Lignin-degrading Fungus Phanerochaete Chrysosporium Research Articles

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5′-adenosyl)-l-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.

Mycelial growth and solid-state fermentation of lignocellulosic waste by white-rot fungus Phanerochaete chrysosporium under lead stress

Lignocellulosic biomass is an abundant renewable resource difficult to degrade. Its bioconversion plays important roles in carbon cycles in nature, which may be influenced by heavy metals in environment. Mycelial growth and the degradation of lignocellulosic waste by lignin-degrading fungus Phanerochaete chrysosporium under lead stress were studied. It was shown that P. chrysosporium could grow in liquid media with 400 mg L −1 Pb(II), and mycelial dry weight was reduced by 54% compared to the control. Yellow mycelia in irregular short-strip shape formed in Pb-containing media, whereas the control showed ivory-white regular mycelial pellets. Two possible responses to Pb stress were: dense hyphae, and secretion from mycelia to resist Pb. During solid-state fermentation of straw, fungal colonization capability under Pb stress was positively correlated with the removal efficiency of soluble–exchangeable Pb when its content was higher than 8.2 mg kg −1 dry mass. Carboxymethyl cellulase activity and cellulose degradation were inhibited at different Pb concentrations, whereas low Pb concentrations increased xylanase and ligninolytic enzyme activities and the hemicellulose and lignin degradation. Cluster analyses indicated that Pb had similar effects on the different microbial indexes related to lignin and hemicellulose degradation. The present findings will advance the understandings of lignocellulose degradation by fungi under Pb pollution, which could provide useful references for developing metal-polluted waste biotreatment technology.

Diversity and evolution of LTR retrotransposons in the genome of Phanerochaete chrysosporium (Fungi: Basidiomycota)

The diversity of mobile elements, in particular LTR retrotransposons, in basidiomycetes fungi has been poorly studied. Using complete genomic sequences, we have for the first time screened the genome of the lignin-degrading fungus Phanerochaete chrysosporium for LTR retrotransposons. A surprisingly high diversity of LTR retrotransposons was found. Twenty-three novel mobile elements from two superfamilies, Pseudoviridae and Metaviridae, were described. The proportion of LTR retrotransposons in the P. chrysosporium genome is low, constituting only about 3%. Nevertheless, LTR retrotransposons of P. chrysosporium represent a dynamic part of the genome, which is evidenced by the presence of intact copies with signs of recent transposition and numerous solo LTR elements. Phylogenetic and structural analyses detected mobile elements having characteristics that had been previously unknown for other LTR retrotransposons.

Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families.

Fungal cytochrome P450s catalyzing hydroxylation of substituted toluenes to form their hydroxymethyl derivatives

The degradation of a series of nitroaromatic compounds by the lignin-degrading fungus Phanerochaete chrysosporium was examined. From 4-nitrotoluene (4-NT), several metabolic intermediates were identified. Initially, 4-NT was converted to 4-nitrobenzyl alcohol (4-NBA), followed by the oxidation reactions to form 4-nitrobenzaldehyde and 4-nitrobenzoic acid, albeit slowly. Exogenously added 4-nitrobenzaldehyde and 4-nitrobenzoic acid were predominantly reduced to 4-NBA. The fungal formation of 4-NBA was inhibited by piperonyl butoxide, a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450 in the hydroxylation of the methyl group. Similarly, 2-, and 3-nitrotoluenes and 4-chlorotoluene were converted to the corresponding arylalcohols by P. chrysosporium. On the other hand, toluene and 4-methoxytoluene were not converted. Thus, P. chrysosporium possesses an alkyl hydroxylation activity against aromatic compounds substituted with a strong electron-withdrawing group.

Stability of immobilized 2,4,6-trinitrotoluene metabolites in soil under long-term leaching conditions

2,4,6-Trinitrotoluene (TNT)-contaminated soil was remediated by an anaerobic/aerobic slurry process. Prior to treatment, the soil was spiked with [14C]-TNT. Leaching experiments were carried out with the decontaminated soil to determine the degree of binding of the radiolabel under a variety of conditions. To simulate natural degradation processes of soil organic matter, each of three columns was subjected to a different treatment known to enhance biological transformation over a 92-week period. Only minor amounts of radioactivity (1.0% of the bound radioactivity) were released from treated soil incubated in the presence of the lignin-degrading fungus Phanerochaete chrysosporium. Simulation of seasonal variation in temperature, including freezing of the soil, did not cause a significant release of radioactivity (1.4%). Growth and flowering of the bush bean Phaseolus vulgaris only released 0.8% of the bound radioactivity to the eluate; however, during the decomposition phase, an additional 7.7% of the bound radioactivity was released. We propose that this radioactivity was bound to soluble humic material that was mobilized due to a pH shift during the decomposition of the plant organic matter. This is supported by the observation that neither free TNT nor its metabolites were present in the eluate. During the different incubation experiments, 3.9 to 8.5% of the bound radioactivity was found as 14CO2. The results indicate a slow turnover of even strongly bound immobilized metabolites of TNT.

Stability of immobilized 2,4,6-trinitrotoluene metabolites in soil under long-term leaching conditions.

2,4,6-Trinitrotoluene (TNT)-contaminated soil was remediated by an anaerobic/aerobic slurry process. Prior to treatment, the soil was spiked with [14C]-TNT. Leaching experiments were carried out with the decontaminated soil to determine the degree of binding of the radiolabel under a variety of conditions. To simulate natural degradation processes of soil organic matter, each of three columns was subjected to a different treatment known to enhance biological transformation over a 92-week period. Only minor amounts of radioactivity (1.0% of the bound radioactivity) were released from treated soil incubated in the presence of the lignin-degrading fungus Phanerochaete chrysosporium. Simulation of seasonal variation in temperature, including freezing of the soil, did not cause a significant release of radioactivity (1.4%). Growth and flowering of the bush bean Phaseolus vulgaris only released 0.8% of the bound radioactivity to the eluate; however, during the decomposition phase, an additional 7.7% of the bound radioactivity was released. We propose that this radioactivity was bound to soluble humic material that was mobilized due to a pH shift during the decomposition of the plant organic matter. This is supported by the observation that neither free TNT nor its metabolites were present in the eluate. During the different incubation experiments, 3.9 to 8.5% of the bound radioactivity was found as 14CO2. The results indicate a slow turnover of even strongly bound immobilized metabolites of TNT.

Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain.

By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.

A Study of the Microbial Degradation of Metal Phthalocyanine Textile Dyes by High-performance Liquid Chromatography and Atomic Absorption

The investigation of the degradation of two commercially available dyes (Remazol Turquoise Blue and Everzol Turquoise Blue) by the lignin-degrading fungus Phanerochaete chrysosporium and comparison with that of a phthalocyanine whose structure is known (Heligon Blue) are presented. Atomic absorption showed a large release of copper from the biomass at day 7. Polarography served to speciate the copper present in the supernatant. Day 5 sees the complete disappearance of the main dye peak with the release of free copper into the supernatant. Day 7 sees a large increase in the free copper signal with two other electroactive species also present in the supernatant, all of which are seen to decrease at day 8. Visible spectroscopy shows that the main decolourization takes place between day 4 and day 6, with complete decolourization occurring at day 7. HPLC analysis again confirms the above results, with possible degradation products detected at 254 nm occurring at 3.621 and 4.170 min at day 7 which may well correlate with those found in polarographic analysis at –1050 and –1150 mV. Day 7 also sees a large increase in a peak at 2.744 min.

Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.

Effects of fungal pretreatment and steam explosion pretreatment on enzymatic saccharification of plant biomass

The effects of consecutive treatments by a lignin-degrading fungus Phanerochaete chrysosporium and by steam explosion for the enzymatic saccharification of plant biomass were studied experimentally, and the optimal operational conditions for obtaining the maximum saccharification were evaluated. Beech wood-meal was treated by the fungus for 98 days and then by high steam temperatures of 170-230 degrees C with steaming times of 0-10 min. The treatment of the wood-meal by fungus prior to steam explosion enhanced the saccharification of wood-meal. The treated wood-meal was separated into holo-cellulose, water soluble material, methanol soluble lignin, and Klason lignin. The saccharification decreased linearly with the increase in the amount of Klason lignin. It was estimated by the equation for the saccharification of exploded wood-meal expressed as a function of steam temperature and steaming time that the maximum saccharification of wood-meal was obtained by consecutive treatments such as fungal treatment for 28 days and then steam explosion at a steam temperature of 215 degrees C and a steaming time of 6.5 min. (c) 1995 John Wiley & Sons, Inc.

Regulation of manganese peroxidase gene transcription by hydrogen peroxide, chemical stress, and molecular oxygen

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of the lignin-degrading fungus Phanerochaete chrysosporium is regulated at the level of gene transcription by H2O2 and various chemicals, including ethanol, sodium arsenite, and 2,4-dichlorophenol, as well as by Mn(II) and heat shock. Northern (RNA) blot analysis demonstrates that the addition of 1.0 mM H2O2 to 5-day-old cultures grown in the absence of Mn results in the appearance of mnp mRNA within 15 min. Higher levels of mnp mRNA are obtained with simultaneous induction by Mn and H2O2 than with H2O2 alone. Although neither MnP activity nor associated protein is detectable in H2O2-induced cultures grown in the absence of Mn, simultaneous induction with Mn and H2O2 results in a 1.6-fold increase in MnP activity compared with the MnP activity resulting from Mn induction alone. In the presence of Mn, purging of low-nitrogen cultures with 100% O2, in contrast to incubation under air, results in an increase in the accumulation of mnp mRNA and a 13-fold increase in MnP activity on day 5. However, in contrast to the effects of H2O2 and heat shock, O2 purging of Mn-deficient cultures results in negligible accumulation of mnp mRNA.

Aromatic Nitroreductase from the Basidiomycete Phanerochaete chrysosporium

A membrane-associated aromatic nitroreductase activity was identified in cell-free extracts of the lignin-degrading fungus Phanerochaete chrysosporium. The enzyme catalyzed the nitro group reduction of 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, 1-chloro-2,4-dinitrobenzene, and 2,4-dichloro-1-nitrobenzene. The corresponding hydroxylamines and/or amines were identified as reaction products by HPLC and/or GC-MS. 1-Nitroso-3-nitrobenzene and 1-hydroxylamino-3-nitrobenzene also were reduced by the enzyme, suggesting they were intermediates in the reaction. The enzyme required NAD(P)H as a cosubstrate and the optimal pH and temperature for the reaction were 6.5 and 50°C, respectively. Enzyme activity was not observed in the presence of molecular oxygen. The membrane-associated enzyme could be solubilized with the nonionic detergent Triton X-100.

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (Vmax) of the system was 3.86 units (U) mg−1 protein with arabinoxylan as substrate and the substrate concentration giving half Vmax (S0.5) was 0.52 mg ml−1. At concentrations of arabinoxylan greater than 15 mg ml−1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components.

Kinetic analysis of manganese peroxidase. The reaction with manganese complexes.

Manganese peroxidase from the lignin-degrading fungus Phanerochaete chrysosporium catalyzes the H2O2-dependent oxidation of Mn2+ to Mn3+. Presteady-state methods were employed to characterize the reactions of free and chelated Mn2+ with the 2-electron and 1-electron oxidized forms of the enzyme, compounds I and II, respectively. At pH 4.5, the optimum pH for steady-state turnover, the reaction of compound I with Mn2+, either free or complexed, is too rapid to measure by stopped flow methods. The reactions of compound I with Mn2+ can only be monitored under non-optimal conditions of pH 2.5. The reaction of compound II with Mn2+ is much slower than compound I. Chelators such as oxalate, lactate, and malonate facilitated the reaction of Mn2+ with compound II. In contrast, succinate, which does not readily form a complex with Mn2+, and polyglutamate, which is polymeric, were ineffective in stimulating the reaction of Mn2+ with compound II. The 1:1 chelator-Mn2+ complex is the preferred substrate for compound II; this conclusion is based on known formation constants for the various Mn2+ complexes. Steady-state kinetics studies were performed by directly measuring the initial rate of Mn3+ formation. The kcat values for the formation of Mn(3+)-oxalate, Mn(3+)-lactate, and Mn(3+)-malonate are 308, 211, and 220 s-1, respectively. The Km values for Mn(2+)-oxalate, Mn(2+)-lactate, and Mn(2+)-malonate are 13, 41, and 18 microM, respectively. These results collectively indicate that manganese peroxidase does not readily oxidize free (hexa-aquo) Mn2+ as previously proposed (Wariishi, H., Valli, K., and Gold, M. H. (1992) J. Biol. Chem. 267, 23688-23695), but the Mn2+ has to be chelated to support steady-state turnover.

An overview of the recent advances on the physiology and molecular biology of lignin peroxidases of Phanerochaete chrysosporium

The lignin-degrading white-rot fungus Phanerochaete chrysosporium produces two families of extracellular peroxidases designated lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) which are components of the lignin degradation system of this organism. The number and types of LIP and MNP isozymes produced vary dramatically in response to changes in culture conditions. Protease-mediated degradation of LIPs was shown to be the major cause for the decay of LIP activity in idiophasic cultures of P. chrysosporium. Use of biochemical mutants has not only yielded information on the relative importance of LIPs and MNPs in lignin degradation but has given us insights into the regulation of production of LIPs and MNPs. The genes encoding the major LIPs have been cloned and sequenced and were shown to have a high degree of homology to each other. Karyotyping studies indicated that heterokaryotic strains contain ten chromosomes and that the LIP genes are distributed on at least two chromosomes.

Glyoxylate-Supported Reactions Catalyzed by Mn Peroxidase of Phanerochaete chrysosporium: Activity in the Absence of Added Hydrogen Peroxide

Mn peroxidases are H 2O 2-utilizing hemeproteins secreted by the lignin-degrading fungus Phanerochaete chrysosporium. We show here that glyoxylate is capable of supporting Mn peroxidase activity without added H 2O 2. This glyoxylate-supported activity is dependent upon Mn 2+ and dioxygen. The involvement of superoxide is demonstrated by inhibition by superoxide scavenging agents, superoxide dismutase, or tetranitromethane. The addition of catalase resulted in dioxygen evolution, indicating that H 2O 2 is an intermediate in the reaction. Formate is one of the oxidation products of glyoxylate as detected by the formate dehydrogenase assay. The generation of H 2O 2 in the presence of Mn 2+ and Mn peroxidase results in Mn 3+ formation. Consequently, we show that the direct reaction between glyoxylate and Mn 3+ also results in formate formation. The stoichiometry of this reaction approaches 1:1. Electron spin resonance, spin-trapping studies show formation of the formate radical CO 2 in the reaction of Mn 3+ and glyoxylate.

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei

We have identified a genomic DNA fragment from the lignin-degrading fungus Phanerochaete chrysosporium ( P.c.) that hybridises to a DNA probe encoding part of the exo-cellobiohydrolase I (CBHI) gene of Trichoderma reesei ( T.r.). This fragment has been subcloned and its nucleotide sequence determined. We demonstrate that it could encode a 516 residue protein that shows strong homology with the known protein sequence of CBHI from T.r. Comparison of the two nucleotide sequences identifies two regions within the P.c. sequence that are not represented in T.r. Further inspection of these regions reveals sequences closely related to the conserved elements of filamentous fungal introns. We conclude that the P.c. genomic sequence contains the same number of introns (2) as found in T.r. but that these are located at different relative positions within the two genes. The transcript from the P.c. sequence is induced in the presence of cellulose but not by glucose and we therefore conclude that this sequence represents the first cellulase gene to have been described from this organism.

Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle.

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the Mn(II)-dependent oxidation of a variety of phenols. Herein, we spectroscopically characterize the oxidized states of MnP compounds I, II, and III and clarify the role of Mn in the catalytic cycle of the enzyme. Addition of 1 equiv of H2O2 to the native ferric enzyme yields compound I, characterized by absorption maxima at 407, 558, 605, and 650 nm. Addition of 2 or 250 equiv of H2O2 to the native enzyme yields compound II or III, respectively, identified by absorption maxima at 420, 528, and 555 nm or at 417, 545, and 579 nm, respectively. These characteristics are very similar to those of horseradish peroxidase (HRP) and lignin peroxidase (LiP) compounds I, II, and III. Addition of 1 equiv of either Mn(II), ferrocyanide, or a variety of phenols to MnP compound I rapidly reduces it to MnP compound II. In contrast, only Mn(II) or ferrocyanide, added at a concentration of 1 equiv, reduces compound II. The Mn(III) produced by the enzymic oxidation of Mn(II) oxidizes the terminal phenolic substrates. This indicates that compounds I and II of MnP contain 2 and 1 oxidizing equiv, respectively, over the native ferric resting enzyme and that the catalytic cycle of the enzyme follows the path native enzyme----compound I----compound II----native enzyme. In addition, these results indicate that Mn(II) serves as an obligatory substrate for MnP compound II, allowing the enzyme to complete its catalytic cycle. Finally, the Mn(II)/Mn(III) redox couple enables the enzyme to rapidly oxidize the terminal phenolic substrates.