EPR Results Research Articles

Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O 2 with a nonheme diiron center

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen- and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mössbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a (mu-1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein Delta9-desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.

The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrin

The bundling of the N-terminal, partial domain helix (Helix C') of human erythroid alpha-spectrin (alphaI) with the C-terminal, partial domain helices (Helices A' and B') of erythroid beta-spectrin (betaI) to give a spectrin pseudo structural domain (triple helical bundle A'B'C') has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern-Volmer quenching constants of Helix C' of alphaI bound to Helices A' and B' of betaI, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and alphaI mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C' was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation-induced blood disorder.

Self-assembled monolayers of radical molecules physisorbed on HOPG(0 0 0 1) substrate studied by scanning tunnelling microscopy and electron paramagnetic resonance techniques

In this paper, we present a combined STM and EPR study on the adsorption and self-organization of monolayers formed from 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy (16DS) and 4′,4′-Dimethylspiro(5α-cholestane-3,2′-oxazolidin)-3′-yloxy (CSL) adsorbed on a highly oriented pyrolitic graphite HOPG(0 0 0 1) substrate. Both 16DS and CSL molecules are persistent free radicals containing a paramagnetic doxyl group. The STM measurements of 16DS on HOPG(0 0 0 1) were performed at the liquid–solid interface while the studies of CSL on HOPG(0 0 0 1) were carried out under ultrahigh vacuum conditions. It was found that the 16DS molecules on the HOPG(0 0 0 1) surface form a highly-ordered monolayer with a domain structure. The high-resolution STM images show structural details of 16DS molecules on HOPG(0 0 0 1) revealing the paramagnetic doxyl group. In contrast, CSL molecules on HOPG(0 0 0 1) form a well-ordered monolayer without domain structure. The EPR results indicate that both compounds deposited on HOPG(0 0 0 1) substrate are not reduced and retain their paramagnetic character. We believe that the molecular systems described can be used in single spin detection experiments using an electron spin noise-scanning tunnelling microscopy (ESN-STM) technique. In particular, the possibility of obtaining contrast spin signals from the paramagnetic and diamagnetic parts of molecules increases the significance of our results.

Tunable Luminescence in Monodisperse Zirconia Spheres

In this article, monodisperse spherical zirconia (ZrO(2)) particles with a narrow size distribution were prepared by the controlled hydrolysis of zirconium butoxide in ethanol, followed by heat treatment in air at low temperature from 300 to 500 degrees C. X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric and differential thermal analysis (TG/DTA), scanning electron microscopy (SEM), transmission electron microscopy (TEM), photoluminescence (PL) spectra, kinetic decay, and electron paramagnetic resonance were used to characterize the samples. The experimental results indicate that the annealed ZrO(2) samples exhibit broad, intense visible photoluminescence. The annealing temperature is indispensable for the luminescence of the obtained ZrO(2) particles. The emission colors of the ZrO(2) samples can be tuned from blue to nearly white to dark orange by varying the annealing temperature. According to the spectral analysis, luminescence lifetimes, and EPR results, the luminescent centers might be attributed to the carbon impurities in the ZrO(2) samples. The possible luminescence mechanism for ZrO(2) samples has been presented in detail.

Characterization of Photoactive Centers in N-Doped In2O3 Visible Photocatalysts for Water Oxidation

N-doped In2O3 films and powders were synthesized, characterized, and evaluated for photoelectrochemical water splitting. The synthetic process was followed in detail by FTIR and UV−vis spectroscopy and the In complex was characterized by X-ray crystallography. NMR, XPS, and EPR were combined in an effort to track the N speciation at each step of the synthesis. The structural, optical and photoelectrochemical properties of the final products (films and powders) were analyzed. Compared to undoped In2O3, N-doped In2O3 showed an increased absorption in the 350−500 nm range with a red shift in the band gap transition. Electrodes prepared from NH4Cl exhibit higher photoactivity compared to the electrodes prepared from urea. NMR, XPS, and EPR results showed that inert amino- and nitrate-type species adsorbed on the surface were produced from urea and NH4Cl, which count toward the N atomic percent but do not increase the activity of In2O3. However, a nitrate-type species in interstitial sites and a paramagnetic s...

Structural, spectroscopic and magnetic investigation of the LiFe 1−xMn xPO 4 ( x=0–0.18) solid solution

Different solid state and sol–gel preparations of undoped and Mn substituted cathode material LiFePO 4 are investigated. Li 3PO 4, Fe 2P 2O 7 and Li 4P 2O 7 are detected and quantified by XRPD only in solid state synthesis. In addition, micro-Raman spectra reveal low amount of different iron oxides clusters. EPR data, combined with the results of magnetization measurements, evidence signals from Fe 3+ ions in maghemite nanoclusters, and in Li 3Fe 2(PO 4) 3. The sol–gel synthesis, showing the lowest amount of impurity phases, seems the most suitable to obtain a promising cathode material. The structural refinement gives new insights into the cation distribution of the Mn doped triphylite structure: (i) about 85% of Mn 2+ ions substitutes Fe 2+, the remaining 15% being located on the Li site, thus suggesting a structural disorder also confirmed by EPR and micro-Raman results; (ii) Mn ions on the Li site are responsible for the observed slight cell volume expansion.

Poly(3‐hexylthiophene)/Poly(methyl methacrylate) Core‐Shell Electrospun Fibers for Sensory Applications

AbstractNew electrospun (ES) sensory fibers consisted of poly(methyl methacrylate) (PMMA) core and poly(3‐hexylthiophene‐2,5‐diyl) (P3HT) shell were successfully prepared using a two‐fluid coaxial electrospinning process. Field‐emission scanning electron microscope (FE‐SEM) studies showed that the prepared ES fibers had diameters of 500–700 nm and worm‐like surface structure of P3HT on the fiber. Red emission fibers were exhibited from the laser confocal microscope. Upon exposed to air under light for two weeks, significant blue‐shifting on both absorption and luminescence spectra was found on the prepared ES fibers. It was probably due to the chain scission occurred in the P3HT and led to the reduced conjugated length. The sensitivity of the ES fibers was much better than that of the spin‐coated P3HT film from the comparison on the variation of photophysical properties. Besides, the EPR result indicated the formation of the P3HT · O2 charge transfer complex (CTC), leading to the fiber conductivity of 10−6 S/cm without an external doping. The present study demonstrates that conjugated polymer based ES core‐shell fibers may have potential applications for oxygen‐sensing devices.

Structure and Surface Properties of Zirconia-Supported Molybdena Obtained by Slurry Deposition

A series of MoO3/t-ZrO2 catalysts was prepared by slurry deposition. Structural studies revealed that the presence of MoO3 had no influence on the phase composition of zirconia support. IR/RS and EPR results confirmed that both isolated and polymeric (2D) oxomolybdenum species occurred on catalysts surface. Adsorption of NO resulted in the reduction of molybdenum centers with simultaneous formation of surface nitrates.

Effect of Mg Addition on the Physical and Catalytic Properties of Cu/CeO2 for NO + CO Reduction

Copper catalysts supported on magnesia promoted ceria are synthesized and used for NO reduction by CO. The supports and the subsequent Cu catalysts are prepared by citric acid sol–gel and impregnation methods, respectively. The results of N2-physisorption, XRD, EPR and TPR measurements indicate that the Mg addition promotes not only the dispersion of CuO, but also the formation of Cu–O–Ce solid solution. The highest amount of Cu–O–Ce solid solution is found in catalyst modified with 5 wt.% MgO. Based on the catalytic performance, two active sites for the reduction NO by CO are proposed: copper matrix and Cu–O–Ce solid solution. The former does not require the involvement of oxygen vacancy and is more active at low temperature range (T < 200 °C), while the latter shows higher NO conversion at temperature above 200 °C, due to the easy regeneration of oxygen vacancy. Also, it is found that the NO conversion over copper matrix is suppressed, while that over Cu–O–Ce solid solution is stimulated in high CO concentration.

Conformational Flexibility of the GM2 Activator Protein Loop Regions Investigated By Site Directed Spin Labeling EPR Spectroscopy

The GM2 activator protein (GM2AP) is an essential component in the degradation pathway of neuronal gangliosides. GM2AP is a required accessory protein for the hydrolytic conversion of GM2 to GM3 by a water soluble hydrolase. The X-ray structure of GM2AP reveals a β-cup topology with multiple conformations of the protein within the unit cell. Because the crystal structures show different conformations of the putative membrane binding loops, we have utilized site-directed spin labeling to investigate conformational flexibility of these loops for protein in solution and bound with GM2 ligand. As such, a series of single and double CYS mutants (still with original 8 CYS in 4 disulfide bridges) have been generated and spin labeled with MTSL. EPR spectra of spin labeled GM2AP were collected with and without GM2 ligand, and no significant changes in the EPR lineshape were seen. EPR spectra were simulated for spin labels located in the loop regions and reveal multiple component fits, while those in the backside of the β-cup beta strands have single component fits. For certain sites in the mobile loops, spectra were acquired as a function of temperature. From these lineshape simulations, the activation energy for the conformational change has been determined. The SDSL EPR results indicate that the multiple conformations observed in the crystallographic unit cell are populated in solution and represent conformational flexibility of the protein; which is not necessarily altered by binding to lipid ligands. Additionally, spin labeled protein was analyzed by mass spectrometry to confirm proper formation of the four disulfide linkages and addition of only one spin label at the mutant cysteine.

Coordination ability of insect kinin analogs

Spectroscopic data, including electronic absorption, CD and EPR results, as well as theoretical calculations have shown that the insertion of 4-aminopyroglutamate, a novel cis-peptide bond mimic, in the insect kinin peptide leads to an effective ligand towards Cu(II) ions at basic pH ranges. The 4-aminopyroglutamate motif induces a specific peptide conformation which favors the formation of one or two five-membered chelating rings stabilizing a bent structure, with the coordination of 3N-type or 4N-type in the metallopeptide molecule involving in metal bonding C-terminal of modified peptide. It is worth noting that the copper(II) bonding does not undergo hydrolysis even at a very high pH range.

Infrared, optical absorption, and EPR spectroscopic studies on natural gypsum

The natural gypsum has been investigated by infrared, Raman, X-ray diffraction, optical absorption and electron paramagnetic resonance spectroscopy. The fundamental stretching and bending vibrations observed in the infrared region for SO 4 2− and H 2O are compared with the near-infrared overtones and combinations of these vibrations. MIR and Raman spectral features are attributed to sulfate fundamentals and lattice vibrations of H 2O, SO 4 2−. The charge transfer and ligand field transition bands were observed near 490, 630, and 800–900 nm and were compared to those of iron oxides. The optical absorption spectrum indicates the presence of ferric and ferrous ions in the mineral. The site symmetry of Fe(III) in the sample is tetragonally distorted. EPR results indicate the presence of the ferric ion in a tetragonally distorted state.

Acetato, formato and chloride copper(II) complexes with 2-(aminomethyl)pyridine: Synthesis, crystal structure, magnetic properties and EPR results

By the reactions of Cu(OAc) 2 · H 2O and Cu(HCOO) 2 · 4H 2O with 2-(aminomethyl)pyridine in different proportions, the compounds Cu(OAc) 2(2-amp) ( 1), Cu(HCOO) 2(2-amp) ( 2), Cu(HCOO) 2(2-amp) 1/2 ( 5), Cu(OAc) 2(2-amp) 2 · H 2O ( 6) and Cu(HCOO) 2(2-amp) 2 · H 2O ( 7) were obtained. In 1 the copper shows an elongated rhombic octahedral stereochemistry determined by a 2-amp molecule and two asymmetrical bidentate acetate groups. The hydrogen bonds between the NH 2 groups and O atoms yield to the formation of a double chain. Compound 2 instead consists in monodimensional chains of Cu(2-amp)(HCOO) units, with monodentate formate groups, linked by syn–anti bridging formate groups. Sheets are formed by hydrogen bonds between the chains. By crystallization of a solution of 6 in chloroform, CuCl 2(2-amp) 2 ( 3) was obtained. It presents a highly distorted square pyramidal geometry around the copper atom. The sheets, formed by the hydrogen bonds between NH 2 and Cl, are interpenetrated and shows π stacking. Magnetic properties and EPR spectra for these new compounds have been studied. Also the magnetic behaviour of Cu(OAc) 2(2-amp) 1/2 ( 4) is described.

THE PROPERTIES AND IDENTIFICATION OF MARBLE FROM PROCONNESOS (MARMARA ISLAND, TURKEY): A NEW DATABASE INCLUDING ISOTOPIC, EPR AND PETROGRAPHIC DATA*

This paper reports an updated and enlarged archaeometric database of Proconnesos marbles with the aim of attaining more reliable inter‐ and intra‐site identification. Quarry and artefact samples (n = 397) from 23 different quarrying areas were characterized using petrographic, isotopic and EPR results. The most distinct property of Proconnesos marbles is their extremely weak EPR intensity, associated with the low concentration of Mn2+impurities. This feature, supported by the contribution of other important isotopic and petrographic variables, forms the basis for the correct identification of Proconnesos marbles. Within the site the distribution of properties is relatively homogeneous; only limited intra‐site discrimination is possible. All the experimental results, as well as the quarry samples are made available to other researchers willing to measure additional experimental properties, thus increasing the rate of within the site discrimination. Extensive survey has proved that, although about 10% of the samples exhibit maximum grain size below 1.0 mm, no locations exist where fine grained marbles could be intentionally quarried. The historical aspects of Proconnesos marble quarrying and usage are also discussed.

Biochemical and spectroscopic studies of human melanotransferrin (MTf): Electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6 M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.

Platinum species in the pores of NaX, NaY and NaA zeolites studied using EPR, XAS and FTIR spectroscopies

The results of X-band EPR, X-ray absorption and Fourier transform infrared spectroscopy on Pt(NH(3))(4)(2+) exchanged NaX, NaY and NaA zeolites reveal after oxygen calcination at 573 K that diamagnetic Pt(2+) is not the only product. Calcination provides Pt(3+) cations, but depending on the heating rate, the decomposition of amino groups during calcination also produces hydrogen that reduces Pt(3+) to Pt(2+) and Pt(+). NaX (Si/Al = 1.23) has a more negative framework charge than NaY (Si/Al = 2.31), so Pt(3+) can be stabilized only in NaX, whereas lower oxidation states of Pt such as Pt(+) can be stabilized in both, NaX and NaY, and neither of the paramagnetic Pt cations are stabilized in NaUSY (Si/Al = 3). The autoreduction process allows controlling the number of Pt(3+) and Pt(+) in the NaX zeolite by changing the calcination heating rate: a heating rate of 1.25 K min(-1) gives only Pt(+), but 0.5 K min(-1) gives a Pt(3+)/Pt(+) ratio close to 1. The structure of the support is also important for the synthesis of Pt species. While isolated paramagnetic Pt ions were stabilized in faujasite zeolites (NaX and NaY), a paramagnetic Pt dimer was obtained in a Linde type A zeolite (LTA, Si/Al = 1) by applying the same preparation methods. The fraction of paramagnetic Pt species which were characterized by X-band EPR spectroscopy amounts to 2-18% of the total Pt in the zeolites, the remaining Pt must be diamagnetic.

Spectroscopic, electrochemical and computational study of Pt–diimine–dithiolene complexes: rationalising the properties of solar cell dyes

A series of [Pt(ii)(diimine)(dithiolate)] complexes of general formula [Pt{X,X'-(CO(2)R)(2)-bpy}(mnt)] (where X = 3, 4 or 5; R = H or Et, bpy = 2,2'-bipyridyl and mnt = maleonitriledithiolate), have been spectroscopically, electrochemically and computationally characterised and compared with the precursors [Pt{X,X'-(CO(2)R)(2)-bpy}Cl(2)] and X,X'-(CO(2)R)(2)-bpy. The study includes cyclic voltammetry, in situ EPR spectroelectrochemical studies of fluid solution and frozen solution samples, UV/Vis/NIR spectroelectrochemistry, hyrid DFT and TD-DFT calculations. The effect of changing the position of the bpy substituents from 3,3' to 4,4' and 5,5' is discussed with reference to electronic changes seen within the different members of the family of molecules. The performance of the mnt complexes in dye-sensitised solar cells has been previously described and the superior performance of [Pt{3,3'-(CO(2)R)(2)-bpy}(mnt)] is now explained in terms of decreased electronic delocalisation through twisting of the bipyridyl ligand as supported by the EPR and computational results.

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and 2H NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, 2H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order ( S mol) and dynamics ( T 1) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2–8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a “transient site”, which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel.

Stabilisation of reactive intermediates in molecular sieves

This paper presents studies on paramagnetic intermediates, free atoms and radicals produced in γ-irradiated molecular sieves and their reactions with adsorbate molecules or exchangeable cations. Four different systems have been investigated using EPR spectroscopy, Na-A/CH4, AgNa-A/CH3OH, Ag-SAPO-11/C2H4 and AgCs-rho/NH3. It was found that methyl radicals are formed in two different sites in Na-A/CH4 and in one of them they are stable at room temperature. The formation of Ag·CH2OH+ radical cation with one-electron bond between silver and carbon has been established in AgNa-A/CH3OH by EPR experiments with [13C]CH3OH and DFT calculations. In Ag-SAPO-11/C2H4 the stabilisation of biligand silver/ethylene complex, Ag0(C2H4)2 was postulated based on EPR and DFT results. Tetrameric silver clusters (Ag 4 3+ ) produced radiolytically in AgCs-rho/NH3 strongly interact with two ammonia molecules as was deduced from the changes in superhyperfine structure of high-field EPR line of Ag 4 3+ pentet for zeolite exposed to [14N]NH3 and [15N]NH3. The presented examples clearly show that the combination of radiation methods with EPR technique is very useful to study the structure and reactivity of paramagnetic intermediates.

An EPR and Optical Study of VO2+ in Bis (Glycine) Cadmium Chloride Single Crystals

Results of EPR and optical studies of VO2+ doped in bis (glycine) cadmium(II) chloride, belonging to a third site as a substitutional one are reported. The spin Hamiltonian parameters obtained for the site are gzz = 1.9159, gyy = 1.9695, gxx = 1.9853, Azz = 210.4 G, Ayy = 109.8 G, and Axx = 107.0 G. By correlating the EPR and spectral data, the molecular orbital bonding parameters have been evaluated.