Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom

Caenorhabditis elegans globin GLB-26 (expressed from gene T22C1.2) has been studied in comparison with human neuroglobin (Ngb) and cytoglobin (Cygb) for its electron transfer properties. GLB-26 exhibits no reversible binding for O(2) and a relatively low CO affinity compared to myoglobin-like globins. These differences arise from its mechanism of gaseous ligand binding since the heme iron of GLB-26 is strongly hexacoordinated in the absence of external ligands; the replacement of this internal ligand, probably the E7 distal histidine, is required before binding of CO or O(2) as for Ngb and Cygb. Interestingly the ferrous bis-histidyl GLB-26 and Ngb, another strongly hexacoordinated globin, can transfer an electron to cytochrome c (Cyt-c) at a high bimolecular rate, comparable to those of inter-protein electron transfer in mitochondria. In addition, GLB-26 displays an unexpectedly rapid oxidation of the ferrous His-Fe-His complex without O(2) actually binding to the iron atom, since the heme is oxidized by O(2) faster than the time for distal histidine dissociation. These efficient mechanisms for electron transfer could indicate a family of hexacoordinated globin which are functionally different from that of pentacoordinated globins.     

Structural basis of human cytoglobin for ligand binding

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.     

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.     

Hyperthermal stability of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb), recent additions to the globin family, display a hexa-coordinated (bis-histidyl) heme in the absence of external ligands. Although these proteins have the classical globin fold they reveal a very high thermal stability with a melting temperature (Tm) of 100 degrees C for Ngb and 95 degrees C for Cygb. Moreover, flash photolysis experiments at high temperatures reveal that Ngb remains functional at 90 degrees C. Human Ngb may have a disulfide bond in the CD loop region; reduction of the disulfide bond increases the affinity of the iron atom for the distal (E7) histidine, and leads to a 3 degrees C increase in the T(m) for ferrous Ngb. A similar Tm is found for a mutant of human Ngb without cysteines. Apparently, the disulfide bond is not involved directly in protein stability, but may influence the stability indirectly because it modifies the affinity of the distal histidine. Mutation of the distal histidine leads to lower thermal stability, similar to that for other globins. Only globins with a high affinity of the distal histidine show the very high thermal stability, indicating that stable hexa-coordination is necessary for the enhanced thermal stability; the CD loop which contains the cysteines appears as a critical region in the neuroglobin thermal stability, because it may influence the affinity of the distal histidine.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Heme-hemopexin: a 'chronosteric' heme-protein

Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed beta-propeller domains, resembling two thick disks that lock together at a 90 degrees angle. The heme is bound between the two beta-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, (*)NO, and cyanide by detaching His213; however, O(2) induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates (*)NO/O(2) and (*)NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a 'chronosteric' heme-protein.     

Cytoglobin is a stress-responsive hemoprotein expressed in the developing and adult brain.

Cytoglobin (Cygb) is a novel tissue hemoprotein relatively similar to myoglobin (Mb). Because Cygb shares several structural features with Mb, we hypothesized that Cygb functions in the modulation of oxygen and nitric oxide metabolism or in scavenging free radicals within a cell. In the present study we examined the spatial and temporal expression pattern of Cygb during murine embryogenesis. Using in situ hybridization, RT-PCR, and Northern blot analyses, limited Cygb expression was observed during embryogenesis compared with Mb expression. Cygb expression was primarily restricted to the central nervous system and neural crest derivatives during the latter stages of development. In the adult mouse, Cygb is expressed in distinct regions of the brain as compared with neuroglobin (Ngb), another globin protein, and these regions are responsive to oxidative stress (i.e., hippocampus, thalamus, and hypothalamus). In contrast to Ngb, Cygb expression in the brain is induced in response to chronic hypoxia (10% oxygen). These results support the hypothesis that Cygb is an oxygen-responsive tissue hemoglobin expressed in distinct regions of thenormoxic and hypoxic brain and may play a key role in the response of the brain to ahypoxic insult.     

Do neuroglobin and myoglobin protect Toxoplasma gondii from nitrosative stress?

Toxoplasma gondii is a Apicomplexa obligate intracellular protozoan parasite that infects up to a third of the world's population. In most humans infected with T. gondii, the disease toxoplasmosis is asymptomatic. However, T. gondii causes blindness, severe neurological disorders, hepatitis, and pneumonia in immunocompromised patients, and severe damage to the fetus. Here, we postulate that the colonization of the retina, heart, and skeletal muscle by T. gondii may reflect the role of neuroglobin (Ngb) and myoglobin (Mb) to protect the parasite from the toxoplasmacidal effects of nitric oxide (NO). This is based on the knowledge that Ngb and Mb catalyzes NO oxidation yielding the harmless nitrate. The postulated protective role of Ngb and Mb on the viability of T. gondii is reminiscent of that postulated for hemoglobin (Hb) and Mb in protecting intraerythrocytic Plasmodia and T. cruzi in cardiomyocytes, respectively, from the parasiticidal effect of NO. Therefore, undesirable pathogen protection by pseudo-enzymatic NO scavenging may represent a new unexpected function of members of the Hb superfamily.     

Neuroglobin and cytoglobin: genes, proteins and evolution

Hemoglobin and myoglobin are oxygen transport and storage proteins of most vertebrates. Neuroglobin (Ngb) and cytoglobin (Cygb)--two recent additions to the vertebrate globin superfamily--have still disputed functions. Combining the data from all available resources, we investigate the evolution of these novel globins. Both Ngb and Cygb show little sequence variation in vertebrate evolution, suggesting conserved structures and functions, and an important role in the animal's metabolism. Exon-intron patterns remained unchanged in Ngb and Cygb, with the exception of the addition of a 3' exon to Cygb early in mammalian evolution. In phylogenetic analyses, Ngb forms a common branch with globin X, another recently identified globin with undefined function in lower vertebrates, and with some invertebrate nerve globins. This shows an early divergence of this branch in animal evolution. Cygb is related to myoglobin, and associated with an eye-specific globin from birds. The pattern of globin evolution shows that proteins with clear respiratory roles evolved independently from intracellular globins with uncertain functions. This result suggests either multiple independent functional changes or a yet undefined respiratory role of tissue globins like Ngb and Cygb.     

Modulation of oxygen binding to insect hemoglobins: the structure of hemoglobin from the botfly Gasterophilus intestinalis

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 A crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O(2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).     

Structure-function relationships in the growing hexa-coordinate hemoglobin sub-family

Hemoglobin and related heme proteins, generally referred to as 'globins', reversibly bind gaseous diatomic ligands (O2, NO, and CO) to a penta-coordinate heme iron atom, the ligand filling the sixth coordination site. Over the last decade, several new globins have been reported to display a functionally-relevant hexa-coordinate heme iron atom, whose sixth coordination site is taken by an endogenous protein ligand. The reversible intramolecular hexa- to penta-coordination process at the heme-Fe atom modulates exogenous ligand binding properties of hexa-coordinate globins. Here, we review current knowledge on hexa-coordinate globins in terms of their structural and functional properties.     

Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

Module M1 of zebrafish neuroglobin acts as a structural and functional protein building block for a cell-membrane-penetrating activity

Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins.     

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the “hemoglobin (Hb) superfamily.” The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His¹¹⁷ and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His¹¹⁷ is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.     

Characterization of the function of cytoglobin as an oxygen-dependent regulator of nitric oxide concentration

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an O(2)-dependent manner. This regulates NO levels in the vascular wall; however, the underlying molecular basis of this O(2)-dependent NO consumption remains unclear. While cytoglobin (Cygb) was discovered a decade ago, its physiological function remains uncertain. Cygb is expressed in the vascular wall and can consume NO in an O(2)-dependent manner. Therefore, we characterize the process of the O(2)-dependent consumption of NO by Cygb in the presence of the cellular reductants and reducing systems ascorbate (Asc) and cytochrome P(450) reductase (CPR), measure rate constants of Cygb reduction by Asc and CPR, and propose a reaction mechanism and derive a related kinetic model for this O(2)-dependent NO consumption involving Cygb(Fe(3+)) as the main intermediate reduced back to ferrous Cygb by cellular reductants. This kinetic model expresses the relationship between the rate of O(2)-dependent consumption of NO by Cygb and rate constants of the molecular reactions involved. The predicted rate of O(2)-dependent consumption of NO by Cygb is consistent with experimental results supporting the validity of the kinetic model. Simulations based on this kinetic model suggest that the high efficiency of Cygb in regulating the NO consumption rate is due to the rapid reduction of Cygb by cellular reductants, which greatly increases the rate of consumption of NO at higher O(2) concentrations, and binding of NO to Cygb, which reduces the rate of consumption of NO at lower O(2) concentrations. Thus, the coexistence of Cygb with efficient reductants in tissues allows Cygb to function as an O(2)-dependent regulator of NO decay.     

Molecular dynamics simulation of carboxy and deoxy human cytoglobin in solution

Cytoglobin (Cgb), the fourth member of the vertebrate heme globin family, is widely expressed in mammalian tissues, and reversibly binds to CO, O₂ and other small ligands. The diverse functions of Cgb may include ligand transport, redox reactions and enzymatic catalysis. Recent studies indicate that Cgb is a potential gene medicine for fibrosis and cancer therapy. In the present work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamic characteristics in carboxy and deoxy human Cgb. The simulation results showed that the loop regions and internal cavities were significantly affected through the binding of an exogenous ligand. The AB, GH and EF loops were found to undergo significant rearrangement and this led to distinct cavity adjustments in Xe2, Xe4 and the distal pocket. In addition, solvent accessibility and torsion angle analyses revealed an interactive distal network comprised of His⁸¹(E7), Leu⁴⁶(B10) and Arg⁸⁴(E10). The MD study of carboxy and deoxy human Cgb revealed that CO-ligated Cgb modulates the protein conformation primarily by loop and cavity rearrangements rather than the heme sliding mechanism found in neuroglobin (Ngb). The significant differences between Cgb and Ngb in the loop and cavity properties are presumably linked to their various biological functions.     

Searching for neuroglobin's role in the brain

Neuroglobin is a small globin that plays an important role in the protection of brain neurons from ischemic and hypoxic injuries. The molecular mechanisms by which Ngb performs its physiological function are still under debate. Suggestions include oxygen storage and delivery, scavenging of NO and/or reactive oxygen species, oxygen sensing and signal transduction. In recent years, the molecular structures of Ngb with carbon monoxide bound to the heme iron and without an exogenous ligand have been solved, and interesting structural changes have been noticed upon ligand binding. Moreover, equilibrium and kinetic properties of the reactions with ligands have been examined in great detail. Here we summarize the molecular properties of Ngb and discuss them in relation to the potential physiological functions.     

Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases

Microbial flavohemoglobins (flavoHbs) and hemoglobins (Hbs) show large *NO dioxygenation rate constants ranging from 745 to 2900 microM(-1) s(-1) suggesting a primal *NO dioxygenase (NOD) (EC 1.14.12.17) function for the ancient Hb superfamily. Indeed, modern O2-transporting and storing mammalian red blood cell Hb and related muscle myoglobin (Mb) show vestigial *NO dioxygenation activity with rate constants of 34-89 microM(-1) s(-1). In support of a NOD function, microbial flavoHbs and Hbs catalyze O2-dependent cellular *NO metabolism, protect cells from *NO poisoning, and are induced by *NO exposures. Red blood cell Hb, myocyte Mb, and flavoHb-like activities metabolize *NO in the vascular lumen, muscle, and other mammalian cells, respectively, decreasing *NO signalling and toxicity. HbFe(III)-OO*, HbFe(III)-OONO and protein-caged [HbFe(III)-O**NO2] are proposed intermediates in a reaction mechanism that combines both O-atoms of O2 with *NO to form nitrate and HbFe(III). A conserved Hb heme pocket structure facilitates the dioxygenation reaction and efficient turnover is achieved through the univalent reduction of HbFe(III) by associated reductases. High affinity flavoHb and Hb heme ligands, and other inhibitors, may find application as antibiotics and antitumor agents that enhance the toxicity of immune cell-derived *NO or as vasorelaxants that increase *NO signalling.