Biomedical subjects

Luc Moens

Publications and source records attributed to Luc Moens.

At least 37 records · Page 2Linked to original sources

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Evaluation of an accurate calibration and spectral standardization procedure for Raman spectroscopy.

Due to modern developments Raman spectroscopy has evolved into a fast vibrational technique. Detailed fingerprints in combination with non-destructivity and minimal sample preparation has allowed the construction of reference libraries in a variety of research fields. Long-term stability and comparability are important characteristics when developing reference libraries. In addition, small shifts in highly similar spectra of different samples may limit the full potential of Raman spectroscopy. Since libraries often contain a large number of different and/or highly similar spectra, it is important that each data point in all the spectra corresponds to the exact Raman wavenumber. This is often not the case, due to shifts in optical pathway and/or shifts in laser wavelength. This paper describes a complete calibration protocol (wavelength and intensity) and evaluates the procedure for both short and long term stability, by means of 60 randomly selected measurement sessions spread over a period of nine months. A two-step standardization procedure is proposed to deal with spectral shifts.

Calibration↗

PubMed2005

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.

Automation↗

PubMed2005

Bishistidyl heme hexacoordination, a key structural property in Drosophila melanogaster hemoglobin.

Hemoglobins at high concentration have been isolated long ago from some insect larvae living in hypoxic environments. Conversely, a monomeric hemoglobin has been discovered recently in the fruit fly Drosophila melanogaster as intracellular protein expressed both in larvae and in the adult fly. Such a finding indicates that the oxygen supply in insects may be more complex than previously thought, relying not only on O2 diffusion through the tubular tracheal system, but also on carrier-mediated transport and storage. We present here the crystal structure of recombinant D. melanogaster hemoglobin at 1.20 A resolution. Spectroscopic data show that the protein displays a hexacoordinated heme, whose axial ligands are the proximal and distal His residues. Such bis-His ligation of the heme has sizable effects on the protein local structure. Three protein matrix cavities, comparable in size but not in topological locations with those of sperm whale myoglobin, are spread through the protein matrix; one of these can host a xenon atom. Additionally, D. melanogaster hemoglobin binds one molecule of 3-(cyclohexylamino)propanesulfonic acid (CAPS) buffer at a surface pocket, next to the EF hinge. Despite the high resolution achieved, no sequence/structure features specifically supporting the heme hexa- to pentacoordination transition required for diatomic ligand binding could be recognized.

Alkanesulfonic Acids↗

PubMed2005

Nongovernmental valorization of carbon dioxide.

Carbon dioxide (CO2) is considered the largest contributor to the greenhouse gas effect. Most attempts to manage the flow of CO2 or carbon into our environment involve reducing net emissions or sequestering the gas into long-lived sinks. Using CO2 as a chemical feedstock has a long history, but using it on scales that might impact the net emissions of CO2 into the atmosphere has not generally been considered seriously. There is also a growing interest in employing our natural biomes of carbon such as trees, vegetation, and soils as storage media. Some amelioration of the net carbon emissions into the atmosphere could be achieved by concomitant large withdrawals of carbon. This report surveys the potential and limitations in employing carbon as a resource for organic chemicals, fuels, inorganic materials, and in using the biome to manage carbon. The outlook for each of these opportunities is also described.

Bioelectric Energy Sources↗

PubMed2005

Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the detection of chromium species after incubation of chromium in simulated sweat.

The presence of chromium in chromium-tanned leather represents a considerable health problem since it can lead to chronic allergic contact dermatitis. Apart from trivalent chromium (Cr(III)), which is used for tanning, leather often contains hexavalent chromium (Cr(VI)), resulting from the oxidation of Cr(III) during the tanning process. This study deals with the chromium compounds in simulated sweat when brought into contact with Cr(III) or Cr(VI) and with chromium-tanned leathers. A capillary electrophoresis (CE) method was developed, with inductively coupled plasma-sector field-mass spectrometry (ICP-SF-MS) for element-specific detection. Two different electrophoretic runs, applying once the positive and once the negative polarity mode, were necessary for the detection of positively and negatively charged chromium species. Although sometimes described in the literature, a pre-run derivatization of the chromium-species was not performed here to prevent species transformation. 50 mmol.L(-1) sodium phosphate at a pH of 2.5 was used as CE separation buffer and as make-up liquid for the CE-ICP-SF-MS interface. When applied to simulated sweat samples incubated with Cr(VI), this method showed that methionine is responsible for the reduction of Cr(VI) into Cr(III), which, at its turn, forms a complex with lactic acid. In the case of sweat plus Cr(III), the latter step was also seen. Applied to simulated sweat in contact with leather samples, the method developed showed the presence of the former species among a much more complex pattern.

Chromium↗

PubMed2005

Determination of the acaricide fenbutatin oxide in water samples by automated headspace-SPME-GC/MS.

The analysis of the acaricide fenbutatin oxide (FBTO) having a molecular weight of 1052.66 g mol(-1) in water samples by capillary GC/MS after in-situ derivatization with sodium tetraethylborate (NaBEt4) and headspace-SPME enrichment is described. Automated SPME is performed at 80 degrees C for 30 min. Detection is carried out in the ion monitoring mode with deuterated triphenyltin (TPhTd15) as internal standard. Good linearity (R2 = 0.9993) was obtained in the dynamic range 20 to 1000 ng L(-1) with a limit of detection of 16 ng L(1) (LOD at 3 S/N) and a limit of quantitation of 50 ng L(-1) (LOQ at 10 S/N). Intra-day RSD% for n=6 was 8.9 at the LOQ level.

Animals↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.

Amino Acid Sequence↗

PubMed2005

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.

Animals↗

PubMed2004

Raman spectroscopic study of Lactarius spores (Russulales, Fungi).

Fungi are important organisms in ecosystems, in industrial and pharmaceutical production and are valuable food sources as well. Classical identification is often time-consuming and specialistic. In this study, Raman spectroscopy is applied to the analysis of fungal spores of Lactarius, an economically and ecologically important genus of Basidiomycota. Raman spectra of spores of Lactarius controversus Pers.: Fr., Lactarius lacunarum (Romagn.) ex Hora, Lactarius quieticolor Romagn. and Lactarius quietus (Fr.: Fr.) Fr. are reported for the first time. The spectra of these species show large similarity. These spectra are studied and compared with the Raman spectra of reference substances known to occur in macrofungi, including saccharides, lipids and some minor compounds that may serve as specific biomarkers (adenine, ergosterol and glycine). Most Raman bands could be attributed to specific components. In agreement with the biological role of fungal spores, high amounts of lipids were observed, the main fatty acid being oleate. In addition to different types of lipids and phospholipids, the polysaccharides chitin and amylopectin could be detected as well. The presence of trehalose is not equivocally shown, due to overlapping bands. Raman band positions are reported for the observed bands of the different species and reference products.

Agaricales↗

PubMed2004

Temperature dependence of NO binding modes in human neuroglobin.

Both the ferrous and ferric forms of wild-type neuroglobin are found to be hexacoordinated with axial ligation of the F8-His and E7-His. Rapidly growing Escherichia coli cell cultures with low O2 concentration generate nitric oxide (NO). Combined electron paramagnetic resonance (EPR) and optical measurements show that wild-type human recombinant neuroglobin, overexpressed in such E. coli cells, still favors the F8His-Fe2+ -E7His conformation, whereby only a small fraction of the protein binds NO. Upon mutation of the E7-His to Leu and Gln, the competition with the distal histidine disappears and the nitrosyl ferrous form is readily observed. At low temperature, the EPR spectra of the NO-ligated Ngb proteins consist of contributions from two geometrically different NO-heme conformations. In combination with EPR data of vertebrate hemoglobins and myoglobins, the temperature dependence of the EPR spectra of the NO adducts of ferrous hNgb and its E7-mutants proves a strong stabilization of one isomer by the E7-histidine in wt hNgb. It is shown that this is not related to the polarity of histidine, but to its specific binding characteristics.

Electron Spin Resonance Spectroscopy↗

PubMed2004

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.

Allosteric Site↗

PubMed2004

Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin.

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s(-1)) and moderate O2 affinity (KO2) approximately 1 microm(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 degrees C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 --> Val CerHbO2 at 1.70 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). The dramatic functional and spectral effects of the Thr-E11 --> Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm(-1) decrease in nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.

Animals↗

PubMed2004

Mapping protein matrix cavities in human cytoglobin through Xe atom binding.

Cytoglobin is the fourth recognized globin type, almost ubiquitously distributed in human tissues; its function is still poorly understood. Cytoglobin displays a core region of about 150 residues, structurally related to hemoglobin and myoglobin, and two extra segments, about 20 residues each, at the N- and C-termini. The core region hosts a large apolar cavity, held to provide a ligand diffusion pathway to/from the heme, and/or ligand temporary docking sites. Here we report the crystal structure (2.4A resolution, R-factor 19.1%) of a human cytoglobin mutant bearing the CysB2(38) --> Ser and CysE9(83) --> Ser substitutions (CYGB*), treated under pressurized xenon. Three Xe atoms bind to the heme distal site region of CYGB* mapping the protein matrix apolar cavity. Despite the conserved globin fold, the cavity found in CYGB* is structured differently from those recognized to play a functional role in myoglobin, neuroglobin, truncated hemoglobins, and Cerebratulus lacteus mini-hemoglobin.

Binding Sites↗

PubMed2004

Structural change of the heme pocket due to disulfide bridge formation is significantly larger for neuroglobin than for cytoglobin.

Human neuroglobin (hNgb) and human cytoglobin (hCygb), two recently discovered members of the vertebrate globin family, are known to be able to form an intramolecular disulfide bridge. Using electron paramagnetic resonance (EPR), we show that formation of a disulfide bridge in ferric hNgb causes a considerable change in the heme pocket structure, whereas this is not so clear for ferric hCygb. The structural results can be related nicely to earlier histidine and dioxygen affinity studies of the ferrous proteins.

Cytoglobin↗

PubMed2004

Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuroglobin reveal a potential role against oxidative stress.

Neuroglobin, recently discovered in the brain and in the retina of vertebrates, belongs to the class of hexacoordinate globins, in which the distal histidine coordinates the iron center in both the Fe(II) and Fe(III) forms. As for most other hexacoordinate globins, the physiological function of neuroglobin is still unclear, but seems to be related to neuronal survival following acute hypoxia. In this study, we have addressed the question whether human neuroglobin could act as a scavenger of toxic species, such as nitrogen monoxide, peroxynitrite, and hydrogen peroxide, which are generated at high levels in the brain during hypoxia; we have also investigated the kinetics of the reactions of its Fe(III) (metNGB) and Fe(II)NO forms with several reagents. Binding of cyanide or NO* to metNGB follows bi-exponential kinetics, showing the existence of two different protein conformations. In the presence of excess NO*, metNGB is converted into NGBFe(II)NO by reductive nitrosylation, in analogy to the reactions of NO* with metmyoglobin and methemoglobin. The Fe(II)NO form of neuroglobin is oxidized to metNGB by peroxynitrite and dioxygen, two reactions that also take place in hemoglobin, albeit at lower rates. In contrast to myoglobin and hemoglobin, metNGB unexpectedly does not generate the cytotoxic ferryl form of the protein upon addition of either peroxynitrite or hydrogen peroxide. Taken together, our data indicate that human neuroglobin may be an efficient scavenger of reactive oxidizing species and thus may play a role in the cellular defense against oxidative stress.

Ferric Compounds↗

PubMed2004

Crystal structure of cytoglobin: the fourth globin type discovered in man displays heme hexa-coordination.

Cytoglobin is a recently discovered hemeprotein belonging to the globin superfamily together with hemoglobin, myoglobin and neuroglobin. Although distributed in almost all human tissues, cytoglobin has not been ascribed a specific function. Human cytoglobin is composed of 190 amino acid residues. Sequence alignments show that a protein core region (about 150 residues) is structurally related to hemoglobin and myoglobin, being complemented by about 20 extra residues both on the N and C termini. In the absence of exogenous ligands (e.g. O2), the cytoglobin distal HisE7 residue is coordinated to the heme Fe atom, thus decreasing the ligand affinity. The crystal structure of human cytoglobin (2.1 A resolution, 21.3% R-factor) highlights a three-over-three alpha-helical globin fold, covering residues 18-171; the 1-17 N-terminal, and the 172-190 C-terminal residue segments are disordered in both molecules of the crystal asymmetric unit. Heme hexa-coordination is evident in one of the two cytoglobin chains, whereas alternate conformation for the heme distal region, achieving partial heme penta-coordination, is observed in the other. Human cytoglobin displays a large apolar protein matrix cavity, next to the heme, not related to the myoglobin cavities recognized as temporary ligand docking stations. The cavity, which may provide a heme ligand diffusion pathway, is connected to the external space through a narrow tunnel nestled between the globin G and H helices.

Amino Acid Sequence↗