Electron Paramagnetic Spectroscopy Research Articles

Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study.

BackgroundThe growth and development of plants is deleteriously affected by various biotic and abiotic stress factors. Wounding in plants is caused by exposure to environmental stress, mechanical stress, and via herbivory. Typically, oxidative burst in response to wounding is associated with the formation of reactive oxygen species, such as the superoxide anion radical (O2•−), hydrogen peroxide (H2O2) and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O2•− formation in plant tissues have been performed using various techniques including electron paramagnetic resonance spin-trap spectroscopy, epinephrine-adrenochrome acceptor methods, staining with dyes such as tetrazolium dye and nitro blue tetrazolium (NBT); however, kinetic measurements have not been performed. In the current study, we provide evidence of O2•− generation and its kinetics in the leaves of spinach (Spinacia oleracea) subjected to wounding.MethodsReal-time monitoring of O2•− generation was performed using catalytic amperometry. Changes in oxidation current for O2•− was monitored using polymeric iron-porphyrin-based modified carbon electrodes (φ = 1 mm) as working electrode with Ag/AgCl as the reference electrode.ResultThe results obtained show continuous generation of O2•− for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O2•− to H2O2, significantly suppressed the oxidation current.ConclusionCatalytic amperometric measurements were performed using polymeric iron-porphyrin based modified carbon electrode. We claim it to be a useful tool and a direct method for real-time monitoring and precise detection of O2•− in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O2•−.

Crystal structure and functional analysis of Leishmania major pseudoperoxidase.

Leishmania major pseudoperoxidase (LmPP) is a recently discovered heme protein expressed by the human pathogen. Previous in vivo and in vitro studies suggest that LmPP is a crucial element of the pathogen's defense mechanism against the reactive nitrogen species peroxynitrite produced during the host immune response. To shed light on the potential mechanism of peroxynitrite detoxification, we have determined the 1.76-Å X-ray crystal structure of LmPP, revealing a striking degree of homology with heme peroxidases. The most outstanding structural feature is a Cys/His heme coordination, which corroborates previous spectroscopic and mutagenesis studies. We also used a combination of stopped-flow and electron paramagnetic spectroscopies that together suggest that peroxynitrite is not a substrate for LmPP catalysis, leaving the function of LmPP an open question.

Revisiting the mechanism of hexavalent chromium ion reduction: The parallel photodecomposition and photocatalytic reduction of chromate(VI) ester

The formation and homogeneous photodecomposition of chromate(VI) ester are often overlooked during the studies of photocatalytic reduction of hexavalent chromium, Cr(VI), in the presence of sacrificial electron donors such as methanol. When photoexcited (λ≤525nm), the chromate(VI) ester, formed by the spontaneous reaction of aqueous Cr(VI) with methanol, undergoes homogeneous stepwise decomposition to trivalent chromium, Cr(III). The addition of semiconductor photocatalysts, i.e., TiO2 and WO3, at relatively low concentrations (<0.5gL−1) was detrimental to the overall rate of Cr(VI) reduction. This was traced to the occurrence of interfacial electron injection from chromate ester to the photocatalyst conduction band, that in turn interrupted inner sphere electron transfer, i.e., photodecomposition. Since it is an interfacial process, the extent of suppression of Cr(VI) reduction is more significant for highly adsorbing TiO2 than on weakly adsorbing WO3. At the same time, even though chromate ester adsorbs strongly on Al2O3, the interfacial electron injection to the insulator particle was unfavorable, and hence no suppression in Cr(VI) reduction was observed. When the TiO2 photocatalyst concentration was increased to 0.5gL−1 and beyond, the photocatalytic reduction path became dominant. Further increase in the photocatalyst concentration saw the linear dependency in the apparent rate of Cr(VI) reduction, as expected for reaction in the non-mass diffusion and non-light scattering limited region. As followed by the electron paramagnetic spectroscopy, both the photodecomposition and photocatalytic reactions follow the stepwise reduction from Cr(VI) to Cr(V) and eventually to Cr(III). For non-photoexcited TiO2 (λ≥420nm), suppression in the rate of Cr(VI) reduction was observed at all concentrations tested. Importantly, the work showcases the parallel but non-independent effects of homogeneous photodecomposition of chromate(VI) ester and its photocatalytic reduction over semiconductor photocatalysts.

Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2

The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol.

In vivo electron paramagnetic resonance oximetry and applications in the brain.

Molecular oxygen (O2) is essential to brain function and mechanisms necessary to regulate variations in delivery or utilization of O2 are crucial to support normal brain homeostasis, physiology and energy metabolism. Any imbalance in cerebral tissue partial pressure of O2 (pO2) levels may lead to pathophysiological complications including increased reactive O2 species generation leading to oxidative stress when tissue O2 level is too high or too low. Accordingly, the need for oximetry methods, which assess cerebral pO2 in vivo and in real time, is imperative to understand the role of O2 in various metabolic and disease states, including the effects of treatment and therapy options. In this review, we provide a brief overview of the common in vivo oximetry methodologies for measuring cerebral pO2. We discuss the advantages and limitations of oximetry methodologies to measure cerebral pO2 in vivo followed by a more in-depth review of electron paramagnetic resonance oximetry spectroscopy and imaging using several examples of current electron paramagnetic resonance oximetry applications in the brain.

Effect of circular permutation on the structure and function of type 1 blue copper center in azurin.

Type 1 copper (T1Cu) proteins are electron transfer (ET) proteins involved in many important biological processes. While the effects of changing primary and secondary coordination spheres in the T1Cu ET function have been extensively studied, few report has explored the effect of the overall protein structural perturbation on active site configuration or reduction potential of the protein, even though the protein scaffold has been proposed to play a critical role in enforcing the entatic or "rack-induced" state for ET functions. We herein report circular permutation of azurin by linking the N- and C-termini and creating new termini in the loops between 1st and 2nd β strands or between 3rd and 4th β strands. Characterization by electronic absorption, electron paramagnetic spectroscopies, as well as crystallography and cyclic voltammetry revealed that, while the overall structure and the primary coordination sphere of the circular permutated azurins remain the same as those of native azurin, their reduction potentials increased by 18 and 124 mV over that of WTAz. Such increases in reduction potentials can be attributed to subtle differences in the hydrogen-bonding network in secondary coordination sphere around the T1Cu center.

Cu(II)-amino acid–CaAl-layered double hydroxide complexes, recyclable, efficient catalysts in various oxidative transformations

Intercalated composite materials were prepared with CaAl-layered double hydroxide as host and Cu(II)-amino acid (l-cysteine, l-histidine and l-tyrosine) complex anions as guests. Two methods (intercalation of the ligand first followed by constructing the complex; preforming the complex first, then introducing it among the layers of the host) and optimization of the synthesis conditions were performed to obtain composites having the complex exclusively among the layers. The composite materials were structurally characterized by powder X-ray diffractometry, mid infrared (IR) spectroscopy with ATR (attenuated total reflectance) or photoacoustic detections, transmission and scanning electron microscopies and X-ray photoelectron spectroscopy. Structural features of the intercalant (coordination number, coordination sites) were elucidated by classical chemical and energy dispersive X-ray analyses, EPR (electron paramagnetic spectroscopy), X-ray absorption and far IR spectroscopies. Structural models based on these methods are also given. Catalytic activities, selectivities and recycling abilities of the substances were studied in the oxidation reactions of cyclohexene with peracetic acid and in situ formed iodosylbenzene as oxidants in the liquid phase. The catalysts were active in the Ullmann coupling reaction as well. The intercalated substances were found to be efficient and highly selective catalysts with very good recycling abilities.

Editorial: Optogenetic Tools in the Molecular Spotlight.

Editorial: Optogenetic Tools in the Molecular Spotlight.

Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.

Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.

Singlet oxygen production in Chlamydomonas reinhardtii under heat stress.

In the current study, singlet oxygen formation by lipid peroxidation induced by heat stress (40 °C) was studied in vivo in unicellular green alga Chlamydomonas reinhardtii. Primary and secondary oxidation products of lipid peroxidation, hydroperoxide and malondialdehyde, were generated under heat stress as detected using swallow-tailed perylene derivative fluorescence monitored by confocal laser scanning microscopy and high performance liquid chromatography, respectively. Lipid peroxidation was initiated by enzymatic reaction as inhibition of lipoxygenase by catechol and caffeic acid prevented hydroperoxide formation. Ultra-weak photon emission showed formation of electronically excited species such as triplet excited carbonyl, which, upon transfer of excitation energy, leads to the formation of either singlet excited chlorophyll or singlet oxygen. Alternatively, singlet oxygen is formed by direct decomposition of hydroperoxide via Russell mechanisms. Formation of singlet oxygen was evidenced by the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl detected by electron paramagnetic resonance spin-trapping spectroscopy and the imaging of green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Suppression of singlet oxygen formation by lipoxygenase inhibitors indicates that singlet oxygen may be formed via enzymatic lipid peroxidation initiated by lipoxygenase.

Comparison of catalytic properties of systems based on nickel complexes with 1,4-diaza-1,3-butadiene ligands in reactions of styrene hydrogenation and ethylene polymerization

Turnover frequencies of catalytic systems based on nickel complexes with 1,4-diaza-1,3-butadiene (α-diimine) ligands in the reactions of styrene hydrogenation and ethylene polymerization were determined. Results are presented of a study by the electron paramagnetic resonance method and IR spectroscopy of 1,4-diaza-1,3-butadiene complexes of nickel(II) and anion radicals formed in the interaction of the starting components under the conditions of catalysis. It was shown that the paramagnetic Ni(I) complexes are precursors of complexes catalytically active in the hydrogenation and polymerization reactions.

Single-Step Synthesis of Halogenated Graphene through Electrochemical Exfoliation and Its Utilization as Electrodes for Zinc Bromine Redox Flow Battery

We have demonstrated a single step synthesis of halogen functionalized graphenes (HGs) through electrochemical exfoliation of graphite in aqueous potassium halide solutions. Few layers of as-synthesized HGs are characterized using powder X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Electron paramagnetic spectroscopy (EPR), Brunauer-Emmett-Teller (BET) surface area analysis, Field emission scanning electron microscopy (FESEM), and Transmission electron microscopy (TEM). The degree of halogenation is found to vary between 2.32 and 0.26 atomic % in fluorinated graphene (FG) and iodinated graphene (IG) respectively, which could be attributed to the difference in reactivity of the halogen species generated during the exfoliation process. A suitable mechanism is proposed in accordance with the exfoliation phenomena and functionalization of halogens on the graphene sheets based on the experimental observation. As synthesized HGs are utilized as electrode materials for zinc bromine flow batteries (ZBB) and the electrocatalytic effect of HGs electrodes for the 2Br−/Br2 redox couple has been investigated in detail. Cyclic voltammetry (CV), and charge-discharge studies were carried out to study the electrochemical performance of the HGs. Among all HGs, FG has shown the superior electrocatalytic behavior for 2Br−/Br2 redox reaction. The anodic (11.2 mA cm−2) and cathodic (10.7 mA cm−2) peak current densities are higher for FG than that of other halogenated graphenes. ZBB flow cell fabricated with FG as bromine electrode exhibits enhanced electrochemical performance in terms of efficiency (81% of voltaic efficiency and 72% energy efficiency) and durability up to 350 cycles.

Low reduction potential cytochrome b5 isotypes of Giardia intestinalis

Despite lacking mitochondria and a known pathway for heme biosynthesis the micro-aerotolerant anaerobic protozoan parasite Giardia intestinalis encodes four members of the cytochrome b5 family of electron transfer proteins, three of which are small, single-domain proteins. While these are similar in size and fold to their better-known mammalian counterparts the Giardia proteins have distinctly lower reduction potentials, ranging from −140 to −171 mV compared to +6 mV for the bovine microsomal protein. This difference is accounted for by a more polar heme environment in the Giardia proteins, as mutation of a conserved heme pocket tyrosine residue to phenylalanine in the Giardia cytochrome b5 isotype-I (gCYTb5-I Y61F) raises its reduction potential by nearly 100 mV. All three isotypes have UV–visible spectra consistent with axial coordination of the heme by a pair of histidine residues, but electron paramagnetic spectroscopy indicates that the planes of their imidazole rings are nearly perpendicular rather than coplanar as observed in mammalian cytochrome b5, which may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia proteins. Although no function has yet to be ascribed to any Giardia cytochrome b5, the presence of similar sequences in many other eukaryotes indicates that these represent an under-characterized class of low reduction potential family members.

Enhanced pharmacological activity of vitamin B₁₂ and penicillin as nanoparticles.

Sonochemistry has become a well-known technique for fabricating nanomaterials. Since one of the advantages of nanomaterials is that they have higher chemical activities compared with particles in the bulk form, efforts are being made to produce nano organic compounds with enhanced biological activities that could be exploited in the medical area. This study uses the sonication technique to prepare nano Vitamin B12 and nano Penicillin, and demonstrates their enhanced biological and pharmacological activity. The size and morphology of the nano Penicillin and nano Vitamin B12 were investigated using electron microscopy as well as dynamic light scattering techniques. The sizes of Penicillin and Vitamin B12 nanoparticles (NPs) were found to be 70 and 120–180 nm, respectively. The bactericidal effect of nano Penicillin was studied and found to be higher than that of the bulk form. Reducing the size of Vitamin B12 resulted in their enhanced antioxidative activity as observed using the electron paramagnetic spectroscopy technique. The penetration depth of these organic NPs can be detected by an optical iterative method. It is believed that nano organic drugs fabrication will have a great impact on the medical field.

N‐Acetyl Cysteine Attenuates Hypoxia Induced Sympathoexcitation in Human Subjects

This investigation tested the hypothesis that central and peripheral reactive oxygen species (ROS) mediate hypoxia induced sympathoexcitation, which is a central feature of the Obstructive Sleep Apnea‐Hypopnea Syndrome (OSAS). 10 healthy human subjects were recruited. One hour prior to experimentation, each subject randomly ingested either 70 mg.kg‐1 of N‐Acetyl Cysteine (NAC, n=5) or vehicle placebo (n=5). ECG, BP, muscle sympathetic nerve activity (MSNA) and plasma ROS and catecholamines were measured. Subjects underwent a 20 minute intermittent hypoxia training (IHT) protocol consisting of cyclical end‐expiratory apneas with 100% N2. Venous blood was analyzed pre/post IHT for ROS by electron paramagnetic spectroscopy and for catecholamines by ELISA. In the placebo group, MSNA was increased between pre vs. post IHT (P=0.01). In the NAC group, however, MSNA was unchanged (P=0.26). NAC reduced the percent change (% Δ) of ROS observed from pre‐ vs. post‐IHT compared to placebo (P=0.02). The % Δ of ROS was directly related to increasing MSNA (R2=0.83, p=0.01). The % Δ of norepinephrine was lower in the NAC vs. placebo group after IHT (P=0.05), whereas the % Δ of epinephrine was unchanged (P=0.40). These data indicate that NAC reduces central sympathetic outflow in response to IHT, and thereby could reduce cardiovascular risk in OSAS patients.

The relationship of physicochemical properties to the antioxidative activity of free amino acids in Fenton system.

Herein we compared antioxidative activities (AA) of 25 free L-amino acids (FAA) against Fenton system-mediated hydroxyl radical (HO(•)) production in aqueous solution, and examined the relation between AA and a set of physicochemical properties. The rank order according to AA was: Trp > norleucine > Phe, Leu > Ile > His >3,4-dihydroxyphenylalanine, Arg > Val > Lys, Tyr, Pro > hydroxyproline > α-aminobutyric acid > Gln, Thr, Ser > Glu, Ala, Gly, Asn, Asp. Sulfur-containing FAA generated different secondary reactive products, which were discriminated by the means of electron paramagnetic resonance spin-trapping spectroscopy. AA showed a general positive correlation with hydrophobicity. However, when taken separately, uncharged FAA exhibited strong positive correlation of AA with hydrophobicity whereas charged FAA showed negative or no significant correlation depending on the scale applied. A general strong negative correlation was found between AA and polarity. Steric parameters and hydration numbers correlated positively with AA of nonpolar side-chain FAA. In addition, a decrease of temperature which promotes hydrophobic hydration resulted in increased AA. This implies that HO(•)-provoked oxidation of FAA is strongly affected by hydrophobic hydration. Our findings are important for the understanding of oxidation processes in natural and waste waters.

The formation of electronically excited species in the human multiple myeloma cell suspension.

In this study, evidence is provided on the formation of electronically excited species in human multiple myeloma cells U266 in the growth medium exposed to hydrogen peroxide (H2O2). Two-dimensional imaging of ultra-weak photon emission using highly sensitive charge coupled device camera revealed that the addition of H2O2 to cell suspension caused the formation of triplet excited carbonyls 3(R = O)*. The kinetics of 3(R = O)* formation in the real time, as measured by one-dimensional ultra-weak photon emission using low-noise photomultiplier, showed immediate enhancement followed by a slow decay. In parallel to the formation of 3(R = O)*, the formation of singlet oxygen (1O2) in U266 cells caused by the addition of H2O2 was visualized by the imaging of 1O2 using the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Additionally, the formation of 1O2 after the addition of H2O2 to cell suspension was detected by electron paramagnetic resonance spin-trapping spectroscopy using 2,2,6,6-tetramethyl-4-piperidone. Presented results indicate that the addition of H2O2 to cell suspension results in the formation of 3(R = O)* and 1O2 in U266 cell suspension. The contribution of the cell-free medium to the formation of electronically excited species was discussed.

Electron paramagnetic resonance imaging for real-time monitoring of Li-ion batteries.

Batteries for electrical storage are central to any future alternative energy paradigm. The ability to probe the redox mechanisms occurring at electrodes during their operation is essential to improve battery performances. Here we present the first report on Electron Paramagnetic Resonance operando spectroscopy and in situ imaging of a Li-ion battery using Li2Ru0.75Sn0.25O3, a high-capacity (>270 mAh g−1) Li-rich layered oxide, as positive electrode. By monitoring operando the electron paramagnetic resonance signals of Ru5+ and paramagnetic oxygen species, we unambiguously prove the formation of reversible (O2)n− species that contribute to their high capacity. In addition, we visualize by imaging with micrometric resolution the plating/stripping of Li at the negative electrode and highlight the zones of nucleation and growth of Ru5+/oxygen species at the positive electrode. This efficient way to locate ‘electron’-related phenomena opens a new area in the field of battery characterization that should enable future breakthroughs in battery research.

Spectroscopic and computational characterization of laccases and their substrate radical intermediates.

Laccases are multicopper oxidases which oxidize a wide variety of aromatic compounds with the concomitant reduction of oxygen to water as by-product. Due to their high stability and biochemical versatility, laccases are key enzymes to be used as eco-friendly biocatalyst in biotechnological applications. The presence of copper paramagnetic species in the catalytic site paired with the substrate radical species produced in the catalytic cycle makes laccases particularly attractive to be studied by spectroscopic approaches. In this review, the potentiality of a combined multifrequency electron paramagnetic spectroscopy /computational approach to gain information on the nature of the catalytic site and radical species is presented. The knowledge at molecular level of the enzyme oxidative process can be of great help to model new enzymes with increased efficiency and robustness.

An ELIC-GLIC Chimera Reveals Distinct Pathways of Activation in the Cys-Loop Family of Receptors

The Cys-loop family of pentameric ligand-gated ion channels is expressed throughout the human brain and is a target for a wide number of therapeutics including local and general anesthetics, alcohols, and neurosteroids. Recently, high-resolution structures of several members of the family have become available, but allosteric mechanisms governing gating and modulation remain unclear. Prokaryotic homologues (ELIC and GLIC) have the same overall structure as the family of ion channels, and are highly amenable to structural studies in a native environment. We investigated the conformational dynamics of ligand-binding and channel gating in GLIC and ELIC by electron paramagnetic spectroscopy (EPR) and found that the two channels exhibit distinct activation mechanisms. To further understand how the two events are coupled, we engineered a functional chimera that included the extracellular domain of ELIC and the transmembrane domain of GLIC. By using a combination of electrophysiology, EPR spectroscopy, and X-ray crystallography we show the molecular details of coupling between the ligand-binding domain and the channel gate.