The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p‐coumaryl, coniferyl and sinapyl) alcohols in a reaction mediated by peroxidases (EC 1.11.1.7) and laccases (EC 1.10.3.2), yielding H, G and S units, respectively. Although both acidic and basic peroxidases can oxidize p‐coumaryl and coniferyl alcohol, only basic peroxidases are able to oxidize sinapyl alcohol. The AtPrx52 from Arabidopsis is a basic peroxidase that has been reported to be highly homologous to the basic peroxidase of Zinnia elegans, the only peroxidase which has been unequivocally linked to lignin formation. Here, we show how the suppression of AtPrx52 causes a change in lignin composition, mainly at the level of stem interfascicular fibers. Quantification of lignins in two different atprx52 knock‐out mutants revealed a decrease of lignin amount compared with wild type. The S/G ratio, obtained by both nitrobenzene oxidation and thioacidolysis, indicated a decrease in S units in the atprx52 mutants. As deduced from Wiesner and mainly Mäule staining, this reduction in S unit content appears to be restricted to the interfascicular fibers. Moreover, quantitative polymerase chain reaction analysis in atprx52 plants showed a general downregulation of genes involved in lignin biosynthetic pathway, as well as genes related to secondary cell wall. On the other hand, other routes from phenylpropanoid metabolism were induced. Taken together, our results indicate that AtPrx52 is involved in the synthesis of S units in interfascicular fibers at late stages of the lignification process.     

Catalytic Profile of Arabidopsis Peroxidases,AtPrx-2, 25 and 71, Contributing to Stem Lignification

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.     

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in Φ_PSII and q _L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.     

Cell Wall Lignin is Polymerised by Class Ⅲ Secretable Plant Peroxidases in Norway Spruce

Class Ⅲ secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class Ⅲ plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.     

Gene structure,immune response and evolution: Comparative analysis of three 2-Cys peroxiredoxin members of miiuy croaker,Miichthys miiuy

Peroxiredoxin family was a superfamily of selenium independent peroxidases. It was divided into six subtypes: Prx1-4 (typical 2-Cys), Prx5 (atypical 2-Cys) and Prx6 (1-Cys). This study reports the isolation and characterization three 2-Cys peroxiredoxin members of full cDNA and genomic clones from miiuy croaker (Miichthys miiuy). The genetic structure analysis showed that the C-terminal catalytic Cys positioned within GEVCPAXW. This sequence was different between Prx3 and Prx4, but was conservative in different species of the same gene, the X base was S in Prx3 but G in Prx4. Tissues expression analysis showed that the expressions of Prx3 in liver and brain were much higher than other tissues; the values of Prx4 in spleen, intestine and kidney were significantly higher than others; and the expression of Prx5 in muscle was higher than that of other tissues. Real-time PCR results showed that there were highest values of these three Prxs emerging with the time post challenge of Vibrio anguillarum in liver, spleen and kidney although the highest value time differed from each other and the expression of these three genes also changed with the change of infection time. These results indicated that expression analysis of these three genes play some positive function against pathogenic bacteria infection in miiuy croaker.     

Glutathionylation of peroxiredoxin I induces decamer to dimers dissociation with concomitant loss of chaperone activity

Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays a key role in cellular regulation and cell signaling. Peroxiredoxins (Prxs), a family of peroxidases that is involved in removing H(2)O(2) and organic hydroperoxides, are known to undergo a functional change from peroxidase to molecular chaperone upon overoxidation of its catalytic cysteine. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess molecular chaperone activity. We reported earlier that Prx I can be glutathionylated at three of its cysteine residues, Cys52, -83, and -173 [Park et al. (2009) J. Biol. Chem., 284, 23364]. In this study, using analytical ultracentrifugation analysis, we reveal that glutathionylation of Prx I, WT, or its C52S/C173S double mutant shifted its oligomeric status from decamers to a population consisting mainly of dimers. Cys83 is localized at the putative dimer-dimer interface, implying that the redox status of Cys83 may play an important role in stabilizing the oligomeric state of Prx I. Studies with the Prx I (C83S) mutant show that while Cys83 is not essential for the formation of high molecular weight complexes, it affects the dimer-decamer equilibrium. Glutathionylation of the C83S mutant leads to accumulation of dimers and monomers. In addition, glutathionylation of Prx I, both the WT and C52S/C173S mutants, greatly reduces their molecular chaperone activity in protecting citrate synthase from thermally induced aggregation. Together, these results reveal that glutathionylation of Prx I promotes changes in its quaternary structure from decamers to smaller oligomers and concomitantly inactivates its molecular chaperone function.     

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.     

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

A model system to study the lignification process in Eucalyptus globulus

Recalcitrance of plant biomass is closely related to the presence of the phenolic heteropolymer lignin in secondary cell walls, which has a negative effect on forage digestibility, biomass‐to‐biofuels conversion and chemical pulping. The genus Eucalyptus is the main source of wood for pulp and paper industry. However, when compared to model plants such as Arabidopsis thaliana and poplar, relatively little is known about lignin biosynthesis in Eucalyptus and only a few genes were functionally characterized. An efficient, fast and inexpensive in vitro system was developed to study lignification in Eucalyptus globulus and to evaluate the potential role of candidate genes in this biological process. Seedlings were grown in four different conditions, in the presence or absence of light and with or without sucrose in the growth medium, and several aspects of lignin metabolism were evaluated. Our results showed that light and, to a lesser extent, sucrose induced lignin biosynthesis, which was followed by changes in S/G ratio, lignin oligomers accumulation and gene expression. In addition, higher total peroxidase activity and differential isoperoxidase profile were observed when seedlings were grown in the presence of light and sucrose. Peptide sequencing allowed the identification of differentially expressed peroxidases, which can be considered potential candidate class III peroxidases involved in lignin polymerization in E. globulus.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9

Lignin-modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white-rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long-standing issue, we examined the lignin-degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple-gene mutant was generated from a monokaryotic wild-type strain PC9 using plasmid-based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple-gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple-gene mutants were generated. The lignin-degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple-gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple-gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.     

The overexpression of AtPrx37, an apoplastic peroxidase, reduces growth in Arabidopsis

Understanding peroxidase function in plants is difficult because of the lack of substrate specificity, the high number of genes and their diversity in structure. In the present study, the relative expression of 22 genes coding putative peroxidases (E.C 1.11.1.x) in Arabidopsis was studied. The relative expression of AtPrx37 showed a correlation with the cessation of growth in rosette leaves as well as with the growth capacity along the flower stem. Using AtPrx37::GUS construction, its expression was associated with the vascular bundles. Furthermore, the overexpression of AtPrx37 under the control of CaMV 35S promoter rendered a dwarf phenotype with smaller plants and delayed development. The plants overexpressing AtPrx37 also showed an increase in the amount of esterified phenolic material associated with their walls. A role in the growth cessation and phenolic cross-linking during lignin deposition is postulated.     

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.     

Influence of iNOS and COX on peroxiredoxin gene expression in primary macrophages

Peroxiredoxins (Prxs) are a family of multifunctional antioxidant thiol-dependent peroxidases. This study aimed to examine the regulatory mechanisms of Prx gene expression in murine bone marrow-derived macrophages (BMMs) using standardized serum-free conditions. Stimulation with LPS and IFNγ increased mRNA levels of Prx 1, 2, 4, 5, and 6 in BMMs of both C57BL/6 and BALB/c mice, with Prx 1, 2, 4, and 6 more strongly induced in C57BL/6 BMMs. Further investigations on signaling pathways in C57BL/6 BMMs demonstrated that up-regulation of Prx 5 and 6 by LPS and IFNγ was associated with the activation of multiple protein kinases, most notably JAK2, PI3K, and p38 MAPK. Our experiments also revealed a contribution of inducible NO synthase-derived nitric oxide to the increase in Prx 1, 2, 4, and 6 mRNA expression, whereas NADPH oxidase-derived superoxide was not involved. Furthermore, we could show that LPS- and IFNγ-induced gene expression of Prx 6 was also regulated in an NO-independent manner by cyclooxygenases and prostaglandin E₂. Taken together our results indicate a possible role for Prxs in defense mechanisms of activated macrophages against oxidative stress during inflammation or infection.     

Peroxiredoxins: a less studied component of hydrogen peroxide detoxification in photosynthetic organisms

Peroxiredoxins (Prx) are ubiquitous thiol-dependent peroxidases capable of reducing a broad range of toxic peroxides and peroxinitrites. A cysteinyl residue of peroxiredoxins reacts with the peroxides as primary catalytic center and oxidizes to sulfenic acid. The regeneration of the reduced form of Prx is required as a next step to allow its entry into next catalytic cycle. Several proteins, such as thioredoxin, glutaredoxin, cyclophilin, among others, are known to facilitate the regeneration of the reduced (catalytically active) form of Prx in plants. Based on the cysteine residues conserved in the deduced amino acid sequence and their catalytic mechanisms, four groups of peroxiredoxins have been distinguished in plants, namely, 1-Cys Prx, 2-Cys Prx, Type II Prx and Prx Q. Peroxiredoxins are known to play an important role in combating the reactive oxygen species generated at the level of electron transport activities in the plant exposed to different types of biotic and abiotic stresses. In addition to their role in antioxidant defense mechanisms in plants, they also modulate redox signaling during development and adaptation. Besides these general properties, peroxiredoxins have been shown to protect DNA from damage in vitro and in vivo. They also regulate metabolism in thylakoids and mitochondria. The present review summarizes the most updated information on the structure and catalysis of Prx and their functional importance in plant metabolism.     

Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3–4 Å or ∼6–7 Å away from the active site peroxidative cysteine (C_p), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C_p sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H₂O₂ and may have wider implications for protein thiol reactivity toward peroxides.     

Purification,characterization and accumulation of three virus-induced cucumber peroxidases

Three anionic peroxidases (EC 1.11.1.7), named Prx1, 2, and 3, which are rapidly accumulated in cucumber (Cucumis sativus L., cv. Laura) reacting hypersensitively to tobacco necrosis virus, were purified to homogeneity. The three enzymes had an isoelectric point about 4.3, and the relative molecular masses of Prx1, 2, and 3 estimated by SDS-PAGE were 40 700, 38 000, and 37 100, respectively. These peroxidases had a similar pH stability, but differed in their specific activity, pH optimum, and thermal stability By Ouchterlony double diffusion tests with antisera raised against the three purified enzymes, close serological relationships have been demonstrated between the three peroxidases.     

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.     

Lignin peroxidase H2 from Phanerochaete chrysosporium: purification, characterization and stability to temperature and pH

The wood-destroying fungus Phanerochaete chrysosporium secretes extracellular enzymes known as lignin peroxidases that are involved in the biodegradation of lignin and a number of environmental pollutants. Several lignin peroxidases are produced in liquid cultures of this fungus. However, only lignin peroxidase isozyme H8 has been extensively characterized. In agitated nutrient nitrogen-limited culture, P. chrysosporium produces two lignin peroxidases in about equal proportions. The molecular weights of these two major proteins (H2 and H8) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 38,500 (H2) and 42,000 (H8). The isoelectric points of these enzymes were 4.3 for H2 and 3.65 for H8. All subsequent experiments in this study were performed with H2 as it contributed the most (42%) to total activity and had the highest specific activity (57.3 U/mg). The Km values of lignin peroxidase H2 for H2O2 and veratryl alcohol were calculated to be 47 microM and 167 microM at pH 3.5, respectively. The pH optima for veratryl alcohol oxidase activity were pH 2.5 at 25 degrees C, pH 3.0 at 35 degrees C, and pH 3.5 at 45 degrees C. In the same manner the temperature optimum shifted from 25 degrees C at pH 2.5 to 45 degrees C at pH 3.5 and approximately 45-60 degrees C at pH 4.5. During storage the resting enzyme was relatively stable for 48 h up to 50 degrees C. Above this temperature the enzyme lost all activity within 6 h at 60 degrees C. At 70 degrees C all activity was lost within 10 min. The resting enzyme retained approximately 80% of its initial activity when stored at 40 degrees C for 21 h at a pH range of 4.0-6.5. Above pH 7.5 and below 4.0, the enzyme lost all activity in less than 5 h. During turnover the enzyme remained active at pH 5.5 for over 2 h whereas the enzyme activity was lost after 45 min at pH 2.5. The oxidation of veratryl alcohol was inhibited by EDTA, azide, cyanide, and by the catalase inhibitor 3-amino-1,2,4-triazole, but not by chloride. In the absence of another reducing substrate incubation of lignin peroxidase H2 with excess H2O2 resulted in partial and irreversible inactivation of the enzyme. The spectral characteristics of lignin peroxidase H2 are similar to those of other peroxidases. The suitability of lignin peroxidases for industrial applications is discussed.