GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

Structural and Functional Characterization of the Globin-Coupled Sensors of Azotobacter vinelandii and Bordetella pertussis.

Aims: Structural and functional characterization of the globin-coupled sensors (GCSs) from Azotobacter vinelandii (AvGReg) and Bordetella pertussis (BpeGReg). Results: Ultraviolet/visible and resonance Raman spectroscopies confirm the presence in AvGReg and BpeGReg of a globin domain capable of reversible gaseous ligand binding. In AvGReg, an influence of the transmitter domain on the heme proximal region of the globin domain can be seen, and k'CO is higher than for other GCSs. The O2 binding kinetics suggests the presence of an open and a closed conformation. As for BpeGReg, the fully oxygenated AvGReg show a very high diguanylate cyclase activity. The carbon monoxide rebinding to BpeGReg indicates that intra- and intermolecular interactions influence the ligand binding. The globin domains of both proteins (AvGReg globin domain and BpeGRegGb with cysteines (Cys16, 45, 114, 154) mutated to serines [BpeGReg-Gb*]) share the same GCS fold, a similar proximal but a different distal side structure. They homodimerize through a G-H helical bundle as in other GCSs. However, BpeGReg-Gb* shows also a second dimerization mode. Innovation: This article extends our knowledge on the GCS proteins and contributes to a better understanding of the GCSs role in the formation of bacterial biofilms. Conclusions:AvGReg and BpeGReg conform to the GCS family, share a similar overall structure, but they have different properties in terms of the ligand binding. In particular, AvGReg shows an open and a closed conformation that in the latter form will very tightly bind oxygen. BpeGReg has only one closed conformation. In both proteins, it is the fully oxygenated GCS form that catalyzes the production of the second messenger.

Loss of Neuroglobin Expression Alters Cdkn1a/Cdk6-Expression Resulting in Increased Proliferation of Neural Stem Cells.

In the quest to unravel its functional significance, neuroglobin (Ngb), a brain-specific neuroprotective protein, has recently been proposed as an actor in neurodevelopment. As neural stem cells (NSCs) are fundamental during brain development, the present study aimed at investigating the role of Ngb in the growth and proliferation of NSCs by comparing an Ngb-floxed (Ngbfl-)NSC line, equivalent to the wild-type cellular situation, with an in-house created Ngb knockout (NgbKO-)NSC line. NgbKO-NSCs were characterized by an increased growth and proliferation capacity in vitro, supported by RNA sequencing and western blot results reporting the downregulation of Cdkn1a and the upregulation of Cdk6, both enhancing the cell cycle. Based on additional gene ontology enrichment and pathway analyses, we hypothesize that the loss of Ngb affects multiple cellular signaling pathways with the most important being the Akt-Tp53 axis.

Is the heme pocket region modulated by disulfide-bridge formation in fish and amphibian neuroglobins as in humans?

Neuroglobin, a globin characterized by a bis-histidine ligation of the heme iron, has been identified in mammalian and non-mammalian vertebrates, including fish, amphibians and reptiles. In human neuroglobin, the presence of an internal disulfide bond in the CD loop (CD7-D5) is found to modulate the ligand binding through a change in the heme pocket structure. Although the neuroglobin sequences mostly display conserved Cys at positions CD7, D5 and G18/19, a number of exceptions are known. In this study, neuroglobins from amphibian (Xenopus tropicalis) and fish (Chaenocephalus aceratus, Dissostichus mawsoni and Danio rerio) are investigated using electron paramagnetic resonance and optical absorption spectroscopy. All these neuroglobins differ from human neuroglobin in their Cys-positions. It is demonstrated that if disulfide bonds are formed in fish and amphibian neuroglobins, the reduction of these bonds does not result in alteration of the heme pocket in these globins. Furthermore, it is shown that mutagenesis of the Cys residues of X. tropicalis neuroglobin influences the protein structure. The amphibian neuroglobin is also found to be more resistant to H2O2-induced denaturation than the other neuroglobins under study, although all show an overall large stability in high concentrations of this oxidant. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

An N-myristoylated globin with a redox-sensing function that regulates the defecation cycle in Caenorhabditis elegans.

Globins occur in all kingdoms of life where they fulfill a wide variety of functions. In the past they used to be primarily characterized as oxygen transport/storage proteins, but since the discovery of new members of the globin family like neuroglobin and cytoglobin, more diverse and complex functions have been assigned to this heterogeneous family. Here we propose a function for a membrane-bound globin of C. elegans, GLB-26. This globin was predicted to be myristoylated at its N-terminus, a post-translational modification only recently described in the globin family. In vivo, this globin is found in the membrane of the head mesodermal cell and in the tail stomato-intestinal and anal depressor muscle cells. Since GLB-26 is almost directly oxidized when exposed to oxygen, we postulate a possible function as electron transfer protein. Phenotypical studies show that GLB-26 takes part in regulating the length of the defecation cycle in C. elegans under oxidative stress conditions.

Development of a dedicated peptide tandem mass spectral library for conservation science.

In recent years, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.

EPR investigation of the role of B10 phenylalanine in neuroglobin - evidence that B10Phe mediates structural changes in the heme region upon disulfide-bridge formation.

The function of neuroglobin, a member of the vertebrate globin family, is still unknown. In human neuroglobin (NGB), the formation of a disulfide bridge between the CysCD7 and CysD5 is known to affect the heme environment and its ligand-binding kinetics. Here, we show by means of EPR that the PheB10 residue plays a key role in transmitting the structural information from the disulfide bridge to the heme-pocket region. While formation of a disulfide bridge in ferric wild-type NGB leads to a considerable change of its EPR parameters, only minor changes are observed in the case of ferric PheB10Leu NGB. Furthermore, wild-type NGB is found to be much more stable in the presence of H(2)O(2) than its PheB10Leu or its HisE7Leu mutants. While tyrosyl radicals are induced in HisE7Leu NGB by the addition of H(2)O(2), this is not the case for wild-type and PheB10Leu NGB. The results will be discussed in terms of the protein's putative functions.

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry.

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).

Unusual stability of human neuroglobin at low pH--molecular mechanisms and biological significance.

Neuroglobin (Ngb) is a recently discovered globin that is predominantly expressed in the brain, retina and other nerve tissues of human and other vertebrates. Ngb has been shown to act as a neuroprotective factor, promoting neuronal survival in conditions of hypoxic-ischemic insult, such as those occurring during stroke. In this work, the conformational and functional stability of Ngb at acidic pH was analyzed, and the results were compared to those obtained with Mb. It was shown by spectroscopic and biochemical (limited proteolysis) techniques that, at pH 2.0, apoNgb is a folded and rigid protein, retaining most of the structural features that the protein displays at neutral pH. Conversely, apoMb, under the same experimental conditions of acidic pH, is essentially a random coil polypeptide. Urea-mediated denaturation studies revealed that the stability displayed by apoNgb at pH 2.0 is very similar to that of Mb at pH 7.0. Ngb also shows enhanced functional stability as compared with Mb, being capable of heme binding over a more acidic pH range than Mb. Furthermore, Ngb reversibly binds oxygen at acidic pH, with an affinity that increases as the pH is decreased. It is proposed that the acid-stable fold of Ngb depends on the particular amino acid composition of the protein polypeptide chain. The functional stability at low pH displayed by Ngb was instead shown to be related to hexacoordination of the heme group. The biological implications of the unusual acid resistance of the folding and function of Ngb are discussed.

Identification of protein binders in works of art by high-performance liquid chromatography-diode array detector analysis of their tryptic digests.

Proteins in works of art are generally determined by the relative amounts of amino acids. This method, however, implies a loss of information on the protein structure and its modifications. Consequently, we propose a method based on the analysis of trypsin digests using high-performance liquid chromatography (HPLC) UV diode array detection (DAD) for painting binder studies. All reaction steps are done in the same vial; no extraction methods or sample transfer is needed, reducing the risk of sample losses. A collection of pure binders (collagen, ovalbumin, yolk and casein) as well as homemade and historical paint samples have been investigated with this method. Chromatograms of unknowns at 214 nm and 280 nm are compared with those of the reference samples as a fingerprint. There is a good agreement between many peptides, but others seem to have been lost or their retention time shifted due to small compositional changes because of ageing and degradation of the paint. The results are comparable with the results of other techniques used for binder identification on the same samples, with the additional advantage of differentiation between egg yolk and glair.

The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification.

BACKGROUND: Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. RESULTS: In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. CONCLUSION: Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.

The proteome of the human neuroblastoma cell line SH-SY5Y: an enlarged proteome.

The human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) is widely used as a neural cellular model system. The hitherto existing proteome data (115 proteins) are here extended. A total of 1103 unique proteins of this cell line were identified using 2D-LC combined with MALDI-TOF/TOF-MS, SDS-PAGE with nano-LC-MS/MS, N-terminal COFRADIC analysis with nano-LC-MS/MS and 2D-PAGE with MALDI-TOF/TOF-MS peptide mass fingerprinting. The obtained proteome profile of this cell line is discussed.

Raman spectroscopic study of bacterial endospores.

Endospores and endospore-forming bacteria were studied by Raman spectroscopy. Raman spectra were recorded from Bacillus licheniformis LMG 7634 at different steps during growth and spore formation, and from spore suspensions obtained from diverse Bacillus and Paenibacillus strains cultured in different conditions (growth media, temperature, peroxide treatment). Raman bands of calcium dipicolinate and amino acids such as phenylalanine and tyrosine are more intense in the spectra of sporulating bacteria compared with those of bacteria from earlier phases of growth. Raman spectroscopy can thus be used to detect sporulation of cells by a characteristic band at 1,018 cm(-1) from calcium dipicolinate. The increase in amino acids could possibly be explained by the formation of small acid-soluble proteins that saturate the endospore DNA. Large variations in Raman spectra of endospore suspensions of different strains or different culturing conditions were observed. Next to calcium dipicolinate, tyrosine and phenylalanine, band differences at 527 and 638 cm(-1) were observed in the spectra of some of the B. sporothermodurans spore suspensions. These bands were assigned to the incorporation of cysteine residues in spore coat proteins. In conclusion, Raman spectroscopy is a fast technique to provide useful information about several spore components.

Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger?

NGB (human neuroglobin), a recently discovered haem protein of the globin family containing a six-co-ordinated haem, is expressed in nervous tissue, but the physiological function of NGB is currently unknown. As well as playing a role in neuronal O2 homoeostasis, NGB is thought to act as a scavenger of reactive species. In the present study, we report on the reactivity of metNGB (ferric-NGB), which accumulates in vivo as a result of the reaction of oxyNGB (oxygenated NGB) with NO, towards NO2- and H2O2. NO2- co-ordination of the haem group accounts for the activity of metNGB in the nitration of phenolic substrates. The two different metNGB forms, with and without the internal disulfide bond between Cys46 (seventh residue on the inter-helix region between helices C and D) and Cys55 (fifth residue on helix D), exhibit different reactivity, the former being more efficient in activating NO2-. The kinetics of the reactions, the NO2--binding studies and the analysis of the nitrated products from different substrates all support the hypothesis that metNGB is able to generate an active species with the chemical properties of peroxynitrite, at pathophysiological concentrations of NO2- and H2O2. Without external substrates, the targets of the reactive species generated by the metNGB/NO2-/H2O2 system are endogenous tyrosine (resulting in the production of 3-nitrotyrosine) and cysteine (oxidized to sulfinic acid and sulfonic acid) residues. These endogenous modifications were characterized by HPLC-MS/MS (tandem MS) analysis of metNGB after reaction with NO2- and H2O2 under various conditions. The internal S-S bond affects the functional properties of the protein. Therefore metNGB acts not only as scavenger of toxic species, but also as a target of the self-generated reactive species. Self-modification of the protein may be related to or inhibit its postulated neuroprotective activity.

Anoxia or oxygen and glucose deprivation in SH-SY5Y cells: a step closer to the unraveling of neuroglobin and cytoglobin functions.

Several studies support the hypothesis that neuroglobin and cytoglobin play a protective role against cell death when cellular oxygen supply is critical. Although the underlying molecular mechanisms are unknown, previous reports suggest that this protection can be realised by the fact that they act as ROS scavengers. In this study, expression of neuroglobin and cytoglobin was evaluated in a human neuroblastoma cell line (SH-SY5Y) under conditions of anoxia or oxygen and glucose deprivation (OGD). The cells could survive prolonged anoxia without significant loss of viability. They became anoxia sensitive when deprived of glucose. OGD induced significant cell death after 16 h resulting in 54% dead cells after 32 h. Necrosis was the main process involved in OGD-induced cell death. After reoxygenation, apoptotic neurons became more abundant. Real-time quantitative PCR and Western blotting revealed that neuroglobin and cytoglobin were upregulated, the former under OGD and the latter under anoxic conditions. Under OGD, cell survival was significantly reduced after inhibiting cytoglobin expression by transfection with antisense ODN. Moreover, cell survival was significantly enhanced by neuroglobin or cytoglobin overexpression. When neuroglobin or cytoglobin protein expression increased or decreased, the H(2)O(2) level was found to be lower or higher, respectively. We conclude that neuroglobin or cytoglobin act as ROS scavengers under ischemic conditions.

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death.

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

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.

P2Y12 receptor signalling towards PKB proceeds through IGF-I receptor cross-talk and requires activation of Src, Pyk2 and Rap1.

Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J. Neurochem. 89, 442.]. In the present study, the mechanisms involved in this response were further elucidated. In cells transfected with the Gbetagamma-scavenger beta-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gbetagamma-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCzeta decreased basal and 2MeSADP-stimulated phosphorylation of PKB indicating a role for this PKC isoform in PKB signalling. Although the increased PKB phosphorylation was abolished in the presence of the IGF-I receptor tyrosine kinase inhibitor AG 1024, 2MeSADP did not significantly increase receptor phosphorylation. Nevertheless, phosphorylation of a 120 kDa IGF-I receptor-associated protein was observed. The latter protein was identified by MALDI-TOF/TOF-MS as the proline-rich tyrosine kinase 2 (Pyk2) that co-operates with Src in a PLD2-dependent manner. Consistent with the signalling towards Rap1 and PKB, activation of Pyk2 was abrogated by Ca2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y12 and IGF-I receptors that proceeds through Gbetagamma-, Ca2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.

Automated headspace-solid-phase micro extraction-retention time locked-isotope dilution gas chromatography-mass spectrometry for the analysis of organotin compounds in water and sediment samples.

An automated method for the simultaneous determination of six important organotin compounds namely monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), monophenyltin (MPhT), diphenyltin (DPhT) and triphenyltin (TPhT) in water and sediment samples is described. The method is based on derivatization with sodium tetraethylborate followed by automated headspace-solid-phase micro extraction (SPME) combined with GC-MS under retention time locked (RTL) conditions. Home-synthesized deuterated organotin analogues were used as internal standards. Two high abundant fragment ions corresponding to the main tin isotopes Sn118 and Sn120 were chosen; one for quantification and one as qualifier ion. The method was validated and excellent figures of merit were obtained. Limits of quantification (LOQs) are from 1.3 to 15 ng l(-1) (ppt) for water samples and from 1.0 to 6.3 microg kg(-1) (ppb) for sediment samples. Accuracy for sediment samples was tested on spiked real-life sediment samples and on a reference PACS-2 marine harbor sediment. The developed method was used in a case-study at the harbor of Antwerp where sediment samples in different areas were taken and subsequently screened for TBT contamination. Concentrations ranged from 15 microg kg(-1) in the port of Antwerp up to 43 mg kg(-1) near a ship repair unit.

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.