Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC–EPR and HPLC–EPR–MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography–electron paramagnetic resonance spectrometry (HPLC–EPR) and high performance liquid chromatography–electron paramagnetic resonance–mass spectrometry (HPLC–EPR–MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (α^N = 1.58 mT and α^Hβ = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC–EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC–EPR–MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.     

Electron paramagnetic resonance (EPR) spectroscopy of the stable-free radical in the native metallo-cofactor of the manganese-ribonucleotide reductase (Mn-RNR) of Corynebacterium glutamicum

Ribonucleotide reductases (RNR; EC 1.17.4.1) provide the 2′-deoxyribonucleotides for DNA replication of proliferating cells by a uniform radical mechanism using diverse metals. The native metallo-cofactor of the Corynebacterium glutamicum RNR contains manganese and is sensitive to EDTA and radical scavengers. Hybrid holoenzymes, capable of ribonucleotide reduction, were composed of the small manganese-containing (R2F) and the large catalytic subunit (R1E) from either of the two corynebacterial RNRs. A synthetic peptide deduced from the C-terminal region of the nrdF gene inhibited the C. glutamicum-RNR non-competitively and cross-reacted with the C. ammoniagenes-RNR. The C. glutamicum-R2F has a saturable organic radical signal at g=2.005 detected by electron paramagnetic resonance (EPR) spectroscopy and shows a distinct absorption at 408 nm indicative of a tyrosyl-like organic radical (Y·). Quantification of the metal content revealed 0.06 mol Fe but 0.8 mol Mn per mol R2F-monomer and would thus assign two manganese atoms bound to the dimeric metallo-cofactor, while a distinct enzymatic activity (32 µmol×mg^?1×min^?1) was observed in the biochemical complementation assay. Divergence of the C. glutamicum-RNR studied here from the prototypical Salmonella typhimurium class 1b enzyme and the Chlamydia trachomatis class Ic enzyme is discussed below.     

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

Temperature-sensitive mutants of Corynebacterium ammoniagenes ATCC 6872 with a defective large subunit of the manganese-containing ribonucleotide reductase

Chemical mutagenesis of the nucleotide-producing strain Corynebacterium ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine followed by an enrichment protocol yielded 46 temperature-sensitive (ts) clones. A rapid assay for the allosterically regulated Mn-ribonucleotide reductase (RRase) was developed with nucleotide-permeable cells of C. ammoniagenes in order to screen for possible defects in DNA precursor biosynthesis at elevated temperature. Three mutants (CH 31, CH 32, and CH 33) grew well at 30° C but did not proliferate at 40° C because they did not reduce ribonucleotides to 2′-deoxyribonucleotides. They were designated nrd ^ts (nucleotide reduction defective). When the cultures were shifted from 30 to 40° C, the nrd ^ts mutants immediately ceased to incorporate radiolabeled nucleic acid precursors into the DNA fraction, while DNA chain elongation was barely affected. Thus, exhaustion of the deoxyribonucleotide pool ultimately inhibited cell division, leading to a filamentous growth morphology. In contrast to the wild-type, all three nrd ^ts mutants displayed a distinctly enhanced sensitivity of ribonucleotide reduction towards hydroxyurea (in permeabilized cells and in vitro) at 30° C. The results from assays for biochemical complementation of heat-inactivated (2 min, 37° C) mutant enzyme with either the small or the large subunit of wild-type Mn-RRase located the mutational defect on the large subunit. Received: 28 December 1995 / Revision received: 22 January 1997 / Accepted: 29 January 1997     

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.     

Photodegradation of hyaluronic acid: EPR and size exclusion chromatography study.

Photochemically induced radical reactions involving the lateral sequences and the end macromolecular chain groups of hyaluronic acid in aqueous solutions at 293K were studied by EPR spin trapping technique with DMPO (5,5-dimethylpyrroline-1-oxide). In the first 1-10 minutes of irradiation EPR indicates spin adducts of two carbon centered radicals with the splitting constants of aN = 1.60 mT, aH = 2.51 mT and aN = 1.56 mT, aH = 2.28 mT. After longer irradiation time (over 10 minutes) dominate two further DMPO adducts of radicals centered on hetero-atoms with splitting constants of aN = 1.44 mT, aH = 1.60 mT and of aN = 1.49 mT, aH = 1.49 mT. Simultaneously, molecular weight followed by SEC decreases, suggesting that UV irradiation leads to the breaking of interglycosidic bonds of hyaluronic acid main macromolecular chain.     

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.     

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.     

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.     

Deuteration effects on the in vivo EPR spectrum of the reduced secondary photosystem I electron acceptor A1 in cyanobacteria

The photoreduction of the secondary PSI electron acceptor A1 in vivo has recently been detected via X-band EPR spectroscopy in intact spinach chloroplasts and in marine cyanobacteria Synechococcus PCC 7002 [Klughammer, C., and Pace, R. J. (1997) Biochim. Biophys. Acta 1318, 133-144]. A further study of the A1- EPR spectrum of Synechococcus PCC 7002 at room temperature with higher-field resolution revealed partially resolved hyperfine structure which was dominated by 0.4 mT splittings of three equivalent protons. The hyperfine splitting was not significantly affected by incubation of the cyanobacteria in 2H2O medium for 20 h, but was absent in fully deuterated cyanobacteria that were grown in 2H2O medium. Anisotropic g-factors consistent with a phylloquinone radical were derived by spectra simulation. Biosynthetic protonation of quinones via the CH3 donor L-methionine in deuterated cells maintained hyperfine structure in the A1- spectrum, indicating the incorporation of CH3 groups in 60% of the deuterated, photoactive A1 molecules. Conversely, biosynthetic quinone deuteration via L-[methyl-d3]methionine in protonated cells led to the loss of the 0. 4 mT splittings in 54% of the A1 molecules. These observations confirm the conclusion of Heathcote et al. [(1996) Biochemistry 35, 6644-6650] of the identity of EPR-detected, photoreduced A1- in vivo with a phylloquinone (vitamin K1) radical in PSI. The partially resolved hyperfine structure of the A1- spectrum indicates an altered spin distribution in the bound vitamin K1- radical in vivo compared to that of unbound vitamin K1- in vitro.     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

Magnetic nanoparticles in five plant species growing on the Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and half-width of the signal of 32 mT at room temperature to g = 2.71 and 50–55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170‐220 μR/h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2′, 5′-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134?kDa for G6PD, 107?kDa for 6PGD and 121?kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63?kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, K_M and V_max values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC₅₀ values in the range of 0.07–30.13?mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.     

M?ssbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli. A new iron-to-protein stoichiometry

57Fe-enriched ribonucleotide reductase subunit B2 from Escherichia coli strain N6405/pSPS2 has been characterized by M?ssbauer and EPR spectroscopy in its native diferric state and in a new differous form. The native protein exhibits two M?ssbauer doublets in a 1:1 ratio with parameters that are in excellent agreement with those reported for the wild-type protein (Atkin, C. L., Thelander, L., Reichard, P., and Lang, G. (1983) J. Biol. Chem. 248, 7464-7472); in addition, our studies show the absence of adventitiously bound iron. The iron content in the present samples approached 4 per B2 subunit, and the tyrosyl radical content exceeded 1 per B2 subunit. The higher values are attributed to the use of a new epsilon 280 for the protein and more efficient methods for iron extraction. We thus propose that subunit B2 has two binuclear iron clusters, each associated with its own tyrosyl radical, in contradistinction from the prevailing model. Reduction of the native protein with dithionite or reconstitution of the apoprotein with Fe(II) afforded a protein complex with M?ssbauer parameters, delta EQ = 3.13 mm/s and delta = 1.26 mm/s at 4.2 K, and a low field EPR signal associated with an integer spin system. These spectral properties resemble those of methane monooxygenase in its diferrous form. Upon exposure to O2, the reduced subunit B2 readily converts to the diferric state and yields active enzyme.     

Reaction of the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate with human biofluids

Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), an oxidant was previously detected in the plasma of patients with renal failure and the synovial tissue of rheumatoid arthritis patients. This oxidant has been shown to react with DBNBS to give a 3-line electron paramagnetic resonance (EPR) spectrum, previously assigned to the DBNBS radical cation (DBNBS*(+). However, confusion has arisen as to whether this paramagnetic species is indeed DBNBS*(+) or, rather, the DBNBS sulfite radical adduct (DBNBS-SO(3)*(-)). In the present study, DBNBS*(+) (a(N)=1.32 mT) was distinguished from DBNBS-SO(3)*(-) (a(N)=1.32 mT with an additional splitting of a(H)=0.06 mT) by (a) using different EPR parameters, (b) determining the effect of addition of sulfite on the EPR spectrum resulting from the incubation of DBNBS with either human biofluids or the horseradish peroxidase (HRP)-hydrogen peroxide (H(2)O(2)) system, and (c) replacing DBNBS with its analogues (DBNBS-d(2,) DBNBS-15N and DBNBS-d(2)-15N) in the two systems.     

Characterization of the radical product formed from the reaction of nitric oxide with the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate.

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO(*)). The reaction between DBNBS and NO(*) yields a radical product which gives rise to an EPR signal consisting of three lines with an A(N) = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO(*) reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment-mass spectrometry (FAB-MS). Collectively, the FAB-MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO(*). A reaction scheme consistent with these results is presented.     

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.     

Purification of Major Hemolymph Protein of Great Wax Moth, Galleria mellonella

Major hemolymph protein (MHP) was purified from larval hemolymph of Galleria mellonella by KBr density gradient ultracentrifugation, ion exchange chromatography (DEAE‐Trisacryl M), YM‐50 ultrafiltration and gel permeation chromatography (Sephadex G‐100). MHP is composed of two subunit (MHP‐1 and MHP‐2). The molecular weights of each subunit were determined (MHP‐1 = 86 kDa and MHP‐2 = 84 kDa). MHP is present in both hemolymph and fat body during developmental stages, indicating this protein is carrying out some functions different from other major protein such as storage protein and lipophorin.     

Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC-EPR and HPLC-EPR-MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography-electron paramagnetic resonance spectrometry (HPLC-EPR) and high performance liquid chromatography-electron paramagnetic resonance-mass spectrometry (HPLC-EPR-MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC-EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC-EPR-MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.