Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.     

Hybrid Mn-peroxidases from basidiomycetes: a review

The Mn-peroxidase from the fungus Panus tigrinus 8/18 is a hybrid enzyme. It catalyzes both Mn2+-dependent and Mn2+-independent oxidation of organic substrates. The spectral properties of intermediates and the pathway of the catalytic cycle are typical of hybrid Mn-peroxidases. The enzyme catalyzes the "oxidase" reaction (NADH oxidation) without peroxide and with the presence of Mn2+, which takes part in hydrogen peroxide production via Mn3+ and preserves the enzyme from inactivation. With the presence of organic mediators, the hybrid Mn-peroxidase oxidizes nonphenolic compounds: aromatic alcohols and a nonphenolic lignin model compound. The degree of conversion of 2,4,6-trichlorophenol is higher with the presence of l-hydroxybenzotriazole.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata

A homogeneous Mn-dependent peroxidase (MnP) was purified from the extracellular culture fluid of the lignin-degrading white rot fungus Phlebia radiata by anion exchange chromatography. The enzyme had a molecular weight of 49,000 and pI 3.8. It was a glycoprotein, containing carbohydrate moieties accounting for 10% of the molecular weight. Mn-peroxidase was capable of oxidizing phenolic compounds in the presence of H2O2, whereas the effect on nonphenolic lignin model compounds was insignificant. MnP contained protoporphyrin IX as a prosthetic group. During enzymatic reactions H2O2 converted the native MnP to compound II. Mn2+ was essential in completing the catalytic cycle by returning the enzyme to its native state. The oxidation of ultimate substrates was dependent on superoxide radicals, O2- and probably on Mn3+ generated during the catalytic cycle. MnP exhibited high activity of NADH oxidation without exogenously added H2O2. It was shown to produce H2O2 at the expense of NADH.     

Characterization of reactions catalyzed by manganese peroxidase from Phanerochaete chrysosporium

Manganese peroxidase (MnP) is one of two extracellular peroxidases believed to be involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. The enzyme oxidizes Mn(II) to Mn(III), which accumulates in the presence of Mn(III) stabilizing ligands. The Mn(III) complex in turn can oxidize a variety of organic substrates. The stoichiometry of Mn(III) complex formed per hydrogen peroxide consumed approaches 2:1 as enzyme concentration increases at a fixed concentration of peroxide or as peroxide concentration decreases at a fixed enzyme concentration. Reduced stoichiometry below 2:1 is shown to be due to Mn(III) complex decomposition by hydrogen peroxide. Reaction of Mn(III) with peroxide is catalyzed by Cu(II), which explains an apparent inhibition of MnP by Cu(II). The net decomposition of hydrogen peroxide to form molecular oxygen also appears to be the only observable reaction in buffers that do not serve as Mn(III) stabilizing ligands. The nonproductive decomposition of both Mn(III) and peroxide is an important finding with implications for proposed in vitro uses of the enzyme and for its role in lignin degradation. Steady-state kinetics of Mn(III) tartrate and Mn(III) malate formation by the enzyme are also described in this paper, with results largely corroborating earlier findings by others. Based on a comparison of pH effects on the kinetics of enzymatic Mn(III) tartrate and Mn(III) malate formation, it appears that pH effects are not due to ionizations of the Mn(III) complexing ligand.     

Catalytic mechanisms and regulation of lignin peroxidase.

Lignin peroxidase (LiP) is a fungal haemoprotein similar to the lignin-synthesizing plant peroxidases, but it has a higher oxidation potential and oxidizes dimethoxylated aromatic compounds to radical cations. It catalyses the degradation of lignin models but in vitro the outcome is net lignin polymerization. LiP oxidizes veratryl alcohol to radical cations which are proposed to act by charge transfer to mediate in the oxidation of lignin. Phenolic compounds are, however, preferentially oxidized, but transiently inactivate the enzyme. Analysis of the catalytic cycle of LiP shows that in the presence of veratryl alcohol the steady-state turnover intermediate is Compound II. We propose that veratryl alcohol is oxidized by the enzyme intermediate Compound I to a radical cation which now participates in charge-transfer reactions with either veratryl alcohol or another reductant, when present. Reduction of Compound II to native state may involve a radical product of veratryl alcohol or radical product of charge transfer. Phenoxy radicals, by contrast, cannot engage in charge-transfer reactions and reaction of Compound II with H2O2 ensues to form the peroxidatically inactive intermediate, Compound III. Regulation of LiP activity by phenolic compounds suggests feedback control, since many of the products of lignin degradation are phenolic. Such control would lower the concentration of phenolics relative to oxygen and favour degradative ring-opening reactions.     

Oxidation of methoxybenzenes by manganese peroxidase and by Mn3+

Manganese peroxidase, produced by some white-rot fungi during lignin degradation, catalyzes the oxidation of Mn2+ to Mn3+. Whereas Mn3+ is known to oxidize phenolic compounds, its role in lignin degradation is not clear. We have used a series of methoxybenzenes with E1/2 values of 1.76-0.81 V (vs saturated calomel electrode) to investigate the oxidizing ability of Mn3+ chelates generated chemically and enzymatically. Although lignin peroxidase has been shown to oxidize high potential congeners, our results show that manganese peroxidase, or physiological concentrations of Mn3+, oxidize only the lower potential congeners. In addition, Mn3+ increased the rate of decay of the cation radical of 1,2,4,5-tetramethoxybenzene. The kinetics of decay continued to be first order, so Mn3+ does not oxidize the cation radical itself, but probably oxidizes a neutral dienyl radical derived from the cation radical. This indicates a possible role for Mn3+ in lignin degradation, as neutral dienyl radicals are proposed to be products of lignin peroxidase action.     

Induction, isolation, and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH(2)). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH(2) extended the reactions catalyzed by these enzymes to the production of H(2)O(2), the oxidation of Mn(2+), the reduction of Fe(3+), and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

Extracellular laccases and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension cultures

We have investigated the abilities of extracellular enzymes from dark-grown cell-suspension cultures of sycamore maple (Acer pseudoplatanus L.) to oxidize monolignols, the precursors for lignin biosynthesis in plants, as well as a variety of other lignin-related compounds. Laccase and peroxidase both exist as a multiplicity of isoenzymes in filtrates of spent culture medium, but their abilities to produce water-insoluble, dehydrogenation polymers (DHPs) from the monolignols (in the presence of hydrogen peroxide for the peroxidase reaction) appear identical whether or not the enzymes are purified from the concentrated filtrates or left in a crude mixture. The patterns of bonds formed in these DHPs are identical to those found in DHPs synthesized using horseradish peroxidase or fungal laccase, and many of these bonds are found in the natural lignins extracted from different plant sources. On the other hand, sycamore maple laccase is very much less active on phenolic substrates containing multiple aromatic rings than is sycamore maple peroxidase. We suggst that whereas laccase may function during the early stages of lignification to polymerize monolignols into oligo-lignols, cell-wall peroxidases may function when H₂O₂ is produced during the later stages of xylem cell development or in response to environmental stresses.Abbreviations DHP dehydrogenation polymer - IEF isoelectric focuring - NMR nuclear magnetic resonance - PAGE polyacrylamide gel electrophoresis The authors wish to thank Dr. Masahiro Samejima (University of Tokyo) for provision of lignin model compounds and Dr. Göran Gellerstadt (Royal Institute of Technology, Sweden) for helpful suggestions regarding stilbene formation and light spectroscopy. Monolignols were prepared by Mr. Nate Weymouth with help from Dr. Herb Morrison (USDA/ARS, Richard B. Russell Research Center, Athens, GA). Thanks also to Ms. Izabella Poppe of the Complex Carbohydrate Research Center (CCRC) for assistance with carbohydrate analyses, and Mr. Vincent Sorrentino for help with the growth of cell-suspension cultures.     

Purification and properties of NADH oxidase from Bacillus megaterium

NADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD+ and hydrogen peroxide was purified from Bacillus megaterium by 5'-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr = 52,000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mM KPi buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH:oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.     

Hybrid Mn-peroxidases from basidiomycetes

The Mn-peroxidase from the fungus Panus tigrinus 8/18 is a hybrid enzyme. It catalyzes both Mn²⁺-dependent and Mn²⁺-independent oxidation of organic substrates. The spectral properties of intermediates and the pathway of the catalytic cycle are typical of hybrid Mn-peroxidases. The enzyme catalyzes the “oxidase” reaction (NADH oxidation) without peroxide and with the presence of Mn²⁺, which takes part in hydrogen peroxide production via Mn³⁺ and preserves the enzyme from inactivation. With the presence of organic mediators, the hybrid Mn-peroxidase oxidizes nonphenolic compounds: aromatic alcohols and a nonphenolic lignin model compound. The degree of conversion of 2,4,6-trichlorophenol is higher with the presence of 1-hydroxybenzotriazole.     

Purification and Characterization of Cellobiose Dehydrogenases from the White Rot Fungus Trametes versicolor

The white rot fungus Trametes versicolor degrades lignocellulosic material at least in part by oxidizing the lignin via a number of secreted oxidative and peroxidative enzymes. An extracellular reductive enzyme, cellobiose dehydrogenase (CDH), oxidizes cellobiose and reduces insoluble Mn(IV)O(inf2), commonly found as dark deposits in decaying wood, to form Mn(III), a powerful lignin-oxidizing agent. CDH also reduces ortho-quinones and produces sugar acids which can promote manganese peroxidase and therefore ligninolytic activity. To better understand the role of CDH in lignin degradation, proteins exhibiting cellobiose-dependent quinone-reducing activity were isolated and purified from cultures of T. versicolor. Two distinct proteins were isolated; the proteins had apparent molecular weights of 97,000 and 81,000 and isoelectric points of 4.2 and 6.4, respectively. The larger CDH (CDH 4.2) contained both flavin and heme cofactors, whereas the smaller contained only a flavin (CDH 6.4). These CDH enzymes were rapidly reduced by cellobiose and lactose and somewhat more slowly by cellulose and certain cello-oligosaccharides. Both glycoproteins were able to reduce a very wide range of quinones and organic radical species but differed in their ability to reduce metal ion complexes. Temperature and pH optima for CDH 4.2 were affected by the reduced substrate. Although CDH 4.2 showed rather high substrate specificity among the ortho-quinones, it could also rapidly reduce a structurally very diverse collection of other species, from negatively charged triiodide ions to positively charged hexaquo ferric ions. CDH 6.4 showed a higher K(infm) and a lower V(infmax) and turnover number than did CDH 4.2 for all substrates tested. Furthermore, CDH 6.4 did not reduce the transition metals Fe(III), Cu(II), and Mn(III) at concentrations likely to be physiologically relevant, while CDH 4.2 was able to rapidly reduce even very low concentrations of these ions. The reduction of Fe(III) and Cu(II) by CDH 4.2 may be important in sustaining a Fenton's-type reaction, which produces hydroxyl radicals that can cleave both lignin and cellulose. Unlike the CDH proteins from Phanerochaete chrysosporium, CDH 4.2 and CDH 6.4 are unable to produce hydrogen peroxide.     

A search for ligninolytic peroxidases in the fungus pleurotus eryngii involving alpha-keto-gamma-thiomethylbutyric acid and lignin model dimers

Because there is some controversy concerning the ligninolytic enzymes produced by Pleurotus species, ethylene release from alpha-keto-gamma-thiomethylbutyric acid (KTBA), as described previously for Phanerochaete chrysosporium lignin peroxidase (LiP), was used to assess the oxidative power of Pleurotus eryngii cultures and extracellular proteins. Lignin model dimers were used to confirm the ligninolytic capabilities of enzymes isolated from liquid and solid-state fermentation (SSF) cultures. Three proteins that oxidized KTBA in the presence of veratryl alcohol and H2O2 were identified (two proteins were found in liquid cultures, and one protein was found in SSF cultures). These proteins are versatile peroxidases that act on Mn2+, as well as on simple phenols and veratryl alcohol. The two peroxidases obtained from the liquid culture were able to degrade a nonphenolic beta-O-4 dimer, yielding veratraldehyde, as well as a phenolic dimer which is not efficiently oxidized by P. chrysosporium peroxidases. The former reaction is characteristic of LiP. The third KTBA-oxidizing peroxidase oxidized only the phenolic dimer (in the presence of Mn2+). Finally, a fourth Mn2+-oxidizing peroxidase was identified in the SSF cultures on the basis of its ability to oxidize KTBA in the presence of Mn2+. This enzyme is related to the Mn-dependent peroxidase of P. chrysosporium because it did not exhibit activity with veratryl alcohol and Mn-independent activity with dimers. These results show that P. eryngii produces three types of peroxidases that have the ability to oxidize lignin but lacks a typical LiP. Similar enzymes (in terms of N-terminal sequence and catalytic properties) are produced by other Pleurotus species. Some structural aspects of P. eryngii peroxidases related to the catalytic properties are discussed.     

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.     

31P-NMR and ESR studies of the oxidation states of manganese in Staphylococcus aureus

High-resolution 31P-NMR and ESR spectroscopies are used to probe the role of manganese in oxygen metabolism, in vivo, by Staphylococcus aureus. The linewidth of the intracellular orthophosphate resonance in the 31P-NMR spectrum and the amplitude of the ESR sextet of signals due to Mn2+ hexaquo ions are found to be sensitive to the oxygenation state of the cells. These results are attributed to changes in the oxidation state of the manganese. It is concluded that manganous ions are oxidized to Mn3+ in oxygenated cells. Mn3+ is in turn reduced to Mn2+ under anaerobic conditions. The Mn2+ is also oxidized to Mn3+ by hydrogen peroxide probably as a result of the disproportionation of H2O2 to H2O and O2 by an active catalase in S. aureus. Addition of mercaptoethanol to a suspension of oxygenated cells results in the reduction of Mn3+ to Mn2+.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Enzymatic and hydrolytic degradation of benzylaryl ethers related to hardwood lignins

A number of 4-hydroxybenzylphenyl ethers and their acetates were synthesized as models for hardwood lignin and used as substrates in acid hydrolysis and enzymatic oxidation reactions. Under hydrolytic conditions, the acetates underwent ether cleavage at a slower rate than the free phenols. Evidence for carbonium ion intermediates is presented. Cleavage of the ether substrates by peroxidase—peroxide oxidation was much faster than by acid hydrolysis for all substrates except the acetates which did not react. Subsequent oxidation of the component parts of the ether substrates was selective: the syringyl moieties were oxidized in preference to the guaiacyl moieties. Electron spin resonance studies of the oxidation reaction showed that removal of the phenolic hydrogen atom was the first step, followed by quinone—methide formation. A mechanism is proposed to account for the oxidative degradation of the lignin models.     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.