Tryptophan radicals as reaction intermediates in versatile peroxidases: Multifrequency EPR, ENDOR and density functional theory studies

Versatile peroxidases are a new class of ligninolytic enzymes secreted by fungi from the group of white-rot basidiomycetes. Versatile peroxidase (VP) is a structural hybrid between lignin and manganese peroxidase. This hybrid combines the catalytic properties of the two above peroxidases being able to catalyze Mn(II)-dependent and Mn(II)-independent reactions. A long-range electron transfer mechanism has been inferred for the oxidation of big substrate molecules starting from an exposed tryptophan to the heme cofactor. A neutral tryptophan radical has been identified in VP fromBjerkandera adusta andPleurotus eryngii, after H₂O₂ activation and assigned to a specific tryptophan residue using multifrequency electron paramagnetic resonance and electron-nuclear double resonance spectroscopy. Comparative density functional theory calculations were performed for tryptophan neutral and cation radical species, considering also the effect of the polar environment surrounding the radical. The functional role of the radicals is discussed in the context of mechanistic models for VP.     

乙腈-水和乙二醇-水均相溶液中光诱导杜醌激发三重态与色氨酸、酪氨酸的氢原子转移反应

本文用激光闪光光解技术研究了光诱导生物醌杜醌激发三重态(³DQ^*)和色氨酸(Trp)与酪氨酸(Tyr)在乙腈-水(MeCN-H₂O)及乙二醇-水(EG-H₂O)均相溶液中的光化学反应,分析了反应的机理,并基于Stern-Volmer方法测量了反应速率常数. 光解DQ体系可以生成³DQ^*,³DQ^*与Trp、Tyr发生的氢原子转移反应占主导地位. 对于DQ/Trp/MeCN-H₂O和DQ/Trp/EG-H₂O溶液,³DQ^*与Trp反应生成杜醌中性自由基DQH^·、以碳为中心的色氨酸中性自由基Trp^·/NH和以氮为中心的色氨酸中性自由基Trp/N^·. 对于DQ/Tyr/MeCN-H₂O和DQ/Tyr/EG-H₂O溶液,³DQ^*与Tyr反应生成DQH^·和酪氨酸中性自由基Tyr/O^·. ³DQ^*与Trp、Tyr的氢原子转移反应速率常数都在10⁹ L·mol^-1·s^-1量级,反应近似受扩散控制. MeCN/H₂O均相溶液中³DQ^*与Trp、Tyr的反应速率常数要明显高于EG/H₂O均相溶液中的反应速率常数,这与Stokes-Einstein方程定性一致.     

The interaction between hydrogen peroxide and metal oxides: EPR investigations

The interaction of hydrogen peroxide with various metal oxides has been followed by EPR in the aim of investigating the paramagnetic species formed along the complex processes of H₂O₂ decomposition and interaction with the oxide. Data are reported about three systems (MgO, ZrO₂ and Me²⁺/Y zeolite) which are representative of three classes of oxides (insulators, semiconductors and aluminosilicate molecular sieves). Several radical species have been put into evidence in the various cases. The superoxide O ₂ ^? ion has been observed in all cases while in the case of MgO also the OH radical and the O^? radical ion have been identified. In the case of Zn²⁺/Y zeolite, beside the superoxide radical anion, a new signal is observed which shows up upon treating the sample at 640 K and which is preliminarily assigned to a species in the triplet state in the zeolitic cavity.     

Synthesis, crystal structure, Cu doped EPR and voltammetric studies of bis[N-(2-hydroxyethyl)ethylenediamine]zinc(II) squarate monohydrate

Crystal structure of [Zn(hydet-en)₂]·C₄O₄·H₂O (ZHES) (hydet-en is N-(2-hydroxyethyl)ethylenediamine) complex has been synthesized and characterized by analytical, spectroscopic (IR, UV/Vis) and voltammetric techniques. After doping Cu²⁺ ion, its magnetic environment has been identified by electron paramagnetic resonance (EPR) technique. The title complex crystalizes in monoclinic system with space group P2₁/c and with Z=4. Each hydet-en ligand acts as a tridentate ligand through the two N atoms and the hydroxyl O atom, resulting in a six coordinate Zn(II) ion. The EPR spectra were recorded in three perpendicular planes of Cu²⁺ doped ZHES single crystal. The calculated g and A values indicated that the paramagnetic center is rhombic symmetry with the Cu²⁺ ion having distorted octahedral environment. The molecular orbital bond coefficients of the Cu(II) ion in d⁹ state is also calculated by using EPR and optical absorption parameters. The dianion SQ²⁻ is oxidized reversibly in two consecutive steps to the corresponding radical monoanion and neutral form.     

The FT-EPR spectra of the anthraquinone-2,6-disulfonic acid radicals as probe for local proton concentrations in electron transfer reactions

The radical anion of anthraquinone-2,6-disulfonic acid (2,6-AQDS) and different donor radical cations are formed by electron transfer from various pyrimidines to the laser-induced triplet state of 2,6-AQDS. The electron paramagnetic resonance (EPR) spectrum of the radical anion changes drastically with pH due to different protonated forms of this radical. These EPR spectra were used to define an effective pH value as a measure of the local proton (H₃O⁺) concentration in the surroundings of the 2,6-AQDS radical anion in a unbuffered solution. In some cases this effective pH value was found to be time-dependent reflecting the evolution of the protonation and deprotonation processes of acceptor and donor.     

Radiation effect studies on some glycine derivatives in solid state

Five glycyl derivatives, glycyl-L-histidine, L-alanyl-glycine, glycine hydrochloride, gly-gly hydrochloride and, gly-gly-glycine in powder form were exposed to 20?kGy doses of ⁶⁰Co gamma radiation to study the effects of ionizing radiation. In these compounds, the paramagnetic centers formed after irradiation were attributed to the R─NHCH─?O^?OH, ?H₂CHNH, H₂NCH₂?O^?OHHCl, NH₂?HCONHCH₂COOHHCl and HNCH₂?O^?OH radicals, respectively. The effect of gamma irradiation to the radical structures and time stability of the radicals were investigated by EPR spectroscopy. The spectra were computer simulated and the hyperfine coupling constants were determined.     

Detection of vortex excitations in a Bi<Subscript>2</Subscript>Sr<Subscript>2</Subscript>Ca<Subscript>2</Subscript>Cu<Subscript>3</Subscript>O<Subscript>10</Subscript> crystal at temperatures above the critical temperature by EPR of the surface layer

Vortex excitations have been detected at temperatures both below and above the critical temperature when investigating local magnetic fields on the surface of a Bi₂Sr₂Ca₂Cu₃O₁₀ single crystal by means of an electron paramagnetic resonance (EPR) probe. A thin layer of a diphenyl picrylhydrazyl organic radical deposited on the crystal surface is used as the EPR probe. A narrow EPR signal makes it possible to detect weak distortions of the magnetic field appearing at T ≿ T _c. The analysis of the temperature dependences of the resonance field and the EPR linewidth is thebasis of the assumption of the vortex nature of magnetic excitations in this temperature range.     

EPR and XPS measurements of polymeric catalysts doped with hereopolyacids in oxygen adsorption studies

The goal of our studies was to determine the nature of interactions between catalytic active samples, such as polypyrrole (PPy) doped with: chloride anions, heteropolyacids such as H₅PMo₁₀V₂O₄₀, or H₄SiW₁₂O₄₀, and oxygen particles. In order to reveal the mechanism of the linkage between the catalysts and oxygen we provided the electron paramagnetic resonance (EPR) measurements of synthesized samples in O₂ and N₂ flow. Moreover, the X-ray photoelectron spectroscopy (XPS) measurements were performed to show the properties of doped heteropolyacids and the state of nitrogen in polypyrrole matrix. The results of EPR studies of PPy(Cl), PPy(H₄SiW₁₂O₄₀), PPy(H₅PMo₁₀V₂O₄₀) samples, show that the polypyrrole doped with chloride anions interacts much easier with oxygen than polypyrrole doped with heteropolyacids (in the above given order, of preference with the most active polypyrrole system first). It correlates with XPS results, which show that a molybdenium-vanadium anion is more reduced than a silicon-tungsten one. The redox reactions of heteropolyacids involve the oxidation-reduction of the conjugated polymer chain, leaving no or little place for interactions with oxygen. Polypyrrole doped with H₄SiW₁₂O₄₀ shows some oxygen sensitivity as observed in the EPR studies, as opposed to polypyrrole doped with H₅PMo₁₀V₂O₄₀, which is in line with the XPS results.     

EPR and optical absorption studies of Cu<Superscript>2+</Superscript> ions in [ZnPd(CN)<Subscript>4</Subscript>(C<Subscript>4</Subscript>H<Subscript>12</Subscript>N<Subscript>2</Subscript>O<Subscript>2</Subscript>)] single crystals

A Cu²⁺-doped single crystal of catena-trans-bis(N-(2-hydroxyethyl)-ethylenediamine) zinc(II)-tetra-m-cyanopaladate(II) [ZnPd(CN)₄(C₄H₁₂N₂O₂)] complex has been investigated by electron paramagnetic resonance (EPR) technique at room temperature. EPR spectra indicate that Cu²⁺ ions substitute for magnetically equivalent Zn²⁺ ions and form octahedral complexes in [ZnPd(CN)₄(C₄H₁₂N₂O₂)] hosts. The crystal field affecting the Cu²⁺ ion is nearly axial. The optical absorption studies show two bands at 322 nm (30864 cm⁻¹) and 634 nm (15337 cm⁻¹) which confirm the axial symmetry. The spin Hamiltonian parameters and the relevant wave function are determined.     

Impact of combined ultrasound-microwave treatment on structural and functional properties of golden threadfin bream (Nemipterus virgatus) myofibrillar proteins and hydrolysates

The effects of microwave, ultrasound and combined ultrasound-microwave (UM) treatment with different intensities on structural and hydrolysis properties of myofibrillar protein (MP) were investigated. Free radical scavenging ability, angiotensin-I-converting enzyme (ACE) inhibitory activity, and cellular antioxidant and anti-inflammatory abilities of the related bioactive peptides were also evaluated. Raman spectroscopic analysis indicated that MP molecule tended to unfold and stretch with increasing in β-turn and random coil content under mild microwave (100 W), ultrasound (100–200 W) and combined UM treatments. Meanwhile, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) revealed these treatments could also improve the thermal stability against heat-induced denaturation and degeneration. The 200 W ultrasound treatment clearly increased MP solubility by disrupting the highly-ordered aggregates into smaller filament and fragment structures. The 300 W ultrasound coupled with 100 W microwave treatment further enhanced these effects. The resulting partially denatured structure induced by suitable ultrasound and combined UM treatments increased the susceptibility of MP to exogenous enzymes, thereby accelerating hydrolytic process and yielding a high peptide concentration in MP hydrolysates. MP peptides could effectively inhibit free radical and ACE activity, which also improved the ability of antioxidant defence system, and suppressed the production of proinflammatory cytokines in RAW 264.7 cells stimulated by H₂O₂. The combination of 100 W microwave and 300 W ultrasound treatment was optimal method for generating bioactive MP peptides with the strongest multi-activity effects against H₂O₂-induced cell damage.     

EPR study of radiation damage in gamma-irradiated 3-nitroacetophenone single crystal

This paper presents the results of the electron paramagnetic resonance (EPR) study of the anion radical formed from 3-nitroacetophenone (C₈H₇NO₃) (3NAP) single crystal, by gamma irradiation. The EPR spectra of gamma-irradiated single crystals of 3NAP have been recorded at 10-degree intervals for different orientations of crystals in a magnetic field, at room temperature. The EPR analysis of gamma-irradiated crystals of 3NAP has shown that the radiation damage center produced by gamma irradiation is the carbon-centered 3NAP anion radical. One-electron reduction of 3NAP results in general bond loosening. The single crystals have been investigated between 120 and 450?K. The spectra have been found to be temperature-dependent. The EPR parameters of the 3NAP anion radical have been evaluated.     

Study of the structure of FeSiF6·6H2O by EPR and optical spectra

The method for determining the crystalline structure from relevant EPR zero-field splitting and optical spectra data is applied to ferrous fluosilicate (FeSiF₆·6H₂O. The results, consistent with data from Mossbauer and IR magnetic resonance, show that the effective symmetry of the Fe₂₊ site in FeSiF₆·6H₂O is a rhombic distortion rather than a trigonal one (D_3d).     

Pathogen-targeted hydroxyl radical generation during melanization in insect hemolymph: EPR study of a probable cytotoxicity mechanism

The main component of the insect immune system is melanotic encapsulation of pathogenic organisms. Molecular mechanisms of destruction of an encapsulated pathogen are poorly understood. Reactive oxygen species (ROS) are considered as probable cytotoxic agents responsible for destruction of pathogenic organisms in insect hemolymph. In the present work the formation of H₂O₂ during melanization in Galleria mellonella hemolymph in the presence of catalase inhibitor NaN₃ was detected. Enhanced rates of H₂O₂ generation were observed in the hemolymph of insects activated by injection of bacterial cells. Using spin trapping technique in combination with electron paramagnetic resonance spectroscopy we demonstrated that production of H₂O₂ in the hemolymph causes the formation of highly toxic reactive oxygen species, hydroxyl radical. However, neither H₂O₂ nor hydroxyl radical were detected in the absence of NaN₃ in agreement with the high catalase activity in the hemolymph. These observations allow us to propose a unique mechanism of pathogen-targeted cytotoxicity based on localized hydroxyl radical generation within a melanotic capsule.     

Effects of ultrasound-assisted H2O2 on the solubilization and antioxidant activity of yeast β-glucan

Yeast β-glucan (YG) possess an extensive range of biological activities, such as the inhibition of oxidation, but the poor water solubility of macromolecular YG limits its application. In this study, through the combined degradation of ultrasonic waves and H₂O₂, and the optimization of the main process parameters for solubilizing YG by response surface methodology (RSM), a new product of YG_UH was generated. The molecular weight, structural characteristics and degradation kinetics before and after solubilization were evaluated. The results showed that the optimal solubilization conditions were reaction time: 4 h, ultrasonic power: 3 W/mL, H₂O₂ concentration: 24 %. Under these conditions, ultrasound-assisted H₂O₂ increased the solubility (from 13.60 % to 70.00 %) and reduced molecular weight (from 6.73 × 10⁶ Da to 1.22 × 10⁶ Da). Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), Congo red (CR), scanning electron microscopy (SEM) revealed that ultrasound-assisted H₂O₂ increased the conformation's flexibility greatly, without changing the main structure of YG. More importantly, solubilization of YG improved free radical scavenging activity with YG_UH exhibiting the highest levels of DPPH and ABTS⁺ free radical scavenging activity. These results revealed that ultrasound-assisted H₂O₂ degradation could be a suitable way to increase the solubility of YG for producing value-added YG.     

Evidence for the development of persistent carbon-centered radicals from a benzyl phenolic antioxidant: an electron paramagnetic resonance study

Radicals have been generated from the benzyl phenolic antioxidant 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl) benzene, carrying out oxidative and hydrogen abstraction reactions. Transient phenoxyl radicals were directly visualized but only persistent carbon-centered radicals were monitored by electron paramagnetic resonance (EPR). The experimental EPR results let us rationalize our analysis as the sum of two different radicals. One, called the methylene radical, developed from the loss of a benzylic hydrogen gave place to a doublet of triplets witha _CH^H≅2.7 mT anda _PH^H=0.165 mT. Besides, the methyl radical, developed after an intramolecular hydrogen transfer involving a methyl group on the central aromatic ring of the molecule, formed a triplet of triplets, witha ₁^H around 0.060 mT anda ₂^H=0.169 mT. All the contact interactions were tested by EPR simulation of the experimental data.     

The role of metal cation in electron-induced dissociation of tryptophan

The fragmentation of tryptophan (Trp) – metal complexes [Trp+M]⁺, where M = Cs, K, Na, Li and Ag, induced by 22 eV energy electrons was compared to [Trp+H]⁺. Additional insights were obtained through the study of collision-induced dissociation (CID) of [Trp+M]⁺ and through deuterium labelling. The electron-induced dissociation (EID) of [Trp+M]⁺ resulted in the formation of radical cations via the following pathways: (i) loss of M to form Trp^+?, (ii) loss of an H atom to form [(Trp-H)+M]^+?, and (iii) bond homolysis to form C₂H₄NO₂M^+?. Deuterium labelling suggests that H atom loss can occur from heteroatom and/or C–H positions. Other types of fragment ions observed include: C₉H₇NM⁺, C₉H₈N⁺, M⁺, C₂H₃NO₂M⁺, CO₂M⁺, C₁₀H₁₁N₂M⁺, C₁₀H₉NOM⁺. Formation of C₂H₄NO₂M^+? and C₉H₇NM⁺ cations suggests that the metal interacts with both the backbone and aromatic side chain, thus implicating π-interactions for all M. CID of [Trp+M]⁺ resulted in: loss of metal cation (for M = Cs and K); successive loss of NH₃ and CO as the dominant channel for M = Na, Li and Ag; formation of C₂H₃NO₂M⁺. Preliminary DFT calculations were carried out on [Trp+Na]⁺ and [(Trp-H)+Na]^+? which reveal that: the most stable conformation involves chelation by the backbone together with a $\pi $ -interaction with the indole side chain; loss of H atom from $\alpha $ -CH of the side chain is thermodynamically favoured over losses from other positions, with the resultant radical cation maintaining a (N, O, ring) chelated structure which is stabilized by conjugation.     

单晶电子顺磁共振研究中零场分裂张量主轴的确定

用电子顺磁共振二维旋转测角器确定量子化轴即零场分裂张量的一个主轴,根据矢量的正交关系计算与量子化轴垂直的主轴平面上的取向二维角组、以便按照计算的二维角组测量电子顺磁共振谱,确认零场分裂张量的另外两个主轴,同时确定了主轴座标系(X,Y,Z)相对实验座标系(x,y,z)的Euler角。以渗Cr³⁺的Na₃(AlMo₆O₂₄H₆)·8H₂O单晶的电子顺磁共振测量作为上述方法的一个应用例子。 关键词：     

Simulation of EPR Spectra as a Tool for Interpreting the Degradation Pathway of Hyaluronan

Electron paramagnetic resonance (EPR) spin trapping is one of the choice techniques for identifying free radicals and is often used in the study of biological systems. However, its sensitivity can result in a typical complicated EPR spectrum. The accurate simulation of these systems is essential for correct identification of the radical species, whenever more than one species contributes to the spectrum. Programs implementing the linear combination of single simulations allow the interpretation of EPR spectra without modifying experimental conditions. In this study, this approach was used to investigate the influence of the ferrous ion and the role of oxygen, as well on the formation of transient radical species, in the whole mechanism of hyaluronan degradation. Degradation was carried out under different environmental conditions (air, O₂, Ar, N₂, N₂ + CO₂) and EPR spin trapping studies were performed. The advantages of the simulation of multiple species EPR spectra were applied to the obtained results and some aspects of hyaluronan degradation mechanism were elucidated. The depolymerization reaction pathway has been defined according to two possible subsequent steps: the first is consistent with formation of an amidyl radical that induces a series of strand scissions, which stabilize at two different levels of molecular weight. The second step occurs when the molecular weight is lower than before and two different adducts are generated.     

Enhanced blue emission and EPR study of LaMgAl11O19:Eu phosphors

Europium doped LaMgAl₁₁O₁₉ phosphor was prepared by the combustion method. The as-prepared and post-treated (1350 °C 10 h 5% H₂+95% N₂) phosphors were investigated by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), photoluminescence (PL) and electron paramagnetic resonance (EPR) techniques. XRD patterns show that LaMgAl₁₁O₁₉:Eu phosphors have hexagonal structure. FT-IR spectrum exhibits absorption bands corresponding to the stretching vibration of AlO₄ and AlO₆. Morphological studies reveal that this phosphor has faceted plates of varying sizes and shapes. The as-prepared LaMgAl₁₁O₁₉:Eu phosphor consists of both Eu³⁺ and Eu²⁺ ions. The phosphor exhibits a bright blue emission at 450 nm (4f⁶5d→4f⁷ transition of Eu²⁺). On post-treating the phosphor we are able to enhance the blue emission efficiency by 330%. The process was detected from the evolution of excitation, emission and EPR spectra and the results are discussed.     

Involvement of triplet state in the photodissociation of hydrogen peroxide: experimental evidence from time-resolved EPR study

The dissociation of photoexcited hydrogen peroxide to generate a pair of hydroxyl radicals is generally believed to take place in a repulsive electronic singlet state. The results presented here, based on time-resolved EPR experiments on the spin polarisation pattern of the acetone ketyl radical (CH₃)₂C^?OH, generated on photodissociation of H₂O₂ in 2-propanol with a 248?nm laser light, strongly indicate significant involvement of a repulsive triplet state of excited hydrogen peroxide.