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

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

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.     

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.     

Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site

The three-dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu-35, Glu-39, and Asp-179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley-Liss, Inc.     

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58–60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C_α-distance 1.2 Å). However, this peroxidase includes a Mn²⁺ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn²⁺. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

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.     

The status of half-cystine residues and locations of N-glycosylated asparagine residues in human eosinophil peroxidase

The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase

Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its oxidation of Mn2+ at a second catalytic site but also in its ability to directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared in LiP and VP crystal structures, and six residues near VP Trp164 were modified by site-directed mutagenesis. Oxidation of Mn2+ was practically unaffected. However, several mutations modified the oxidation kinetics of the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residues contribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophan environment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA was added as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260F and R257A mutations strongly increased (20-50-fold) the apparent second-order rate constants for reduction of VP compounds I and II by VA to values similar to those found in LiP. Dissociation of the enzyme-product complex seemed to be the limiting step in the turnover of this improved variant. Nonexposed residues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. This was a direct effect since no modification of the surrounding residues was found in the crystal structure of this variant.     

Understanding lignin-degrading reactions of ligninolytic enzymes: binding affinity and interactional profile

Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity.     

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases

Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.     

The crystal structure of lignin peroxidase at 1.70 A resolution reveals a hydroxy group on the cbeta of tryptophan 171: a novel radical site formed during the redox cycle

The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP.     

Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.     

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.     

Molecular dynamics simulations of lignin peroxidase in solution

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.     

Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium

Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pK_a values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pK_a value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.     

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.     

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to LiP of Phanerochaete chrysosporium and a total inhibition of LiP was observed.     

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium. Role of veratryl alcohol in polymer oxidation.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.