Structural determinants of ligand migration in Mycobacterium tuberculosis truncated hemoglobin O

Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, truncated hemoglobin N (trHbN) and truncated hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand-binding characteristics. The X-ray structure of trHbO has been solved and shows several internal cavities and secondary-docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer-simulation techniques. We have computed the free-energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple-ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand-migration regulation.     

Unique ligand-protein interactions in a new truncated hemoglobin from Mycobacterium tuberculosis

A new truncated hemoglobin (HbO) from Mycobacterium tuberculosis has been expressed and purified. Sequence alignment of HbO with other hemoglobins suggests that the proximal F8 residue is histidine and the distal E7 and the B10 positions are occupied by alanine and tyrosine, respectively. The highly conserved residue at the CD1 position, surprisingly, is tyrosine, making HbO the first exception in the hemoglobin family that does not contain phenylalanine at this position. Resonance Raman data suggest that a strong hydrogen bonding network, involving the B10 Tyr and the CD1 Tyr, stabilizes the heme-bound O2 and CO as evidenced by the relatively low frequency of the Fe-O2 stretching mode (559 cm(-1)) and the high frequency of the Fe-CO stretching mode (527 cm(-1)). The presence of this hydrogen bonding network is supported by mutagenesis studies with the B10 tyrosine or the CD1 tyrosine mutated to phenylalanine. Taken together, these data demonstrate a rigid and polar distal pocket in HbO, which is significantly different from that of HbN, the other hemoglobin from M. tuberculosis. The distinct features in the heme active site structures and the temporal expression patterns of HbO and HbN suggest that these two hemoglobins may have very different physiological functions.     

NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr --> Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm(-1), respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm(-1). These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y(8a) mode of the B10 Tyr shifted from 1616 to 1622 cm(-1), confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y(8a) mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.     

Ligand interactions in the distal heme pocket of Mycobacterium tuberculosis truncated hemoglobin N: roles of TyrB10 and GlnE11 residues

The crystallographic structure of oxygenated trHbN from Mycobacterium tuberculosis showed an extended heme distal site hydrogen-bonding network that includes Y(B10), Q(E11), and the bound O(2) (Milani, M., et al. (2001) EMBO J. 20, 3902-3909). In the present work, we analyze the effects that substitutions at the B10 and E11 positions exert on the heme and its coordinated ligands, using steady-state resonance Raman spectroscopy, absorption spectroscopy and X-ray crystallography. Our results show that (1) residues Y(B10) and Q(E11) control the binding and the ionization state of the heme-bound water molecules in ferric trHbN and are important in keeping the sixth coordination position vacant in deoxy trHbN; (2) residue Q(E11) plays a role in maintaining the integrity of the proximal Fe-His bond in deoxy trHbN; (3) in wild-type oxy-trHbN, the size and hydrogen-bonding capability of residue E11 is important to sustain proper interaction between Y(B10) and the heme-bound O(2); (4) CO-trHbN is in a conformational equilibrium, where either the Y(B10) or the Q(E11) residue interacts with the heme-bound CO; and (5) Y(B10) and Q(E11) residues control the conformation (and likely the dynamics) of the protein matrix tunnel gating residue F(E15). These findings suggest that the functional processes of ligand binding and diffusion are controlled in trHbN through the dynamic interaction of residues Y(B10), Q(E11), F(E15), and the heme ligand.     

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.     

Structural characterization of the tunnels of Mycobacterium tuberculosis truncated hemoglobin N from molecular dynamics simulations

The structure of oxygenated trHbN from Mycobacterium tuberculosis shows an extended heme distal hydrogen‐bond network that includes Tyr33(B10), Gln58(E11), and the bound O₂. In addition, trHbN structure shows a network of hydrophobic cavities organized in two orthogonal branches. In the present work, the structure and the dynamics of oxygenated and deoxygenated trHbN in explicit water was investigated from 100 ns molecular dynamics (MD) simulations. Results show that, depending on the presence or the absence of a coordinated O₂, the Tyr33(B10) and Gln58(E11) side chains adopt two different configurations in concert with hydrogen bond network rearrangement. In addition, our data indicate that Tyr33(B10) and Gln58(E11) control the dynamics of Phe62(E15). In deoxy‐trHbN, Phe62(E15) is restricted to one conformation. Upon O₂ binding, the conformation of Gln58(E11) changes and residue Phe62(E15) fluctuates between two conformations. We also conducted a systematic study of trHbN tunnels by analyzing thousands of MD snapshots with CAVER. The results show that tunnel formation is the result of the dynamic reshaping of short‐lived hydrophobic cavities. The analyses indicate that the presence of these cavities is likely linked to the rigid structure of trHbN and also reveal two tunnels, EH and BE, that link the protein surface to the buried distal heme pocket and not present in the crystallographic structure. The cavities are sufficiently large to accomodate and store ligands. Tunnel dynamics in trHbN was found to be controlled by the side‐chain conformation of the Tyr33(B10), Gln58(E11), and Phe62(E15) residues. Importantly, in contrast to recently published works, our extensive systematic studies show that the presence or absence of a coordinated dioxygen does not control the opening of the long tunnel but rather the opening of the EH tunnel. In addition, the data lead to new and distinctly different conclusion on the impact of the Phe62(E15) residue on trHbN tunnels. We propose that the EH and the long tunnels are used for apolar ligands storage. The trajectories bring important new structural insights related to trHbN function and to ligand diffusion in proteins. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water

The survival of Mycobacterium tuberculosis requires detoxification of host *NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k'(NOD) approximately 745 x 10(6) m(-1) x s(-1)), which is approximately 15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O(2) binding is very rapid with rates approaching 1-2 x 10(9) m(-1) x s(-1). These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the *NO derivative of met-trHbN, where both the *NO and water can be directly followed, revealed that water rebinding is quite fast (approximately 1.49 x 10(8) s(-1)) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).     

Viscosity-dependent relaxation significantly modulates the kinetics of CO recombination in the truncated hemoglobin TrHbN from Mycobacterium tuberculosis

Kinetic traces were generated for the nanosecond and slower rebinding of photodissociated CO to trHbN in solution and in porous sol-gel matrices as a function of viscosity, conformation, and mutation. TrHbN is one of the two truncated hemoglobins from Mycobacterium tuberculosis. The kinetic traces were analyzed in terms of three distinct phases. These three phases are ascribed to rebinding: (i) from the distal heme pocket, (ii) from the adjacent apolar tunnel prior to conformational relaxation, and (iii) from the apolar tunnel subsequent to conformational relaxation. The fractional content of each of these phases was shown to be a function of the viscosity and, in the case of the sol-gel-encapsulated samples, sample preparation history. The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand. Implications of these finding with respect to biological function are discussed.     

Reactions of Mycobacterium tuberculosis truncated hemoglobin O with ligands reveal a novel ligand-inclusive hydrogen bond network

Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O(2) and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 microM(-1) s(-1)) and dissociation (<0.006 s(-1)) of O(2) are among the slowest known, precluding transport or diffusion of O(2) as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants for O(2) and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the chi((2,1)) dihedral angle for the indole ring increases progressively from approximately 93 degrees to at least 100 degrees in going sequentially from the deoxy to CO to O(2) derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O(2) dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.     

Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of the dimeric hemoglobin of Scapharca inaequivalvis (HbI) after photodissociation. By combining these studies with X-ray crystallography, three transient ligand docking sites were identified: a primary docking site B in close vicinity to the heme iron, and two secondary docking sites C and D corresponding to the Xe4 and Xe2 cavities of myoglobin. To assess the relevance of these findings for physiological binding, we also performed flash photolysis experiments on HbICO at room temperature and equilibrium binding studies with dioxygen. Our results show that the Xe4 and Xe2 cavities serve as transient docking sites for unbound ligands in the protein, but not as way stations on the entry/exit pathway. For HbI, the so-called histidine gate mechanism proposed for other globins appears as a plausible entry/exit route as well.     

Peroxynitrite scavenging by ferrous truncated hemoglobin GlbO from Mycobacterium leprae

Mycobacterium leprae GlbO has been proposed to represent merging of both O(2) uptake/transport and scavenging of nitrogen reactive species. Peroxynitrite reacts with M. leprae GlbO(II)-NO leading to GlbO(III) via the GlbO(III)-NO species. The value of the second order rate constant for GlbO(III)-NO formation is >1x10(8)M(-1)s(-1) in the absence and presence of CO(2) (1.2x10(-3)M). The CO(2)-independent value of the first order rate constant for GlbO(III)-NO denitrosylation is (2.5+/-0.4)x10(1)s(-1). Furthermore, peroxynitrite reacts with GlbO(II)-O(2) leading to GlbO(III) via the GlbO(IV)O species. Values of the second order rate constant for GlbO(IV)O formation are (4.8+/-0.5)x10(4) and (6.3+/-0.7)x10(5)M(-1)s(-1) in the absence and presence of CO(2) (=1.2x10(-3)M), respectively. The value of the second order rate constant for the peroxynitrite-mediated GlbO(IV)O reduction (= (1.5+/-0.2)x10(4)M(-1)s(-1)) is CO(2)-independent. These data argue for a role of GlbO in the defense of M. leprae against nitrosative stress.     

Ligand migration and hexacoordination in type 1 non-symbiotic rice hemoglobin

Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Structure and dynamics of Mycobacterium tuberculosis truncated hemoglobin N: insights from NMR spectroscopy and molecular dynamics simulations

The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe2?-O? + (?)NO → trHbN-Fe3?-OH? + NO??) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (?)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (?)NO and O? to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.     

Ligand-induced dynamical regulation of NO conversion in Mycobacterium tuberculosis truncated hemoglobin-N

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of "truncated hemoglobin" N (trHbN), formally acting as NO-dioxygenase, yielding the harmless nitrate ion. X-ray crystal structures have shown that trHbN hosts a two-branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate-limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 micros), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy-trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual-path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification.     

Reaction of Mycobacterium tuberculosis truncated hemoglobin O with hydrogen peroxide: evidence for peroxidatic activity and formation of protein-based radicals

In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H(2)O(2) to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme pi-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr(55) and Tyr(115), which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H(2)O(2) and the one-electron donor 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs.     

Ligand specificity of group I biotin protein ligase of Mycobacterium tuberculosis

Background

Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC.

Methodology/Principal Findings

BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the ∼29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5′AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5′AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5′AMP. Docking simulations also suggest that bio-5′AMP hydrogen bonds to the conserved ‘GRGRRG’ sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K_m for BCCP was ∼5.2 µM and ∼420 nM for biotin. MtBPL has low affinity (K_b = 1.06×10⁻⁶ M) for biotin relative to EcBirA but their K_m are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5′AMP by EcBirA is channeled for its repressor activity.

Conclusions/Significance

These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.     

H2O2 and (.)NO scavenging by Mycobacterium leprae truncated hemoglobin O

Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H₂O₂ and of trHbOFe(IV)O reduction by NO and NO₂⁻ are reported. The value of the second-order rate constant for H₂O₂-mediated oxidation of trHbOFe(III) is 2.4 × 10³ M⁻¹ s⁻¹. The value of the second-order rate constant for NO-mediated reduction of trHbOFe(IV)O is 7.8 × 10⁶ M⁻¹ s⁻¹. The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 × 10¹ s⁻¹. The value of the second-order rate constant for NO₂⁻-mediated reduction of trHbOFe(IV)O is 3.1 × 10³ M⁻¹ s⁻¹. As a whole, trHbOFe(IV)O, generated upon reaction with H₂O₂, catalyzes NO reduction to NO₂⁻. In turn, NO and NO₂⁻ act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H₂O₂ and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration.     

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.     

Modeling truncated hemoglobin vibrational dynamics

We present a study on the near equilibrium dynamics of two small proteins in the family of truncated hemoglobins, developed under the framework of a Gaussian network approach. Effective beta carbon atoms are taken into account besides Calphas for all residues but glycines in the coarse-graining procedure, without leading to an increase in the degrees of freedom (beta Gaussian Model). Normalized covariance matrix and deformation along slowest modes with collective character are analyzed, pointing out anticorrelations between functionally relevant sites for the proteins under study. In particular, we underline the functional motions of an extended tunnel-cavity system running inside the protein matrix, which provide a pathway for small ligands binding with the iron in the heme group. We give a rough estimate of the order of magnitude of the relaxation times of the slowest two overdamped modes and compare results with previous studies on globins.     

Ligand Binding in the Conserved Interhelical Loop of CorA, a Magnesium Transporter from Mycobacterium tuberculosis

CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg²⁺ and Co²⁺) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved “MPEL” motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.The heterogeneous membrane environment is very challenging to mimic for structural, dynamic, and functional studies of membrane proteins. It is not surprising, therefore, that different aspects of the structure can be brought to light under different conditions. Recently, an excellent set of crystal structures of the CorA Mg²⁺ transporter have been published (1–3), and whereas many CorA mysteries were solved, the highly conserved periplasmic interhelical loops in the pentameric structure were not well resolved. Here, solution nuclear magnetic resonance (NMR) spectroscopy of the transmembrane domain resolves these loops, and the secondary structure and ion binding in this domain are characterized.The 2TM-GxN family of transporters is a large group of integral membrane proteins responsible for metal ion transport (especially for magnesium) across membranes (4, 5). CorA is a prototypical member in this family responsible for magnesium influx as well as efflux in some cases (5, 6). In the extensive phylogenetic analysis, it was shown that CorA is characterized by a universally conserved “GMN” motif in an interhelical loop connecting two conserved transmembrane helices at the C terminus of the full-length protein (4). As the only constitutively expressed magnesium transporter, CorA can play an important role in the viability of pathogens, such as Helicobacter pylori (7).Pentameric CorA from Thermotoga maritima forms two distinguishable domains; that is, a large cytoplasmic domain and a small transmembrane domain (1–3). In this latter domain there are two transmembrane helices connected by an interhelical periplasmic loop. The first transmembrane helix (TM1)2 lines the pore, whereas the second transmembrane helix (TM2) forms an outer ring of helices, which appears to have only weak interactions with the TM1 helices.Different mechanisms of substrate transport for CorA have been proposed (1–3, 6, 8), whereas the structure and function of the conserved loop is still an open issue. Based on phylogenetic analyses, it has been shown that there were two conserved sequences in the interhelical loop. One is the GMN motif that is universally conserved, and the other is the MPEL motif that is conserved throughout most bacterial genomes. The glutamic acid residue in the MPEL sequence is almost universally conserved in CorA and CorA homologs in eukaryotic cells, including yeast and humans (4). However, it is not conserved in Methanococcus jannaschii for which CorA has been functionally characterized (9). Although the Mycobacterium tuberculosis protein, studied here, has not been functionally characterized in detail, it has been shown to transport magnesium ions across the membrane.³ Because the interhelical loop is the only portion of the protein exposed to the periplasmic side and because there is a highly conserved negatively charged residue, it has been suggested that the loop could act as an initial magnesium binding site (1). This could result in enhancing Mg²⁺ concentration at the mouth of the pore, enhancing substrate selection and generating partial cation dehydration (1). Recent functional studies of CorA homologs from yeast have shown that this negatively charged residue in the loop plays an important role in function (10–12). Substitution of Glu by Lys results in a dramatic reduction in transport activity in yeast (10), and substitution of a positively charged residue (Arg in A1r2p, a CorA homolog in yeast) by a Glu increases channel activity (11). However, the CorA from M. jannaschii does not contain either this residue or a negatively charged loop. Based on these results, the negatively charged loop appears to be functionally important and may act as an initial substrate binding site for increasing the local substrate concentration to facilitate ion transport (10–12), whereas it may not be functionally essential at least for some CorA members. However, it has also been argued, based on limited crystallographic data, that the loop may not form a binding site for substrate selection and dehydration (3). Instead, it was speculated that the loop could mediate the relative movement of the two transmembrane helices (3). Accordingly, further investigation of this functionally important loop is warranted.In the present work we characterize the interaction between the isolated transmembrane domain of CorA (CorA-TMD) and its substrates as well as an inhibitor. Such a “divide and conquer” strategy has been successfully applied to several membrane proteins, such as the M2 protein (a proton channel from influenza A) (13–15), Vpu (a membrane protein encoded by human immunodeficiency virus involved in the budding of new viral particles from the host cell) (16, 17), GlpG (a rhomboid intra-membrane protease) (18, 19), and S2P (a intra-membrane metalloprotease) (20). In addition, the electron density map from the crystal structure of the isolated CorA soluble domain was used to solve the structure of full-length CorA by molecular replacement (1). This suggests that the structural influence of the transmembrane domain on the soluble domain is limited, at least for the conformational state that was crystallized (1). Hence, a divide and conquer strategy for CorA appears to be justified and provides an opportunity to characterize this domain by solution NMR.It has been shown that NMR has a unique ability of characterize weak interactions that are not readily characterized by other methods (21, 22). In the present work we characterize the binding of two substrates (Mg²⁺ and Co²⁺) to the loop as well as an inhibitor (Cobalt(III) hexamine chloride, HexCo³⁺) by NMR. Our data indicate that the ligands of CorA can weakly but specifically bind to the interhelical loop of CorA-TMD through their interaction with the conserved negatively charged glutamic acid residue in the MPEL motif, supporting the hypothesis that the loop acts as an initial binding site.