Low-temperature Electron Paramagnetic Resonance Research Articles

A desamidination process forming a novel guanidine derivative during the complexation between copper(II) and guanidinoacetic acid

A novel trianionic disubstituted guanidine, ( 1 N-carboxymethylguanidino)hydroxyacetic acid-C 5H 6N 3O 5), oag 3−, has been formed through a desamidination process occurred during copper(II) complexation with guanidinoacetic acid, C 3H 7N 3O 2 (Gaa), which produced [Cu 2(oag)(Gaa)(H 2O)]NO 3 · 2H 2O ( 1). A proposal mechanism of formation of 1 is presented based on the low temperature electron paramagnetic resonance and previous potentiometric studies, which have indicated the presence of CuH 2Gaa as the precursor species.

The Copper Clusters in the Particulate Methane Monooxygenase (pMMO) from Methylococcus Capsulatus (Bath)

AbstractThe particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) is a multi‐copper protein that hydroxylates methane and other small n‐alkanes to their corresponding alcohols with high regiospecificity and stereoselectivity. The copper ions appear to be arranged into ˜5 trinuclear copper clusters. Two of these clusters are thought to be involved in the dioxygen chemistry and alkane hydroxylation mediated by the enzyme. Accordingly, these catalytic clusters (C‐clusters) are typically oxidized, as the protein is isolated. The remaining copper ions are normally reduced, and it has been suggested that they provide a reservoir of reducing equivalents needed for the turnover of the enzyme. In this study, we have oxidized the protein to different levels of oxidation of the copper ions using dioxygen, hydrogen peroxide, ferricyanide, and dioxygen in the presence of the suicide substrate acetylene, and have characterized the oxidized copper centers using low temperature electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy. The results are consistent with the grouping of the copper ions into catalytic clusters and electron transfer clusters (E‐clusters). Quantification indicates that there are, indeed, two C‐clusters. A model in which the two C‐clusters are activated by dioxygen starting from their fully reduced states and mediate the hydroxylation chemistry of methane will be presented and discussed.

Direct Probing of Copper Active Site and Free Radical Formed during Bicarbonate-dependent Peroxidase Activity of Bovine and Human Copper,Zinc-superoxide Dismutases: LOW-TEMPERATURE ELECTRON PARAMAGNETIC RESONANCE AND ELECTRON NUCLEAR DOUBLE RESONANCE STUDIES

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.

Analysis of Different Vanadium Charge States in Vanadium Doped 6H-SiC by Low Temperature Optical Absorption and Electron Paramagnetic Resonance

Analysis of Different Vanadium Charge States in Vanadium Doped 6H-SiC by Low Temperature Optical Absorption and Electron Paramagnetic Resonance

Magnetic properties of zigzag ladder with spatially anisotropic exchange coupling

Low-temperature specific heat, susceptibility, magnetization, and electron paramagnetic resonance studies of Cu(phen)(H 2 O) 2 SO 4 have been performed to characterize the magnetic interactions. In contrast to expectations based on structural considerations, the investigations revealed surprisingly weak magnetic correlations which can be ascribed to the non-trivial magnetochemistry of the hydrogen bonds present in the structure.

The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins.

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.

Comparison of Laurentian Fulvic Acid luminescence with that of the hydroquinone/quinone model system: Evidence from low temperature fluorescence studies and EPR spectroscopy

Fluorescence techniques have been used to identify humic substances, (e.g. aquatic fulvic acids from different origins). Synchronous scans have proven adequate for distinguishing detail and differentiating samples. As well, fluorescence has often been used to probe humic interactions with metals and xenobiotic organics. In this study, the fluorescence of a well-characterized material, Laurentian fulvic acid (LFA) was compared with that of a simple hydroquinone/quinone (H2Q/Q) model system. Synchronous fluorescence (room T) and laser-excited fluorescence experiments at 10°K were carried out to characterize the fluorophore. The synchronous fluorescence behavior of LFA displayed similarities to that of an equilibrated, aerated, H2Q/Q system, but only at higher pH. (Higher pH favours a shift of the redox equilibrium towards quinone.) In contrast, the spectra of LFA suggest the role of “quinone” groups, even at lower pH. A significant feature of these spectra is a lowest energy band. At low temperature this band was more selectively excited at 470 nm, but vibrationally resolved line narrowed spectra were not observed. Fluorescence lifetimes were very short compared to the laser pulse width of ca. 10 ns. Under the low T condition the spectra of LFA and the model are essentially identical. We suggest that the principal luminophore in LFA is a quinone-hydroquinone type, perhaps a charge transfer complex consistent with the absence of the fluorescence line-narrowing phenomenon. The spectra imply a lowest energy component of LFA emission from a fluorophore very similar to that of the simplest H2Q/Q. The significant similarities of the model system to LFA are underscored by striking parallels in the radicals in the two systems seen in well-resolved electron paramagnetic resonance (EPR) spectra that could be obtained at pH = 6.

Optical spin polarisation of the N-V centre in diamond

Low-temperature electron paramagnetic resonance (EPR) spectroscopy has been used to establish the ordering of the ground state spin levels of the nitrogen-vacancy colour centre in diamond. EPR spectroscopy with laser illumination of the sample is used to show that the optically induced spin polarisation observed in the ground state arises due to preferential population of the S z =0 state.

Detection of nitrogen incorporation in nm-thin HfO 2 layers on (1 0 0)Si by electron spin resonance

We report on a low-temperature electron spin resonance (ESR) study of (1 0 0)Si/HfO 2 entities with ultrathin layers of amorphous (a)- HfO 2 deposited by distinct chemical vapor deposition (CVD) techniques using chemically different precursors. The incorporation of N is revealed in (1 0 0)Si/HfO 2 structures with ultrathin a-HfO 2 films deposited by CVD using Hf(NO 3) 4 as precursor: Upon 60Co γ-irradiation, a prominent ESR powder pattern is observed, which via ESR measurements at two observational frequencies has been incontrovertibly identified as originating from NO 2 radicals (density ⩾55 at ppm). The molecules are found to be stabilized and likely homogeneously distributed in the a-HfO 2 network. Based on symmetry considerations, it is suggested that during deposition, N is incorporated in the HfO 2 network as neutral N≡O 3 precursors, which are transformed into ESR-active NO 2 radicals upon γ-irradiation. The N incorporation appears inherent to the particular nitrado CVD process, an aspect that may bear on the electrical properties of the insulator, such as, e.g., introducing charge traps.

Effect of Eu(3+) on characterization of photosystem II particles from spinach by spectroscopy.

Photosystem II (PSII) particles were purified from Eu(3+)-treated spinach and studied by spectroscopy. The results showed that electron transport rate of PS II was accelerated by Eu3+ treating, that violet shift of the PSII Soret band or Q-band was 6 nm or 2 nm for the ultraviolet-visible (UV-Vis) spectrum, that the violet shift of the PSII fluorescence emission peak was 9 nm for fluorescence emission spectrum, that the PSII Signal II's of low-temperature electron paramagnetic resonance (EPR) spectrum was intensified under light, and that the PSII CD spectrum was similar to that of control. It is suggested that Eu(3+) might bind to the PSII reaction center complex and enhance the electron transport rate of PSII CD; however, Eu(3+) treatment does not change the configuration of the PSII reaction center complex.

Magnetic properties of S= zigzag ladder with spatially anisotropic exchange coupling

Abstract Low-temperature specific heat, susceptibility, magnetization, and electron paramagnetic resonance studies of Cu(phen)(H 2 O) 2 SO 4 have been performed to characterize the magnetic interactions. In contrast to expectations based on structural considerations, the investigations revealed surprisingly weak magnetic correlations which can be ascribed to the non-trivial magnetochemistry of the hydrogen bonds present in the structure.

Two redox-active beta-carotene molecules in photosystem II.

Photosystem II (PS II) contains secondary electron-transfer paths involving cytochrome b(559) (Cyt b(559)), chlorophyll (Chl), and beta-carotene (Car) that are active under conditions when oxygen evolution is blocked such as in inhibited samples or at low temperature. Intermediates of the secondary electron-transfer pathways of PS II core complexes from Synechocystis PCC 6803 and Synechococcus sp. and spinach PS II membranes have been investigated using low temperature near-IR spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. We present evidence that two spectroscopically distinct redox-active carotenoids are formed upon low-temperature illumination. The Car(+) near-IR absorption peak varies in wavelength and width as a function of illumination temperature. Also, the rate of decay during dark incubation of the Car(+) peak varies as a function of wavelength. Factor analysis indicates that there are two spectral forms of Car(+) (Car(A)(+) has an absorbance maximum of 982 nm, and Car(B)(+) has an absorbance maximum of 1027 nm) that decay at different rates. In Synechocystis PS II, we observe a shift of the Car(+) peak to shorter wavelength when oxidized tyrosine D (Y(D)*) is present in the sample that is explained by an electrostatic interaction between Y(D)* and a nearby beta-carotene that disfavors oxidation of Car(B). The sequence of electron-transfer reactions in the secondary electron-transfer pathways of PS II is discussed in terms of a hole-hopping mechanism to attain the equilibrated state of the charge separation at low temperatures.

Various intracellular compartments cooperate in the release of nitric oxide from glycerol trinitrate in liver.

1. Glycerol trinitrate (GTN) has been used in therapy for more than 100 years. Biological effects of GTN are due to the release of the biomediator nitric oxide (NO). However, the mechanism by which GTN provides NO, in particular in liver, is still unknown. In this study, we provide experimental evidence showing that cytoplasm, endoplasmic reticulum, and mitochondria are required for the release of NO from GTN in the liver. 2. NO and nitrite (NO(2)(-)) were determined using low-temperature electron paramagnetic resonance and the Griess reaction, respectively. 3. The first step of GTN biotransformation is the release of NO(2)(-). This step is performed in cytoplasm and catalyzed by glutathione-S-transferase. The second step is the rate-limiting step where NO(2)(-) is slowly reduced to NO. This is mainly catalyzed by cytochrome P-450. The second phase can be significantly enhanced by decreasing the pH value, a situation which occurs during ischemia. At high NADPH concentrations exceeding physiological values, cytochrome P-450 catalyzes GTN biotransformation without the involvement of cytoplasmic glutathione-S-transferase. 4. In conclusion, our data show that NO(2)(-) derived from the first step of biotransformation of GTN in the liver is the precursor of NO but not a product of NO degradation; consequently, NO(2)(-) levels are not likely to be a marker of NO release from GTN as earlier suggested.

Adenosyl coenzyme and pH dependence of the [4Fe–4S] 2+/1+ transition in lysine 2,3-aminomutase

5 ′-[ N-[(3 S)-3-Amino-carboxypropyl]- N-methylamino]-5 ′-deoxyadenosine ( azaSAM), an analog of S-adenosyl- l-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe–4S] 2+ center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with K ds of 1.4±0.3 μM at pH 8.0 and 2.2±0.6 μM at pH 6.5. Reduction of the [4Fe–4S] 2+ center in the presence of this analog gives a 10K [4Fe–4S] 1+ electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl- l-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (p K a=7.08) might affect reduction of the [4Fe–4S] 2+ center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe–4S] 2+ center. The signal shape of the low-temperature EPR signal for the [4Fe–4S] 1+ center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.

THz Magneto-Spectroscopy in Megagauss Fields

Recent progress of megagauss field generation and its application to terahertz (THz) spectroscopy are reviewed. A new device feed gap compensator is introduced, which is found to improve the implosion symmetry of electromagnetic flux compression. 600-T fields can be generated reproducibly by this device. Cyclotron resonance (CR) experiments of p-In 1-x Mn x As, n-Cd 1-x Mn x Te and n-ZnO in the THz range have been performed. It has been found that the electron mass is strongly dependent on the magnetic ion concentration. The heavy hole CR is observed at around 460 T; its cyclotron mass (0.455m 0 ) is much larger than the calcuclted value (0.28m 0 ) or the literature value (0.35m 0 ). This fact may indicate the break down of the effective mass theory. The electron cyclotron mass obtained at 10.6 μm (28.3 THz) in n-ZnO is found to be explained by the resonant polaron effect. We also performed the low-temperature THz electron spin resonance experiment up to 150 T. We found the spin-phonon coupled mode in CuGeO 3 and a collective excitation in NaV 2 O 5 above the critical field where the spin gap closes.

Hemin: A Possible Cause of Oxidative Stress in Blood Circulation of β-Thalassemia/Hemoglobin E Disease

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of g -thalassemia/hemoglobin E disease ( g -thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 w M in serum of g -thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of g -thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of g -thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum hemin, r =0.778 (p <0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p =0.259) and 0.519 (p =0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of g -thal/Hb E.

EPR study of the structural phase transitions in chromium doped thallium indium disulphide

The results of low-temperature electron paramagnetic resonance (EPR) investigations of Cr 3+ doped TlInS 2 single crystals in the temperature range 5–300 K are presented. Remarkable temperature dependences of the linewidth and resonance field of the main absorption peak in EPR spectra of Cr 3+ ion were observed. These peculiarities are attributed to the well-known successive phase transitions between 200 and 220 K. Considerable changes of EPR spectra were observed at the temperatures below 80 K, which are associated with strong line broadening, splitting, and appearance of additional peaks. Such transformations are considered as results of tetragonal distortions of InS 4 tetrahedra. An active group responsible for the previously reported phase transitions at the temperatures 216, 204 and 79 K has been determined.

Radical para-benzoic acid derivatives: transmission of ferromagnetic interactions through hydrogen bonds at long distances.

Investigation of the transmission of magnetic interactions through hydrogen bonds has been carried out for two different benzoic acid derivatives which bear either a tert-butyl nitroxide (NOA) or a poly(chloro)triphenylmethyl (PTMA) radical moiety. In the solid state, both radical acids formed dimer aggregates by the complementary association of two carboxylic groups though hydrogen bonding. This association ensured that atoms with most spin density are separated from one another by more than 15 A. Thus, no competing through-space magnetic exchange interactions are expected in these dimers and, hence, they provide good models to investigate whether noncovalent hydrogen bonds play a role in the long-range transmission of magnetic interactions. The nature of the magnetic exchange interaction and their strengths within similar dimer aggregates in solution was assessed by electron spin resonance (ESR) spectroscopy. In the case of radical NOA, low-temperature ESR experiments showed a weak ferromagnetic interaction between the two radicals in the dimer aggregates (which have the same geometry as in the solid state). In contrast, the corresponding solution ESR study performed with radical PTMA did not lead to any conclusive results, as aggregates were formed by noncovalent interactions other than hydrogen bonds. However, the bulkiness of the poly(chloro)triphenylmethyl radical prevented interdimer contacts in the solid state between regions of high spin density. Hence, solid-state measurements of the alpha phase of PTMA radical provided evidence of the intradimer interaction to confirm the transmission of a weak ferromagnetic interaction through the carboxylic acid bridges, as found for the NOA radical. Moreover, crystallization of the PTMA radical in presence of ethanol to form the beta phase of PTMA radical prevented the dimer formation; this resulted in the suppression of this interaction and provides further evidence of the magnetic exchange mechanism through noncovalent hydrogen bonds at long distances.

Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H 2O 2, and Cu(II)/H 2O 2/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H 2O 2, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.

Characterization of nitrides by electron paramagnetic resonance (EPR) and optically detected magnetic resonance (ODMR)

We will highlight our recent work on the properties of residual defects and dopants in GaN heteroepitaxial layers and on the nature of recombination from InGaN single quantum well (SQW) light emitting diodes (LEDs) through magnetic resonance techniques. Electron paramagnetic resonance (EPR) and optically detected magnetic resonance (ODMR) were performed on undoped (highly resistive and n-type) and intentionally doped (Si, Mg, or Be) GaN films grown by a variety of techniques (MOCVD, MBE, and HVPE) in order to obtain general trends and behavior. Through the spin-Hamiltonian parameters, these methods can reveal symmetry information, the character of the wave function and (ideally) the chemical identity of the defect. In addition, low temperature EPR intensities can be used to determine the neutral acceptor or donor concentrations without the need for contacts or the high temperatures required for Hall effect measurements. The ODMR was performed on both bandedge (mainly shallow donor–shallow acceptor recombination) and deep (visible and near-IR) PL bands. In spite of the radically different (non-equilibrium) growth techniques, many of the same defects were found in the various samples. Finally, earlier ODMR studies of recombination from Nichia InGaN ‘green’ and ‘blue’ LEDs were extended to include shorter (‘violet’) and longer (‘amber’) wavelength LEDs and an undoped 30 Å In 0.3Ga 0.7N/GaN heterostructure. The results provide evidence for spatially separated electrons and holes in the optically-active 30 Å InGaN layers under low photoexcitation conditions, likely due to localization at different potential minima in the x– y planes and/or the large strain-induced piezoelectric fields parallel to the growth direction.