negative clinical isolates overexpressed the thiol-dependent peroxidase alkyl hydroperoxidase reductase C (AhpC), indicating the need of another peroxidase to assure protection of the pathogen against oxidizing species (Sherman et al., 1996).More recently, a mechanism of INH resistance in M. tuberculosis through downregulation of KatG was proposed based on the observation that mutations in the furA2 -katG intergenic region conferred INH resistance (Ando et al., 2011).The protein has been identified in the cytosol, membrane fraction and culture filtrates of M. tuberculosis (Gu et al., 2003;Mawuenyega et al., 2005;Malen et al., 2007).It displays a broad peroxidase activity, as well as a high catalase activity (k cat /K M = 2 x 10 6 M -1 s -1 ) (Johnsson et al., 1997), catalyzing the dismutation of H 2 O 2 into dioxygen and water.It also reduces peroxynitrite (k = 1.4 x 10 5 M -1 s -1 at pH 7.4 and 25 ºC (Wengenack et al., 1999)).The catalytic mechanism of H 2 O 2 reduction by KatG involves the initial two-electron oxidation of the enzyme to compound I ((Fe IV=O) • ).KatG contains a unique post-translational modification in the form of a three amino acid adduct (Met255-Tyr229-Trp107) with a specific role in the catalase reaction since mutation of any of the three residues virtually eliminates catalase but not peroxidase activity (Jakopitsch et al., 2004;Ghiladi et al., 2005).It has been proposed that catalase activity in KatG is associated with a radical formation in the Met-Tyr-Trp adduct, whereas during the peroxidase activity a tyrosyl radical is formed (Zhao et al., 2010).In the case of peroxynitrite reduction, oxidation of resting state KatG to compound II (Fe IV=O) plus • NO 2 has been proposed (Wengenack et al., 1999).

Thiol-dependent peroxidases
Peroxidases with catalytic activities dependent on critical cysteine residues are called thioldependent peroxidases.These enzymes catalyze the reduction of H 2 O 2 , 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., 2003;Wood et al., 2003;Trujillo et al., 2007). (1 where ROOH is organic peroxide; ONOOH is peroxynitrous acid; NO 2 -is nitrite; ROH is organic alcohol; Trx(SH) 2 is reduced thioredoxin and TrxS 2 is oxidized thioredoxin.
The oxidizing part of the catalytic cycle involves a SN 2 reaction occurring through a nucleophilic attack of the deprotonated thiol at the so called peroxidatic cysteine residue (C P ) 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 C P suffers a two-electron oxidation to sulfenic acid (E-SOH). (2) 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 C P and another cysteine residue, which is called the resolving cysteine residue (C R ), 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 (pK 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 C P 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).
Other intriguing aspect related to thiol-dependent peroxidase catalytic mechanism is the molecular mechanisms of the oxidizing substrate specificity: although in most cases thioldependent peroxidases can catalyze the reduction of a broad range of peroxides, preferential substrates vary, and do not reflect the expected trend that correlates thiolate reactivity with leaving group pK a3 (Trujillo et al., 2007) that was reported for the reactivities of other thiolate with peroxides (Trindade et al., 2006;Trujillo et al., 2007).
Thiol-dependent peroxidases can be classified into two main groups4 based on sequence homology: glutathione peroxidases (Gpxs) and peroxiredoxins (Prxs).Since there are not genes for enzymes of the GPx type in M. tuberculosis genome, but there are several members of the Prx family, we will focus in the latter group of enzymes through the rest of this chapter.

Peroxiredoxins (EC 1.11.1.15)
Prxs are a family of thioredoxin-scaffold enzymes with thiol-dependent peroxidase activity (Chae et al., 1994).They are ubiquitous, present in all living kingdoms and in different cellular compartments.They are also abundant, with concentrations usually in the µM range (Hofmann et al., 2002).Due to their peroxidase activity, these enzymes play a role in antioxidant defenses.Moreover, at the light of the signaling role ascribed to H 2 O 2 and other peroxides, Prxs are also regarded as key players in redox signaling processes and regulation of transcription factors (Rhee et al., 2005;Hall et al., 2009;Brigelius-Flohe & Flohe, 2011;Rhee & Woo, 2011).Peroxiredoxins have been functionally classified into 1-Cys Prxs and 2cysteine Prxs according to the number of cysteine residues that participate in catalysis (Poole, 2007).The first part of the catalytic cycle is common for all kinds of Prxs and consists on the reduction of the peroxide substrate with concomitant oxidation of the C P to a sulfenic acid derivative.In the case of 1-Cys Prxs, this sulfenic acid is reduced by different reducing pathways that depend on the particular 1-Cys Prx and that for most of them are still unclear.In 2-Cys Prxs, the sulfenic acid in C P reacts with another Cys residue also required for catalysis, C R that can be either in the same or in a different protein subunit (atypical or typical 2-Cys Prxs, respectively), forming a disulfide bridge that is reduced by Trx or a Trx-related protein.More recently, a Prx classification base on sequence homology has been proposed in the peroxiredoxin classification index (PREX) database (http://csb.wfu.edu/prex/index.php)(Nelson et al., 2011;Soito et al., 2011).Subfamilies thus identified are denoted by the name of one or more canonical member, as indicated below: Further information regarding this sequence-based classification of Prxs can be found in the PREX database and references therein.
Peroxiredoxins are now known to be, at least in some cases, very efficient peroxidases (Trujillo et al., 2007;Parsonage et al., 2008;Manta et al., 2009).The local sequence motif at the active site, ProXXXThrXXCys, is very conserved among different Prx subfamilies, although Thr is replaced by Ser in few known Prx sequences and a peroxidatic selenocysteine (Sec) instead of Cys has been reported in a Prx from Eubacterium acidamidophilum (Sohling et al., 2001;Hofmann et al., 2002;Poole, 2007;Nelson et al., 2011).Prxs also contain a highly conserved Arg.These conserved residues along with several backbone interactions determine a low pK a value of C P and contribute to the catalytic mechanism of Prxs, in which transition state stabilization has been proposed to be involved (Hall et al., 2010), although the precise mechanism of catalysis is still to be unraveled.Prx concentrations in different cells and tissues are frequently regulated, and usually increase under conditions of oxidative stress.Moreover, their catalytic activities are also regulated by different mechanisms, including protein phosphorylation (Chang et al., 2002;Woo et al., 2010) and inactivation due to overoxidation of the C P , which involves the two-electron oxidation of the sulfenic form of the enzyme to sulfinic acid (Yang et al., 2002).Recent data from our group indicated that the mechanism of C P overoxidation is similar to that of oxidation, with the deprotonated sulfenate (or its tautomeric sulfoxide form) and the protonated peroxide as the reacting species (Hugo et al., 2009;Reyes et al., 2011). (3) In 2-Cys Prxs, the susceptibility to overoxidation depends on the structural GGLG and YF motifs present mostly in eukaryotic 2-Cys Prxs (Yang et al., 2002) but also in some prokaryotic organisms including cyanobacteria (Pascual et al., 2010).These structural motifs make disulfide formation with C R to occur at a slower rate and thus, 2-Cys Prxs that possess them are more prone to oxidative inactivation (Wood et al., 2003).Cysteine sulfinic acid, previously considered as an irreversible post-transductional modification, is now known to be reversed by enzymatic mechanisms in different 2-Cys Prxs (Chang et al., 2004;Iglesias-Baena et al., 2011) and has been suggested to be involved in signaling processes (Iglesias-Baena et al., 2010).Moreover, overoxidized forms of some members of the Prx family gained function as molecular chaperones (Moon et al., 2005;Lim et al., 2008).

Peroxiredoxins from M. tuberculosis
The genome of M. tuberculosis codifies for different thiol-dependent peroxidases of the Prx type, namely AhpC, TPx, AhpE, and two putative Bcps proteins (Cole et al., 1998), which have been detected in the cytosolic, membrane and culture medium fractions (Figure 2).We will describe below the main functional characteristics of M. tuberculosis Prxs as well as reported evidences of their participation in peroxide detoxification in cellular or animal models of tuberculosis disease.
MtAhpC is functionally classified as a typical 2-Cys Prx, although site directed mutagenesis experiments revealed that it has three instead of two Cys residues involved in catalysis: C P (Cys 61), the putative C R (Cys 174) and a third Cys (Cys 176) whose role in catalysis is not completely clear but could provide an alternative route of disulfide bond formation (Guimaraes et al., 2005).Whereas Cys 61 plays a central role in catalysis, the ehzyme remains partially functional in the absence of Cys 174 and 176 and possible adopts a 1-Cys-like mechanism (Chauhan & Mande, 2002;Koshkin et al., 2004).MtAhpC has been detected both in the bacterial cytosol (Covert et al., 2001) and as a membrane associated protein (Gu et al., 2003).AhpC forms part of bacterial alkyl hydroperoxide reductase (Ahp) system (Storz et al., 1987).In enterobacteria, this system commonly consists of two components, AhpC and a flavin-containing disulfide reductase (AhpF) that reduces AhpC at NADH expense, and both enzymes are jointly up-regulated under oxidative stress conditions targeting the oxyR regulon (Tartaglia et al., 1989).However, AhpF is lacking in all mycobacteria.In this context, two reducing systems for M. tuberculosis AhpC (MtAhpC) have been proposed.Firstly, alkyl hydroperoxide reductase D (AhpD), that contains a CXXC motif, can reduce MtAhpC.The ahpD gene is found immediately downstream of ahpC, in the position occupied by ahpF in S. typhimurium genome, and both proteins are controlled by the same promoter (Hillas et al., 2000).Oxidized AhpD is regenerated by dihydrolipoamide acyltransferase (DlaT); in turn, dihydrolipoamide dehydrogenase (Lpd) mediates the reduction of DlaT at NADH expense and completes the catalytic cycle (Bryk et al., 2002).dlaT (Rv2215) encodes the E2 component of the piruvate deshydrogenase complex, and lpdC (Rv0462), the only functional Lpd in M. tuberculosis (Argyrou & Blanchard, 2001), most probably codifies the E3 components of the piruvate deshydrogenase complex (Tian et al., 2005).Secondly, thioredoxin C (TrxC), but not thioredoxin B (TrxB) or A (TrxA), was also able to act as AhpC reducing substrates (Jaeger et al., 2004), and the catalytic cycle is completed by thioredoxin reductase (MtTR) and NADPH.Catalytic efficiency of TrxC-mediated AhpC reduction was ~ 100 fold lower than that measured using AhpD as reducing substrate (2.5 x 10 4 versus 2.7 x 10 6 M -1 s -1 , respectively) (Jaeger et al., 2004).However, the preferential reducing substrate would be determined not only by catalytic efficiencies but also by the steady-state concentrations of

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Thiol-Dependent Peroxidases in Mycobacterium tuberculosis Antioxidant Defense 301 reducing substrates at reduced state.MtTrxC is consistently seen as a major spot in bacterial proteomes while the spot corresponding to MtAhpD is of much lower intensity (Jungblut et al., 1999;Mollenkopf et al., 1999), indicating a lower concentration of MtAhpD compared to MtTrxC in these cells.Moreover, MtTR is also an abundant protein in Mycobacteria (Jungblut et al., 1999;Mollenkopf et al., 1999), and it is expected to keep TrxC at reduced state as long as NADPH is not limiting (Jaeger et al., 2004).These data suggest that, despite the lower catalytic efficiency of MtTrxC compared to MtAhpD in MtAhpC reduction, both enzymatic pathways could be contributing to MtAhpC-mediated peroxide detoxification in vivo.Concerning the oxidizing substrate specificity, AhpC are broadspectrum peroxidases that catalyze the reduction of H 2 O 2 , organic hydroperoxides and peroxynitrite.The catalytic efficiency of t-BuOOH reduction (an artificial hydroperoxide used as a mimic of natural organic hydroperoxides) by MtAhpC was reported as ~ 10 4 M -1 s -1 (Jaeger et al., 2004).The enzyme could also reduce another artificial organic hydroperoxide, cumene hydroxperoxide at similar rates.H 2 O 2 and linoleic acid hydroperoxides, but not phosphatidylcholine hydroperoxide, were also reduced by MtAhpC.This enzyme, together with other bacterial AhpC enzymes, where the first Prxs for which a peroxynitrite reductase activity was demonstrated (k = 1.3 x 10 6 M -1 s -1 at pH 6.85 and RT (Bryk et al., 2000)).H 2 O 2 was a preferential substrate of MtAhpC, although precise activity measurements were difficult to estimate due to the basal activity of the TR/Trx system (Jaeger et al., 2004).In the case of another AhpC protein (from Salmonella typhimurium) the catalytic efficiency for H 2 O 2 reduction was reported as 3.7 x 10 7 M -1 s -1 (Parsonage et al., 2008).Thus, oxidizing substrate selectivity of bacterial AhpC seems to follow the same trend as for other members of the AhpC-Prx1 subfamily, where reduction of peroxynitrite is somewhat slower than that of H 2 O 2 at near-physiological pHs, and occur with similar pH independent rate constants 5 (Manta et al., 2009).There is no data regarding the pK a of C P and redox potential of AhpC from M. tuberculosis.The pK a values of C P in S. tiphymurium AhpC was first determined as < 5 (Bryk et al., 2000) and more recently reported as 5.8 (Nelson et al., 2008), indicating that C P would be mostly deprotonated at physiological pH.The midpoint reduction potential of the enzyme was reported as -178 ± 0.4 mV, somehow lower than that reported for mammalian Prx 3 (Eº' = -290 mV) (Cox et al., 2010) and plant 2-Cys Prxs and PrxQ (Eº' = −288 to −325 mV) (Dietz et al., 2006).Data regarding redox potential of MtAhpD is lacking.In turn, M. tuberculosis Trx redox potentials have not been investigated so far, but redox potential of other bacterial Trxs has been reported to be low ( -270 mV for E. coli Trx (Krause et al., 1991).
Since the standard midpoint reduction potential for H 2 O 2 reduction to water and for ONOOH reduction to nitrite and water are 1.77 and 1.6 V, respectively (Latimer, 1938;Koppenol & Kissner, 1998), the thermodynamic driving forces would highly favor the flux of electrons from Trxs to these peroxides through AhpC.In addition to its peroxidase 5 According to the mechanism of reaction in which the thiolate form of the CP reacts with the protonated peroxide, and considering that all the reported pKa values of Prxs including AhpC are < 6.3 ( 68), ( 70), ( 71), and that the pKa for the first H2O2 deprotonation is far above physiological pH, the pHindependent rate constant for H2O2-mediated Prx oxidation is practically the same as the rate constant determined at physiological pH.However, the pKa value of peroxynitrous acid is around 6.8 (reported values of ONOOH pKa = 6.5-6.8 ( 23), ( 24), ( 25)) and therefore, only 50 % or 20 % of peroxynitrite would be as be protonated at pH 6.8 or 7.4, respectively.Thus, the pH-independent rate constant of Prx oxidation by peroxynitrite would be 2 or 5 times higher than the value determined at pH 6.8 or 7.4.
activity, some bacterial AhpCs have other functions: Helicobacter pylori AhpC can form high molecular weight aggregates with chaperone activity under oxidative stress conditions (Huang et al., 2010).Moreover, AhpC from some Gram negative microorganisms show a deglutathionylating activity that depends on C R rather than on C P (Yamamoto et al., 2008).
Size exclusion chromatography indicated that wild-type MtAhpC performs as a heterogeneous mixture of oligomers under non-reducing conditions, whereas under reduced state the enzyme is a homogeneous oligomer formed by 10-or 12-subunits.The C176S mutant form of AhpC is dimeric under oxidized state, and forms oligomers of 10-12 subunits upon reduction.The crystallographic structure of C176S MtAhpC trapped as an intermediate of its catalytic cycle (where condensation had already occurred but still the enzyme was under its oligomeric form) was consistent with the formation of a ring shaped oligomer of 12 subunits, a hexamer of dimers (Guimaraes et al., 2005).The relationship between MtAhpC oligomerisation and activity has not been addressed.In the case of Salnonella typhimurium AhpC, decameric under reduced state, the analysis of mutated forms of the enzyme at the decamer-building interface indicated that the oligomerization is quite important, but not essential to activity (Parsonage et al., 2005).
The role of MtAhpC in the detoxification of peroxides in vivo was first suggested by the fact that pathogenic, INH-resistant strains lacking KatG over-expressed MtAhpC, which would represent a compensatory mechanism allowing the bacteria to get rid of cytotoxic peroxides (Sherman et al., 1996).Overexpression of MtAhpC in those strains was associated to mutations in the gene promoter (Wilson & Collins, 1996).Thus, MtAhpC was proposed as a potential drug target.However, data obtained using M. tuberculosis strains lacking MtAhpC are not straightforward.AhpC expression in virulent strains of M. tuberculosis grown in vitro was repressed and increased under conditions of static growth, probably reflecting adaptation of the bacterium during its infection cycle (Springer et al., 2001).AhpC expression was also induced by hypoxia (Sherman et al., 2001).S. typhimurim lacking ahpC became hypersusceptible to reactive nitrogen species and MtAhpC complemented the defect.The enzyme also protected human cells from toxicity caused by reactive nitrogen species (Chen et al., 1998).Whereas inactivation of MtAhpC caused no effect on bacterial growth during acute infection in mice and had no effect on in vitro sensitivity to H 2 O 2 , it caused an increase susceptibility to organic hydroperoxide and peroxynitrite-mediated toxicity (Springer et al., 2001;Master et al., 2002).Inactivation of MtAhpC caused a decrease in the survival of M. tuberculosis in non-stimulated macrophages but not in macrophages stimulated with interferon- (Master et al., 2002).Strains lacking DlaT showed retarded growth, were highly susceptible to killing by acidified nitrite in vitro, showed decreased intracellular survival during macrophage infection and were less virulent in a mouse model of tuberculosis (Shi & Ehrt, 2006).Overall, these data indicate the importance of both MtAhpC and MtDlaT, its reductant through MtAhpD, for M. tuberculosis to overcome oxidative stress encountered inside its primary host cells and to establish a successful infection.

Thiol peroxidase (MtTPx, Rv1932)
The second Prx from M. tuberculosis to be identified belonged to the TPx subfamily (Jaeger et al., 2004), enzymes widely distributed among Gram-positive and Gram-negative bacteria.In the case of E. coli Tpx, the enzyme is localized in the periplasmic space.In M. tuberculosis,

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TPx was firstly characterized as an extracellular antigen that induces a strong proliferative response in animals (Weldingh et al., 1998) .MtTPx was repeatedly found in culture filtrates; it has also been found associated to membranes and in cytosolic fractions (Rosenkrands et al., 2000;Covert et al., 2001;Malen et al., 2007;Malen et al., 2010).
TPxs are atypical 2-Cys Prxs.They typically contain three cysteine residues where Cys60 is C P , C93 is C R and Cys806 is catalytically irrelevant.However, site directed mutagenesis studies revealed that MtTPx lacking Cys 93 remained active for a limited period of time before getting inactivated by C P overoxidation to sulfinic acid, and therefore the role of Cys93 is likely the formation of an intramolecular disulfide with the sulfenic acid in C P and to avoid C P overoxidation under conditions of restricted availability of reducing substrates (Trujillo et al., 2006).MtTPx reacts very rapidly with peroxynitrite (k = 1.5 x 107 M -1 s -1 at pH 7.4 and 25 ºC) 7 .Reduction of t-BuOOH was slower (k ~ 1 x 10 5 M -1 s -1 at pH 7.4 and 25 ºC).
Reduction of H 2 O 2 was faster than that of t-BuOOH, although the exact number was difficult to estimate.The enzyme was hardly active towards linolenic acid hydroperoxide and could not reduce phosphatidylcholine hydroperoxide.Concerning the reductive part of the catalytic cycle, both MtTrxB and MtTrxC reduced MtTPx with similar catalytic efficiencies (4.6 and 5.8 x 10 4 M -1 s -1 , respectively).Since according to proteomic data currently available MtTrx C would be much more abundant than MtTrxB, the former would play a major role as MtTPx reducing substrate.Mycothiol plus mycothione reductase/NADPH were not able to reduce MtTPx (Jaeger et al., 2004).
The crystallography structure of MtTpx (Rho et al., 2006) and on the inactive mutant C60S MtPx (Stehr et al., 2006), as for other bacterial TPxs, indicated that Cys60 in MtTPx forms part of a typical catalytic triad with Thr57 and Arg130.The enzyme is dimeric both in the crystal structure and in solution (Rho et al., 2006;Stehr et al., 2006).In C60S MtTPx, a cocrystallized acetate molecule interacted with Ser60, Arg130 and Thr57 (Stehr et al., 2006).
Similarly, the wild type enzyme also showed anions near the active site.Co-crystallization with anions is frequently observed in Prxs; it has been proposed the existence of an anionbinding site in the neighborhood of reactive thiols in proteins, that could participate in transition state stabilization and thus, in the acceleration of peroxides reduction in general (Hall et al., 2010;Ferrer-Sueta et al., 2011).
M. tuberculosis strains lacking functional MtTPx had a lower peroxidase activity than their wild type counterparts, indicating that the enzyme importantly contributes to the total peroxidase activity in M. tuberculosis.Moreover, MtTPx mutants were more sensitive to H 2 O 2 and •NO-mediated toxicity, but the effect was recovered when they were complemented with the tpx gene.Strains lacking MtTPx failed to grow and survive in macrophages, particularly after activation by interferon-.Growth was significantly restored in the macrophages from iNOS knockout mice.This is consistent with the ability of the enzyme to rapidly reduce peroxynitrite in vitro.Moreover, strains lacking MtTPx failed to initiate an acute infection and to maintain a persistent infection, and were less virulent than wild type strains (Hu & Coates, 2009).In the M. bovis strain BCG, TPx is induced in response to exposure to diamide, an agent that causes thiol oxidation (Dosanjh et al., 2005).

Alkyl hydroperoxide reductase E (MtAhpE, Rv2238c)
The genome of M. tuberculosis also codifies for a one-cysteine Prx, alkyl hydroperoxide reductase E, which is highly conserved among many Mycobacteria (Cole et al., 1998;Passardi et al., 2007).MtAhpE belongs to a novel family of Prxs, comprising bacterial and archaean AhpE and AhpE-like enzymes (Passardi et al., 2007;Soito et al., 2011).This protein has been identified in the membrane fraction of M. tuberculosis H37Rv using a proteomics approach (Gu et al., 2003).The expression of MtAhpE increases during the dormant phase of tuberculosis disease (Murphy & Brown, 2007).Although MtAhpE shows greater sequence similarity with mammalian typical two-Cys Prxs than with one-Cys Prxs (Passardi et al., 2007;Soito et al., 2011), it has only one Cys residue and functions by a one-Cys mechanism.Accordingly, in the oxidized form of the enzyme P is as sulfenic acid, as revealed by crystallographic studies and by mass spectrometry analysis (Li et al., 2005;Hugo et al., 2009).We have reported the peroxidase activity of MtAhpE, being the first member of the AhpE family to be functionally characterized (Hugo et al., 2009).The physiological reducing substrate(s) for MtAhpE (as well as AhpE-like Prxs) is/are still unknown, but its catalytic activity was demonstrated using the artificial substrates dithiotreitol (DTT) and thionitrobenzoic acid (TNB).Neither N-acetylcysteine nor glutathione could reduce oxidized MtAhpE but led to mixed disulfides formation.Concerning oxidizing substrate specificity, MtAhpE reduces peroxynitrite three orders of magnitude faster than H 2 O 2 (1.9 x 10 7 versus 8.2 x 10 4 M -1 s -1 at pH 7.4 and 25 ºC, respectively 8 ).These rate constants were measured directly by taking advantage of the decrease in Trp-dependent fluorescence intensity that the enzyme exhibits upon oxidation.Moreover, the kinetics of peroxide-mediated inactivation by overoxidation of C P to sulfinic acid was measured following the increase in the enzyme's intrinsic fluorescence intensity (k = 40 M -1 s -1 for H 2 O 2 -mediated overoxidation) (Hugo et al., 2009).This value was very similar to that previously calculated for mammalian Prx 1 oxidative inactivation by H 2 O 2 (57 M -1 s -1 ) (Wood et al., 2003;Stone, 2004).The pK a of the thiol (in reduced MtAhpE) and of the sulfenic acid (in oxidized MtAhpE) were reported to be 5.2 and 6.6, respectively.Thus, taking into account the intrabacterial pH of wild-type M. tuberculosis (6.8-7.5 (Vandal et al., 2008)), >95 % of the reduced and >50 % of the oxidized form of C P in MtAhpE would be deprotonated, and therefore, at their reactive forms with peroxides (Hugo et al., 2009).More recently, we have performed a comprehensive study on MtAhpE oxidizing substrate specificity as well as on its oxidative inactivation (Reyes et al., 2011).For most peroxides tested, oxidation as well as oxidative inactivation rates 8 Considering a mechanism of reaction where thiolate and sulfenate as well as protonated peroxides are the reactive species, the reported pKa values of the thiol and sulfenic acid in reduced and oxidized MtAhpE (Hugo et al., 2009) and the pK a of the H2O2 and peroxynitrite above indicated, pH independent rates constants can be calculated as very similar (for H2O2) and ~ 5 fold higher (for peroxynitrite) that the corresponding values measured at pH 7.4.correlated with leaving group pK a , indicating that both reactions occur by similar mechanisms, i.e. reaction of the thiolate or sulfenate anion at C P with the protonated peroxide.In contrast, the hydroperoxide at position 15 of arachidonic acid (15-HpETE) and linolenic acid-derived hydroperoxides reacted surprisingly fast, with rate constants of ~10 8 and ~10 5 M -1 s -1 for MtAhpE oxidation and overoxidation, respectively.The molecular basis for the fast reactivity of MtAhpE with fatty acid hydroperoxides is intriguing.The quaternary structure of MtAhpE in solution is tightly regulated by the oxidation state of the C P, the enzyme being a dimer under reduced state and slowly forming high molecular weight aggregates upon oxidation (Hugo et al., 2009).Analysis of the reported crystallographic structure of the protein under reduced state (Li et al., 2005) showed a hydrophobic grove present in the dimeric enzyme, and formed by residues from both subunits, which is proposed as an anchoring site for fatty acid hydroperoxide binding (Reyes et al., 2011).These data set MtAhpE (and probably other AhpE-like Prxs) as potential Prxs specialized for fatty acid hydroperoxide detoxification.However, the roles of MtAhpE in reduction of these or other peroxides in vivo, as well as in macrophage infection or bacterial virulence, remain to be investigated.
Table 1.Functional data on Prxs from M. tuberculosis: acidity constants, reducing substrates and kinetics of peroxide reduction.

Bacterioferritin comigratory proteins (Bcp, Rv2521; BcpB, Rv1608c)
The genome of M. tuberculosis also codifies for two putative Prxs of the Bcp type (Cole & Barrell, 1998).Evidence for the first Bcp (Rv2125) expression at a protein level exists, both in the membrane fraction (Gu et al., 2003) and in the cytosol of H37Rv strains (Mawuenyega et al., 2005).The protein has been shown to be target of modification by the small protein Pup, a post-translational modification that targets proteins for degradation by the M. tuberculosis proteosome (Pearce et al., 2006;Festa et al., 2010).To note, pupylation and proteosome function are essential for the virulence of this bacterium, for reasons still unknown (Darwin et al., 2003;Gandotra et al., 2007).In the case of BcpB (Rv1608c), it was identified associated to the membrane fraction of M. tuberculosis H37Rv (Gu et al., 2003).The genes for both putative Bcps are considered as non-essential according to mutagenesis analysis in H37Rv strain (Sassetti et al., 2003).Structural and functional data regarding both putative Bcps from M. tuberculosis and their role in infection processes await further investigation.

Conclusions
M. tuberculosis is an extremely successful pathogen, despite of being exposed to cytotoxic peroxides formed inside the phagosome of activated macrophages, its primary host cells.
The bacterium expresses a heme-dependent peroxidase, KatG, and various thioldependent peroxidases of the Prx type.From the data reviewed herein, it becomes clear that Prxs from M. tuberculosis differ in cellular location, and have diverse oxidizing and reducing substrate specificities, that may explain in part the presence of different subfamilies of Prxs in the bacterium.Available data indicate that at least two of them (MtAhpC and MtTPx) play a role in pathogenesis.The third one, MtAhpE, has an outstanding reactivity with fatty-acid derived hydroperoxides, but since natural reducing substrate(s) has not been identified so far, its peroxidase catalytic activity in vivo remains to be confirmed.Similarly, further investigation is required to characterize the two putative Bcp proteins from M. tuberculosis.

Fig. 1 .
Fig. 1.Peroxide sources in M. tuberculosis (Mt)-harboring phagosomes of activated macrophages.Details on the pathways leading to the production of peroxides (H 2 O 2 , peroxynitrite and fatty acid hydroperoxides (FA-OOH), in bold) among other reactive nitrogen and oxygen species are given in the text.Dashed lines indicate reactions involving several steps and intermediates.

Fig. 2 .
Fig. 2. Cellular localization and reducing substrates of peroxidases from M. tuberculosis.The five Prxs and the heme peroxidase KatG have distinct, although overlapping cellular distributions.MtKatG (orange) has been found in the cytosol, membrane and extracellular space.MtAhpC (blue) is a cytosolic enzyme also that was also found associated to the bacterial membrane.MtTPx (green) was detected in culture media repeatedly.It has also been found in membrane fractions and in the cytosol.MtAhpE (violet), and the putative BcpB and Bcp (yellow) were detected in cell membrane fractions, and the latter also in the cytosol.Reducing systems for MtAhpC and MtTpx (in grey) are shown without considering their cellular localization.MtAhpE and MtBcps reducing substrates are still unknown.
).This subfamily is both the largest and the most widely distributed, with members found in archaea, bacteria, and all classes of eukaryotes These proteins are functionally classified as typical 2-Cys Prxs.Found in aerobic gram-positive bacteria of the order Actinomycetes and some archaea.AhpE from Mycobacterium tuberculosis has been functionally classified as a 1-Cys Prx, but information regarding the catalytic mechanisms of other members of this group is lacking.