Ascorbic acid radicals induced by the action of radiation in tissues from rat organs frozen at 77 K

Subsequent annealing technique and computer assistant analysis of EPR spectra were used to isolate an asymmetric EPR signal Rs(g = 2,0051; delta H = 0.8 mT) from the EPR spectrum of rat spleen gamma-irradiated at 77 K. Radicals with the same EPR spectrum were registered in: 1) water solution of ascorbic acid (2.10(-2) M, pH 3.4) frozen and irradiated at 77 K and 2) water-glycerol solution of ascorbic acid (10(-2) M, pH 10.3) frozen rapidly at the moment of intensive autooxidation. These model experiments allow to conclude that Rs signal is caused by the radicals of semidehydroascorbic acid. Radiochemical yield of these radicals as well as of all the radicals induced by gamma radiation in the whole rat tissues were measured. The EPR signal (Rs) is equivalent to the well known "artifact" signal of lyophilized tissues. The explanation of the mechanism of the radicals formation taking place under annealing of the frozen and irradiated tissues was suggested.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Effect of inorganic salts on free radicals production by near UV irradiation of frozen aqueous solutions of adenine and its derivatives

The addition of Na and Ca chlorides to adenine (A), adenosine (Ado) and adenosine diphosphate solutions at pH 5.3 has been shown to result in intensification of EPR signals in samples irradiated by near UV at 77 K and appearance of signals of Cl ₂ ^?? and peroxyl radicals. The peroxyl radicals contribution can exceed 30% of total amount of paramagnetic products. The addition of inorganic phosphate reduces the contribution of peroxyl radicals. Possible mechanisms of the processes involved are discussed.     

Electron paramagnetic resonance study of water distribution in starch granules

An electron paramagnetic resonance (EPR) study was performed for potato and wheat starch containing Cu2+ ions as a paramagnetic probe. Distribution of water in the starch granules as well as the interactions between the copper and starch matrix of different crystalline structures were determined. EPR spectra of the native starches consisted of two different centers of Cu2+. One of them, giving at 293 and 77 K an EPR signal of axial symmetry with a well-resolved hyperfine structure (HFS), was assigned to the Cu2+ -starch complex in which Cu2+ ions strongly interacted with oxygen atoms of the starch matrix. Another Cu2+ species, exhibiting an isotropic signal at 293 K and an axial signal with resolved HFS at 77 K, was attributed to a [Cu(H2O)6]2+ complex freely rotating at room temperature and immobilized at low temperatures. Interaction of Cu2+ with the starch matrix and the relative number of the particular copper species depended on the crystallographic type of starch. Dehydration at 393 K resulted in elimination of the rotating complex signal and decrease of the total intensity of the EPR spectrum caused by clustering of the Cu2+ ions. Freezing at 77 K and thawing led to restoring of the spectrum intensity and reappearing of the signal of the [Cu(H2O)6]2+ complex. This effect, related to liberation of water molecules from the granule semicrystalline growth rings on freezing/thawing, was especially visible for wheat starch, indicating differences in the water retention ability of starch granules of different crystallographic structure.     

X- and Q-band electron paramagnetic resonance of CO2- in hydroxyapatite single crystals

Both X- and Q-band electron paramagnetic resonance (EPR) research has been conducted using slightly carbonated hydroxyapatite (HAp) single crystals after exposure to ionizing radiation. Below a temperature of 90 K, O(-) and CO(2-) radicals were detected, whereas at room temperature only CO(2-) spectra could be observed. The O(-) ion has previously been investigated in high-purity HAp single crystals, whereas EPR spectra of CO(2-) in HAp single crystals have not been reported. Both paramagnetic defects exhibit EPR angular variations in planes containing the c axis of the crystal from which spin Hamiltonian parameters were derived. Arguments are given for the presence of two CO(2-) defects in the irradiated HAp single crystals.     

Effect of temperature on the electron paramagnetic resonance spectrum of irradiated alanine.

Polycrystalline samples of the amino acid L-alpha-alanine have been irradiated with X rays at both room temperature and higher temperatures. The electron paramagnetic resonance (EPR) spectra of alanine powder irradiated at room temperature are dominated by the well-known room-temperature-stable alanine radical CH3C*HCOOH. Upon heating of room-temperature-irradiated alanine powder, a strong decay of the signal was observed, and the features of the spectrum recently ascribed to a second stable radical in alanine irradiated at room temperature become more pronounced, providing an experimental isolation of this second alanine radical. In combination with the high-temperature experiments, a multivariate statistical decomposition method, maximum likelihood common factor analysis, was used to determine the number of components in irradiated alanine powder which behave differently as a function of temperature. The EPR components found in the present study are compared with simulations using earlier EPR and ENDOR single-crystal data.     

Powder and single-crystal electron paramagnetic resonance studies of yeast cytochrome c peroxidase and its peroxide and its peroxide compound, Compound ES

Electron paramagnetic resonance absorption spectrum of ferric cytochrome c peroxidase exhibited a mixture of high- and low-spin compounds. The principal values and the eigenvectors of the g-tensor for the low-spin species were determined by single-crystal EPR spectroscopy at 77 K. The powder EPR spectra of the peroxide compound, Compound ES, were measured at S-, X-, and Q-band microwave frequencies. Careful examination at 77 K showed a narrow free radical-like signal at g = 2.004 with hyperfine structures accompanied by a broad signal spreading on both low- and high-field sides. Single-crystal EPR analyses of Compound ES clearly demonstrated that there exist at least two different radical species: one is isotropic with hyperfine structure at g = 2.004 and the other exhibits an axially symmetric signal at 5 K and broad signal centered at g = 2.004 at 77 K, respectively. The principal values and the eigenvectors of the g-tensor for the axially symmetric signal were determined: g(parallel) = 2.034 and g(perpendicular) = 2.006, 1.999. The orientation of the unique axis (g(parallel)) was found to be identical to that of the heme normal. A new radical signal with complicated hyperfine structures in the g = 2.004 region was observed upon illumination of Compound ES at both 5 and 77 K. The photoinduced species grew effectively by the illumination light around 500 nm. On warming to -80 degrees C, the photoinduced signal was reversibly brought back to the original radical species of Compound ES via an intermediate species. From these results, we have proposed the possible sites for the free radical centers in Compound ES.     

Mitochondrial dysfunction in patients with severe sepsis: an EPR interrogation of individual respiratory chain components

Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron-sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ*-SQ*). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219-223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ*-SQ*) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron-sulfur redox centres is linked to mortality.     

ESR spectra of paramagnetic centers of ferricytochrome c, induced by UV-radiation at 77 K

Paramagnetic properties of ferricytochrome c aqueous glyceric solutions exposed to UV-light of various wave lengths at 77K have been investigated. Absorption of longwave UV- and visible light by hemin (5-240 min) does not induce the formation of paramagnetic centres in hemoprotein. However absorption of UV-light by chromophorms of a protein component of ferricytochrome c induces the formation of free radicals with a quantum yield of 0.07. During long-term (40 to 200 min) UV-irradiation (260-380 nm) of protein solutions several types of paramagnetic centres including HCO radicals are formed.     

Comparison of the EPR properties of photosystem I iron-sulphur centres A and B in spinach and barley

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.     

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.     

Retrospective dosimetry after criticality accidents using low-frequency EPR: a study of whole human teeth irradiated in a mixed neutron and gamma-radiation field

In the context of accidental or intentional radiation exposures (nuclear terrorism), it is essential to separate rapidly those individuals with substantial exposures from those with exposures that do not constitute an immediate threat to health. Low-frequency electron paramagnetic resonance (EPR) spectroscopy provides the potential advantage of making accurate and sensitive measurements of absorbed radiation dose in teeth without removing the teeth from the potential victims. Up to now, most studies focused on the dose-response curves obtained for gamma radiation. In radiation accidents, however, the contribution of neutrons to the total radiation dose should not be neglected. To determine how neutrons contribute to the apparent dose estimated by EPR dosimetry, extracted whole human teeth were irradiated at the SILENE reactor in a mixed neutron and gamma-radiation field simulating criticality accidents. The teeth were irradiated in free air as well as in a paraffin head phantom. Lead screens were also used to eliminate to a large extent the contribution of the gamma radiation to the dose received by the teeth. The EPR signals, obtained with a low-frequency (1.2 GHz) spectrometer, were compared to dosimetry measurements at the same location. The contribution of neutrons to the EPR dosimetric signal was negligible in the range of 0 to 10 Gy and was rather small (neutron/gamma-ray sensitivity in the range 0-0.2) at higher doses. This indicates that the method essentially provides information on the dose received from the gamma-ray component of the radiation.     

Mitochondrial dysfunction in patients with severe sepsis: An EPR interrogation of individual respiratory chain components

Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron-sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ^·-SQ^·). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219-223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ^·-SQ^·) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron-sulfur redox centres is linked to mortality.     

EPR studies of 5-bromouracil crystal after irradiation with X rays in the bromine K-edge region

Radicals induced in a single crystal of 5-bromouracil (BrUra) by synchrotron soft X rays in the bromine K-edge region (13.461-13.482 keV) were investigated using the X-band EPR method. The crystal was irradiated at three peak energies of the absorption spectrum at room temperature or at 80 K. A hydrogen abstraction radical derived from N1 of the pyrimidine ring was commonly observed for all of the energies used, though with some variation in quantity. Similar characteristics were also observed in the EPR signal for the off-K-edge low-energy (13.42 keV) and (60)Co gamma rays used for comparison. When irradiated at 80 K, a much larger exposure (roughly 10 times) of soft X rays was needed to obtain the same signal intensity as that observed at room temperature. EPR signals were not detectable with gamma irradiation at liquid nitrogen temperature.     

Analysis of the active center of hydrogenase from Desulfovibrio vulgaris Miyazaki by magnetic measurements

Hydrogenase [hydrogen: ferricytochrome c3 oxidoreductase, EC 1.12.2.1] solubilized and purified from the particulate fraction of Desulfovibrio vulgaris Miyazaki F (IAM 12604) contains 8 iron and 8 labile sulfide ions in one molecule which is composed of two unequal subunits (Mr: 60,000 + 29,000). It does not contain nickel atoms. The EPR (electron paramagnetic resonance) spectrum has an isotropic signal at g = 2.017 which is independent of the temperature. The peak-to-peak width of the signal is about 20 G. The signal intensity is nearly equivalent to 1 unpaired electron per molecule. No other signals can be detected in the field range between 2,240 and 4,240 G (which corresponds to g-values between 2.91 and 1.54). Ferricyanide has only a little effect on the shape and intensity of the EPR signal. The hydrogenase reduced under H2 is EPR silent. The M?ssbauer spectrum has no hyperfine splitting at 4K. The isomer shift and quadrupole splitting at 77K are 0.38 and 0.87 mm/s, respectively. Based on these magnetic measurements, the structure of the active center of hydrogenase was suggested to be [4Fe-4S]3+ + [4Fe-4S]2+.     

The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

M?ssbauer spectroscopic studies of the nature of centre X of photosystem I reaction centres from the cyanobacterium Chlorogloea fritschii

Reduced photosystem I samples, which give the electron paramagnetic resonance (EPR) signals associated with A, A and B, and A, B and X centres, have been studied using M?ssbauer spectroscopy. The M?ssbauer spectra obtained from each type of sample is different, which indicates that iron is associated with all three centres. The spectra are similar to those obtained from ferredoxins with 4Fe-4S centres and were fitted with oxidized and reduced components, the relative proportions depending on the degree of reduction of the sample as monitored by EPR. The sample which gave only the A EPR signal showed about 26% of the reduced component, the sample which gave A and B EPR signals showed about 48% of the reduced component, while the sample which gave A, B and X EPR signals showed about 65% of the reduced component. The measurements are consistent with X being a 4Fe-S4 centre.     

Characterization of the free radical of mammalian ribonucleotide reductase

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.     

Donor function of phosphate ions in photosystem 2

Evidence was obtained for the interaction between the photosystem 2 (PS2) reaction centre (RC) chlorophyll (Chl) P680 and inorganic phosphate, Pi. The light-induced endogenous basal electron transport to ferricyanide in PS2 depended on endogenous Pi. The electron transport in phosphate deficient chloroplasts was absent, and could be resumed upon the addition of exogenous Pi or of the exogenous electron donor, diphenylcarbazide. Some chloroplast Chl molecules were apparently bound with Pi to a complex via the magnesium atom that was detected by the increase in absorbance in the Chl a absorption maximum at 435 nm observed after the consumption of endogenous Pi in the photophosphorylation reactions. The electron paramagnetic resonance (EPR) Signal I, found in the spectra at 77 K after irradiation of frozen samples in chloroplasts poor in endogenous Pi, was the sum of P700+ and P680+ signals. The P680+ signal disappeared after addition of Pi, diphenylcarbazide or diuron to the chloroplasts before freezing. In addition, the EPR doublet signal of the phosphate anion radicals was recorded at 77 K after irradiation in the ethanol solutions of Chl a containing potassium phosphate. The same doublet signal was discovered in the difference EPR spectrum "chloroplasts minus chloroplasts with diuron" at 77 K after irradation. The results are a possible evidence of the participation of phosphate ions in the primary light reactions of PS2. This revised version was published online in June 2006 with corrections to the Cover Date.