Mycobacterial truncated hemoglobins: From genes to functions

Gene 398 (2007) 42 – 51 www.elsevier.com/locate/gene

Mycobacterial truncated hemoglobins: From genes to functions Paolo Ascenzi a,b,⁎, Martino Bolognesi c , Mario Milani c , Michel Guertin d , Paolo Visca a,b National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italy Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘Roma Tre’, Viale Guglielmo Marconi 446, I-00146 Roma, Italy c Department of Biomolecular Sciences and Biotechnology, University of Milano, and C.N.R.-I.N.F.M., Research Unit of Milano, Via Celoria 26, I-20131 Milano, Italy d Departement de Biochimie et de Microbiologie, Pavillon Marchand, Université Laval, Faculté des Sciences et de Génie, Quebec, Canada, G1K 7P4 a

b

Received 10 December 2006; received in revised form 29 January 2007; accepted 13 February 2007 Available online 29 April 2007

Abstract Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging threats for human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification mechanisms. Mycobacterial truncated hemoglobins (trHbs) have recently been implicated in scavenging of reactive nitrogen species. Individual members from each trHb family (N, O, and P) can be present in the same mycobacterial species. The distinct features of the heme active site structure combined with different ligand binding properties and in vivo expression patterns of mycobacterial trHbs suggest that these globins may accomplish diverse functions. Here, recent genomic, structural and biochemical information on mycobacterial trHbs is reviewed, with the aim of providing further insights into the role of these globins in mycobacterial physiology. © 2007 Elsevier B.V. All rights reserved. Keywords: Mycobacterial truncated hemoglobin; Genome; Structure; Scavenging of reactive nitrogen species

1. Introduction The genus Mycobacterium (Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales; Corynebacterineae; Mycobacteriaceae; Mycobacterium) designates a group of widespread organisms, most of which living in water, soil and food sources. The taxonomy of the genus is rather complicate and still based on a combination of several phenotypic and genotypic characteristics. Some species, including Mycobacterium tuberculosis and Mycobacterium leprae, are facultative or obligate Abbreviations: trHb, truncated hemoglobin; trHbN, trHb belonging to group I or N; trHbO, trHb belonging to group II or O; trHbP, trHb belonging to group III or P; flavoHb, flavohemoglobin; Hb, hemoglobin; iNOS, inducible nitric oxide synthase; Lb, leghemoglobin; Mb, myoglobin; Ngb, neuroglobin; Hb(II)– O2, ferrous oxygenated Hb; Hb(II)–NO, ferrous nitrosyated Hb; Hb(III), ferric Hb; Hb(III)–NO, ferric nitrosylated Hb; Hb(II)–O2, ferrous oxygenated Hb; Hb (IV)_O, ferryl Hb. ⁎ Corresponding author. National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italy. Tel.: +39 06 5517 3200(2); fax: +39 06 5517 6321. E-mail address: [email protected] (P. Ascenzi). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.02.043

pathogens, respectively, while other species are either opportunistic pathogens or harmless environmental saprophytes (for a review see Tortoli, 2003, and references cited therein). Tuberculosis and leprosy, caused by M. tuberculosis and M. leprae, respectively, have a global prevalence of ∼ 15 million cases. More than 14.5 million cases are tuberculosis, and these have an incidence of ∼9 million new cases per year, mostly in South-East Asia and sub-Saharan Africa regions. Both diseases are curable, but although effective antimicrobial strategies have been established, ∼ 1.7 million people still die every years from tuberculosis. Recently, the emergence of antibiotic resistant strains of tuberculosis and the high incidence of new mycobacterial diseases among immunocompromised patients has led to new research priorities to combat these pathogens (http://www.who.int/mediacentre/factsheets/). Mycobacteria typically cause granulomatous disease during which they are faced with the multiple antimicrobial functions of activated macrophages, also including the release of nitrogen monoxide U ( NO) (see Ratledge and Dale 1999). This article will combine recent genomic, structural and functional information to address

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Fig. 1. Structure-based sequence alignment of micobacterial trHbN, trHbO, and trHbP. The globin fold topological positions, as defined in Physeter catodon (sperm whale) Mb, are shown on the top of the sequence alignment, together with the amino acid sequence. Conserved residues that are relevant for globin fold and function are highlighted in black boxes. Alignments were obtained using CLUSTAL_X (Thompson et al., 1997) and manually adjusted for optimal matching of critical residues.

the role of mycobacterial truncated hemoglobins (trHbs)1 in detoxification of reactive nitrogen species. 2. Truncated hemoglobins in mycobacteria TrHbs display amino acid sequences significantly shorter than classical (non)vertebrate hemoglobins (Hbs) and myoglobins (Mbs), to which they are related by very low sequence similarity. Three main trHb groups have been identified based on sequence clustering; these are group I (also named trHbN), group II (trHbO), and group III (trHbP) (Wittenberg et al., 2002; Vinogradov et al., 2006; Vuletich and Lecomte, 2006) (Figs. 1 and S1). Until now the occurrence of trHbs has been documented in a limited number of mycobacterial species, namely trHbN in M. tuberculosis, Mycobacterium bovis, Mycobacterium smegmatis and Mycobacterium avium, trHbO in M. leprae and all the above species, while trHbP in M. avium only (Wittenberg et al., 2002; Vinogradov et al., 2006; Vuletich and Lecomte, 2006). The regression in content of trHb paralogues from M. avium (three trHbs), through M. tuberculosis (two trHbs), to M. leprae (one trHb) has been proposed to reflect an adaptation from saprophytic lifestyle to obligate intracellular parasitism which paralleled the loss of functions provided by trHbN and trHbP (Wittenberg et al., 2002; Vuletich and Lecomte, 2006). 1 Although for some prokariotic trHbs the alternative names of GlbN, GlbO, and GlbP have been proposed to designate N-, O-, and P-type truncated globins, the conventional definition of trHb is still currently adopted and will be used hereafter.

To gain further insight into the occurrence and distribution of trHbs in genus Mycobacterium, we expanded previous searches for mycobabecterial trHbs, posing particular emphasis for those pathogenic species in which these proteins could play a role in survival to the nitrosative stress encountered in the host (Nathan and Shiloh, 2000; Poole and Hughes, 2000; Cooper et al., 2002; Frey and Kallio, 2003; Wu et al., 2003; Frey and Kallio, 2005; Poole, 2005). The strategy for trHbs searching in mycobacteria was to consider only taxonomically defined species and well characterized strains whose genome had entirely been sequenced or whose sequence was N 99% complete. In this way, the absence of a trHb could be ascertained when no homologue was found in whole-genome searches, and novel patterns of trHb homologues could be retrieved from the complete genomes of several pathogenic and non-pathogenic Mycobacterium species. The former species include: (i) all members of the M. tuberculosis complex, namely M. tuberculosis, M. bovis, Mycobacterium africanum, and Mycobacterium microti, that are the causative agents of tuberculosis in humans and various animals; (ii) the opportunistic pathogen M. avium which causes invasive infection in immunosuoppressed patients and Johne's disease in cattle and other ruminants, and has been associated with Crohn's disease in humans; (iii) M. leprae, the causative agent of leprosy; (iv) Mycobacterium marinum and Mycobacterium ulcerans (the agent of Buruli ulcer) both of which are found in salt and fresh water and cause human granulomatous disease in temperate, tropical and subtropical regions; and (v)

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M. smegmatis which is generally considered a non-pathogenic species but has recently been incriminated in cases of disease. However, trHbs could also be detected in the genome of environmentally useful species, as in case of Myocobacterium vanbalenii and Mycobacterium flavescens, which have a potential role in bioremediation processes since they can degrade a wide range of toxic chemicals (Ratledge and Dale, 1999). Pseudogenes for trHbs were not detected except for a 351-nt sequence corresponding to the M. leprae putative trHbN pseudogene (Table S1). Mycobacterial trHb length varies from 121 amino acid residues for M. smegmatis trHbN to 215 amino acid residues for M. vanbalenii trHbN, with the majority being ≤136 amino acid residues (Fig. S1). Homology between members of different classes is insignificant (b 20% identity). Fig. 2 illustrates the phylogenetic tree corresponding to the trHb alignment for selected mycobacterial species (reported in Fig. S1). TrHbNs appear to form a relatively heterogeneous group, while trHbOs are more homogeneous. Within trHbN and trHbO groups, extremely high identity was observed between groups of phylogenetically related species, including the four pathogenic members of the M. tuberculosis complex, the M. marinum-M. ulcerans group, and the M. vanbalenii-M. flavescens group (Devulder et al., 2005). Interestingly, M. vanbalenii trHbN is predicted to contain a ∼ 80 amino acid residue N-terminal extension which lacks significant homology with any protein from the NCBI database (http://www.ncbi.nlm. nih.gov/), while both M. smegmatis and M. flavescens trHbN lack the N-terminal 11 amino acid residues forming the pre-A helix (Fig. S1 and Table S1). Such a deletion has been shown to U reduce NO-dioxygenase activity of M. smegmatis trHbN and U to result in less protection from NO toxicity compared with the M. tuberculosis counterpart (Lama et al., 2006). Minor straindependent sequence length variation was also observed for M. tuberculosis trHbO, due to the presence of a 6 amino acid residue N-terminal extension in strain CDC1551 (Fig. S1). Analysis of co-occurrence of the three classes of trHbs indicates that all individual species examined have trHbO and all but one have trHbN, while only 3/11 species analyzed have trHbP (Table S1). These results are consistent with the notion that a trHbO-like globin provided the progenitor structure from which trHbPs and trHbNs as well as the classical 3-on-3 structural fold originated (Nakajima et al., 2005; Vinogradov et al., 2006; Vuletich and Lecomte, 2006). Remarkably, M. marinum and M. ulcerans trHbP homologues escaped previous database searches which nearly exclusively detected this class of globins in α and β Proteobacteria (Vinogradov et al., 2006; Vuletich and Lecomte, 2006). Hence, present analysis expands the number of Mycobacterium species carrying individual members of all three trHb lineages. In this regard, it was also suggested that the trHbP protein lineage originated in Proteobacteria and that horizontal gene transfer events may have led to acquisition by M. avium. However, the presence of closely related trHbPs in taxonomically distant species, as in the case of M. avium and the M. marinum–M. ulcerans group (Devulder et al., 2005), suggests that such events occurred early before species divergence, and that all

Fig. 2. Minimum evolution tree of mycobacterial trHb sequences constructed using the MEGA 3 program (Kumar et al., 2004). Robustness of phylogeny was determined by bootstrap analysis based on 1000 resamplings of data, and expressed as percentages. TrHbN, trHbO and TrHbP homologues are designated with N, O and P monograms, respectively, following strain abbreviations. Maf, M. africanum GM041182; Mav M. avium subsp. paratuberculosis K-10; Mbo, M. bovis AF2122/97; Mfl, M. flavescens PYR-GCK; Mle, M. leprae TN; Mma, M. marinum BAA-535; Mmi, M. microti OV254; Msm, M. smegmatis MC2 155; MtuH, M. tuberculosis H37Rv; M. tuberculosis CDC1551; Mul, M. ulcerans Agy99; Mva, M. vanbaalenii PYR-1. P. catodon (sperm whale) Mb (PcaMb) was used as outgroup. Numbers following the slash indicate the length of sequence used for phylogenetic analysis.

three classes of trHbs independently evolved to accomplish different functions. 3. The 2-on-2 fold of mycobacterial trHbs Downsizing of trHbs compared with classical (non)vertebrate Hbs and Mbs (Fig. 1) mirrors their reduced structural complexity. The trHb tertiary structure is based on the typical 2on-2 α-helical sandwich (Fig. 3), that is conserved with group-

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Fig. 3. The trHb fold. Overlay of M. tuberculosis trHbN (PDB code: 1IDR; green) (Milani et al., 2001) and M. tuberculosis trHbO (PDB code: 1NGH; blue) (Milani et al., 2003). N- and C-terminal regions are labeled, together with α-helices, following the conventional globin fold nomenclature (Perutz, 1979). The heme groups of both trHbs are shown at the center of the picture. The oxygen atoms of the heme propionates are in red.

specific local modifications throughout the family. The 2-on-2 fold can be seen essentially as a subset of the well known 3-on-3 α-helical globin fold, strongly conserved in (non)vertebrate Hbs and Mbs. Such fold is achieved in trHbs through deletion/ truncation of α-helices and specific residue substitutions, resulting in conservation of only the B, E, G, and H α-helices of the classical globin fold (see Pesce et al., 2000; Milani et al., 2003; Milani et al., 2005; Nardini et al., 2006) (Fig. 3). Note that the heme-proximal F-helix, carrying the invariant HisF8 proximal residue, is replaced for the most part by an extended polypeptide loop (Fig. 3). A short segment linking the C- and Ehelices forces the heme distal E-helix close to the porphyrin ring, resulting in close packing of the E7, E10, and E11 residues in the distal pocket, a feature particularly evident in trHbN and trHbO (Pesce et al., 2000; Milani et al., 2001; Milani et al., 2003; Nardini et al., 2006). Such clustering may preclude, at least in trHbN, ligand diffusion to/from the heme distal site via the ‘E7 gate’ previously observed in sperm whale Mb (Bolognesi et al., 1982; Perutz, 1989; Milani et al., 2001; Milani et al., 2003; Nardini et al., 2006). Despite their reduced size, trHbNs display a remarkable tunnel/cavity system through the protein matrix that may support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/ or multiligand reactions (Milani et al., 2001; Wittenberg et al., 2002; Milani et al., 2003; Milani et al., 2004a; Milani et al., 2005; Nardini et al., 2006). In M. tuberculosis trHbN, such inner tunnel/cavity system displays two branches, about 20 Å and about 8 Å long, respectively; overall, the tunnel volume is about 265 Å3 (Milani et al., 2001). On the other hand, because of residue substitutions, in M. tuberculosis trHbO the tunnel long branch is restricted to two neighboring cavities, and the tunnel short branch is virtually absent (Milani et al., 2003). Analysis of Campylobacter jejuni trHbP, the only trHbP for

which a crystal structure is known, indicates that the tunnel system is virtually absent (Nardini et al., 2006), leaving a proper protein matrix tunnel system as a structural feature typical of trHbNs. In trHbs, the heme group is stabilized by the HisF8-Fe coordination bond, and complemented by van der Waals contacts to neighboring amino acid residues (among which C7, CD1, E14, EF7, F4, FG5, G5, G8, and H18), hydrogen bonds (involving E2 and F1 residues), and salt bridge interactions to heme propionates (involving E6, E10, and F1 Arg/Lys residues) (Milani et al., 2001; Milani et al., 2003; Nardini et al., 2006). Altogether, the trHb scaffold locates the heme properly and supports the coordination of the heme Fe atom that is in excellent agreement with the heme stereochemistry reported for (non)vertebrate Hbs and Mbs (Perutz, 1979; Bolognesi et al., 1997; Pesce et al., 2000; Milani et al., 2003; Milani et al., 2005; Nardini et al., 2006). In particular, the proximal HisF8 residue is observed in a staggered azimuthal orientation relative to the heme pyrrole N atoms in both M. tuberculosis trHbN and trHbO crystal structures. Such a feature is indicative of an unstrained proximal HisF8, supporting heme inplane location of the Fe atom. This has been related to fast oxygen association and electron donation to the heme-Fe bound distal ligand (see Wittenberg et al., 2002; Milani et al., 2005), that may facilitate (pseudo-)enzymatic functions (see Mukai et al., 2001; Samuni et al., 2004; Ascenzi et al., 2007). The mycobacterial trHb heme distal site cavity is characterized by an array of unusual residues, as compared to classical (non) vertebrate Hbs and Mbs (Fig. 1). The distal B10 residue is almost fully conserved as Tyr. The CD1 site, invariantly Phe in most (non)vertebrate globins, may host Tyr or Leu. The distal E7 residue, His in most (non)vertebrate Hbs, can be Gln, Thr, His, Ser, Leu or Ala, in trHbs. The E11 residue is mostly Gln/Thr. Other relevant sites are E14 (invariantly Phe), E15 (mostly Leu), and G8 (Trp, Val or Ile). Such a selection of distal site residues,

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varying in size and polarity, is instrumental in achieving trHbspecific hydrogen-bonded distal site networks that stabilize the heme-bound ligand through interactions with residues TyrB10 and the neighboring CD1, E7, E11 and G8 side chains. It is noteworthy that in trHbNs and trHbOs a Trp residue invariably occupies the G8 position (Fig. 1). The existing evidences suggest this as a major difference between trHbs and (non)vertebrate Hbs and Mbs (see Milani et al., 2001; Wittenberg et al., 2002; Milani et al., 2005; Nardini et al., 2006). The fine details of the hydrogen-bonded networks vary in the different trHbs, maintaining, however, a strongly intertwined nature (see Pesce et al., 2000; Milani et al., 2001; Falzone et al., 2002; Wittenberg et al., 2002; Milani et al., 2003; Hoy et al., 2004; Milani et al., 2004b, 2005; Nardini et al., 2006). In ferrous M. tuberculosis trHbN, the heme-bound O2 is stabilized by a network of hydrogen bonds that include TyrB10 and GlnE11. As a result, the O2 molecule is tightly buried in the distal site, and may be polarized to a partial superoxide character through the hydrogen bond to TyrB10 (Milani et al., 2001; Ouellet et al., 2006). Comparable ligand stabilizing interactions (including residues TyrB10, GlnE11, and the ligand) are observed in the cyanide derivative of ferric M. tuberculosis trHbN, where the cyanide anion is bound in an orientation perpendicular to the heme plane (Milani et al., 2004b). Raman spectroscopy indicates that in M. tubercolosis trHbN the hemeU Fe-bound O2, CO, NO, and OH– ligands are stabilized by hydrogen bonding to TyrB10 (Couture et al., 1999; Yeh et al., 2000; Mukai et al., 2002, 2004). In M. tuberculosis trHbO two distal site residues appear particularly relevant for ligand recognition and binding. On one hand, the CD1 site is occupied by a Tyr residue that is part of a post-translational covalent modification in half of the molecules of the dodecameric M. tuberculosis trHbO observed in the crystals. Specifically, the TyrB10 phenolic O atom is covalently linked to the aromatic ring of TyrCD1, providing a rigid di-Tyr assembly in contact and hydrogen-bonded to the heme-Fecoordinated cyanide. The second relevant distal residue is TrpG8, that fills a significant fraction of the heme distal site. The TrpG8 indole ring is parallel and in contact with the porphyrin ring, providing a bifurcated hydrogen bond to the heme-bound ligand and to TyrCD1 OH group. These, together with a direct hydrogen bond from TyrCD1 to the ligand, lock the cyanide heme ligand in ferric M. tuberculosis trHbO (Milani et al., 2003). In keeping with these observations, resonance Raman data suggest that the heme-Fe-bound O2 and CO are at hydrogen bonding distance to TyrB10, TyrCD1, and TrpG8 in ferrous M. tuberculosis trHbO (Mukai et al., 2002; Ouellet et al., 2003; Mukai et al., 2004). Stabilization of the heme-Fe-bound ligand by distal G8 and CD1 residues is unprecedented. In trHbs, hydrogen-bonded networks may modulate the positioning and dynamics of distal pocket residues that not only stabilize the heme-Fe-bound ligand but also participate in the control of: (i) ligand access to the heme-Fe atom; (ii) ligand diffusion within the distal heme pocket; and (iii) ligand diffusion into and out of the distal heme pocket. The hydrogen bonding networks may also confer an added degree of stability to the heme-Fe-bound ligand by making ligand dissociation

dependent on multiple, simultaneous hydrogen bond ruptures (see Milani et al., 2005). Heme hexacoordination has been observed in the ferrous derivative of M. leprae trHbO (Visca et al., 2002a). The empty inner space created by protein matrix tunnel/cavities and the nonhelical pre-F region may facilitate the conformational transitions required to achieve a hexacoordinated heme (see Wittenberg et al., 2002; Milani et al., 2005). As observed for other hexacoordinated globins (e.g., human neuroglobin and cytoglobin as well as Drosophila melanogaster Hb (Pesce et al., 2003; de Sanctis et al., 2004; Vallone et al., 2004; de Sanctis et al., 2006), heme-Fe hexato-penta-coordination equilibrium may modulate exogenous ligand binding properties (Visca et al., 2002a). 4. Scavenging of reactive nitrogen species by mycobacterial trHbs During infection, mycobacteria are faced with the toxic effects U of reactive nitrogen species, primarily NO, produced by U activated macrophages expressing inducible NO-synthase (iNOS) (MacMicking et al., 1997; Nathan and Shiloh, 2000; Cooper et al., 2002; Visca et al. 2002b; Ohno et al., 2003; Schnappinger et al., 2006). The distinct features of the heme U active site structure of NO-responsive mycobacterial trHbs (see Milani et al., 2001; Visca et al., 2002a; Milani et al., 2003, 2004b, 2005) and their ligand binding properties (see Milani et al., 2005) combined with co-occurrence of multiple trHb classes in individual mycobacterial species and the temporal expression patterns of trHbs in vivo (Ouellet et al., 2002, 2003; Fabozzi et al., 2006) suggest that these globins play different physiological functions, including scavenging of reactive nitrogen and oxygen species, O2 uptake/transport, cellular respiration, and (pseudo-) enzymatic reactions. Interestingly, having retained only one trHb, M. leprae trHbO has been proposed to represent merging of multiple functions (Couture et al., 1999; Mukai et al., 2002; Ouellet et al., 2002; Pathania et al., 2002a; Visca et al., 2002a,b; Wittenberg et al., 2002; Ouellet et al., 2003; Liu et al., 2004; Mukai et al., 2004; Samuni et al., 2004; Milani et al., 2005; Ascenzi et al., 2006a,b; Fabozzi et al., 2006; Lama et al., 2006). U Heme-proteins could detoxify NO by the rapid and irreversible reaction of their ferrous oxygenated derivative with UNO. The efficiency of this process, which is postulated to depend on the superoxide character of the heme-Fe-bound O2, may be U enhanced by intramolecular diffusion of NO, momentarily trapped within cavities of the protein matrix. Alternatively, O2 can react with the ferrous nitrosylated heme-proteins, however this very slow process is physiologically irrelevant. Furthermore, ferrous deoxygenated, oxygenated, and nitrosylated as well as ferric heme-proteins facilitate peroxynitrite2 scavenging. All these processes lead to the formation of the ferric heme-protein species. Subsequently, the ferric heme-proteins could be reduced to the ferrous species by specific reductases (see Gow et al., 1999; 2 The recommended IUPAC nomenclature for peroxynitrite is oxoperoxonitrate(1-); for peroxynitrous acid, it is hydrogen oxoperoxonitrate. The term peroxynitrite is used in the text to refer generically to both ONOO- and its conjugate acid HOONO (see Herold, 2004a).

P. Ascenzi et al. / Gene 398 (2007) 42–51 Table 1 Kinetic parameters for NO-mediated oxidation of ferrous oxygenated hemeproteins a kon Fe(II)–O2 + NO → Fe(III)–OONO → Fe(III) + NOU− 3 kon (M− 1 s− 1)

Heme-protein b

M. tuberculosis trHbN M. tuberculosis trHbO c M. leprae trHbO d E. coli flavoHb e Horse heart Mb f Murine Ngb g Human Hb

8

7.5 × 10 6.0 × 105 2.1 × 106 ≥6 × 108 4.4 × 107 N7.0 × 107 8.9 × 107 h

h h (s− 1) n.d. n.d. 3.4 ∼ 2 × 102 N3.4 × 102 ≈ 3.0 × 102 N5.8 × 101 i N 3.3 × 101 i

n.d., not determined. a In the reaction scheme, the notation Hb refers to all globins. b pH = 7.5 and 23.0 °C. From (Ouellet et al., 2002). c pH = 7.5 and 23.0 °C. From (Ouellet et al., 2003). d pH = 7.3 and 20.0 °C. From (Ascenzi et al., 2006a). e pH = 7.0 and 20.0 °C. From (Gardner et al., 2000). f pH = 7.0 and 20.0 °C. From (Herold et al., 2001). g pH = 7.0 and 20.0 °C. From (Brunori et al., 2005). h pH = 7.0 and 20.0 °C. From (Herold et al., 2001). i The two values represent the decay rates for Fe(III)OONO α- and β-Hb subunits. pH = 7.5 and 20.0 °C. From (Herold, 1999).

Brunori, 2001a,b; Brunori and Gibson, 2001; Flögel et al., 2001; Frauenfelder et al., 2003; Frey and Kallio, 2003; Wu et al., 2003; Brunori et al., 2004; Frey and Kallio, 2005; Herold and Fago, 2005; Poole, 2005). As reported for several heme-proteins (see Herold, 1999; Gardner et al., 2000; Herold et al., 2001; Brunori et al., 2005), ferrous oxygenated M. tuberculosis trHbN and trHbO as well as U M. leprae trHbO have been shown to facilitate NO scavenging U (Ouellet et al., 2002, 2003; Ascenzi et al., 2006a). The fast NO oxidation catalyzed by oxygenated trHbN in comparison to that U of trHbO (Table 1) makes the NO detoxification role for trHbN likely (Ouellet et al., 2002, 2003; Ascenzi et al., 2006a), trHbO playing only a marginal role in the protection of mycobacteria U against NO (Visca et al., 2002a,b; Ouellet et al., 2003; Ascenzi et al., 2006a; Pawaria et al., 2007). The involvement of trHbN in protection from reactive nitrogen U species, particularly NO, has been documented in vivo using both reverse genetic approaches and homologous or heterologous expression systems (Ouellet et al., 2002; Pathania et al., 2002a; Lama et al., 2006; Pawaria et al., 2007), and this has substantially U been ascribed to NO-dioxygenase activity. In fact, a M. bovis U mutant lacking trHbN does not oxidize NO to NO3− and shows U decreased respiration upon exposure to NO (Ouellet et al., 2002). Moreover, heterologous expression of M. tuberculosis trHbN, which is identical to the M. bovis counterpart, significantly protects M. smegmatis and flavohemoglobin mutants of both Escherichia coli and Salmonella enterica Typhimurium from UNO damage through O -sustained detoxification mechanism 2 (Pathania et al., 2002b; Pawaria et al., 2007). Although to a lesser extent, a similar protective effect was also reported for M. smegmatis trHbN in the homologous system (Lama et al., 2006). The physiological role of M. tuberculosis trHbO has been primarily related to O2 metabolism. In fact, trHbO is a membrane-associated protein which interacts with the CyoB

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subunit of E. coli terminal oxydase cytochrome o complex, and sustains aerobic respiration under microaerobic conditions by facilitating O2 delivery to component(s) of the electron transfer chain. Hence, trHbO was hypothesized to be endowed with O2 uptake or delivery properties during mycobacterial hypoxia and latency (Pathania et al., 2002a; Liu et al., 2004). This hypothesis is in apparent contrast with the low O2 association and dissociation rates reported for trHbO (Ouellet et al., 2003) and with its constitutive expression under aerobic conditions during the whole growth cycle of M. bovis (Mukai et al., 2002; Pathania et al., 2002b). TrHb(II)–O2 could still be able to U sustain bacterial aerobic respiration by scavenging NO or other reactive species that would block the respiratory chain. In this context, the high stability of trHb(II)–O2 would secure the U reaction with NO even at very low O2 tensions, as those that may exist in infected or necrotic tissue (Fabozzi et al., 2006). Intriguingly, trHbO(II)-O2 from M. tuberculosis and M. leprae U was also shown to oxidize NO, though the slow rate of this reaction argued against a primary role in direct protection from UNO (Ouellet et al., 2002; Ascenzi et al., 2006a), consistent with U the low NO consumption activity and the poor protection U against NO toxicity recently documented for M. tuberculosis trHbO, as compared with M. tuberculosis trHbN (Pawaria et al., 2007). U Several secondary reactions have been postulated for NO, primarily the formation of peroxynitrite following reaction with the superoxide radical (O2U−),which is concomitantly produced by activated macrophages (Beckman and Koppenol, 1996; Nathan and Shiloh, 2000; Zahrt and Deteric, 2002; Goldstein et al., 2005; Ascenzi et al., 2006c). Then, peroxynitrite could Table 2 Kinetic parameters for peroxynitrite-mediated oxidation of ferrous nitrosylated heme-proteins a

Heme-protein M. leprae trHbO b Glycine max Lb c Horse heart Mb Human Ngb f Human Hb g a b c d e f g

[CO2] (M) – 1.2 × 10− 3 – 1.0 × 10− 3 –d 1.2 × 10− 3 e – – 1.2×10− 3

lon (M− 1 s− 1) N1 × 108 N1 × 108 8.8 × 103 1.2 × 105 3.1 × 104d 1.7 × 105 e 1.3 × 105 6.1 × 103 5.3 × 104

In the reaction scheme, the notation Hb refers to all globins. pH 7.3 and 20.0 °C. From (Ascenzi et al., 2006b). pH 7.3 and 20.0 °C. From (Herold and Puppo, 2005a). pH 7.5 and 20.0 °C. From (Herold and Boccini, 2006). pH 7.0 and 20.0 °C. From (Herold and Boccini, 2006). pH 7.2 and 20.0 °C. From (Herold et al., 2004). pH 7.2 and 20.0 °C. From (Herold, 2004a).

b (s− 1) 2.6 × 101 2.4 × 101 2.0 2.5 ∼ 1.2 × 101d 1.1 × 101 e 1.2 × 10− 1 ∼1 ∼1

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rapidly react with CO2 at the site of inflammation leading to the formation of strong oxidant and nitrating species (e.g., CO3U− U and NO2) (Goldstein et al., 2005; Ascenzi et al., 2006c). In analogy to Glycine max Lb, horse heart Mb, human Ngb, and human Hb (see Exner and Herold, 2000; Herold et al., 2002, 2003; Boccini and Herold, 2004; Herold, 2004a; Herold et al., 2004; Herold and Fago, 2005; Herold and Puppo, 2005a,b; Herold and Boccini, 2006), M. leprae trHbO(II)–NO and trHbO(II)–O2 catalyze peroxynitrite scavenging (Ascenzi et al., 2006b). M. leprae trHbO(II)–NO displays the most favorable parameters for peroxynitrite scavenging both in the absence and presence of CO2 (Table 2). CO2 facilitates the Fe(II)–NO to Fe (III)–NO transition without affecting the dissociation of the Fe (III)–NO complex which represents the rate limiting step. In contrast, the catalytic parameters for peroxynitrite scavenging by Fe(II)–O2 heme-proteins considered are similar (Herold et al., 2003; Boccini and Herold, 2004; Herold and Puppo, 2005b; Ascenzi et al., 2006b) (Table 3). CO2 facilitates the conversion of the Fe(II)–O2 derivative of M. leprae trHbO, Glycine max Lb, horse heart Mb, and human Hb to Fe(IV) _ O, and the reduction of the Fe(IV) _ O species of Glycine max Lb and human Hb to Fe(III) derivative.3 M. tuberculosis trHbO(IV) _ O has recently been reported to induce the formation of Trp and Tyr radicals, resulting in cross-linked protein oligomers (Ouellet et al., in press). Furthermore, the Fe(IV) _ O species appears to be involved in the formation of Tyr radicals which evolve to diTyr adducts (Herold, 2004b). Scavenging of reactive nitrogen species by mycobacterial trHbs can be of physiopathological relevance provided that (i) the trHb concentration in vivo is high enough to support detoxification and (ii) the trHb(III) reductase system(s) is efficient to restore trHb(II). Interestingly, Mycobacterium bovis trHbO concentration was observed to range between 0.2% and 0.5% of total proteins (Pathania et al., 2002b), i.e. grossly between 2 × 10− 5 M and 1 × 10− 4 M; the trHbO concentration (ranging between 1 × 10− 4 M and 4 × 10− 4 M) is similar to that of Mb, which proved to be an efficient scavenger of reactive nitrogen species in different models (Brunori, 2001a; Brunori, 2001b; Flögel et al., 2001). Lastly, search for oxidoreductase genes in mycobacterial genomes identified, among others, the products of trx (thioredoxin), trxB (bifunctional thioredoxin reductase/thioredoxin), fdxA (ferredoxin), fprA (NADPH-ferredoxin reductase), and fprB (ferredoxin/ferredoxin NADPreductase) as putative candidates for trHb(III) reduction (Visca et al., 2002b; Fabozzi et al., 2006). As a whole, mycobacterial trHbs seem to be suited for different physiological functions, including scavenging of reactive nitrogen and oxygen species, O2 uptake/transport, cellular respiration U and (pseudo-)enzymatic reactions. NO detoxification by M. tuberculosis trHbN has been demonstrated in vivo and in vitro, while M. tuberculosis trHbO is less likely to perform this In the presence of CO2, heme-protein oxidation is facilitated by COU-3 . In fact, CO2 reacts rapidly with peroxynitrite leading to ONOOC(O)O- which decays very rapidly to COU-3 and UNO2. COU-3 is a stronger oxidant than UNO2 and peroxynitrite. In contrast to peroxynitrite, UNO2 nitrates with preference Tyr and Trp residues (see Goldstein et al., 2005; Ascenzi et al., 2006c). 3

Table 3 Kinetic parameters for peroxynitrite-mediated oxidation of ferrous oxygenated heme-proteins a

Heme-protein M. leprae trHbO b Glycine max Lb c Horse heart Mb d Human Hb e a b c d e

[CO2] (M) – 1.2 × 10− 3 – 1.2 × 10− 3 – 1.2 × 10− 3 – 1.2 × 10− 3

don (M− 1 s− 1) 4.8 × 104 6.3 × 105 5.5 × 104 8.8 × 105 5.4 × 104 4.1 × 105 3.3 × 104 3.5 × 105

fon (M− 1 s− 1) 1.3 × 104 1.7 × 104 2.1 × 104 3.6 × 105 2.2 × 104 3.2 × 104 3.3 × 104 1.1 × 105

In the reaction scheme, the notation Hb refers to all globins. pH 7.3 and 20.0 °C. (Ascenzi et al., 2006b). pH 7.3 and 20.0 °C. From (Herold and Puppo, 2005b). pH 7.5 and 20.0 °C. From (Herold et al., 2003). pH 7.4 and 20.0 °C. From (Boccini and Herold, 2004).

function. Peroxynitrite detoxification by M. leprae trHbO has been shown to be rapid and this trHbO might function in this role. 5. Concluding remarks TrHbs have been shown to build a minimal but efficient 2on-2 α-helical sandwich fold around the heme group. The ensuing proteins display structural and functional properties which are comparable to those of classical (non)vertebrate Mbs and Hbs. In addition, trHbs may support functional U roles other than O2 storage or diffusion, such as NO and peroxynitrite scavenging, under physico-chemical conditions mirroring different cellular environments. The occurrence of trHbs in mycobacterial genomes varies depending on the species, and this in part reflects the ecology of the genus. Species that are commonly found in variable natural environments (soil and water) and cause infection as facultative parasites have all three or at least two trHb types, irrespective of whether they are classified as fast or slow growers. In contrast, adaptation to obligate parasitism resulted in loss of trHb genes, as in the case of M. leprae which retained the ancestral (O-type) trHb. Since M. leprae has retained a minimalistic repertoire of functional genes (Cole et al., 2001; Brosch et al., 2001), trHbO can be regarded as essential product for intracellular parasitism, while trHbN and trHbP appear to be dispensable. This unique requirement makes trHbO a candidate target for novel antimycobacterial drugs capable of inhibiting heme reactivity of bacterial Hbs. Lastly, in vivo expression of trHbs by mycobateria could have implications in laboratory diagnosis and clinical follow-up of mycobacterial infection.

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