The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes

The hemoprotein ligninase of Phanerochaete chrysosporium catalyzes, in the presence of H2O2, a variety of seemingly different oxidations in lignin and lignin model compounds. Here we show that the enzyme also catalyzes the oxidation of various methoxybenzenes. ESR spectroscopy shows that the compounds are oxidized to aryl cation radicals. These decompose, evidently by H2O addition. Thus, 1,4-dimethoxybenzene is converted to p-benzoquinone and methanol. We propose a unified mechanism, based on formation of aryl cation radicals, to explain the various reactions catalyzed by the ligninase.     

Ligninase-mediated phenoxy radical formation and polymerization unaffected by cellobiose:quinone oxidoreductase

Phanerochete chrysosporium ligninase (+ H2O2) oxidized the lignin substructure-related compound acetosyringone to a phenoxy radical which was identified by ESR spectroscopy. Cellobiose:quinone oxidoreductase (CBQase) + cellobiose, previously suggested to be a phenoxy radical reducing system, was without effect on the radical. Ligninase polymerized guaiacol and it increased the molecular size of a synthetic lignin. These polymerizations, reflecting phenoxy radical coupling reactions, were also unaffected by the CBQase system. We conclude that ligninase catalyzes phenol polymerization via phenoxy radicals, which CBQase does not affect. The CBQase system also did not produce H2O2, and its physiological role remains obscure. Glucose oxidase + glucose did produce H2O2 as expected, but, like CBQase, it did not reduce the phenoxy radical of acetosyringone. Because intact cultures of P. chrysosporium depolymerize lignins, it is likely that phenol polymerization by ligninase is prevented or reversed in vivo by an as yet undescribed system.     

Oxidation of benzo(a)pyrene by extracellular ligninases of Phanerochaete chrysosporium. Veratryl alcohol and stability of ligninase

Benzo(a)pyrene was oxidized with crude and purified extracellular ligninase preparations from Phanerochaete chrysosporium. Both the crude enzyme and the purified fractions oxidized the substrate to three organic soluble products, namely benzo(a)pyrene 1,6-, 3,6-, and 6,12-quinones. These findings support the recent proposition that lignin-degrading enzymes are peroxidases, mediating oxidation of aromatic compounds via aryl cation radicals. The ligninase which was unstable in the presence of hydrogen peroxide could be stabilized by addition of 3,4-dimethoxy benzyl alcohol to the reaction mixture. The oxidation of benzo(a)pyrene was enhanced in the presence of this alcohol.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Non-K-region o-quinones as enzyme-generated inactivators of dihydrodiol dehydrogenase

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on compound I formation of the lignin peroxidase from Phanerochaete chrysosporium

Ligninase, isolated from the wood-destroying fungus Phanerochaete chrysosporium, catalyzes the oxidation of lignin and lignin-related compounds. Ligninase reacts with H2O2 to form the classical peroxidase intermediates Compounds I and II. We have determined the activation energy of ligninase Compound I formation to be 5.9 kcal/mol. The effect of pH and ionic strength on the rate of ligninase Compound I formation was studied. In contrast to all other peroxidases, no pH effect was observed. This is despite homology of active-site amino acids residues (Tien, M., and Tu, C.-P. D. (1987) Nature 326, 520-523) which are proposed to affect the pH profile of Compound I formation. Ligninase Compound I formation can also be supported by organic peroxides. The second-order rate constants with the organic peroxides are lower, suggesting that H2O2 is the preferred substrate.     

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.     

Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes.

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.     

Mechanism of oxidative C alpha-C beta cleavage of a lignin model dimer by Phanerochaete chrysosporium ligninase. Stoichiometry and involvement of free radicals

The hemoprotein ligninase of Phanerochaete chrysosporium Burds. catalyzes the oxidative cleavage of lignin model dimers between C alpha and C beta of their propyl side chains. The model dimers hitherto used give multiple products and complex stoichiometries upon enzymatic oxidation. Here we present experiments with a new model dimer, 1-(3,4-dimethoxyphenyl)-2-phenylethanediol (dimethoxyhydrobenzoin, DMHB) which is quantitatively cleaved by ligninase in air to give benzaldehyde and veratraldehyde according to the stoichiometry: 2DMHB + O2----2PhCHO + 2Ph(OMe)2CHO. Catalytic amounts of H2O2 are required for this aerobic reaction. Under anaerobic conditions, ligninase uses H2O2 as the oxidant for cleavage: DMHB + H2O2----PhCHO + Ph(OMe)2CHO. Electron spin resonance experiments done in the presence of spin traps, 2-methyl-2-nitrosopropane or 5,5-dimethyl-1-pyrroline-N-oxide, show that C alpha-C beta cleavage yields alpha-hydroxybenzyl radicals as intermediate products. Under anaerobic conditions, these radicals react further to give the final aldehyde products. In air, O2 adds to the carbon-centered radicals, probably giving alpha-hydroxybenzylperoxyl radicals which fragment to yield superoxide, benzaldehyde, and veratraldehyde. These results lead us to propose a mechanism for C alpha-C beta cleavage in which attack by ligninase and H2O2 on the methoxylated ring of DMHB yields a cation radical, which then cleaves to give either benzaldehyde and an alpha-hydroxy(dimethoxybenzyl) radical or veratraldehyde and an alpha-hydroxybenzyl radical (cf. Kersten, P. J., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612; Snook, M. E., and Hamilton, G. A. (1974) J. Am. Chem. Soc. 96, 860-869). Similar mechanisms probably apply to the enzymatic C alpha-C beta cleavage of natural lignin.     

Characterization of mercapturic acid and glutathionyl conjugates of benzo[a]pyrene-7,8-dione by two-dimensional NMR.

Non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones represent alternative metabolites of PAH trans-dihydro diol proximate carcinogens. These PAH o-quinones react readily with glutathione and N-acetyl-L-cysteine, and these adducts may be responsible for their detoxication. Reactions between benzo[a]pyrene-7,8-dione and either N-acetyl-L-cysteine or glutathione gave three predominant products which were purified by semipreparative reverse-phase high-pressure liquid chromatography and characterized by homonuclear two-dimensional correlation spectroscopy (COSY). The first product corresponded to a Michael type, 1,4-addition product isolated at the level of quinone oxidation. The second product converted to the first and is a presumptive 1,4-addition product isolated at the level of hydroquinone oxidation. The third product was 7,8-dihydroxybenzo[a]pyrene (a hydroquinone) and was formed as a result of the reductive potential of the thiol. Additional proof for the catechol structure was obtained by its conversion to its diacetate and its identity with authentic 7,8-diacetoxybenzo[a]pyrene. The structures of these adducts and intermediates confirm that thiol addition involves formation of the ketol and rearrangement to give a catechol followed by oxidation to yield the quinone adduct. No evidence was obtained for the formation of either bisphenol or bisglutathionyl adducts. The COSY spectra provide the first complete structure of a benzo[a]pyrenyl-peptide conjugate.     

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.     

Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol beta-aryl ether substructure of lignin.

This study examined the ligninase-catalysed degradation of lignin model compounds representing the arylglycerol beta-aryl ether substructure, which is the dominant one in the lignin polymer. Three dimeric model compounds were used, all methoxylated in the 3- and 4-positions of the arylglycerol ring (ring A) and having various substituents in the beta-ether-linked aromatic ring (ring B), so that competing reactions involving both rings could be compared. Studies of the products formed and the time courses of their formation showed that these model compounds are oxidized by ligninase (+ H2O2 + O2) in both ring A and ring B. The major consequence with all three model compounds is oxidation of ring A, leading primarily to cleavage between C(alpha) and C(beta) (C(alpha) being proximal to ring A), and to a lesser extent to the oxidation of the C(alpha)-hydroxy group to a carbonyl group. Such C(alpha)-oxidation deactivates ring A, leaving only ring B for attack. Studies with C(alpha)-carbonyl model compounds corresponding to the three basic model compounds revealed that oxidation of ring B leads in part to dealkoxylations (i.e. to cleavage of the glycerol beta-aryl ether bond and to demethoxylations), but that these are minor reactions in the model compounds most closely related to lignin. Evidence is also given that another consequence of oxidation of ring B in the C(alpha)-carbonyl model compounds is formation of unstable cyclohexadienone ketals, which can decompose with elimination of the beta-ether-linked aromatic ring. The mechanisms proposed for the observed reactions involve initial formation of aryl cation radicals in either ring A or ring B. The cation radical intermediate from one of the C(alpha)-carbonyl model compounds was identified by e.s.r. spectroscopy. The mechanisms are based on earlier studies showing that ligninase acts by oxidizing appropriately substituted aromatic nuclei to aryl cation radicals [Kersten, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 2609-2612; Hammel, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 8348-8353].     

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.     

Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine

Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.     

Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.     

Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions.

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.     

First synthesis of a polysaccharide-supported lignin model compound and study of its oxidation promoted by lignin peroxidase

Veratrylchitosan, a polysaccharide-supported lignin model compound, has been synthesised by covalently attaching 3-(3,4-dimethoxybenzyloxy)propionic acid to the polysaccharide chitosan through an amide linkage. When this polymer was used as a substrate in the oxidation promoted by lignin peroxidase (LiP), significant decomposition of the lignin model resulted in the formation of veratraldehyde. The oxidation mechanism involves an initial transfer of one electron from chitosan to the active species of LiP (LiP I) followed by C(alpha)-H deprotonation of an aromatic cation radical. A benzylic radical is then formed which is further oxidised to a benzyl cation. Reaction with water and hydrolysis of the hemiacetal then lead to veratraldehyde formation. An increase in the yields of the oxidation product is observed in the presence of the mediator 2-chloro-1,4-dimethoxybenzene, thus indicating that a more efficient degradation results from the transfer of an electron from the polymer to the radical cation of the mediator.     

Enzymatic oxidative activation and transformation of the antitumor agent mitoxantrone

Ambient temperature incubation of the anticancer agent mitoxantrone with horseradish peroxidase and hydrogen peroxide converts it into a hexahydronaphtho[2,3-f]quinoxaline-7,12-dione in which one side chain has cyclized to the chromophore. The structure of this cyclic metabolite was secured by independent synthesis. This peroxidative conversion of mitoxantrone, the progress of which can be followed spectrophotometrically, is accompanied by formation of a free radical species. The EPR characteristics, and dependence on pH of the latter, suggest it exists as a radical cation. The enzymatic oxidation of mitoxantrone is totally irreversible. The purified cyclic metabolite is a substrate for the peroxidase affording the unstable fully oxidized diimino compound and this reaction is fully reversible upon addition of ascorbate or other biological reductants. Admixture of the fully oxidized diimino product with the reduced cyclic metabolite generates the corresponding radical cation species by disproportionation-comproportionation processes. Independent kinetic studies confirm that reaction of the peroxidase with the cyclic metabolite proceeds more rapidly than with mitoxantrone itself. A derivative of mitoxantrone, in which the side-chain secondary amine functions are acylated, generates a radical cation upon treatment with the peroxidase-H2O2 system but does not cyclize subsequently. Derivatives without phenolic hydroxyls or those in which the phenolic hydroxyls are blocked also undergo peroxidative reaction. These observations suggest that initial peroxidative attack occurs at the aromatic nitrogens of mitoxantrone. The possible relevance of these results to the anticancer action of mitoxantrone and the implications for suppression of lipid peroxidation in vivo are discussed.     

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.