The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein

The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein.     

Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.     

14-3-3 binding and phosphorylation of neuroglobin during hypoxia modulate six-to-five heme pocket coordination and rate of nitrite reduction to nitric oxide

Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1-5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.     

Neuroglobin Promotes Neurite Outgrowth via Differential Binding to PTEN and Akt

Neuroglobin, the third mammalian globin with a hexa-coordinated heme, exists predominantly in neurons of the brain. Neuroglobin plays an important role in neuronal death upon ischemia and oxidative stress. The physiological function of neuroglobin remains unclear. Here, we report a novel function of neuroglobin in neurite development. Knocking-down neuroglobin exhibited a prominent neurite-deficient phenotype in mouse neuroblastoma N2a cells. Silencing neuroglobin prevented neurite outgrowth, while ectopic expression of neuroglobin but not homologous cytoglobin promoted neurite outgrowth of N2a cells upon serum withdrawal. In primary cultured rat cerebral cortical neurons, neuroglobin was upregulated and preferentially distributed in neurites during neuronal development. Overexpression of neuroglobin but not cytoglobin in cultured cortical neurons promoted axonal outgrowth, while knocking-down of neuroglobin retarded axonal outgrowth. Neuroglobin overexpression suppressed phosphatase and tensin homolog (PTEN) but increased Akt phosphorylation during neurite induction. Bimolecular fluorescence complementation and glutathione S-transferase pull-down assays revealed that neuroglobin and various mutants (E53Q, E118Q, K119N, H64A, H64L, and Y44D) bound with Akt and PTEN differentially. Neuroglobin E53Q showed a prominent reduced PTEN binding but increased Akt binding, resulting in decreased p-PTEN, increased p-Akt, and increased neurite length. Taken together, we demonstrate a critical role of neuroglobin in neuritogenesis or development via interacting with PTEN and Akt differentially to activate phosphatidylinositol 3-kinase/Akt pathway, providing potential therapeutic applications of neuroglobin for axonopathy in neurological diseases.     

Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O(2) supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a hexacoordinated heme. O(2) and CO bind to the heme iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold adapted to host the reversible bis-histidyl heme complex and an elongated protein matrix cavity, held to facilitate O(2) diffusion to the heme. The neuroglobin structure suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.     

Molecular dynamics simulation of a carboxy murine neuroglobin mutated on the proximal side: heme displacement and concomitant rearrangement in loop regions

Neuroglobin, a member of vertebrate globin family, is distributed primarily in the brain and retina. Considerable evidence has accumulated regarding its unique ligand-binding properties, neural-specific distribution, distinct expression regulation, and possible roles in processes such as neuron protection and enzymatic metabolism. Structurally, neuroglobin enjoys unique features, such as bis-histidyl coordination to heme iron in the absence of exogenous ligand, heme orientational heterogeneity, and a heme sliding mechanism accompanying ligand binding. In the present work, molecular dynamics (MD) simulations were employed to reveal functional and structural information in three carboxyl murine neuroglobin mutants with single point mutations F106Y, F106L and F106I, respectively. The MD simulation indicates a remarkable proximal effect on detectable displacement of heme and a larger tunnel in the protein matrix. In addition, the mutation at F106 confers on the CD region a very sensitive mobility in all three model structures. The dynamic features of neuroglobin demonstrate rearrangement of the inner space and highly active loop regions in solution. These imply that the conserved residue at the G5 site plays a key role in the physiological function of this unusual protein.     

Allosteric regulation and temperature dependence of oxygen binding in human neuroglobin and cytoglobin. Molecular mechanisms and physiological significance

Two new globin proteins have recently been discovered in vertebrates, neuroglobin in neurons and cytoglobin in all tissues, both showing heme hexacoordination by the distal His(E7) in the absence of gaseous ligands. In analogy to hemoglobin and myoglobin, neuroglobin and cytoglobin are supposedly involved in O2 storage and delivery, although their physiological role remains to be solved. Here we report O2 equilibria of recombinant human neuroglobin (NGB) and cytoglobin (CYGB) measured under close to physiological conditions and at varying temperature and pH ranges. NGB shows both alkaline and acid Bohr effects (pH-dependent O2 affinity) and temperature-dependent enthalpy of oxygenation. O2 and CO binding equilibrium studies on neuroglobin mutants strongly suggest that the bound O2 is stabilized by interactions with His(E7) and that this residue functions as a major Bohr group in the presence of Lys(E10). As shown by the titration of free thiols with 4,4'-dithiodipyridine and by mass spectrometry, this mechanism of modulating O2 affinity is independent of formation of an internal disulfide bond under the experimental conditions used, which stabilize thiols in the reduced form. In CYGB, O2 binding is cooperative, consistent with its proposed dimeric structure. Similar to myoglobin but in contrast to NGB, O2 binding to CYGB is pH-independent and exothermic throughout the temperature range investigated. Our data support the hypothesis that CYGB may be involved in O2-requiring metabolic processes. In contrast, the lower O2 affinity in NGB does not appear compatible with a physiological role involving mitochondrial O2 supply at the low O2 tensions found within neurons.     

A role for human neuroglobin in apoptosis

Over the past decade, following the discovery of the human heme protein neuroglobin, many studies have searched for evidence for this protein's mechanism of action. Much data has accrued showing that high levels of neuroglobin will protect cells from apoptotic cell death, following a wide range of challenges. Various explanations of its actions, based on measured reactivity with oxygen, nitric oxide, or free radicals, have been proposed, but none have, as yet, been substantiated in vivo. Following preliminary experiments, it was previously hypothesised that "the central role of neuroglobin in highly metabolically active cells and retinal and brain neurons is to reset the trigger level of mitochondrial cytochrome c release necessary to commit the cells to apoptosis" (I.U.M.B.M. Life (2008) 60, 398). In this article, we review the evidence, which has accumulated to support this hypothesised mechanism of action of neuroglobin and integrate this data, with other reported intracellular functions of neuroglobin, to suggest a plausible central role for neuroglobin in the control of apoptosis.     

The binding of cytochrome c to neuroglobin: a docking and surface plasmon resonance study

It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45muM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9kcalmol(-1)) and a moderately large, favourable entropy change (14.8calmol(-1)deg(-1)). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.     

Human neuroglobin functions as a redox-regulated nitrite reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ～2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.     

Reactions of peroxynitrite with globin proteins and their possible physiological role

It is now widely accepted that, besides their well-established function in O(2) transport, hemoglobin and myoglobin also undergo several redox reactions aimed to scavenge toxic free radicals and reactive oxygen and nitrogen species. At least some of these reactions are believed to play an important physiological role in the defense against oxidative stress. This aspect is exemplified by the recently discovered neuroglobin, a globin expressed in the brain. Rather than being considerably involved in reversible O(2) binding, neuroglobin is likely to undergo redox reactions to protect neurons against oxidative and potentially pathogenic pathways, as those operating after episodes of tissue hypoxia or ischemia. A major part of the cellular damage occurring under such conditions has been ascribed to formation of peroxynitrite, that originates from the reaction between two biologically important free radicals, nitric oxide (NO ) and superoxide. Here we review the current knowledge of the reactions of different forms of hemoglobin, myoglobin, and neuroglobin with peroxynitrite and discuss their physiological role on the basis of measured rate constants and on the probability of occurrence of these reactions in vivo.     

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.     

Ligand binding, reactivity and biological activity of a distal pocket mutant of neuroglobin

We have generated the Lys67Glu mutant form of neuroglobin. Experimental spectral studies are consistent with a six coordinate heme in which the distal histidine bond is stretched compared to the wild type protein. Carbon monoxide binding to the ferrous form of the mutant follows a hyperbolic concentration dependence limiting at the histidine dissociation rate of 0.7 s(-1). Further analysis indicates a significantly lowered histidine binding constant. Oxygen binding kinetic studies confirm the higher heme ligand dissociation level and indicate a p50 value for oxygen binding<1 mmHg. The ferrous form of the protein yields an oxygenated intermediate on reaction with oxygen. The rate of oxidation, by oxygen, follows a complex concentration dependence, consistent with the presence of two distinct oxidation mechanisms. A quantitative model for the two oxidation processes has been developed, which is consistent with a lowered distal histidine binding constant in the mutant form of the protein. These data suggest that the protein structure surrounding the heme site in neuroglobin limits access to external ligands and provides an energy barrier to the structural changes following ligand binding in this protein. However, the mutation does not appear to affect reactivity with cytochrome c and the anti-apoptotic activity of the mutant in human cells of neuronal origin is increased as compared to the wild type protein.     

Functional properties of neuroglobin and cytoglobin. Insights into the ancestral physiological roles of globins

Neuroglobin and cytoglobin are two recently discovered vertebrate globins, which are expressed at low levels in neuronal tissues and in all tissues investigated so far, respectively. Based on their amino acid sequences, these globins appear to be phylogenetically ancient and to have mutated less during evolution in comparison to the other vertebrate globins, myoglobin and hemoglobin. As with some plant and bacterial globins, neuroglobin and cytoglobin hemes are hexacoordinate in the absence of external ligands, in that the heme iron atom coordinates both a proximal and a distal His residue. While the physiological role of hexacoordinate globins is still largely unclear, neuroglobin appears to participate in the cellular defence against hypoxia. We present the current knowledge on the functional properties of neuroglobin and cytoglobin, and describe a mathematical model to evaluate the role of mammalian retinal neuroglobin in supplying O2 supply to the mitochondria. As shown, the model argues against a significant such role for neuroglobin, that more likely plays a role to scavenge reactive oxygen and nitrogen species that are generated following brain hypoxia. The O2 binding properties of cytoglobin, which is upregulated upon hypoxia, are consistent with a role for this protein in O2-requiring reactions, such as those catalysed by hydroxylases.     

Nitrite Reductase Activity of Nonsymbiotic Hemoglobins from Arabidopsis thaliana

Plant nonsymbiotic hemoglobins possess hexacoordinate heme geometry similar to that of the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2 (AHb1 and AHb2, respectively) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for reduction of nitrite to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M(-1) s(-1), respectively, at pH 7.4 and 25 °C. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. The release of free NO gas during the reaction under anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin, and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in species submerged in water, nonsymbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction.     

The endogenous production of hydrogen sulphide in intrauterine tissues

Background  

Hydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro.     

Neuroglobin,an oxygen-binding protein in the mammalian nervous system (localization and putative functions)

The review summarizes current data on neuroglobin, the heme-containing protein discovered in mammalian nerve cells in 2000. It presents general characteristics of neuroglobin as well as data on its evolutionary changes and expression across different taxa. Neuroglobin distribution in specific brain structures and outside the brain is described. The issue of the occurrence of neuroglobin not only in neurons but also in astroglial cells is discussed. Subcellular localization of neuroglobin is characterized with a special focus on its detection in the nucleus of nerve cells, suggesting its involvement in nuclear functions. Current ideas on the probable functional significance of neuroglobin are reported. Neuroglobin is presumed to be involved in metabolism of reactive nitrogen and oxygen species as well as in intracellular signaling pathways. Besides, neuroglobin has neuroprotective and antiapoptotic functions. Since its expression changes during ontogenesis, its neuroprotective role in ageing is specifically highlighted. Changes in expression and localization of neuroglobin are suggested to influence the adaptive potential of an organism.     

Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family

Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O(2) supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe(2+)) form, indicating a His-Fe(2+)-His binding scheme. O(2) or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe(3+)) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k(on)) and a slow dissociation rate (k(off)) for both O(2) and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O(2) and CO binding is suggested to be slow in vivo. Because of this competition, the observed O(2) affinity of recombinant human neuroglobin is average (1 torr at 37 degrees C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 degrees C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E'(o) = -129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.     

Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

In the past few years, overwhelming evidence has accrued that a high level of expression of the protein neuroglobin protects neurons in vitro, in animal models, and in humans, against cell death associated with hypoxic and amyloid insult. However, until now, the exact mechanism of neuroglobin’s protective action has not been determined. Using cell biology and biochemical approaches we demonstrate that neuroglobin inhibits the intrinsic pathway of apoptosis in vitro and intervenes in activation of pro-caspase 9 by interaction with cytochrome c. Using systems level information of the apoptotic signalling reactions we have developed a quantitative model of neuroglobin inhibition of apoptosis, which simulates neuroglobin blocking of apoptosome formation at a single cell level. Furthermore, this model allows us to explore the effect of neuroglobin in conditions not easily accessible to experimental study. We found that the protection of neurons by neuroglobin is very concentration sensitive. The impact of neuroglobin may arise from both its binding to cytochrome c and its subsequent redox reaction, although the binding alone is sufficient to block pro-caspase 9 activation. These data provides an explanation the action of neuroglobin in the protection of nerve cells from unwanted apoptosis.