Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.     

H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin

The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN⁻ have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. Ligand binding in heme-containing proteins is determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. Since both the RR Fe–OH⁻ and Fe–CN⁻ frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH⁻ ligand, CN⁻ binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

The quantum mechanically mixed-spin state in a non-symbiotic plant hemoglobin: the effect of distal mutation on AHb1 from Arabidopsis thaliana

Non-symbiotic hemoglobins are hexacoordinated heme proteins found in all plants. To gain insight into the importance of the heme hexacoordination and the coordinated distal histidine in general for the possible physiological functions of these proteins, the distal His(E7) of Arabidopsis thaliana hemoglobin (AHb1) was substituted by a leucine residue. The heme properties of the wild-type and mutant proteins have been characterized by electronic absorption, resonance Raman and electron paramagnetic resonance spectroscopic studies at room and low temperatures. Significant differences between the wild-type and mutant proteins have been detected. The most striking is the formation of an uncommon quantum mechanically mixed-spin heme species in the mutant. This is the first observation of such a spin state in a plant hemoglobin. The proportion of this species, which at room temperature coexists with a minor pentacoordinated high-spin form, increases markedly at low temperature.     

Purification and spectroscopic characterization of Ctb, a group III truncated hemoglobin implicated in oxygen metabolism in the food-borne pathogen Campylobacter jejuni

Campylobacter jejuni is a food-borne bacterial pathogen that possesses two distinct hemoglobins, encoded by the ctb and cgb genes. The former codes for a truncated hemoglobin (Ctb) in group III, an assemblage of uncharacterized globins in diverse clinically and technologically significant bacteria. Here, we show that Ctb purifies as a monomeric, predominantly oxygenated species. Optical spectra of ferric, ferrous, O(2)- and CO-bound forms resemble those of other hemoglobins. However, resonance Raman analysis shows Ctb to have an atypical nu(Fe)(-)(CO) stretching mode at 514 cm(-)(1), compared to those of the other truncated hemoglobins that have been characterized so far. This implies unique roles in ligand stabilization for TyrB10, HisE7, and TrpG8, residues highly conserved within group III truncated hemoglobins. Because C. jejuni is a microaerophile, and a ctb mutant exhibits O(2)-dependent growth defects, one of the hypothesized roles of Ctb is in the detoxification, sequestration, or transfer of O(2). The midpoint potential (E(h)) of Ctb was found to be -33 mV, but no evidence was obtained in vitro to support the hypothesis that Ctb is reducible by NADH or NADPH. This truncated hemoglobin may function in the facilitation of O(2) transfer to one of the terminal oxidases of C. jejuni or, instead, facilitate O(2) transfer to Cgb for NO detoxification.     

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.     

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.     

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-.     

Characterization of nonsymbiotic tomato hemoglobin

The nonsymbiotic tomato hemoglobin SOLly GLB1 (Solanum lycopersicon) is shown to form a homodimer of approximately 36 kDa with a high affinity for oxygen. Furthermore, our combined ultraviolet/visible, resonance Raman, and continuous wave electron paramagnetic resonance (EPR) measurements reveal that a mixture of penta- and hexacoordination of the heme iron is found in the deoxy ferrous form, whereas the ferric form shows predominantly a bis-histidine ligation (F8His-Fe(2+/3+)-E7His). This differs from the known forms of vertebrate hemoglobins and myoglobins. We have successfully applied our recently designed pulsed-EPR strategy to study the low-spin ferric form of tomato hemoglobin. These experiments reveal that, in ferric SOLly GLB1, one of the histidine planes is rotated 20 degrees (+/-10 degrees ) away from a N(heme)-Fe-N(heme) axis. Additionally, the observed g-values indicate a quasicoplanarity of the histidine ligands. From the HYSCORE (hyperfine sublevel correlation) measurements, the hyperfine and nuclear quadrupole couplings of the heme and histidine nitrogens are identified and compared with known EPR/ENDOR data of vertebrate Hbs and cytochromes. Finally, the ligand binding kinetics, which also indicate that the ferrous tomato Hb is only partially hexacoordinated, will be discussed in relation with the heme-pocket structure. The similarities and differences with other known nonsymbiotic plant hemoglobins will be highlighted.     

Two distinct heme distal site states define Cerebratulus lacteus mini-hemoglobin oxygen affinity

The nerve tissue hemoglobin of Cerebratulus lacteus (CerHb) is the smallest naturally occurring known hemoglobin. Stabilization of the diatomic bound species (e.g., O(2)) is achieved through a network of hydrogen bonds based on three key residues TyrB10, GlnE7, and ThrE11. The first two residues are typically associated in hemoglobins with enhanced O(2) affinity, related to hydrogen bond stabilization of the heme-bound O(2) resulting in a decrease of the ligand dissociation rates. In contrast to the above observations, the affinity of CerHb for O(2) is only moderate, and the rate of O(2) dissociation is unexpectedly high. To gain insight on the diverse molecular mechanisms controlling ligand affinities, we have analyzed w.t. CerHb and its ThrE11-->Val mutant by means of joint molecular dynamics and quantum mechanics simulation techniques, complementing recent site-directed mutagenesis experiments. Our results suggest that the observed O(2) dissociation rates can only be explained through a dynamic equilibrium between high and low affinity states of the w.t. CerHb heme distal site.     

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. (1)H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00ppm, GlnE7N(ε1)H/N(ε2)H at 10.66ppm/-3.27ppm, and PheE11 C(δ)H at 11.75ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen bonding network loop between the 6-propionate, the heme ligand and nearby amino acids, tailoring in this way the electron density in the heme-ligand moiety.     

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme

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

Tyrosine B10 and heme-ligand interactions of Lucina pectinata hemoglobin II: control of heme reactivity

The distal pocket of hemoglobin II (HbII) from Lucina pectinata is characterized by the presence of a GlnE7 and a TyrB10. To elucidate the functional properties of HbII, biophysical studies were conducted on HbII and a HbI PheB10Tyr site-directed mutant. The pH titration data at neutral conditions showed visible bands at 486, 541, 577 and 605 nm for both proteins. This suggests the possible existence of a conformational equilibrium between an open and closed configuration due to the interactions of the TyrB10, ligand, and heme iron. The kinetic behavior for the reaction of both ferric proteins with H2O2 indicates that the rate for the formation of the ferryl intermediates species varies with pH, suggesting that the reaction is strongly dependent on the conformational states. At basic pH values, the barrier for the reaction increases as the tyrosine adopts a closed conformation and the ferric hydroxyl replaces the met-aquo species. The existence of these conformers is further supported by resonance Raman (RR) data, which indicate that in a neutral environment, the ferric HbII species is present as a possible mixture of coordination and spin states, with values at 1558 and 1580 cm(-1) for the nu2 marker, and 1479, 1492, and 1503 cm(-1) for the nu3 mode. Moreover, the presence of the A3 and A(o) conformers at 1924 and 1964 cm(-1) in the HbII-CO infrared spectra confirms the existence of an open and closed conformation due to the orientation of the TyrB10 with respect to the heme active center.     

The amino acid sequence of the alpha chain of HB 2 completes the primary structure of the hemoglobins of the Antarctic fish Notothenia coriiceps neglecta

1. The blood of Notothenia coriiceps neglecta (a cold-adapted notothenioid fish, widely distributed in Antarctic waters, and characterized by a relatively low content of erythrocytes and hemoglobin), contains two hemoglobin components, Hb 1 and Hb 2; the amino acid sequences of the beta chain of Hb 1 and Hb 2 are identical. 2. The amino acid sequence of the alpha chain of Hb 2 has been established, thus completing the elucidation of the primary structure of the two hemoglobins.     

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.     

Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.     

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3₁₀-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3₁₀-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.     

Cyanide binding and heme cavity conformational transitions in Drosophila melanogaster hexacoordinate hemoglobin

The reason for the presence of hemoglobin-like molecules in insects, such as Drosophila melanogaster, that live in fully aerobic environments has yet to be determined. Heme endogenous hexacoordination (where HisE7 and HisF8 axial ligands to the heme Fe atom are both provided by the protein) is a recently discovered mechanism proposed to modulate O(2) affinity in hemoglobins from different species. Previous results have shown that D. melanogaster hemoglobin 1 (product of the glob1 gene) displays heme endogenous hexacoordination in both the ferrous and ferric states. Here we present kinetic data characterizing the exogenous cyanide ligand binding process, and the three-dimensional structure (at 1.4 A resolution) of the ensuing cyano-met D. melanogaster hemoglobin. Comparison with the crystal structure of the endogenously hexacoordinated D. melanogaster hemoglobin shows that the transition to the cyano-met form is supported by conformational readjustment in the CD-D-E region of the protein, which removes HisE7 from the heme. The structural and functional features of D. melanogaster hemoglobin are examined in light of previous results achieved for human and mouse neuroglobins and for human cytoglobin, which display heme endogenous hexacoordination. The study shows that, despite the rather constant value for cyanide association rate constants for the ferric hemoproteins, different distal site conformational readjustments and/or heme sliding mechanisms are displayed by the known hexacoordinate hemoglobins as a result of exogenous ligand binding.     

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.     

NMR studies of the heme pocket conformations of monomeric hemoglobins from Glycera dibranchiata. Implications for ligand binding

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.     

Ligand orientation of human neuroglobin obtained from solution NMR and molecular dynamics simulation as compared with X-ray crystallography

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in many biological processes such as enzymatic reaction, signal transduction and the mitochondria function. Different from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imidazole of proximal His⁹⁶(F8) and distal His⁶⁴(E7) directly bound to the metal ion. In the present work, solution ¹H NMR spectroscopy was employed to investigate the electronic structure of heme center of wild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocket were assigned. Two heme orientations resulting from a 180° rotation about the α-γ-meso axis with a population ratio about 2:1 were observed. Then the ¹H NMR chemical shifts of the ferriheme methyl groups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle φ is consistent with that from the molecular dynamics simulation but not with those from the crystal data. Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobin reflects the changeability of heme environment for the Ngb family.