Identification and quantification of radical reaction intermediates by electron spin resonance spectrometry of laccase-catalyzed oxidation of wood fibers from beech (Fagus sylvatica)

During laccase-catalyzed oxidation of beech wood fibers in an aqueous suspension, phenoxy radicals were detected in steady-state concentrations by electron-spin resonance (ESR) spectrometry of the suspension liquid, suggesting that colloidal lignin functions as a mediator between laccase and the fiber lignin matrix. Phenoxy radicals were observed directly, whereas ESR spin-trapping techniques gave no evidence for reduced oxygen species, such as the superoxide or hydroxyl radical. A reaction mechanism involving parallel direct oxidation of the lignin on fiber surfaces and a phenol/phenoxy cyclic mediator process in the suspension liquid could accordingly describe laccase-catalyzed oxidation of beech wood fibers. Cytochrome c assays for detection of superoxide in systems involving lignin oxidized by oxidoreductases should be used with caution, as cytochrome c may be reduced by species other than superoxide. Received: 24 March 1997 / Received revision: 27 May 1997 / Accepted: 1 June 1997     

Generation of phenoxy radicals by methemoglobin-hydrogen peroxide studied by electron paramagnetic resonance

The free radical intermediates of phenol derivatives, produced by the methemoglobin-hydrogen peroxide system at pH 5 and 7, are detected by electron paramagnetic resonance equipped with a continuous-flow apparatus. All the radicals from phenols are the phenoxy radicals, as identified by analyzing the observed hyperfine structures of the spectra with the aid of SCF-LCAO MO calculations. Comparing with the reaction of Fenton's reagent, it is concluded that free OH radical, even if it exists, is not liberated into the solution in the methemoglobin-hydrogen peroxide system.     

Long range electron transfer between tyrosine and tryptophan in hen egg-white lysozyme

The azide, dibromide and dichloride radicals oxidize one or more tryptophan side chains in hen egg-white lysozyme. The indolyl radical produced in this second-order 1-electron oxidation subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction with a rate constant of 130 +/- 10 s-1 at pH 7, 25 degrees C. The final indolyl and phenoxy equilibrium mixture then decays with a t1/2 approximately 2 s. The faster intramolecular reaction exhibits a pH dependence; on decreasing the pH from 9 the first-order rate constant increases to a maximum near pH 5.4 and then declines as the pH is lowered further. In contrast, the first-order rate constant for the intramolecular electron transfer between the tyrosine and tryptophan of the peptide trpH-pro-tyrOH remains unchanged between approx. pH 11 and 6.5 and then increases as the pH is lowered further. This difference in the observed pH dependence suggests that changes in structure or ionization state influence the protein electron transfer rate. We also discuss the radiation inactivation of lysozyme in light of these observations.     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

The effect of pH on the oxidation of bovine serum albumin by hypervalent myoglobin species

Bovine serum albumin (BSA) was used as a probe for the oxidation of proteins by hypervalent myoglobin species in solutions with pH from 5.3 to 7.7. The reaction between perferrylmyoglobin, *MbFe(IV)=O, and BSA was studied by activating metmyoglobin with equimolar amounts of hydrogen peroxide in the presence of BSA. A minor pH dependence was observed as judged from the formation of BSA-centered radicals, which were monitored at room temperature by electron spin resonance spectroscopy, and the formation of dityrosine. The reaction between ferrylmyoglobin, MbFe(IV)=O, and BSA was pH-dependent. BSA-centered radicals and dityrosine were formed in low levels at neutral pH and increased at low pH to the same levels as observed in the reaction of *MbFe(IV)=O with BSA. The present results demonstrate that protein-centered radicals can be formed from the non-radical MbFe(IV)=O under mildly acidic conditions, and this should be taken into account when considering oxidation in cellular compartments of low pH and in meat-related products.     

ESR study of electron transfer reactions between gamma-irradiated pyrimidines, adriamycin and oxygen

Solid pyrimidine nucleic acid bases (cytosine, thymine, and uracil) were gamma-irradiated (50 KGy) and dissolved in deaerated solutions of adriamycin in water and dimethylsulfoxide (DMSO). Analogous experiments using unirradiated pyrimidines as controls were also performed. In water only gamma-irradiated cytosine showed a reaction with the adriamycin yielding a single ESR peak (g = 2.0033) consistent with the adriamycin semiquinone radical. Since the unirradiated cytosine gave no reaction, the result suggests an electron transfer from cytosine radicals (generated by gamma-radiolysis) to adriamycin. In DMSO the three gamma-irradiated and unirradiated pyrimidines reacted with adriamycin yielding the adriamycin semiquinone radical observed by ESR. These results suggest that in DMSO an electron is transferred to adriamycin from the pyrimidine radicals and from the parent pyrimidine molecules. However, the process is on the order of 10(5) times more efficient for the pyrimidine radicals. Superoxide radicals (O2-.) were formed following addition of oxygen to the deaerated DMSO solutions containing adriamycin semiquinone radicals. O2-. was spin trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results show a possible reaction sequence in which an electron transferred to adriamycin, by pyrimidine radicals and parent pyrimidine molecules, is subsequently transferred to dissolved oxygen.     

Peroxidase catalyzed oxidation of naturally-occurring phenols and hardwood lignins

Horseradish peroxidase and hydrogen peroxide form phenoxy radicals from 4-substituted-2,6-dimethoxyphenols, milled wood lignin and alkali lignins. A number of factors governing this reaction are examined. Side chain cleavage to quinones is the principal disproportionation reaction of these radicals. Catalysis by UV light and inhibition by quinones is observed. Aerobic oxidation of phenols is catalyzed by small amounts of hydrogen peroxide. Lignin substrates are degraded by the same oxidation mechanism as are the simple phenolic substrates.     

Characterization of the inhibitor complexes of cobalt carboxypeptidase A by electron paramagnetic resonance spectroscopy

The metal coordination sphere of cobalt-substituted carboxypeptidase A and its complexes with inhibitors has been characterized by X-band electron paramagnetic resonance (EPR) spectroscopy. The temperature dependence of the EPR spectrum of cobalt carboxypeptidase and the g anisotropy are consistent with a distorted tetrahedral geometry for the cobalt ion. Complexes with L-phenylalanine, a competitive inhibitor of peptide hydrolysis, as well as other hydrophobic L-amino acids all exhibit very similar EPR spectra described by three g values that differ only slightly from that of the cobalt enzyme alone. In contrast, the EPR spectra observed for the cobalt enzyme complexes with 2-(mercaptoacetyl)-D-Phe, L-benzylsuccinate, and L-beta-phenyllactate all indicate an approximately axial symmetry of the cobalt atom in a moderately distorted tetrahedral metal environment. Phenylacetate, beta-phenylpropionate, and indole-3-acetate, which exhibit mixed modes of inhibition, yield EPR spectra indicative of multiple binding modes. The EPR spectrum of the putative 2:1 inhibitor to enzyme complex is more perturbed than that of the 1:1 complex. For beta-phenylpropionate, partially resolved hyperfine coupling (122 x 10(-4) cm-1) is observed on the g = 5.99 resonance, possibly indicating a stronger metal interaction for this binding mode. The structural basis for the observed EPR spectral perturbations is discussed with reference to the existing crystallographic kinetic and electronic absorption, nuclear magnetic resonance, and magnetic circular dichroic data.     

A direct electron spin resonance and spin-trapping investigation of peroxyl free radical formation by hematin/hydroperoxide systems

Direct electron spin resonance was used to detect tert-alkylperoxyl radicals generated by hematin and the corresponding hydroperoxides at near-physiological pH values. The spin-trapping method was necessary to detect the less persistent primary ethylperoxyl radical. Under a nitrogen atmosphere, the electron spin resonance signal of the tert-alkylperoxyl radicals decreased, and the ethylperoxyl spin-adduct concentration did not change. Concomitant studies, using a Clark oxygen electrode, show that oxygen was consumed by the hematin-tert-alkyl hydroperoxide systems, but was released by the hematin-ethyl hydroperoxide reaction. Thus, molecular oxygen seems to play a subsidiary role in the hematin-catalyzed decomposition of hydroperoxides. Based on the electron spin resonance and oxygen electrode results, a mechanism for the continuous production of the peroxyl free radicals is proposed for hematin/hydroperoxide systems. The present spectroscopic methodology can be used to search for peroxyl free radical formation by hemoprotein/hydroperoxide systems.     

Electron spin resonance and pulse radiolysis studies on the spin trapping of sulphur-centered radicals

Thiyl radicals are shown to be readily trapped with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO) giving characteristic spin adducts with hyperfine coupling constants aN 1.52-1.58, aH 1.52-1.80 mT, and g values in the range 2.0065-2.0067 for the DMPO adducts and aN 1.50-1.56, aH 1.70-1.92 mT, g 20049-2.0051 for the TMPO adducts. Kinetic data obtained from pulse radiolysis studies show that, in general, thiyl radicals react rapidly with these spin traps with rate constants of the order of 10(7)-10(8) dm3 mol-1 s-1. The tetramethylated spin trap TMPO though giving slightly less intense electron spin resonance (ESR) spectra, produces longer lived adducts, and is suggested to be of greater utility due to the more characteristic nature of the coupling constants of the observed adducts; reaction of certain thiyl radicals with DMPO produces adducts which are superficially similar to the hydroxyl radical adduct to the same trap.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light and electron microscopic demonstration of phospholipase B activity in the mouse eosinophil

Phospholipase B (EC 3.1.1.5) which hydrolyzes phospholipids in the alpha and beta positions was demonstrated in murine leukocytes using light and electron microscopic histochemical techniques. Leukocytes (neutrophils, lymphocytes, macrophages, eosinophils) were harvested from peritoneal exudates of mice. Cells were fixed in 4% calcium-formol fixative for 10 min at 4 degrees C for light microscopy and 30 min at room temperature for electron microscopy, after which they were incubated at 37 degrees C in medium at pH 6.6 containing 2 microM lysolecithin and CaCl2. The fatty acids released during the hydrolytic reaction were trapped as a calcium precipitate and were converted to a cobalt precipitate for light microscopy by treatment with cobalt acetate or to a lead precipitate for electron microscopy by treatment with lead nitrate. The reaction products were observed to be present in eosinophils and absent in neutrophils, lymphocytes and macrophages. It is concluded that the eosinophilic leukocyte is the carrier cell for phospholipase B in inflammatory reactions.     

An ESR investigation of the reactions of glutathione, cysteine and penicillamine thiyl radicals: competitive formation of RSO., R., RSSR-., and RSS(.)

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.     

Free radical scavenging actions of metallothionein isoforms I and II

By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15mM of MT-I and 0.3mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 × 10¹⁵ spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 × 10¹⁵ spins/ml) generated in a Fenton reaction. Similarly, 0.3mM of MT-I scavenged almost 90% of the superoxide (2.22 × 10¹⁵ spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a “spin-trap” for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM μl/ml concentrations could completely scavenge all the reactive species (2.15 × 10¹⁵ spins/ml) generated.

The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.     

Mechanism of resistance of Pediococcus cerevisiae strains to 5-fluorodeoxyuridine.

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

Cadaverine protects Vibrio vulnificus from superoxide stress

An electron paramagnetic resonance (EPR) signal characteristic of the 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spin adduct, which is formed from the reaction of DMPO with superoxide radicals generated by xanthine oxidase-mediated reaction, was significantly reduced by the cadaverine or Escherichia coli Mn-containing superoxide dismutase (MnSOD). Likewise, cytochrome c reduction by superoxide was inhibited by cadaverine, and the inhibition level increased in proportion to the level of cadaverine. The cadA mutant of Vibrio vulnificus, which does not produce cadaverine because of the lack of lysine decarboxylase, exhibits less tolerance to superoxide stress in comparison with wild type. The results indicate that cadaverine scavenges superoxide radicals, and protects cells from oxidative stress.     

Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide

Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.