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.     

NADH oxidation by manganese peroxidase with or without alpha-hydroxy acid

NADH oxidation by manganese peroxidase (MnP) was done in a reaction mixture including either alpha-hydroxy acid or acetate. The oxidation in the former reaction mixture was inhibited by a catalase and was accelerated by exogenous H2O2, while the oxidation in the latter reaction mixture was inhibited by a superoxide dismutase and was not accelerated by the exogenous H2O2. These results indicated that there are significant differences between the two reaction systems, particularly, in the active oxygen species involved in the reactions. Additionally, the experiment of MnP reduction with Mn(II) suggests that MnP has a separate catalytic activity other than an oxidation of Mn(II) to Mn(III) in the reaction mixture including acetate.     

裂褶菌F17锰过氧化物酶酶活力影响因素的响应面优化(英文)

锰过氧化物酶是真菌分泌的一种糖基化的含有血红素辅基的胞外蛋白,在染料降解和脱色过程中起着重要作用。本实验利用本实验室保存的的白腐真菌裂褶菌Schizophyllum sp.F17产锰过氧化物酶(MnP),研究MnP的酶学性质,并对酶活条件进行优化。实验通过超滤浓缩、DEAE-纤维素、DE52离子交换层析和Sephadex G-75凝胶过滤等步骤,分离纯化得到电泳纯的锰过氧化物酶。该酶蛋白含量为23μg/mL,分子量大小为49.2kDa,在0.1mmol/L H2O2中半衰期为5~6min。Mn2+、H2O2以及酶的用量可以影响MnP酶促反应的效率,在单因子分析法的基础上,通过全因子中心组合设计响应面分析表明:H2O2以及H2O2与酶用量之间的交互作用对酶促反应的作用是最显著的。在优化条件下,酶对偶氮染料金橙G、刚果红显示出较强的脱色能力。     

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.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

The catalytic site of manganese peroxidase. Regiospecific addition of sodium azide and alkylhydrazines to the heme group.

Manganese peroxidase (MnP), which normally oxidizes Mn2+ to Mn3+, is rapidly and completely inactivated in an H2O2-dependent reaction by 2 equivalents of sodium azide. The inactivation is paralleled by formation of the azidyl radical and high yield conversion of the prosthetic heme into a meso-azido adduct. The meso-azido enzyme is oxidized by H2O2 to a Compound II-like species with the Soret band red-shifted 2 nm relative to that of native Compound II. The time-dependent decrease in this Compound II-like spectrum (t1/2 = 2.3 h) indicates that the delta-meso azido heme is more rapidly degraded by H2O2 than the prosthetic heme of control enzyme (t1/2 = 4.8 h). MnP is also inactivated by phenyl-, methyl-, and ethylhydrazine. The phenylhydrazine reaction is too rapid for kinetic analysis, but KI = 402 microM and kinact = 0.22/min for the slower inactivation by methylhydrazine. Reaction with phenylhydrazine at pH 4.5 does not yield iron-phenyl, N-phenyl, or meso-phenyl heme adducts. Ethylhydrazine inactivates the enzyme both at pH 4.5 and 7.0, but only detectably produces delta-meso-ethyl-heme at pH 7.0. Reconstitution of apo-MnP with hemin or delta-meso-ethylheme yields enzyme with, respectively, 50 and 5% of the native activity. The delta-meso-alkyl group thus suppresses most of the catalytic activity of the enzyme even though a Compound II-like species is still formed with H2O2. Finally, Co2+ inhibits the enzyme competitively with respect to Mn2+ but does not inhibit its inactivation by azide or the alkylhydrazines. The results argue that substrates interact with the heme edge in the vicinity of the delta-meso-carbon. They also suggest that Mn2+ and Co2+ bind to a common site close to the delta-meso-carbon without blocking the approach of small molecules to the heme edge. An active site model is proposed that accommodates these results.     

Oxidative degradation of azo dyes by manganese peroxidase under optimized conditions

The application of enzyme-based systems in waste treatment is unusual, given that many drawbacks are derived from their use, including low efficiency, high costs and easy deactivation of the enzyme. The goal of this study is the development of a degradation system based on the use of the ligninolytic enzyme manganese peroxidase (MnP) for the degradation of azo dyes. The experimental work also includes the optimization of the process, with the objective of determining the influence of specific physicochemical factors, such as organic acids, H(2)O(2) addition, Mn(2+) concentration, pH, temperature, enzyme activity and dye concentration. A nearly total decolorization was possible at very low reaction times (10 min) and at high dye concentration (up to 1500 mg L(-)(1)). A specific oxidation capacity as high as 10 mg dye degraded per unit of MnP consumed was attained for a decolorization higher than 90%. Among all, the main factor affecting process efficiency was the strategy of H(2)O(2) addition. The continuous addition at a controlled flow permitted the progressive participation of H(2)O(2) in the catalytic cycle through a suitable regeneration of the oxidized form of the enzyme, which enhanced both the extent and the rate of decolorization. It was also found that, in this particular case, the presence of a chelating organic acid (e.g., malonic) was not required for an effective operation. Probably, Mn(3+) was chelated by the dye itself. The simplicity and high efficiency of the process open an interesting possibility of using of MnP for solving other environmental problems.     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.     

Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators.

Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.     

Enhanced catalytic properties of MnP by exogenous addition of manganese and hydrogen peroxide

The efficiency of the application of crude and purified MnP in processes such as degradation of hazardous compounds is greatly dependent of the Mn and HO concentrations. Mn exerted a positive effect on the reaction rate whereas excessively high HO concentrations caused a partial inactivation of MnP, and as a result additional increases of Mn did not positively affect DMP oxidation rate. According to our results, concentrations around 5,000mM Mn and 100mM HO would maximize the catalytic MnP properties for the oxidation of 2,6-dimethoxyphenol. The presence of other cofactors in the crude enzyme such as organic acids stabilize formed Mn and the oxidation rate was higher than the corresponding to the purified one.     

Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from "Chelatobacter" strain ATCC 29600.

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H2O2. Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg(2+)-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 microM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.     

Purification and characterization of manganese peroxidase of the white-rot fungus Irpex lacteus

The production of manganese peroxidase (MnP) by Irpex lacteus, purified to electrophoretic homogeneity by acetone precipitation, HiPrep Q and HiPrep Sephacryl S-200 chromatography, was shown to correlate with the decolorization of textile industry wastewater. The MnP was purified 11.0-fold, with an overall yield of 24.3%. The molecular mass of the native enzyme, as determined by gel filtration chromatography, was about 53 kDa. The enzyme was shown to have a molecular mass of 53.2 and 38.3 kDa on SDS-PAGE and MALDI-TOF mass spectrometry, respectively, and an isoelectric point of about 3.7. The enzyme was optimally active at pH 6.0 and between 30 and 40 degrees C. The enzyme efficiently catalyzed the decolorization of various artificial dyes and oxidized Mn (II) to Mn (III) in the presence of H(2)O(2). The absorption spectrum of the enzyme exhibited maxima at 407, 500, and 640 nm. The amino acid sequence of the three tryptic peptides was analyzed by ESI Q-TOF MS/MS spectrometry, and showed low similarity to those of the extracellular peroxidases of other white-rot basidiomycetes.     

Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2

Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.     

Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study

The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.     

Heterologous expression of active manganese peroxidase from Phanerochaete chrysosporium using the baculovirus expression system

The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was recombined into a baculovirus and heterologously expressed in Sf9 cells. The recombinant Mn peroxidase has the same molecular weight as the native enzyme as determined by SDS-PAGE and cross-reacts with a Mn peroxidase-specific antibody. The recombinant enzyme has a slightly lower pI than the native fungal isozyme H4 indicating some differences in post-translational modification. Phenol red, guaiacol, and vanillylacetone, substrates of the native Mn peroxidase, are oxidized by the recombinant enzyme. All of the activities are dependent on both Mn (II) and H2O2.     

Preparation and catalytic performance of surfactant-manganese peroxidase-Mn(II) ternary complex in organic media

A novel preparation method for surfactant-MnP-Mn(II) ternary complex utilizing water-in-oil emulsions has been developed. The surfactant-MnP complex was spectroscopically characterized, strongly suggesting that the heme environment of the surfactant-MnP complex in benzene is identical to that of native MnP in the aqueous buffer. o-Phenylenediamine oxidation catalyzed by the surfactant-MnP-Mn(II) ternary complex was performed in benzene. The ternary complex efficiently catalyzed the oxidation, and the complex was catalytically stable. Kinetic experiments revealed that the reaction mechanism was as follows: MnP is oxidized by H(2)O(2) and the oxidized intermediate catalyzes the oxidation of Mn(II) to Mn(III) and the latter, after complexed with malonate, readily oxidizes o-PDA inside the complex. Thus, the organic substrate o-PDA, but not Mn(III), shuttled between the surfactant-MnP-Mn(II) ternary complex and organic solvent.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

Laccase-catalyzed oxidation of Mn(2+) in the presence of natural Mn(3+) chelators as a novel source of extracellular H(2)O(2) production and its impact on manganese peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn(2+) to Mn(3+), as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn(3+) complexes were produced at 0.15, 0.05, and 0.10 micromol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H(2)O(2) formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H(2)O(2) production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn(2+) oxidation and abiotic decomposition of these organic chelators by the resulting Mn(3+), which leads to formation of superoxide and its subsequent reduction to H(2)O(2). A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn(3+) complexes in assays containing 1 mM Mn(2+) and 100 mM oxalate or malonate, but omitting an additional H(2)O(2) source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn(2+) oxidation in providing H(2)O(2) for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.