Electron Paramagnetic Resonance Resonance Research Articles

EPR and ENDOR studies of Fe(II) hemoproteins reduced and oxidized at 77 K

gamma-irradiation of frozen solutions of Fe(II) hemoproteins at 77 K generates both electron paramagnetic resonance (EPR) active singly reduced and oxidized heme centers trapped in the conformation of the Fe(II) precursors. The reduction products of pentacoordinate (S = 2) Fe(II) globins, peroxidases and cytochrome P450cam show EPR and electron-nuclear double resonance (ENDOR) spectra characteristic of (3d 7) Fe(I) species. In addition, cryoreduced Fe(II) alpha-chains of hemoglobin and myoglobin exhibit an S = 3/2 spin state produced by antiferromagnetic coupling between a porphyrin anion radical and pentacoordinate (S = 2) Fe(II). The spectra of cryoreduced forms of Fe(II) hemoglobin alpha-chains and deoxymyoglobin reveal that the Fe(II) precursors adopt multiple conformational substates. Reduction of hexacoordinate Fe(II) cytochrome c and cytochrome b5 as well as carboxy complexes of deoxyglobins produces only Fe(II) porphyrin pi-anion radical species. The low-valent hemoprotein intermediates produced by cryoreduction convert to the Fe(II) states at T > 200 K. Cryogenerated Fe(III) cytochrome c and cytochrome b5 have spectra similar to these for the resting Fe(III) states, whereas the spectra of the products of cryooxidation of pentacoordinate Fe(II) globins and peroxidases are different. Cryooxidation of CO-Fe(II) globins generates Fe(III) hemes with quantum-mechanically admixed S = 3/2, 5/2 ground states. The trapped Fe(III) species relax to the equilibrium ferric states upon annealing at T > 190 K. Both cryooxidized and reduced centers provide very sensitive EPR/ENDOR structure probes of the EPR-silent Fe(II) state.

EPR Free Induction Decay Coherence Observed after a Single Pulse in Saturation Recovery Experiments for Samples with Resolved Multiline CW Spectra

An electron paramagnetic resonance (EPR) spin-coherence signal has been observed following a single pulse for rapidly tumbling radicals with well-resolved nuclear hyperfine splitting in fluid solution when B1 is large enough to excite multiple hyperfine lines. This signal, which has the shape of a spin echo, arises from constructive interference of overlapping free induction decays (FIDs) from the hyperfine lines. It has been observed for 2,6-di-t-butyl-1,4-benzosemiquinone, 2,5-di-t-butyl-1,4-benzosemiquinone, 2,3,5,6-tetramethoxy-1,4-benzosemiquinone, 2,4,6-tri-t-butylphenoxyl radical, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy. It occurs at a time after the pulse that is equal to the inverse of the nuclear hyperfine splitting, independent of EPR resonance frequency from 250 MHz to 9.1 GHz. As the length of the pulse is increased, separate coherence signals can be observed that correspond to the beginning and end of the pulse. This coherence is distinct from the "single-pulse echo" signals discussed in the literature. For 2,6-di-t-butyl-1,4-benzosemiquinone, which has two resolved couplings (1.24 and 0.052 G), FID oscillations with a period that corresponds to the larger hyperfine coupling are observed on the coherence signal that arises from the smaller hyperfine coupling. If phase cycling is not perfect, the coherence signal can interfere with measurements of T1 by saturation recovery.

Continuous Wave EPR Oximetric Imaging at 300 MHz Using Radiofrequency Power Saturation Effects

A novel continuous wave (CW), radiofrequency (RF), electron paramagnetic resonance (EPR) oximetric imaging technique is proposed, based on the influence of oxygen concentration on the RF power saturation of the EPR resonance. A linear relationship is demonstrated between the partial oxygen pressure (pO(2)) and the normalized signal intensity (I(N)), defined as, I(N) = (I(HP) - I(LP))/I(LP), where I(LP) and I(HP) refer to signal intensities at low (P(L)) and high (P(H)) RF power levels, respectively. A formula for the determination of pO(2), derived on the basis of the experimental results, reliably estimated various oxygen concentrations in a five-tube phantom. This new technique was time-efficient and also avoided the missing angle problem associated with conventional spectral-spatial CW EPR oximetric imaging. In vivo power saturation oximetric imaging in a tumor bearing mouse clearly depicted the hypoxic foci within the tumor.

Myosin cleft closure by double electron–electron resonance and dipolar EPR

Dipolar electron paramagnetic resonance (EPR) has been used to determine theconformation of the myosin actin-binding cleft in various states of actomyosin ATPase andbinding to actin. Comparison of the crystal structures of isolated components and electronmicroscopy of the actin–myosin complex suggested that the cleft between the upper andlower 50 kDa subdomains closes on binding to actin. The changes in the myosin cleftmight be coupled to the state of the nucleotide in the active site and providecommunication between ATP hydrolysis and actin binding. In order to detect these changesin solution, we measured the distances between double cysteine mutants of theDictyostelium discoidium myosin II MD by continuous wave EPR and pulsedEPR—double electron–electron resonance (DEER). We have engineered doublecysteine mutants C416/C537 which are labelled with spin probes and measuredthe distances between the upper and lower domains in various nucleotide statesand in the presence of actin. We found two major populations of distances at∼15 Å (conventional EPR) and at 25 Å (DEER) and shifting in the populations as a function ofnucleotide state and actin binding, which suggest that the cleft movement is coupled tonucleotides and actin binding and dissociation.

Properties of nanoporous carbon with pores filled by Ni atoms

AbstractUsing the electron spin resonance (ESR) method, conductance measurements, and small‐angle X‐ray scattering (SAXS), the properties of nanoporous carbon (NPC) containing atoms of Ni in the pores are studied in comparison with the nickel‐free NPC studied before. Although the amount of Ni introduced into the nanopores is small, the NPC properties change significantly. The ESR spectra show that, at low temperatures, the charge carriers are captured at the localized states. The bulk ferromagnetism is not observed at temperatures as low as 3.2 K. Magnetic properties are strongly temperature dependent and are not limited by the paramagnetism of free charge carriers. The temperature dependence of the ESR resonance field indicates the influence of ferromagnetic inclusions. The ferromagnetic shift of the resonance fields is observed up to a temperature of 80 K. The temperature dependence of the conductance reveals an exponential growth with the activation energy close to those obtained from the ESR measurements. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Study of structural evolution in Ce‐ and Nd‐doped aluminoborosilicate glasses under β‐irradiation

AbstractThe structural changes in aluminoborosilicate glasses under β‐irradiation as a function of rare earth oxide content and the dose have been studied using Electron Paramagnetic Resonance (EPR) and luminescence spectroscopies. The EPR measurements at helium temperature (4 K) showed that β‐irradiation affects valence state of cerium ions. According to concentration and dose dependences, the line with g ∼ 2.89 can be attributed to paramagnetic Ce3+ ions resulting from Ce4+ reduction under irradiation. The effect of CeO2 content leads to the modifications of the EPR signal of defects at helium and the change in the relative proportion of electron and hole defects with the increase of the cerium doping level at room temperature. We observed also a decrease of the defect concentration as a function of the Nd doping into these glass compositions but by two orders less than for Ce‐doped samples. Exposure to irradiation induced the appearance of a non‐resolved EPR band in the region of defects. We can assume that possible dipolar interaction between Nd3+ ion and hole can result in not typical EPR resonance shape of paramagnetic defects at g ∼ 2. Moreover, estimation of the ratio between splitted components of the 4F3/2 – 4I9/2 transition depending on the irradiation dose showed that dose increase leads to increase of this ratio suggesting that the nearest environment of Nd ions is expected to be changed. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

High-frequency dynamic nuclear polarization using mixtures of TEMPO and trityl radicals.

In a previous communication [Hu et al., J. Am. Chem. Soc. 126, 10844 (2004)], an approach was demonstrated that improves the efficiency of the cross-effect polarization mechanism employed in high field dynamic nuclear polarization (DNP) experiments. Specifically, it was shown that tethering two TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) radicals increases the electron-electron dipole coupling from approximately 1 MHz in solutions of monomeric TEMPO to approximately 25 MHz in a tethered biradical. The larger coupling resulted in an increase in the DNP enhancements by a factor of approximately 3-4, from 45-50 to approximately 165. Here, a second approach to improving the efficiency of the polarization process is described that involves approximately satisfying the matching condition |omega(2e)-omega(1e)|=omega(n), where omega(2e) and omega(1e) are two frequencies in the electron paramagnetic resonance (EPR) spectrum and omega(n) is the Larmor frequency of the nuclear spins being polarized. Specifically, in a mixture of TEMPO and trityl [tris (8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl) methyl] radicals, the intensity maxima in the EPR spectra of these two species are approximately separated by the (1)H NMR frequency. In this case the frequency difference between the g(yy) value of TEMPO and the narrow pseudo-isotropic g-value of trityl is approximately 224 MHz and the (1)H Larmor frequency is 211 MHz. The optimal magnetic field for DNP using the mixtures was found to coincide with the trityl EPR resonance. At 90 K and 5 T, a mixture of 20 mM TEMPO and 20 mM trityl enhanced the (1)H polarization by a factor of approximately 160, an improvement over the enhancement of approximately 50 with 40 mM TEMPO. The reasons for the improvement are discussed and evidence is presented suggesting that DNP enhancement can be improved further by tethering TEMPO and trityl or two similar radicals.

Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance

The biological function of protein, DNA, and RNA molecules often depends on relative movements of domains with dimensions of a few nanometers. This length scale can be accessed by distance measurements between spin labels if pulsed electron paramagnetic resonance (EPR) techniques such as electron-electron double resonance (ELDOR) and double-quantum EPR are used. The approach does not require crystalline samples and is well suited to biomacromolecules with an intrinsic flexibility as distributions of distances can be measured. Furthermore, oligomerization or complexation of biomacromolecules can also be studied, even if it is incomplete. The sensitivity of the technique and the reliability of the measured distance distribution depend on careful optimization of the experimental conditions and procedures for data analysis. Interpretation of spin-to-spin distance distributions in terms of the structure of the biomacromolecules furthermore requires a model for the conformational distribution of the spin labels.

Multi-frequency ESR in NaCu2O2

NaCu2O2 is regarded as an S = 1/2 zigzag chain with competing nearest neighbor and next nearest neighbor exchange interactions. The spin structure of NaCu2O2 below a Néel temperature TN = 13 K was reported to be helical with a propagation vector q = (0.5, 0.227, 0). To obtain further information on the ordered state of NaCu2O2, we performed multi-frequency electron spin resonance (ESR) and high field magnetization measurements on single crystal samples of NaCu2O2. We observed a large and some small ESR signals and a monotonical increase of magnetization with a change of the slope around 20 T. One of the ESR resonance modes from the large signal is roughly reproduced by one of the helical resonance modes calculated on the assumption of a conical spin structure.

High field magnetization and multi-frequency ESR in the spin-ladder compound Na2Fe2(C2O4)3(H2O)2

We have performed high field magnetization and multi-frequency electron spin resonance (ESR) measurements on single crystal and powder samples of Na2Fe2(C2O4)3(H2O)2, abbreviated as SIO. The Fe2+ ions in SIO are bridged by oxalate groups to make two-leg ladders. The temperature dependence of magnetic susceptibilities parallel and perpendicular to the leg direction show very different and anisotropic behavior. Experimental results of the isomorphous compound Na2Co2(C2O4)3(H2O)2 (SCO) are well explained by isolated magnetic dimers with an Ising-type anisotropy. Therefore, we analyze the frequency dependence of the ESR resonance fields by the same model with a fictitious spin 1 as in SCO. The agreement between experiment and calculation is satisfactorily good. Magnetic susceptibility and magnetization curves calculated with the same parameters obtained in the ESR analyses, however, do not agree well with the experimental ones. This indicates the presence of the exchange interaction between the dimers, namely the interaction along the leg.

Characterization of the Phenoxyl Radical in Model Complexes for the CuBSite of CytochromecOxidase: Steady-State and Transient Absorption Measurements, UV Resonance Raman Spectroscopy, EPR Spectroscopy, and DFT Calculations for M-BIAIP

Physicochemical properties of the covalently cross-linked tyrosine-histidine-Cu(B) (Tyr-His-Cu(B)) unit, which is a minimal model complex [M(II)-BIAIPBr]Br (M = Cu(II), Zn(II)) for the Cu(B) site of cytochrome c oxidase, were investigated with steady-state and transient absorption measurements, UV resonance Raman (UVRR) spectroscopy, X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy, and DFT calculations. The pH dependency of the absorption spectra reveals that the pK(a) of the phenolic hydroxyl is ca. 10 for the Cu(II) model complex (Cu(II)-BIAIP) in the ground state, which is similar to that of p-cresol (tyrosine), contrary to expectations. The bond between Cu(II) and nitrogen of cross-linked imidazole cleaves at pH 4.9. We have successfully obtained UVRR spectra of the phenoxyl radical form of BIAIPs and have assigned bands based on the previously reported isotope shifts of Im-Ph (2-(1-imidazoyl)-4-methylphenol) (Aki, M.; Ogura, T.; Naruta, Y.; Le, T. H.; Sato, T.; Kitagawa, T. J. Phys. Chem. A 2002, 106, 3436-3444) in combination with DFT calculations. The upshifts of the phenoxyl vibrational frequencies for 8a (C-C stretching), 7a' (C-O stretching), and 19a, and the Raman-intensity enhancements of 19b, 8b, and 14 modes indicate that UVRR spectra are highly sensitive to imidazole-phenol covalent linkage. Both transient absorption measurements and EPR spectra suggest that the Tyr-His-Cu(B) unit has only a minor effect on the electronic structure of the phenoxyl radical form, although our experimental results appear to indicate that the cross-linked Tyr radical exhibits no EPR. The role of the Tyr-His-Cu(B) unit in the enzyme is discussed in terms of the obtained spectroscopic parameters of the model complex.

ESR and ENDOR investigations of the degenerate electron exchange reactions of various viologens in solution. Solvent dynamical effects

The temperature dependences of the rates of the degenerate electron transfer of various viologens (1,1′-di(hydrocarbyl)-4,4′-bipyridinium salts) are measured in seven different solvents by means of electron spin resonance (ESR) line broadening. Rates vary between 1.7·108 and 1.1·109 M−1s−1 at room temperature and clearly show a solvent dynamical effect, which is inferred from the dependence of the rate constants on the longitudinal relaxation time of the solvent. Activation energies ranging from 5.3 to 24.4 kJ mol−1 are found. For the first time, hyperfine coupling constants are reported for the radical cations of the hydroxyethyl viologen and the amino viologen based on both continuous-wave ESR and electron-nuclear double resonance spectroscopy. Furthermore, the temperature and the solvent dependence of the hyperfine coupling constants of the methyl viologen radical cation are reported.

LET effects following neutron irradiation of lithium formate EPR dosimeters

Lithium formate electron paramagnetic resonance (EPR) dosimeters were irradiated using 60Co γ-rays or fast neutrons to doses ranging from 5 to 20 Gy and investigated by EPR spectroscopy. Using a polynomial fitting procedure in order to accurately analyze peak-to-peak line widths of first derivative EPR spectra, dosimeters irradiated with neutrons had on average 4.4 ± 0.9% broader EPR resonance lines than γ-irradiated dosimeters. The increase in line width was slightly asymmetrical. Computer simulated first derivative polycrystalline EPR spectra of a CO 2 − radical gave very good reconstructions of experimental spectra of irradiated dosimeters. The spectrum simulations could then be used as a tool to investigate the line broadening observed following neutron irradiation. It was shown that an increase in the simulated Lorentzian line width could explain both the observed line broadening and the asymmetrical effect. The ratio of the peak-to-peak amplitude of first derivative EPR spectra obtained at two different microwave powers (20 and 0.5 mW) was 7.8 ± 1.2% higher for dosimeters irradiated with neutrons. The dependence of the spectrum amplitude on the microwave power was extensively investigated by fitting observations to an analytical non-linear model incorporating, among others, the spin–lattice ( T 1) and spin–spin ( T 2) relaxation times as fitting parameters. Neutron irradiation resulted in a reduction in T 2 in comparison with γ-irradiation, while a smaller difference in T 1 was found. The effects observed indicate increased local radical density following irradiation using high linear energy transfer (LET) neutrons as compared to low LET γ-irradiation. A fingerprint of the LET may thus be found either by an analysis of the line width or of the dependence of the spectrum amplitude on the microwave power. Lithium formate is therefore a promising material for EPR dosimetry of high LET radiation.

Q-Band EPR and ENDOR of Low Temperature X-Irradiated β-d-Fructose Single Crystals

Beta-D-fructose single crystals were in situ X-irradiated at 80 K and measured using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques at Q-band (34 GHz) microwave frequencies. The measurements revealed the presence of at least four carbon-centered radicals stable at 80 K. By means of ENDOR angular variations in the three principal crystallographic planes, six proton hyperfine coupling tensors could be determined and were assigned to four different radicals by the aid of EIE. Two of the radicals exhibit only beta-proton hyperfine couplings and reveal almost identical EIE spectra. For the other two radicals, the major hyperfine splitting originates from a single alpha-proton hyperfine coupling and their EIE spectra were also quite similar. The similarity of the EIE spectra and hyperfine tensors led to the assumption that there are only two essentially different radical structures. The radical exhibiting only beta-proton hyperfine couplings was assigned to a C3 centered radical arising from H3 abstraction and the other radical suggested to be an open-ring species with a disrupted C2-C3 bond and a double C2-O2 bond. A possible formation mechanism for the latter open-ring radical is presented. By means of cluster density functional theory (DFT) calculations, the structures of the two radicals were determined and a fairly good agreement between the calculated and experimental hyperfine tensors was found.

Electron Paramagnetic Resonance Spectroscopy Studies of Oxidative Degradation of an Active Pharmaceutical Ingredient and Quantitative Analysis of the Organic Radical Intermediates Using Partial Least-Squares Regression

Electron paramagnetic resonance (EPR) spectroscopy was used to study the radical species formed during the oxidation of an active pharmaceutical ingredient in the solid state. It was found that the extent of radical generation correlated to the formation of an oxidative degradation product. Multifrequency EPR and electron nuclear double resonance spectroscopy gave additional information on the identity of the organic radical species involved in the oxidation process, and a mechanism was proposed for the degradation, involving the formation of both carbon-centered and peroxy radicals. The multivariate analysis technique of partial least-squares (PLS) regression was then used to determine the extent of oxidation of the active pharmaceutical ingredient from the EPR spectra. The suitability of this approach was demonstrated from its application to a series of standards. The conventional approach for the quantitative analysis of EPR spectra is to measure the peak height or to perform double integration of the spectral region containing the signal of interest. Both of these methods have intrinsic errors associated with them, particularly for weak EPR signals with a poor signal-to-noise ratio or a sloping background response. The results obtained showed that greatly improved quantitation was obtained using the PLS regression approach.

Effect of surfactant coating on magnetic properties of Fe 3O 4 nanoparticles: ESR study

Magnetic properties of surfactant-coated and uncoated superparamagnetic iron oxide nanoparticles, Fe 3O 4 (SPION) were investigated by electron spin resonance (ESR) technique. For all samples, a strong and broad single ESR signal has been observed at all temperatures. A strong temperature dependence of ESR linewidth and resonance field is observed. Also, there is a strong effect of surfactant coating on magnetic properties of Fe 3O 4 nanoparticles. While the resonance field is decreasing by coating, the linewidth of the ESR spectra is increasing. These changes in resonance field and the linewidth are attributed to the decrease in effective magnetic moment due to a non-collinear spin structure originated from the pinning of the surface spins and coated surfactant at the interface of nanoparticles. Also, the changes are due to the contribution of the volume of the diamagnetic coating mass to the sample volume.

Effect of gamma radiation on amlodis and its potential for radiosterilization

In the present work, radiation sensitivity of amlodis (AML) and its active ingredient Amlodipin Besylate (AML-B) were separately investigated by electron spin resonance (ESR) spectroscopy using radiolytic products induced in these drugs. Irradiation in the dose range of 2.5–25 kGy did not create any ESR resonance line in AML-B, but it create five characteristic ESR resonance lines associated with more than one radical species in the case of AML. This signal is attributed to the radical species created upon irradiation of inactive ingredients such as microcrystalline cellulose and sodium starch glycolate of AML. Five resonance lines were observed to be divided into three sub groups of different characteristic behaviors associable with three different radical species. Radical species responsible from observed ESR lines were unstable at room and above room temperatures, however, they conserved their identities over a storage period of 92 days. This permitted to discriminate irradiated AML from unirradiated one. A quadratic function was found to describe best the variations of the intensities of observed resonance lines with applied radiation dose. A model based on three tentative radical species with a pyranose ring formed by the rapture of C H bonds in positions 1 and 4 was proposed to explain the observed five lines experimental ESR spectra. AML was considered not providing the characteristic features of a good dosimetric material due to its low radiation yield and relatively fast decays of the created radical species, but very low radiation sensitivity of its active ingredient, namely AML-B makes AML a good candidate for radiosterilization.

Magnetic phase coexistence in CMR manganites: ESR evidence

The coexistence of magnetic phases in colossal magnetoresistant (CMR) manganites is characteristic of this family of compounds. We studied this phenomenon in a hole doped sample of La 0.75Ca 0.25MnO 3 by means of magnetization measurements and electron spin resonance (ESR) technique. The magnetic transition at T c=220 K was determined as the maximum slope in the M( T) curve. Above 250 K the ESR spectrum consists of a single lorentzian line centered at g=1.99(1). The ESR resonance splits below 250 K and a second resonance, of FM character, is present with a T dependent resonance field ( H r). When T decreases, H r diminishes for the FM line while the PM line remains essentially centered at a constant H r. At the same time, a continuous transference of intensity takes place, from the PM line to the FM one. We observed the coexistence of both, PM and FM lines, down to 220 K. In this extended range of coexistence (250–220 K) the magnetization shows also a peculiar behavior.

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water.

We have examined enzymes in nearly anhydrous organic solvents spanning a wide range of dielectric constants using a combination of electron paramagnetic resonance (EPR) spectroscopy, molecular dynamics simulations, high-pressure kinetic studies and the electrostatic model of Kirkwood. This approach enabled us to investigate the relationship between catalytic activity, protein flexibility and solvent polarity for an enzymatic reaction proceeding through a highly polar transition state in the near absence of water. Further insights into water-protein interactions and the involvement of water in enzyme structure and function have been obtained by EPR and multinuclear nuclear magnetic resonance studies of enzymes suspended and immobilized in organic solvents with and without added water. In these systems, correlations were observed between the water content and enzyme activity, flexibility, and active-site polarity, although the structural properties of suspended and immobilized enzymes differed markedly. These results have helped to elucidate the role of water in molecular events at the enzymic active site leading to improved biocatalysis in low-water environments.

In vivo spin trapping of nitric oxide.

The measurement of nitric oxide (NO) in biological samples has normally required destructive chemical techniques. The ability to detect NO non-invasively in living animals or excised organs has great potential using specialized electron paramagnetic resonance (EPR) methods. Although NO is paramagnetic, it cannot be observed directly unless it is complexed with ferrous iron-dithiocarbamate ligand spin trap complexes. Despite the minimally invasive nature of the technique, highly sensitive localized concentrations of NO may be observed ("trapped") in vivo by both L-band EPR and magnetic resonance imaging.