Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

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.     

Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis

The truncated hemoglobin of Mycobacterium tuberculosis (Mt-trHbO) is a small heme protein belonging to the hemoglobin superfamily. Truncated hemoglobins (trHbs) are believed to have functional roles such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O?/NO chemistry, O? delivery under hypoxic conditions, and long-term ligand storage. Based on sequence similarities, they are classified into three groups. Experimental studies revealed that all trHbs display a 2-on-2 α-helical sandwich fold rather than the 3-on-3 α-helical sandwich fold of the classical hemoglobin fold. Using locally enhanced sampling (LESMD) molecular dynamics, the ligand-binding escape pathways from the distal heme binding cavity of Mt-trHbO were determined to better understand how this protein functions. The importance of specific residues, such as the group II and III invariant W(G8) residue, can be seen in terms of ligand diffusion pathways and ligand dynamics. LESMD simulations show that the wild-type Mt-trHbO has three diffusion pathways while the W(G8)F Mt-trHbO mutant has only two. The W(G8) residue plays a critical role in ligand binding and stabilization and helps regulate the rate of ligand escape from the distal heme pocket. Thus, this invariant residue is important in creating ligand diffusion pathways and possibly in the enzymatic functions of this protein.     

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).     

Theoretical Investigations of Nitric Oxide Channeling in Mycobacterium tuberculosis Truncated Hemoglobin N

Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation of nitric oxide (•NO) to nitrate with a second-order rate constant k ≈ 745 μM⁻¹ s⁻¹ at 23°C (nitric oxide dioxygenase reaction). It was proposed that this high efficiency is associated with the presence of hydrophobic tunnels inside trHbN structure that allow substrate diffusion to the distal heme pocket. In this work, we investigated the mechanisms of •NO diffusion within trHbN tunnels in the context of the nitric oxide dioxygenase reaction using two independent approaches. Molecular dynamics simulations of trHbN were performed in the presence of explicit •NO molecules. Successful •NO diffusion from the bulk solvent to the distal heme pocket was observed in all simulations performed. The simulations revealed that •NO interacts with trHbN at specific surface sites, composed of hydrophobic residues located at tunnel entrances. The entry and the internal diffusion of •NO inside trHbN were performed using the Long, Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by •NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic funnel-shape entrance, covering a large area extending far from the tunnel entrance. This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities capable of hosting up to three •NO molecules, thereby accelerating •NO capture and entry. The importance of hydrophobicity of entrances for •NO capture is highlighted by a comparison with a polar mutant for which residues at entrances were mutated with polar residues. A complete map of •NO diffusion pathways inside trHbN matrix was calculated, and •NO molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect agreement with the three-dimensional free-energy distribution calculated using implicit ligand sampling. The trajectories showed that •NO significantly alters the dynamics of the key amino acids of Phe⁶²(E15), a residue proposed to act as a gate controlling ligand traffic inside the Long tunnel, and also of Ile¹¹⁹(H11), at the entrance of the Short tunnel. It is noteworthy that •NO diffusion inside trHbN tunnels is much faster than that reported previously for myoglobin. The results presented in this work shed light on the diffusion mechanism of apolar gaseous substrates inside protein matrix.     

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.     

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.     

Heme-ligand tunneling in group I truncated hemoglobins

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 alpha-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.     

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 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.     

Cyanide binding to truncated hemoglobins: a crystallographic and kinetic study

Cyanide is one of the few diatomic ligands able to interact with the ferric and ferrous heme-Fe atom. Here, the X-ray crystal structure of the cyanide derivative of ferric Mycobacterium tuberculosis truncated hemoglobin-N (M. tuberculosis trHbN) has been determined at 2.0 A (R-general = 17.8% and R-free = 23.5%), and analyzed in parallel with those of M. tuberculosis truncated hemoglobin-O (M. tuberculosis trHbO), Chlamydomonas eugametos truncated hemoglobin (C. eugametos trHb), and sperm whale myoglobin, generally taken as a molecular model. Cyanide binding to M. tuberculosis trHbN is stabilized directly by residue TyrB10(33), which may assist the deprotonation of the incoming ligand and the protonation of the outcoming cyanide. In M. tuberculosis trHbO and in C. eugametos trHb the ligand is stabilized by the distal pocket residues TyrCD1(36) and TrpG8(88), and by the TyrB10(20) - GlnE7(41) - GlnE11(45) triad, respectively. Moreover, kinetics for cyanide binding to ferric M. tuberculosis trHbN and trHbO and C. eugametos trHb, for ligand dissociation from the ferrous trHbs, and for the reduction of the heme-Fe(III)-cyanide complex have been determined, at pH 7.0 and 20.0 degrees C. Despite the different heme distal site structures and ligand interactions, values of the rate constant for cyanide binding to ferric (non)vertebrate heme proteins are similar, being influenced mainly by the presence in the heme pocket of proton acceptor group(s), whose function is to assist the deprotonation of the incoming ligand (i.e., HCN). On the other hand, values of the rate constant for the reduction of the heme-Fe(III)-cyanide (non)vertebrate globins span over several orders of magnitude, reflecting the different ability of the heme proteins considered to give productive complex(es) with dithionite or its reducing species SO(2)(-). Furthermore, values of the rate constant for ligand dissociation from heme-Fe(II)-cyanide (non)vertebrate heme proteins are very different, reflecting the different nature and geometry of the heme distal residue(s) hydrogen-bonded to the heme-bound cyanide.     

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.     

Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O₂ and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O₂/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.     

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.     

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.     

DevS, a heme-containing two-component oxygen sensor of Mycobacterium tuberculosis

Mycobacterium tuberculosis can exist in the actively growing state of the overt disease or in a latent quiescent state that can be induced, among other things, by anaerobiosis. Eradication of the latent state is particularly difficult with the available drugs and requires prolonged treatment. DevS is a member of the DevS-DevR two-component regulatory system that is thought to mediate the cellular response to anaerobiosis. Here we report the cloning, expression, and initial characterization of a truncated version of DevS (DevS642) containing only the N-terminal GAF sensor domain (GAF-A) and of the full-length protein DevS. The DevS truncated construct quantitatively binds heme in a 1:1 stoichiometry, and the complex of the protein with ferrous heme reversibly binds O2, NO, and CO. UV-vis and resonance Raman spectroscopy of the wild-type protein and the H149A mutant confirm that His149 is the proximal ligand to the heme iron atom. While the heme-CO complex is present as two conformers in the GAF-A domain, a single set of [Fe-C-O] vibrations is observed with the full-length protein, suggesting that interactions between domains within DevS influence the distal pocket environment of the heme in the GAF-A domain.     

A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue

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 --> 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.     

Protein fold and structure in the truncated (2/2) globin family

Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered "truncated hemoglobin" family. The analysis here presented, in agreement with previous surveys, shows that truncated hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 alpha-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 alpha-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the "truncated hemoglobins" term, that does not represent properly the observed 2-on-2 alpha-helical sandwich fold, and adopting the simple "2/2Hb" term to concisely address this protein family.     

Transient ligand docking sites in Cerebratulus lacteus mini-hemoglobin

The monomeric hemoglobin of the nemertean worm Cerebratulus lacteus functions as an oxygen storage protein to maintain neural activity under hypoxic conditions. It shares a large, apolar matrix tunnel with other small hemoglobins, which has been implicated as a potential ligand migration pathway. Here we explore ligand migration and binding within the distal heme pocket, to which the tunnel provides access to ligands from the outside. FTIR/TDS experiments performed at cryogenic temperatures reveal the presence of three transient ligand docking sites within the distal pocket, the primary docking site B on top of pyrrole C and secondary sites C and D. Site C is assigned to a cavity adjacent to the distal portion of the heme pocket, surrounded by the B and E helices. It has an opening to the apolar tunnel and is expected to be on the pathway for ligand entry and exit, whereas site D, circumscribed by TyrB10, GlnE7, and the CD corner, most likely is located on a side pathway of ligand migration. Flash photolysis experiments at ambient temperatures indicate that the rate-limiting step for ligand binding to CerHb is migration through the apolar channel to site C. Movement from C to B and iron-ligand bond formation involve low energy barriers and thus are very rapid processes in the wt protein.