A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis.
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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 degrees C) because of a fast combination (25 microM(-1).s(-1)) and a slow dissociation (0.2 s(-1)) rate. Resonance Raman spectroscopy and ligand association/dissociation 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-Guerin 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. (+info)
Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli.
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Expression of the vhb gene encoding hemoglobin from Vitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant beta-lactamase relative to the VHb-expressing control. (+info)
Ligand binding and hexacoordination in synechocystis hemoglobin.
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A large and phylogenetically diverse group of organisms contain truncated hemoglobins, including the unicellular cyanobacterium Synechocystis (Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L., and Bolognesi, M. (2000) EMBO J. 19, 2424-2434). Synechocystis hemoglobin is also hexacoordinate, with a heme pocket histidine that reversibly coordinates the ligand binding site. Hexacoordinate hemoglobins are ubiquitous in plants and are now being identified in a diverse array of organisms including humans (Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., and Klucas, R. V. (1998) Plant Physiol. 118, 1121-1125; Trent, J. T., III, Watts, R. A., and Hargrove, M. S. (2001) J. Biol. Chem. 276, 30106-30110). Rate constants for association and dissociation of the hexacoordinating amino acid side chain in Synechocystis hemoglobin have been measured along with bimolecular rate constants for association of oxygen and carbon monoxide following laser flash photolysis. These values were compared with ligand binding initiated by rapid mixing. Site-directed mutagenesis was used to determine the roles of several heme pocket amino acids in facilitating hexacoordination and stabilizing bound oxygen. It is demonstrated that Synechocystis hemoglobin contains a very reactive binding site and that ligand migration through the protein is rapid. Rate constants for hexacoordination by His(46) are also large and facilitated by other heme pocket amino acids including Gln(43). (+info)
Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme.
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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 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-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 approximately 28 A 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. (+info)
A hemoglobin from plants homologous to truncated hemoglobins of microorganisms.
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We have identified a nuclear-encoded Hb from plants (GLB3) that has a central domain similar to the "truncated" Hbs of bacteria, protozoa, and algae. The three-dimensional structure of these Hbs is a 2-on-2 arrangement of alpha-helices, distinct from the 3-on-3 arrangement of the standard globin fold [Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. & Bolognesi, M. (2000) EMBO J. 19, 2424-2434]. GLB3-like genes are not found in animals or yeast, but our analysis reveals that they are present in a wide range of Angiosperms and a Bryophyte. Although cyanobacteria and Chlamydomonas have 2-on-2 Hbs (GLBN), GLB3 is more likely related to GLBO-type 2-on-2 Hbs from bacteria. Consequently, GLB3 is unlikely to have arisen from a horizontal transfer between the chloroplast and nuclear genomes. Arabidopsis thaliana GLB3 protein exhibits unusual concentration-independent binding of O(2) and CO. The absorbance spectrum of deoxy-GLB3 is unique; the protein forms a transient six-coordinate structure after reduction and deoxygenation, which slowly converts to a five-coordinate structure. In A. thaliana, GLB3 is expressed throughout the plant but responds to none of the treatments that induce plant 3-on-3 Hbs. Our analysis of the sequence, ligand interactions, and expression profile of GLB3 indicates that this protein has unique biochemical properties, evolutionary history, and, most likely, a function distinct from those of other plant Hbs. (+info)
Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.
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Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host. (+info)
Mycobacterium tuberculosis hemoglobin HbO associates with membranes and stimulates cellular respiration of recombinant Escherichia coli.
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The truncated hemoglobins HbN and HbO of Mycobacterium tuberculosis H37Rv share little sequence similarity and display structural differences in their EF-loop regions, suggesting distinct function(s) for these hemoglobins. HbO of M. tuberculosis was expressed in Escherichia coli and Mycobacterium smegmatis as a 14.5-kDa homodimeric heme protein exhibiting nearly 50-fold (P(50) approximately 0.51) lower oxygen affinity than HbN. 40-50% of HbO remained associated with the cell membranes and significantly enhanced its respiration in comparison with the membrane fractions of control cells or cells overproducing HbN. Oxygen uptake of HbO-associated membranes was decreased by washing and restored by adding HbO. Additionally, membrane vesicles prepared from terminal oxidase-deficient (cyo(-), cyd(-)) mutants of E. coli did not exhibit significant enhancement in oxygen uptake in the presence of HbO, suggesting its interaction(s) with the electron transport chain. Expression of HbO in Mycobacterium bovis bacillus Calmette-Guerin, an experimental model of M. tuberculosis, was observed (0.2-0.5% of total cellular proteins) throughout its aerobic growth. These results provided evidence for the involvement of HbO with the component of aerobic electron transport chain, suggesting that its function may be related to the facilitation of oxygen transfer during aerobic metabolism of M. tuberculosis. Membrane association properties of HbO may thus play a crucial role in sequestering oxygen and facilitating its availability to internalized M. tuberculosis (an obligate aerobe) under the hypoxic conditions of its intracellular habitat. (+info)
Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide.
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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-Guerin 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 (trHbN.O(2)) 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 trHbN.O(2) and NO at 23 degrees C was 745 microM(-1).s(-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 trHbN's ability to detoxify NO. (+info)