Unique Electron Paramagnetic Resonance Research Articles

Iron level changes in the brain with neurodegenerative disease

Nanosized magnetite inside ferritin may control the progression of neurodegenerative disease (ND) by creating an electronic noise in the neural cells. We discovered that brains with ND have a unique electron paramagnetic resonance (EPR) spectrum. Our measurements showed that the collapse of the broad ferritin maximum contained in the EPR spectra possibly relates to the onset and progression of the ND. Ferritin malfunction triggers the perturbation of iron concentration that either increases or decreases over the normal levels in brain without ND. This supports a conjecture that accumulated iron results in an increased volume of magnetite crystals, whose fluctuated magnetic moments may interfere with the normal function of neural synapses and contribute to the neurodegenerative disease. The mechanism of the iron mobility relates to iron canals in the neural cell's membrane by which the iron enters and leaves the neural cells. This gate keeper malfunction may relate to a speculation that this is due to the appearance of 2Fe-2S in EPR spectra of brains with ND. Statement of significanceIn this manuscript we describe feedback between electronic structure of atoms in the brain, easiness of becoming magnetized in a magnetic field and the ability of the brain to hold the magnetic field on its own in cases of neurodegenerative-diseased and healthy brain. This contribution is novel and significant for a number of reasons, as follows: We revealed that diseased brains have a distinct electronic structure from healthy brains. We identified the easiness of brain samples to become magnetized in a magnetic field and the brains’ ability to hold the magnetic field on its own in cases of neurodegenerative-diseased and healthy brains. This paper addresses a new hypothesis, and we consider that it will generate broad that may be of broad interdisciplinary interest and generate further debate.

Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A MnIII(μ-O)(μ-OH)FeIII species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the MnIII(μ-O)(μ-OH)FeIII state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded MnIII(μ-OH)FeIII species. This final intermediate precedes formation of the MnIII/FeIII resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.

Observation and deconvolution of a unique EPR signal from two cocrystallized spin triangles.

A 16-line pattern has been theoretically predicted, but hitherto not reported, for the Electron Paramagnetic Resonance (EPR) spectrum of antiferromagnetically coupled CuII triangles experiencing isotropic exchange of isosceles magnetic symmetry. Now, the crystallization of such a triangular species and its X-ray structure determination in a polar space group, R3 (No. 146), has enabled its single crystal EPR study. Its detailed magnetic susceptibility, and X- and Q-band, powder and single crystal EPR spectroscopic study reveals the effect of molecular structure and of Dzyaloshinskii-Moriya interactions (DMI) on the g‖, g⊥ and A‖ parameters of the spectrum; DMI is considered for the first time in such a context. Moreover, careful analysis of the spectrum allows the deconvolution of two slightly different cocrystallized magnetic species.

Effect of Al3+/Si4+ codoping on the structural, optoelectronic and UV sensing properties of ZnO

Abstract

Electron Paramagnetic Resonance Spectroscopy as a Probe of Hydrogen Bonding in Heme-Thiolate Proteins.

Despite utilizing a common cofactor binding motif, hemoproteins bearing a cysteine-derived thiolate ligand (heme-thiolate proteins) are involved in a diverse array of biological processes ranging from drug metabolism to transcriptional regulation. Though the origin of heme-thiolate functional divergence is not well understood, growing evidence suggests that the hydrogen bonding (H-bonding) environment surrounding the Fe-coordinating thiolate influences protein function. Outside of X-ray crystallography, few methods exist to characterize these critical H-bonding interactions. Electron paramagnetic resonance (EPR) spectra of heme-thiolate proteins bearing a six-coordinate, Fe(III) heme exhibit uniquely narrow low-spin (S = 1/2), rhombic signals, which are sensitive to changes in the heme-thiolate H-bonding environment. To establish a well-defined relationship between the magnitude of g-value dispersion in this unique EPR signal and the strength of the heme-thiolate H-bonding environment, we synthesized and characterized of a series of six-coordinate, aryl-thiolate-ligated Fe(III) porphyrin complexes bearing a tunable intramolecular H-bond. Spectroscopic investigation of these complexes revealed a direct correlation between H-bond strength and g-value dispersion in the rhombic EPR signal. Using density functional theory (DFT), we elucidated the electronic origins of the narrow, rhombic EPR signal in heme-thiolates, which arises from an Fe-S pπ-dπ bonding interaction. Computational analysis of the intramolecularly H-bonded heme-thiolate models revealed that H-bond donation to the coordinating thiolate reduces thiolate donor strength and weakens this Fe-S interaction, giving rise to larger g-value dispersion. By defining the relationship between heme-thiolate electronic structure and rhombic EPR signal, it is possible to compare thiolate donor strengths among heme-thiolate proteins through analysis of low-spin, Fe(III) EPR spectra. Thus, this study establishes EPR spectroscopy as a valuable tool for exploring how second coordination sphere effects influence heme-thiolate protein function.

Uncovering the Role of Oxygen Atom Transfer in Ru-Based Catalytic Water Oxidation.

The realization of artificial photosynthesis carries the promise of cheap and abundant energy, however, significant advances in the rational design of water oxidation catalysts are required. Detailed information on the structure of the catalyst under reaction conditions and mechanisms of O-O bond formation should be obtained. Here, we used a combination of electron paramagnetic resonance (EPR), stopped flow freeze quench on a millisecond-second time scale, X-ray absorption (XAS), resonance Raman (RR) spectroscopy, and density functional theory (DFT) to follow the dynamics of the Ru-based single site catalyst, [RuII(NPM)(4-pic)2(H2O)]2+ (NPM = 4-t-butyl-2,6-di(1',8'-naphthyrid-2'-yl)pyridine, pic = 4-picoline), under the water oxidation conditions. We report a unique EPR signal with g-tensor, gx = 2.30, gy = 2.18, and gz = 1.83 which allowed us to observe fast dynamics of oxygen atom transfer from the RuIV═O oxo species to the uncoordinated nitrogen of the NPM ligand. In few seconds, the NPM ligand modification results in [RuIII(NPM-NO)(4-pic)2(H2O)]3+ and [RuIII(NPM-NO,NO)(4-pic)2]3+ complexes. A proposed [RuV(NPM)(4-pic)2═O]3+ intermediate was not detected under the tested conditions. We demonstrate that while the proximal base might be beneficial in O-O bond formation via nucleophilic water attack on an oxo species as shown by DFT, the noncoordinating nitrogen is impractical as a base in water oxidation catalysts due to its facile conversion to the N-O group. This study opens new horizons for understanding the real structure of Ru catalysts under water oxidation conditions and points toward the need to further investigate the role of the N-O ligand in promoting water oxidation catalysis.

Spectroscopic analysis of catalytic water oxidation by [Ru(II)(bpy)(tpy)H2O]2+ suggests that Ru(V)═O is not a rate-limiting intermediate.

Modern chemistry's grand challenge is to significantly improve catalysts for water splitting. Further progress requires detailed spectroscopic and computational characterization of catalytic mechanisms. We analyzed one of the most studied homogeneous single-site Ru catalysts, [Ru(II)(bpy)(tpy)H2O](2+) (where bpy = 2,2'-bipyridine, tpy = 2,2';6',2″-terpyridine). Our results reveal that the [Ru(V)(bpy)(tpy)═O](3+) intermediate, reportedly detected in catalytic mixtures as a rate-limiting intermediate in water activation, is not present as such. Using a combination of electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy, we demonstrate that 95% of the Ru complex in the catalytic steady state is of the form [Ru(IV)(bpy)(tpy)═O](2+). [Ru(V)(bpy)(tpy)═O](3+) was not observed, and according to density functional theory (DFT) analysis, it might be thermodynamically inaccessible at our experimental conditions. A reaction product with unique EPR spectrum was detected in reaction mixtures at about 5% and assigned to Ru(III)-peroxo species with (-OOH or -OO- ligands). We also analyzed the [Ru(II)(bpy)(tpy)Cl](+) catalyst precursor and confirmed that this molecule is not a catalyst and its oxidation past Ru(III) state is impeded by a lack of proton-coupled electron transfer. Ru-Cl exchange with water is required to form active catalysts with the Ru-H2O fragment. [Ru(II)(bpy)(tpy)H2O](2+) is the simplest representative of a larger class of water oxidation catalysts with neutral, nitrogen containing heterocycles. We expect this class of catalysts to work mechanistically in a similar fashion via [Ru(IV)(bpy)(tpy)═O](2+) intermediate unless more electronegative (oxygen containing) ligands are introduced in the Ru coordination sphere, allowing the formation of more oxidized Ru(V) intermediate.

ZnO Oxygen Vacancies Formation and Filling Followed by in Situ Photoluminescence and in Situ EPR

Oxygen vacancies of zinc oxide were followed by photoluminescence (PL) and electron paramagnetic resonance (EPR) spectroscopies. The green PL emission was associated with oxygen vacancies: its intensity is enhanced upon static thermal treatment under inert or under vacuum, whereas it decreases upon oxygen treatment. A unique EPR signal at g = 1.96 was measured at room temperature after thermal in situ treatment under flow of inert or oxygenated atmospheres, its double integration follows the same trends than the green PL emission and its evolution was shown to probe the oxygen vacancy concentrations. The relative concentration of the related paramagnetic species would be increased/decreased upon trapping/release of the electron associated to the formation/filling of oxygen vacancy. The influence of Ti impurities on the PL and RPE signals was investigated. Finally, it is concluded that the EPR signal is related to oxygen vacancies and its position shift could be explained by the involvement of some mixing ...

Electron paramagnetic resonance as a sensitive tool to assess the iron oxide content in cells for MRI cell labeling studies

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.

Characterization of Two Different Five-Coordinate Soluble Guanylate Cyclase Ferrous–Nitrosyl Complexes

Soluble guanylate cyclase (sGC), a hemoprotein, is the primary nitric oxide (NO) receptor in higher eukaryotes. The binding of NO to sGC leads to the formation of a five-coordinate ferrous-nitrosyl complex and a several hundred-fold increase in cGMP synthesis. NO activation of sGC is influenced by GTP and the allosteric activators YC-1 and BAY 41-2272. Electron paramagnetic resonance (EPR) spectroscopy shows that the spectrum of the sGC ferrous-nitrosyl complex shifts in the presence of YC-1, BAY 41-2272, or GTP in the presence of excess NO relative to the heme. These molecules shift the EPR signal from one characterized by g 1 = 2.083, g 2 = 2.036, and g 3 = 2.012 to a signal characterized by g 1 = 2.106, g 2 = 2.029, and g 3 = 2.010. The truncated heme domain constructs beta1(1-194) and beta2(1-217) were compared to the full-length enzyme. The EPR spectrum of the beta2(1-217)-NO complex is characterized by g 1 = 2.106, g 2 = 2.025, and g 3 = 2.010, indicating the protein is a good model for the sGC-NO complex in the presence of the activators, while the spectrum of the beta1(1-194)-NO complex resembles the EPR spectrum of sGC in the absence of the activators. Low-temperature resonance Raman spectra of the beta1(1-194)-NO and beta2(1-217)-NO complexes show that the Fe-NO stretching vibration of the beta2(1-217)-NO complex (535 cm (-1)) is significantly different from that of the beta1(1-194)-NO complex (527 cm (-1)). This shows that sGC can adopt different five-coordinate ferrous nitrosyl conformations and suggests that the Fe-NO conformation characterized by this unique EPR signal and Fe-NO stretching vibration represents a highly active sGC state.

New Insights into the Interactions of Serum Proteins with Bis(maltolato)oxovanadium(IV):  Transport and Biotransformation of Insulin-Enhancing Vanadium Pharmaceuticals

Significant new insights into the interactions of the potent insulin-enhancing compound bis(maltolato)oxovanadium(IV) (BMOV) with the serum proteins, apo-transferrin and albumin, are presented. Identical reaction products are observed by electron paramagnetic resonance (EPR) with either BMOV or vanadyl sulfate (VOSO4) in solutions of human serum apo-transferrin. Further detailed study rules out the presence of a ternary ligand-vanadyl-transferrin complex proposed previously. By contrast, differences in reaction products are observed for the interactions of BMOV and VOSO4 with human serum albumin (HSA), wherein adduct formation between albumin and BMOV is detected. In BMOV-albumin solutions, vanadyl ions are bound in a unique manner not observed in comparable solutions of VOSO4 and albumin. Presentation of chelated vanadyl ions precludes binding at the numerous nonspecific sites and produces a unique EPR spectrum which is assigned to a BMOV-HSA adduct. The adduct species cannot be produced, however, from a solution of VOSO4 and HSA titrated with maltol. Addition of maltol to a VOSO4-HSA solution instead results in formation of a different end product which has been assigned as a ternary complex, VO(ma)(HSA). Furthermore, analysis of solution equilibria using a model system of BMOV with 1-methylimidazole (formation constant log K1 = 4.5(1), by difference electronic absorption spectroscopy) lends support to an adduct binding mode (VO(ma)2-HSA) proposed herein for BMOV and HSA. This detailed report of an in vitro reactivity difference between VOSO4 and BMOV may have bearing on the form of active vanadium metabolites delivered to target tissues. Albumin binding of vanadium chelates is seen to have a potentially dramatic effect on pharmacokinetics, transport, and efficacy of these antidiabetic chelates.

Electron paramagnetic resonance analysis of liver and serum in Sprague-Dawley rats exposed subchronically to coplanar pentachlorobiphenyl-congener 3,3',4,4',5

A unique electron paramagnetic resonance (EPR) signal was found at g = 2.49 at 77 K in the liver tissues or microsomes of Sprague–Dawley rats that were exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by the oral route. This unique EPR signal was not detected in the liver excised from rats treated with carbon tetrachloride or 3-methyl-cholanthrene. The EPR signal intensity increased in proportion to the dose; no gender-specific difference was noted and the EPR signal at g = 2.49 may have originated from heme-iron in cytochrome P450. Although, several serum parameters of the PCB126-treated rats were slightly higher than those of the control, PCB126 does not induce hepatic dysfunction at the dose range used in this study. 3,3′,4,4′,5-Pentachlorobiphenyl was given daily via the oral route to pregnant rats. The rats were reared until Day 22 of pregnancy, after which they were killed and EPR signals were obtained from the fetal livers. A signal at g = 2.49 was observed for each fetal liver. These results indicate that the EPR signal at g = 2.49 could be appropriate as a biomarker of exposure to PCB126 or TCDD in the situation where hepatic dysfunction is not detected.

Role for the nitrogenase MoFe protein α-subunit in FeMo-cofactor binding and catalysis

Two components constitute Mo-dependent nitrogenase (EC 1.18.6.1)--the Fe protein (a homodimer encoded by nifH) and the MoFe protein (an alpha 2 beta 2 tetramer encoded by nifDK). The MoFe protein provides the substrate-binding site and probably contains six prosthetic groups of two types--four Fe-S centres and two Fe- and Mo-containing cofactors. To determine the distribution and catalytic function of these metalloclusters, we and others are attempting to change the catalytic and spectroscopic features of nitrogenase by substituting specific amino acids targeted as potential metallocluster ligands, particularly those to the FeMo-cofactor, which is responsible for the biologically unique electron paramagnetic resonance signal (S = 3/2) of nitrogenase, and is believed to be the N2-reducing site. Here we describe mutant strains of Azotobacter vinelandii that have single amino-acid substitutions within the MoFe protein alpha-subunit. These substitutions alter both substrate-reduction properties and the unique electron paramagnetic resonance signal, indicating that the FeMo-cofactor is associated with both the alpha-subunit and the substrate-reducing site.

Nitric oxide adducts of the binuclear iron site of hemerythrin: spectroscopy and reactivity.

Nitric oxide forms adducts with the binuclear iron site of hemerythrin (Hr) at [Fe(II),Fe(II)]deoxy and [Fe(II),Fe(III)]semimet oxidation levels. With deoxyHr our results establish that (i) NO binds reversibly, forming a complex which we label deoxyHrNO, (ii) NO forms a similar but distinct complex in the presence of fluoride, which we label deoxyHrFNO, (iii) NO is directly coordinated to one iron atom of the binuclear pair in these adducts, most likely in a bent end-on fashion, and (iv) the iron atoms in the binuclear sites of both deoxyHrNO and deoxyHrFNO are antiferromagnetically coupled, thereby generating unique electron paramagnetic resonance (EPR) detectable species. The novel EPR signal of deoxyHrNO (deoxyHrFNO) with g[[ = 2.77 (2.58) and g = 1.84 (1.80) is explained by the magnetic interaction of the Fe(II) (S' = 2) and [FeNO]7 (S = 3/2) centers observed by Mössbauer spectroscopy. Antiferromagnetic coupling leads to a ground state of Seff = 1/2. Analysis of the EPR parameters using the isotropic spin-exchange Hamiltonian, Hex = 2JS3/2.S2, and including zero-field splitting leads to a coupling constant, -J approximately 23 cm-1, for deoxyHrNO. The resonance Raman spectrum of deoxyHrNO shows features at 433 and 421 cm-1 that shift downward with 15N16O and that are assigned to stretching and bending modes, respectively, of the [FeNO]7 unit. Sensitivity of the bending mode to D2O suggests that bound NO participates in hydrogen bonding. We propose that the terminal oxygen atom of NO is hydrogen bonded to the proton of the mu-hydroxo bridge in the Fe-(OH)-Fe unit. A bent Fe-N-O geometry is supported by spectroscopic and structural comparisons to synthetic complexes and is consistent with a limiting [FeII,FeIIINO-] formulation for deoxyHrNO. Reversibility of NO binding to deoxyHr is demonstrated by bleaching of the optical and EPR spectra of deoxyHrNO upon additions of excess N3- or CNO-. DeoxyHrNO undergoes autoxidation under anaerobic conditions over the course of several hours. The product of this autoxidation appears to be an EPR-silent NO adduct of semimetHr. The formal one-electron oxidations of the binuclear iron site of deoxyHr by NO and by HNO2 can conceivably occur with no net change in charge on the iron site. In contrast, autoxidation of oxy- to metHr requires a change in net charge on the iron site, which may provide a kinetic barrier.

Saturation transfer spectroscopy: signals sensitive to very slow molecular reorientation

A density matrix-transition rate matrix formalism is employed to compute the eight unique electron paramagnetic resonance signals at the first two harmonics of the applied Zeeman modulation. Calculations are carried out for conditions appropriate for investigating spin-labeled biomolecules with partially saturating microwave fields and Zeeman modulation amplitudes comparable to resonance linewidths. Spectra are computed for rotational correlation times ranging from 10 −7 to 10 −3 s and for modulation frequencies of 50 and 100 kHz. These simulations indicate that of the eight signals the in-phase dispersion signal at the first harmonic of the modulation and the out-of-phase dispersion signal at the first harmonic afford the best sensitivity to molecular motion and the largest signal amplitudes. It is suggested that study of these signals is the method of choice for monitoring slowly tumbling spin labels when signal-to-noise considerations are critical. The conclusions derived from computer simulations are borne out by experimental measurements performed on 10 −3 M solutions of the steroid spin label 17β-hydroxy-4′,4′-dimethylspiro-[5α-androstrance-3,2′-oxazolidin]-3′-oxyl in sec-butylbenzene.

Possible higher valence states of cytochrome P-450 during oxidative reactions

The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.

Anion Binding to Bovine Erythrocyte Superoxide Dismutase: EVIDENCE FOR MULTIPLE BINDING SITES WITH QUALITATIVELY DIFFERENT PROPERTIES

Abstract The binding of azide, thiocyanate, cyanate, and cyanide to bovine erythrocyte superoxide dismutase has been studied by means of optical spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and nuclear magnetic relaxation dispersion (NMRD). The results show that there are at least four anion binding sites on the protein, and these are divided equally into two classes. One of these classes of binding sites strongly binds 2 moles of azide producing an altered EPR spectrum, no change in the NMRD, and only a minor change in the visible optical spectrum. These two binding sites are considered to be the Zn2+ ions of the protein. The other class of binding sites binds 2 moles of cyanide, lowering the NMRD to the diamagnetic level, and producing substantial changes in the optical and EPR spectra. These two binding sites are considered to be the 2 Cu2+ ions of the protein. Thiocyanate and cyanate appear to bind only to the Zn2+, having no effect on the NMRD and minor effects on the optical spectrum, although each ion produces a characteristic EPR spectrum. Addition of excess azide to the protein results in the appearance of an intense charge transfer band near 373 nm, produces minor changes in the 2-azide complex EPR spectrum and lowers the NMRD to the diamagnetic level. The presence of excess cyanide produces an absorption band at 530 nm and a unique EPR spectrum (Rotilio, G., Finazzi Agro, A., Calabrese, L., Bossa, F., Guerrieri, P., and Mandovi, B. (1971) Biochemistry, 10, 611). The data have been rationalized in terms of a model in which zinc and copper are relatively close to each other in the protein, perhaps existing in a ligand-bridged bimetal complex.