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Macrophage‐generated oxygen‐ and nitrogen‐reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis ‘truncated hemoglobin’ N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three‐dimensional structure of oxygenated trHbN, solved at 1.9 Å resolution, displays the two‐over‐two α‐helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N‐terminal α‐helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for ∼28 Å through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, and Mycobacterium bovis each express two genes, glbN and glbO, encoding distantly related truncated hemoglobins (trHbs), trHbN and trHbO, respectively. Here we report that disruption of M. bovis bacillus Calmette-Gué rin glbN caused a dramatic reduction in the NO-consuming activity of stationary phase cells, and that activity could be restored fully by complementing knockout cells with glbN. Aerobic respiration of knockout cells was inhibited markedly by NO in comparison to that of wild-type cells, indicating a protective function for trHbN. TyrB10, which is highly conserved in trHbs and interacts with the bound oxygen, was found essential for NO consumption. Titration of oxygenated trHbN (trHbN⅐O2) with NO resulted in stoichiometric oxidation of the protein with nitrate as the major product of the reaction. The second-order rate constant for the reaction between trHbN⅐O2 and NO at 23°C was 745 M ؊1 ⅐s ؊1 , demonstrating that trHbN detoxifies NO 20-fold more rapidly than myoglobin. These results establish a role for a trHb and demonstrate an NO-metabolizing activity in M. tuberculosis or M. bovis. trHbN thus might play an important role in persistence of mycobacterial infection by virtue of trHbNs ability to detoxify NO.

Two putative hemoglobin genes, glbN and glbO, were recently discovered in the complete genome sequence of Mycobacterium tuberculosis H37Rv. Here, we show that the glbN gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P 50 ‫؍‬ 0.013 mmHg at 20°C) because of a fast combination (25 M ؊1 ⅐s ؊1 ) and a slow dissociation (0.2 s ؊1 ) rate. Resonance Raman spectroscopy and ligand association͞dissociation kinetic measurements, along with mutagenesis studies, reveal that the stabilization of the bound oxygen is achieved through a tyrosine at the B10 position in the distal pocket of the heme with a conformation that is unique among the globins. Physiological studies performed with Mycobacterium bovis bacillus Calmette-Guérin demonstrate that the expression of HbN is greatly enhanced during the stationary phase in aerobic cultures but not under conditions of limited oxygen availability. The results suggest that, physiologically, the primary role of HbN may be to protect the bacilli against reactive nitrogen species produced by the host macrophage.

Truncated hemoglobins (trHbs) are small hemoproteins forming a separate cluster within the hemoglobin superfamily; their functional roles in bacteria, plants, and unicellular eukaryotes are marginally understood. Crystallographic investigations have shown that the trHb fold (a two-on-two ␣-helical sandwich related to the globin fold) hosts a protein matrix tunnel system offering a potential path for ligand diffusion to the heme distal site. The tunnel topology is conserved in group I trHbs, although with modulation of its size/structure. Here, we present a crystallographic investigation on trHbs from Mycobacterium tuberculosis, Chlamydomonas eugametos, and Paramecium caudatum, showing that treatment of trHb crystals under xenon pressure leads to binding of xenon atoms at specific (conserved) sites along the protein matrix tunnel. The crystallographic results are in keeping with data from molecular dynamics simulations, where a dioxygen molecule is left free to diffuse within the protein matrix. Modulation of xenon binding over four main sites is related to the structural properties of the tunnel system in the three trHbs and may be connected to their functional roles. In a parallel crystallographic investigation on M. tuberculosis trHbN, we show that butyl isocyanide also binds within the apolar tunnel, in excellent agreement with concepts derived from the xenon binding experiments. These results, together with recent data on atypical CO rebinding kinetics to group I trHbs, underline the potential role of the tunnel system in supporting diffusion, but also accumulation in multiple copies, of low polarity ligands/molecules within group I trHbs. Truncated hemoglobins (trHbs)1 are small oxygen-binding hemoproteins, identified in bacteria, higher plants, and in certain unicellular eukaryotes, building a separate cluster within the hemoglobin superfamily. Based on amino acid sequence analysis, three trHb phylogenetic groups (groups I, II, and III) have been recognized (1). TrHbs display amino acid sequences that are 20 -40 residues shorter than (non)vertebrate hemoglobins, to which they are scarcely related by sequence similarity. Notably, trHbs belonging to the different groups, but also within the same group, may share less then 20% amino acid sequence identity (1) (Fig. 1). TrHbs from more than one group can coexist in some bacteria, suggesting a wide diversification of functions. Possible trHb functions that are consistent with observed biophysical properties include long term ligand or substrate storage, NO detoxification, O 2 /NO sensor, redox reactions, and O 2 delivery under hypoxic conditions (1-3). In Mycobacterium bovis BCG, trHbN promotes an efficient dioxygenase reaction whereby NO is converted to nitrate by the oxygenated heme (4).So far, four group I trHbs from Chlamydomonas eugametos (Ce-trHb), Paramecium caudatum (Pc-trHb), Mycobacterium tuberculosis (Mt-trHbN), and Synechocystis sp. (Ss-trHb) and one group II trHb from M. tuberculosis (Mt-trHbO) have been structurally characterized (5-9). The main s...

Truncated hemoglobins (Hbs) are small hemoproteins, identified in microorganisms and in some plants, forming a separate cluster within the Hb superfamily. Two distantly related truncated Hbs, trHbN and trHbO, are expressed at different developmental stages in Mycobacterium tuberculosis. Sequence analysis shows that the two proteins share 18% amino acid identities and belong to different groups within the truncated Hb cluster. Although a specific defense role against nitrosative stress has been ascribed to trHbN (expressed during the Mycobacterium stationary phase), no clear functions have been recognized for trHbO, which is expressed throughout the Mycobacterium growth phase. The 2.1-Å crystal structure of M. tuberculosis cyano-met trHbO shows that the protein assembles in a compact dodecamer. Six of the dodecamer subunits are characterized by a double conformation for their CD regions and, most notably, by a covalent bond linking the phenolic O atom of TyrB10 to the aromatic ring of TyrCD1, in the heme distal cavity. All 12 subunits display a cyanide ion bound to the heme Fe atom, stabilized by a tight hydrogen-bonded network based on the (globin very rare) TyrCD1 and TrpG8 residues. The small apolar AlaE7 residue leaves room for ligand access to the heme distal site through the conventional ''E7 path,'' as proposed for myoglobin. Different from trHbN, where a 20-Å protein matrix tunnel is held to sustain ligand diffusion to an otherwise inaccessible heme distal site, the topologically related region in trHbO hosts two protein matrix cavities.T runcated hemoglobins (trHbs) are a class of small oxygenbinding hemoproteins, dispersed in eubacteria, cyanobacteria, protozoa, and plants, recently recognized as a separate cluster within the hemoglobin (Hb) superfamily. On the basis of amino acid sequence analysis, three phylogenetic groups (groups I, II, and III) have been identified within the trHb family; some organisms contain genes from more than one group, suggesting different functions for trHbs belonging to the diverse groups (1). Crystal structures of three group I trHbs (2, 3) revealed that trHbs are clearly not just another variation on the motif of vertebrate myoglobin (Mb) and Hb. Neither are they similar to nonvertebrate Hbs, including the heme-containing domain of flavohemoglobins, nor to the plant symbiotic and nonsymbiotic Hbs (4-10). Major structural differences associated with known trHbs are an unprecedented 2-on-2 ␣-helical sandwich fold, resulting from striking editing of the classical 3-on-3 globin ␣-helical sandwich, and an extended hydrophobic tunnel͞cavity network linking the solvent space and the distal heme pocket (1-3). Much smaller and topologically unrelated cavities, known by their ability to incorporate Xe atoms, have been found in Mb and interpreted as temporary ligand-docking sites (11,12). In trHbs, the positioning and size of the hydrophobic tunnel suggest important roles in controlling ligand access to the heme, in ligand storage, and͞or accumulation (1-3).An additional major diffe...

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The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O 2 , CO, and OH ؊ derivatives of the wild type protein and the B10 Tyr 3 Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.