Free hydroxyl radical is not involved in an important reaction of lignin degradation by Phanerochaete chrysosporium Burds.

Hydroxyl radical (HO.) has been implicated in the degradation of lignin by Phanerochaete chrysosporium. This study assessed the possible involvement of HO. in degradation of lignin substructural models by intact cultures and by an extracellular ligninase isolated from the cultures. Two non-phenolic lignin model compounds [aryl-C(alpha)HOH-C(beta)HR-C(gamma)H2OH, in which R = aryl (beta-1) or R = O-aryl (beta-O-4)] were degraded by cultures, by the purified ligninase, and by Fenton's reagent (H2O2 + Fe2+), which generates HO.. The ligninase and the cultures formed similar products, derived via an initial cleavage between C(alpha) and C(beta) (known to be an important biodegradative reaction), indicating that the ligninase is responsible for model degradation in cultures. Products from the Fenton degradation were mainly polar phenolics that exhibited little similarity to those from the biological systems. Mass-spectral analysis, however, revealed traces of the same products in the Fenton reaction as seen in the biological reactions; even so, an 18O2-incorporation study showed that the mechanism of formation differed. E.s.r. spectroscopy with a spin-trapping agent readily detected HO. in the Fenton system, but indicated that no HO. is formed during ligninase catalysis. We conclude, therefore that HO. is not involved in fungal C(alpha)-C(beta) cleavage in the beta-1 and beta-O-4 models and, by extension, in the same reaction in lignin.     

Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum in biodegradation of cellulose

A special low-molecular-weight peptide named Gt factor, was isolated and purified via HPLC from the culture extract of the brown-rot fungus Gloeophyllum trabeum. It had high-affinity Fe(3+)-chelating ability and could reduce Fe(3+) to Fe(2+). In the presence of O(2), it could produce hydroxyl radicals HO*. The effects of Gt factor on cellulose degradation suggested that Gt factor could disrupt inter- and intra- hydrogen bonds in cellulose chains by a HO*-involved mechanism. This resulted in depolymerization of cellulose chains, which produced more reducing and non-reducing ends, thus making cellulose accessible for further degradation. This pathway was quite different from the hydrolytic processes driven by cellulases, and Gt factor might play an important role in the early stage of cellulose depolymerization by brown-rot fungi.     

Tris(picolinato)manganese(II): a chemical model for the mechanism and function of mitochondrial superoxide dismutase

The reaction of HO2. with the allylic groups of lipids initiates their peroxidation and auto-oxidation, and probably represents the most serious biological hazard of O2.- -derived species. The presence of tris(picolinato)manganese(II) [MnII(PA)2(PAH)(H2O)], a model complex for mitochondrial superoxide dismutase, (i) efficiently catalyzes the disproportionation of O2.-, (ii) precludes the formation HO2., and thereby (iii) prevents hydrogen abstraction from allylic and thiol groups. Such protection demonstrates that a primary function of superoxide dismutase is to block the formation of HO2., which is the obligatory intermediate for the nonenzymatic proton-induced disproportionation process. This requires that the primary step for the enzyme-O2.- reaction be kinetically favored and dominant relative to the protonation reaction (HA + O2.-).     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of P. aeruginosa O10 (Lányi) lipopolysaccharides

Mild acid degradation of lipopolysaccharides from Pseudomonas aeruginosa O10a and O10a,b (Lányi classification) resulted in O-specific polysaccharides built up of trisaccharide repeating units containing 2-acetamido-2,6-dideoxy-D-glucose (N-acetylquinovosamine, DQuiNAc), 2-acetamido-2,6-dideoxy-D-galactose (N-acetylfucosamine, DFucNAc), and 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutyramido] -L-glycero-L-manno-nonulosonic acid. The latter is a di-N-acyl derivative of a new sialic-acid-like sugar which was called by us pseudaminic acid (PseN2). A 3-hydroxybutyric acid residue was also found in natural carbohydrates for the first time. In the O10a,b polysaccharide pseudaminic acid carried an O-acetyl group at position 4. For selective cleavage of the O10a polysaccharide, solvolysis with hydrogen fluoride was employed which, owing to the relatively high stability of the glycosidic linkage of pseudaminic acid, led to the disaccharide with this sugar on the non-reducing terminus. Performing the solvolysis in methanol afforded the methyl glycoside of this disaccharide which proved to be more advantageous for further analysis. Carboxyl-reduction made the glycosidic linkage of pseudaminic acid extremely labile, and mild acid hydrolysis of the carboxyl-reduced 010a polysaccharide afforded the trisaccharide with a ketose derivative on the reducing terminus. Establishing the structure of the oligosaccharide fragments obtained and interpreting the 13C nuclear resonance spectra of the polysaccharides allowed to determine the following structure for their repeating units: (formula: see text) In the polysaccharides the N-acetylquinovosamine residue is attached not to pseudaminic acid itself, but to its N-acyl substituent, 3-hydroxybutyryl group, and thus the monomers are linked via both glycosidic and amidic linkages.     

Conformation, hydrogen bonding and aggregate formation of guanosine 5'-monophosphate and guanosine in dimethylsulfoxide.

The tetrabutylammonium salt of guanosine 5'-monophosphate (5'-GMP) dissolves in DMSO-d6 forming aggregated species which exhibit some properties of reverse micelles. 1H NOESY experiments show that the 5'-GMP adopts the syn conformation about the glycosidic bond. Molecular mechanics calculations reveal a stable structure with this conformation in which the phosphate group and the amino group of the base are in close enough proximity to hydrogen bond. In contrast inosine 5'-monophosphate in DMSO-d6, which has no NH2 group for hydrogen bond stabilization of the syn conformation, is shown by NMR to have the anti structure. Guanosine in DMSO-d6 behaves differently from 5'-GMP. Guanosine adopts the anti conformation and forms a symmetric dimer via hydrogen bonding between the N3 and NH2 of the bases.     

The Haber-Weiss cycle--70 years later

The chain reactions HO* + H2O2 --> H2O + O2*- + H+ and O2*- + H+ + H2O2 --> O2 + HO* + H2O, commonly known as the Haber-Weiss cycle, were first mentioned by Haber and Willst?tter in 1931. George showed in 1947 that the second reaction is insignificant in comparison to the fast dismutation of superoxide, and this finding appears to have been accepted by Weiss in 1949. In 1970, the Haber-Weiss reaction was revived by Beauchamp and Fridovich to explain the toxicity of superoxide. During the 1970s various groups determined that the rate constant for this reaction is of the order of 1 M(-1) s(-1) or less, which confirmed George's conclusion. The reaction of superoxide with hydrogen peroxide was dropped from the scheme of oxygen toxicity, and superoxide became the source of hydrogen peroxide, which yields hydroxyl radicals via the Fenton reaction, Fe2+ + H2O2 --> Fe3+ + HO- + HO*. In 1994, Kahn and Kasha resurrected the Haber-Weiss reaction again, but this time the oxygen was believed to be in the singlet (1delta(g)) state. As toxicity arises not from a Fenton-catalysed Haber-Weiss reaction, but from the Fenton reaction, the Haber-Weiss reaction should not be mentioned anymore.     

The action of ionizing radiation on protein. III. Radical formation in L-phenylalanine.HCl.

The radicals produced by X-irradiation in L-phenylalanine.HCl crystals have been analysed by electron-spin-resonance (e.s.r.) spectroscopy. Four radicals have been identified: the radical resulting from electron capture by the carboxylic group, the radical resulting from deamination, an hydrogen addition radical to the benzene ring, and the radical resulting from hydrogen abstraction from the methylene group bonded to Cbeta. These identifications are supported by INDO calculations.     

Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution

The structure of the disaccharide cellulose subunit cellobiose (4-O-β-D-glucopyranosyl-D-glucose) in solution has been determined via neutron diffraction with isotopic substitution (NDIS), computer modeling and nuclear magnetic resonance (NMR) spectroscopic studies. This study shows direct evidence for an intramolecular hydrogen bond between the reducing ring HO3 hydroxyl group and the non-reducing ring oxygen (O5′) that has been previously predicted by computation and NMR analysis. Moreover, this work shows that hydrogen bonding to the non-reducing ring O5′ oxygen is shared between water and the HO3 hydroxyl group with an average of 50% occupancy by each hydrogen-bond donor. The glycosidic torsion angles φ_H and ψ_H from the neutron diffraction-based model show a fairly tight distribution of angles around approximately 22^° and −40^°, respectively, in solution, consistent with the NMR measurements. Similarly, the hydroxymethyl torsional angles for both reducing and non-reducing rings are broadly consistent with the NMR measurements in this study, as well as with those from previous measurements for cellobiose in solution.     

The antibacterial action of lactoperidoxase. The nature of the bacterial inhibitor

Lactoperoxidase (EC 1.11.1.7), an enzyme present in various mammalian glands and in their secretions, catalyses the oxidation of thiocyanate by hydrogen peroxide to form a compound that inhibits the growth, oxygen uptake and acid production of certain bacteria. This compound was found to be too unstable to isolate in pure form, but its properties in dilute aqueous solution were studied with a view to establishing its identity. At thiocyanate concentrations of approximately 1mm, formation of the inhibitor, which took place by a nonstoicheiometric reaction, was maximal when an approximately equimolar amount of hydrogen peroxide was added. Excess of hydrogen peroxide oxidized the inhibitor to sulphate and cyanate. The inhibitor displayed a polarographic reduction wave of which the half-wave potential was pH-dependent. Studies of the variation of the polarographic half-wave potential and of the u.v. extinction with pH indicated that the inhibitor existed in an acid-base equilibrium (pK(a) 5.1+/-0.1). The inhibitor decomposed by a mechanism involving H(+) ions and thiocyanate, the kinetics varying according to whether the inhibitor was in its acidic or basic form. From these studies it was concluded that the inhibitor was either cyanosulphurous acid (HO(2)SCN) or cyanosulphuric acid (HO(3)SCN).     

An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.     

密粘褶菌胞外低分子量多肽在纤维素降解中作用的研究

从褐腐真菌中能强烈降解纤维素的代表菌株密粘褶菌(Gloeophyllum trabeum)的胞外酶液中首次分离纯化得到一低分子量的活性多肽组分(称作Gt因子),此组分能在有O.2和Fe³⁺存在时产生羟基自由基HO·;对纤维素降解的研究表明,Gt因子不同于纤维素酶对纤维素的β1.4糖苷键的水解作用,而以HO·氧化的途径作用于纤维素,导致纤维素中氢键的断裂,降低纤维素的结晶度,使其暴露出更多的末端,从而有利于纤维素的进一步降解。     

The influence of protonation or alkylation of the phosphate group on the e.s.r. spectra and on the rate of phosphate elimination from 2-methoxyethyl phosphate 2-yl radicals.

The e.s.r. spectra of 1-yl, 2-yl and 3'-yl methoxyethyl phosphate radicals derived from CH3OCH2CH2-OPO3H2 by hydrogen abstraction have been measured in aqueous solutions and the hyperfine constants determined. The coupling constants vary strongly with protonation or alkylation of the phosphate group. The 2-yl radicals eliminate phosphate. The rate-constants for the elimination (ke) have been estimated by e.s.r. measurements and by product studies as a function of pH using 60Co gamma-radiolysis. The ke values vary from approximately 0.3 s(-1) for the CH3OCHCH2OPO3--radical and approximately 10(3) s-1 for CH3OCHCH2OPO3H-, to approximately 3 X 10(6) S-1 for CH3OCHCH2OPO3H2. Alkylation of the phosphate group increases the elimination rate-constant to a similar extent as protonation. The results support a recent mechanism which described the OH-radical-induced single-strand breaks of DNA in aqueous solution starting from the C-4' radical of the sugar moiety. It is further concluded the C-4' radical of DNA eliminates the 3'-phosphate group faster than the 5'-phosphate group.     

E.s.r. studies of sulphur-substituted pyrimidines. 2-thio-5-carboxyuracil at 77 K.

Single crystals of 2-thio-5-carboxyuracil were irradiated and studied at 77 K with e.s.r. spectroscopy. Five resonances were observed and related to the sulphur atom in the 2 position of the pyrimidine ring. Three of the resonances have been assigned to three conformations of a radical formed by hydrogen abstraction from N1. The principal values for the nitrogen coupling are 9-7, 0-0 and 0-0 gauss. The g tensor principal values are 2-173, 1-997 and 1-990 for the dominant conformation of this radical. Two other radicals could not be identified unambiguously.     

Solvent dependent photocyclization and photophysics of some 2-ethynylbiphenyls.

The spectroscopic properties and photochemical behavior of molecules having 2-ethynylbiphenyl or 2-phenyldiphenylacetylene structures are reported. These molecules undergo photocyclization reactions to yield phenanthrene and dihydrophenanthrene products via putative isophenanthrene (cyclic allene) intermediates. Phenanthrene formation from the isophenanthrene intermediates does not occur via a unimolecular sigmatropic hydrogen shift, but rather by protonation or hydrogen abstraction mechanisms involving the solvent. Cyclization efficiencies are much lower than is the case for previously-investigated 2-vinylbiphenyl systems. The 2-phenyldiphenylacetylenes have high fluorescence quantum yields and long singlet lifetimes when compared to diphenylacetylene. The 2-ethynylbiphenyls decay via a combination of fluorescence and intersystem crossing.     

QM/MM studies on the calcium‐assisted β‐elimination mechanism of pectate lyase from bacillus subtilis

Pectate lyase utilizes the anti‐β‐elimination chemistry to catalyze the cleavage of α‐1,4 glycosidic bond between _D‐galacturonate regions during the degradation of plant polysaccharide pectin. We report here detailed mechanistic studies of the Bacillus subtilis pectate lyase (BsPel) using QM/MM calculations. It was found that the residue Arg279 serves as the catalytic base to abstract the α‐proton from C₅₂ atom of substrate Ada2 subsite, forming an unstable carbanion intermediate. The glycosidic bond of this intermediate is scissile to generate the 4,5‐unsaturated digalacturonate product and a negatively charged β‐leaving group. Two active site residues (Lys247 and Arg279) and two Ca²⁺ ions (Ca2 and Ca3) form hydrogen‐bonding and coordination interactions with C₅₂? COO^? of Ada2, respectively, which facilitate the proton abstraction and stabilize the generated carbanion intermediates. Arg284 is not the potential proton donor to saturate the leaving group. Actually, the proton source of leaving group is the solvent water molecule rather than any active site acidic residues. In addition, the calculation results suggest that careful selections of QM‐ and Active‐regions are essential to accurately explore the enzymatic reactions. Proteins 2016; 84:1606–1615. © 2016 Wiley Periodicals, Inc.     

Free radicals in dicarboxylic acids: an e.s.r. study of radical conversions in gamma-irradiated single crystals of glutaric acid and glutaric-2,2,4,4-d4 acid

The gamma-radiation-induced free radicals in single crystals of glutaric acid and glutaric-2,2,4,4-d4 acid were studied in the temperature range 77-300 K by e.s.r. techniques. At 77 K the decarboxylation radical and the anion are stabilized. At higher temperatures the decarboxylation radical is found to be converted into a hydrogen abstraction radical with an activation energy of 6.3 +/- 0.5 kcal/mole for the non-deuterated crystal. This radical is stable at room temperature. The anion seems be be converted to an unidentified intermediate radical which in turn is converted to the gamma-acyl radical. An analysis of the g-value anisotropy and of the 13C hyperfine splitting variation for this radical in the deuterated crystal is consistent with the assigned radical structure. By heat treatment the alpha-acyl radical is converted to another form of the hydrogen abstraction radical with an activation energy of 9.6 +/- 0.6 kcal/mole in the deuterated crystal. U.V.-light (gamma = 254 nm) transforms one of the room temperature radicals into the other.     

Product analyses in DNA strand scission by antitumor antibiotic elsamicin A.

Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.     

Crystal structure and n.m.r. analysis of lactulose trihydrate.

The 13C CPMAS n.m.r. spectrum of 4-O-beta-D-galactopyranosyl-D-fructose (lactulose) trihydrate, C12H22O11.3 H2O, identifies the isomer in the crystals as the beta-furanose. This is confirmed by a crystal structure analysis, using CuK alpha X-ray data at room temperature. The space group is P212121, with Z = 4 and cell dimensions a = 9.6251(3), b = 12.8096(3), c = 17.7563(4) A. The structure was refined to R = 0.031 and Rw 0.025 for 1929 observed structure amplitudes. All the hydrogen atoms were unambigously located on difference syntheses. The conformation of the pyranose ring is the normal 4C1 chair and that of the furanose ring is 4T3. The 1----4 linkage torsion angles are O-5'-C-1'-O-1'-C-4 = 79.9(2) degrees and C-1'-O-1'-C-4-C-5 = -170.3(2) degrees. All hydroxyls, ring and glycosidic oxygens, and water molecules are involved in the hydrogen bonding, which consists of infinite chains linked together by water molecules to form a three-dimensional network. There is a three-centered intramolecular, interresidue hydrogen bond from O-3-H to O-5' and O-6'. The n.m.r. spectrum of the amorphous, dehydrated trihydrate suggests the occurrence of a solid-state reaction forming the same isomeric mixture as was observed in crystalline anhydrous lactulose, although the mutarotation of the trihydrate when dissolved in Me2SO is very slow.     

Cyanide-insensitive NADH oxidation by subcellular fractions isolated from human polymorphonuclear blood cells.

The biochemical triad, NADH oxidation, oxygen (O2) uptake and hydrogen peroxide (H2O2) formation, by subcellular fractions of human blood polymorphonuclears (PMNs) was investigated. It was found that this biochemical triad (1) was under the control of the granule-rich fraction (GRF) only; (2) was not inhibited by cyanide; (3) occurred stoichiometrically for its three components, and (4) accounted quantitatively for the respiratory burst of the stimulated PMN. It was also shown that the above biochemical triad (1) involved an enzymatic step; (2) was enhanced by acidic pH (0.5) and Mg++; (3) was inhibited by Cu++ or low concentration of Mn++; (4) was dependent on H2O2, perhydroxyl radical (HO2) and hydroxyl radical (HO) since either catalase or superoxide dismutase or scavengers of HO2 or HO were inhibitor, and (5) involved multistep reactions. Evidence is provided that the sequence of the reactions is first a generation of H2O2, (spontaneously from NADH in our incubation medium), secondly the production of HO from H2O2, thirdly the oxidation of NADH with further production of HO2,O2 uptake and H2O2 formation, probably through a chain reaction. The identification of the enzyme(s) involved in these multistep reactions needs further studies.