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.     

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.     

Multiple active site conformers in the carbon monoxide complexes of trematode hemoglobins

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (approximately 60 cm(-1)) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm(-1), for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm(-1), respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm(-1), respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.     

Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin

We report an unusually high frequency (543 cm(-)(1)) for an Fe-CO stretching mode in the CO complex of Ascaris suum hemoglobin as compared to vertebrate hemoglobins in which the frequency of the Fe-CO mode is much lower. A second Fe-CO stretching mode in Ascaris hemoglobin is observed at 515 cm(-1). We propose that these two Fe-CO stretching modes arise from two protein conformers corresponding to interactions of the heme-bound CO with the B10-tyrosine or the E7-glutamine residues. This postulate is supported by spectra from the B10-Tyr --> Phe mutant in which the 543 cm(-1) line is absent. Thus, a strong polar interaction, such as hydrogen bonding, of the CO with the distal B10 tyrosine residue is the dominant factor that causes this anomalously high frequency. Strong hydrogen bonding between O(2) and distal residues in the oxy complex of Ascaris hemoglobin has been shown to result in a rigid structure, rendering an extremely low oxygen off rate [Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. London B 163, 206-214]. In contrast, the CO off rate in Ascaris hemoglobin is very similar to that in sperm whale myoglobin. The high CO off rate relative to that of O(2) in Ascaris hemoglobin is attributed to a rapid equilibrium between the two conformations of the protein in the CO adduct, with the off rate being determined by the conformer with the higher rate.     

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.     

Mycobacterium tuberculosis hemoglobin HbO associates with membranes and stimulates cellular respiration of recombinant Escherichia coli.

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-Guérin, 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.     

The carbon monoxide derivative of human hemoglobin carrying the double mutation LeuB10-->Tyr and HisE7-->Gln on alpha and beta chains probed by infrared spectroscopy

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10-->Tyr and HisE7-->Gln on the alpha, beta, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm(-1) in both the alpha and the beta mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the alpha and beta chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated alpha or beta chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species.     

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.     

1H NMR investigation of the distal hydrogen bonding network and ligand tilt in the cyanomet complex of oxygen-avid Ascaris suum hemoglobin.

The O(2)-avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O(2) off rates. Solution (1)H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO(2) crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4224-4228) with the exception that the heme in solution is rotated by 180 degrees about the alpha,gamma-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO(2), provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented approximately 30 degrees away from the heme normal and indicates significant steric tilt because of interaction with Tyr(30)(B10). The three side chain labile protons for the distal residues Tyr(30)(B10) and Gln(64)(E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO(2) crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O(2). The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr(30)(B10) side chain supports a crowded and constrained distal pocket.     

Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens

Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens (At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1? resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions

The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.     

Hydrogen bonding in the active site of ketosteroid isomerase: electronic inductive effects and hydrogen bond coupling

Computational studies are performed to analyze the physical properties of hydrogen bonds donated by Tyr16 and Asp103 to a series of substituted phenolate inhibitors bound in the active site of ketosteroid isomerase (KSI). As the solution pK(a) of the phenolate increases, these hydrogen bond distances decrease, the associated nuclear magnetic resonance (NMR) chemical shifts increase, and the fraction of protonated inhibitor increases, in agreement with prior experiments. The quantum mechanical/molecular mechanical calculations provide insight into the electronic inductive effects along the hydrogen bonding network that includes Tyr16, Tyr57, and Tyr32, as well as insight into hydrogen bond coupling in the active site. The calculations predict that the most-downfield NMR chemical shift observed experimentally corresponds to the Tyr16-phenolate hydrogen bond and that Tyr16 is the proton donor when a bound naphtholate inhibitor is observed to be protonated in electronic absorption experiments. According to these calculations, the electronic inductive effects along the hydrogen bonding network of tyrosines cause the Tyr16 hydroxyl to be more acidic than the Asp103 carboxylic acid moiety, which is immersed in a relatively nonpolar environment. When one of the distal tyrosine residues in the network is mutated to phenylalanine, thereby diminishing this inductive effect, the Tyr16-phenolate hydrogen bond becomes longer and the Asp103-phenolate hydrogen bond shorter, as observed in NMR experiments. Furthermore, the calculations suggest that the differences in the experimental NMR data and electronic absorption spectra for pKSI and tKSI, two homologous bacterial forms of the enzyme, are due predominantly to the third tyrosine that is present in the hydrogen bonding network of pKSI but not tKSI. These studies also provide experimentally testable predictions about the impact of mutating the distal tyrosine residues in this hydrogen bonding network on the NMR chemical shifts and electronic absorption spectra.     

Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity

Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure.     

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-.     

Structural and functional properties of a truncated hemoglobin from a food-borne pathogen Campylobacter jejuni

Campylobacter jejuni contains two hemoglobins, Cgb and Ctb. Cgb has been suggested to perform an NO detoxification reaction to protect the bacterium against NO attack. On the other hand, the physiological function of Ctb, a class III truncated hemoglobin, remains unclear. By using CO as a structural probe, resonance Raman data show that the distal heme pocket of Ctb exhibits a positive electrostatic potential. In addition, two ligand-related vibrational modes, nu(Fe-O(2)) and nu(O-O), were identified in the oxy derivative, with frequencies at 542 and 1132 cm(-1), respectively, suggesting the presence of an intertwined H-bonding network surrounding the heme-bound ligand, which accounts for its unusually high oxygen affinity (222 microm(-1)). Mutagenesis studies of various distal mutants suggest that the heme-bound dioxygen is stabilized by H-bonds donated from the Tyr(B10) and Trp(G8) residues, which are highly conserved in the class III truncated hemoglobins; furthermore, an additional H-bond donated from the His(E7) to the Tyr(B10) further regulates these H-bonding interactions by restricting the conformational freedom of the phenolic side chain of the Tyr(B10). Taken together, the data suggest that it is the intricate balance of the H-bonding interactions that determines the unique ligand binding properties of Ctb. The extremely high oxygen affinity of Ctb makes it unlikely to function as an oxygen transporter; on the other hand, the distal heme environment of Ctb is surprisingly similar to that of cytochrome c peroxidase, suggesting a role of Ctb in performing a peroxidase or P450-type of oxygen chemistry.     

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.     

Structure and ligand selection of hemoglobin II from Lucina pectinata

Lucina pectinata ctenidia harbor three heme proteins: sulfide-reactive hemoglobin I (HbI(Lp)) and the oxygen transporting hemoglobins II and III (HbII(Lp) and HbIII(Lp)) that remain unaffected by the presence of H(2)S. The mechanisms used by these three proteins for their function, including ligand control, remain unknown. The crystal structure of oxygen-bound HbII(Lp) shows a dimeric oxyHbII(Lp) where oxygen is tightly anchored to the heme through hydrogen bonds with Tyr(30)(B10) and Gln(65)(E7). The heme group is buried farther within HbII(Lp) than in HbI(Lp). The proximal His(97)(F8) is hydrogen bonded to a water molecule, which interacts electrostatically with a propionate group, resulting in a Fe-His vibration at 211 cm(-1). The combined effects of the HbII(Lp) small heme pocket, the hydrogen bonding network, the His(97) trans-effect, and the orientation of the oxygen molecule confer stability to the oxy-HbII(Lp) complex. Oxidation of HbI(Lp) Phe(B10) --> Tyr and HbII(Lp) only occurs when the pH is decreased from pH 7.5 to 5.0. Structural and resonance Raman spectroscopy studies suggest that HbII(Lp) oxygen binding and transport to the host bacteria may be regulated by the dynamic displacements of the Gln(65)(E7) and Tyr(30)(B10) pair toward the heme to protect it from changes in the heme oxidation state from Fe(II) to Fe(III).     

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.     

The truncated oxygen-avid hemoglobin from Bacillus subtilis: X-ray structure and ligand binding properties

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 A resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand-inclusive hydrogen bond network" described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 A).