Identification of Tyr74 and Tyr177 as substrate oxidation sites in cationic cell wall-bound peroxidase from Populus alba L

Cationic cell wall-bound peroxidase (CWPO-C) has the capability to oxidize sinapyl alcohol, ferrocytochrome c, and synthetic lignin polymers, unlike most peroxidases that have been characterized in flowering plants, such as horseradish peroxidase and Arabidopsis thaliana peroxidase A2. It has been suggested that the oxidation site is located on the CWPO-C surface, and homology modeling and chemically modified CWPO-C studies suggest that Tyr74 and/or Tyr177 are possible participants in the catalytic site. The present study clarifies the importance of these Tyr residues for substrate oxidation, using recombinant CWPO-C and recombinant mutant CWPO-C with phenylalanine substitution(s) for tyrosine. Such recombinant proteins, produced in Escherichia coli as inclusion bodies, were successfully refolded to yield the active form, and purified recombinant protein solutions exhibited typical spectra of high-spin ferric protein and displayed H(2) O(2) -dependent oxidation of guaiacol, 2,6-dimethoxyphenol, and syringaldazine. Measurement of peroxidase activity with these guaiacyl and syringyl compounds as reducing substrates indicated that a single mutation, Y74F or Y177F, resulted in substantial loss of oxidation activity (～ 40-60% and 82%, respectively). Also, over 95% of the oxidation activity was lost with a double mutation, Y74F/Y177F. These results indicated that Tyr74 and Tyr177, rather than the heme pocket, play a central role in the oxidation of these substrates. This is the first report of active residues on an enzyme surface being identified in a plant peroxidase. This study also suggests that sinapyl alcohol incorporation into lignin is performed by a peroxidase that generates Tyr radicals on its surface.     

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.     

Lignin dehydrogenative polymerization mechanism: a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in beta-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO-C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO-C was capable of oxidizing high-molecular-weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO-C characteristics are important to produce polymer lignin. The results suggest that CWPO-C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.     

Role of proximal methionine residues in Leishmania major peroxidase

The active site architecture of Leishmania major peroxidase (LmP) is very similar with both cytochrome c peroxidase and ascorbate peroxidase. We utilized point mutagenesis to investigate if the conserved proximal methionine residues (Met248 and Met249) in LmP help in controlling catalysis. Steady-state kinetics of methionine mutants shows that ferrocytochrome c oxidation is <2% of wild type levels without affecting the second order rate constant of first phase of Compound I formation, while the activity toward a small molecule substrate, guaiacol or iodide, increases. Our diode array stopped-flow spectral studies show that the porphyrin π-cation radical of Compound I in mutant LmP is more stable than wild type enzyme. These results suggest that the electronegative sulfur atoms of the proximal pocket are critical factors for controlling the location of a stable Compound I radical in heme peroxidases and are important in the oxidation of ferrocytochrome c.     

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.     

Balance between anionic and cationic extracellular peroxidase activities in Sedum album leaves after ozone exposure. Analysis by high-performance liquid chromatography

Peroxidase activity was assayed with different electron donors (guaiacol, ascorbate, syringaldazine) in the intercellular fluid of Sedum album L. leaves after ozone exposure. Anionic and cationic peroxidases were separated and purified by high performance ion-exchange and gel permeation chromatography. Both isoperoxidases were tested as regards their molecular weight and apparent kinetic constants with different substrates. Ascorbate peroxidase activity was rapidly stimulated after ozone exposure, whereas syringaldazine peroxidase activity reached its maximum 24 h later. Increases in ascorbate and syringaldazine peroxidase activities occurred simultaneously with increases in cationic and anionic peroxidase activities, respectively. Apparent K_m values indicate a high affinity of cationic peroxidases for ascorbate and of anionic peroxidases for syringaldazine. The metabolic role of this balance between cationic and anionic peroxidases after ozone exposure is discussed.     

Delignification of Wood Chips and Pulps by Using Natural and Synthetic Porphyrins: Models of Fungal Decay

Kraft pulps, prepared from softwoods, and small chips of birch wood were treated with heme and tert-butyl hydroperoxide in aqueous solutions at reflux temperature. Analyses of treated pulps showed decreases in kappa number (a measure of lignin content) from about 36 to less than 2, with concomitant increases in brightness (80% increase in the better samples). Analyses of treated wood chips revealed selective delignification and removal of hemicelluloses. After 48 h of treatment, lignin losses from the wood chips approached 40%, and xylose/mannose (hemicellulose) losses approached 70%, while glucose (cellulose) losses were less than 10%. Examination of delignified chips by transmission electron microscopy showed that the removal of lignin occurred in a manner virtually indistinguishable from that seen after decay by white rot fungi. Various metalloporphyrins, which act as biomimetic catalysts, were compared to horseradish peroxidase and fungal manganese peroxidase in their abilities to oxidize syringaldazine in an organic solvent, dioxane. The metalloporphyrins and peroxidases behaved similarly, and it appeared that the activities of the peroxidases resulted from the extraction of heme into the organic phase, rather than from the activities of the enzymes themselves. We concluded that heme-tert-butyl hydroperoxide systems in the absence of a protein carrier mimic the decay of lignified tissues by white rot fungi.     

A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.     

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.     

Role of C-terminal acidic cluster in stabilization of heme spin state of ascorbate peroxidase from Leishmania major

Architecture of hemoprotein is solely responsible for different nature of heme coordination. Here we report that substitution of the acidic surface residue Glu226 to Ala in ascorbate peroxidase from Leishmania major alters the 5 coordinate high spin (5cHS) to a 6 coordinate low spin (6cLS) form at pH 7.5. Using UV-visible spectrophotometry, we show that the sixth ligand of heme in Glu226Ala at pH 7.5 is hydroxo. When the pH is decreased to 5.5, a new species of Glu226Ala appeared that had a spectrum characteristic of a 6cHS derivative. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I was not seen in the Glu226Ala in presence of H₂O₂. Similarly guaiacol, ascorbate and ferrocytochrome c oxidation rate was 10³ orders less for the Glu226Ala mutants compared to the wild type. These data suggested that surface acidic residue Glu226 might play role in proper maintenance of active site conformation.     

Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers

The oxidation of phenolic oligomers by lignin and manganese peroxidases was studied by transient-state kinetic methods. The reactivity of peroxidase intermediates compound I and compound II was studied with the phenol guaiacol along with a beta-O-4 phenolic dimer, trimer, and tetramer. Compound I of both peroxidases is much more reactive than compound II. The rate constants for these substrates with Mn peroxidase compound I range from 1.0 x 10(5) M-1 s-1 for guaiacol to 1.1 x 10(3) M-1 s-1 for the tetramer. Reactivity is much higher with lignin peroxidase compound I with rate constants ranging from 1.2 x 10(6) M-1s-1 for guaiacol to 3.6 x 10(5) M-1 s-1 for the tetramer. Rate constants with compound II are much lower with Mn peroxidase exhibiting very little reactivity. The rate constants dramatically decreased with both peroxidases as the size of the substrate increased. The extent of the decrease was much more dramatic with Mn peroxidase, leading us to conclude that, despite its ability to oxidize phenols, Mn2+ is the only physiologically significant substrate. The rate decrease associated with increasing substrate size was more gradual with lignin peroxidase. These data indicate that whereas Mn peroxidase cannot efficiently directly oxidize the lignin polymer, lignin peroxidase is well suited for direct oxidation of polymeric lignin.     

Purification and characterization of a basic peroxidase from the medium of cell suspension cultures of chicory

A 34-kDa cationic peroxidase (Cicpx) with a pI of 8.9 was purified to homogeneity (RZ 3.5) from the medium of cell suspension cultures of chicory (Cichorium intybus L.) by a combination of ammonium sulphate precipitation, ultrafiltration, ion exchange and gel filtration chromatography. The partial amino acid sequence presented a low homology with other plant peroxidases. Antibody against spinach peroxidase was shown to cross react with chicory isoperoxidase on immunoblots. Unlike anionic peroxidases, Cicpx displayed a high reactivity towards guaiacol and no reactivity towards syringaldazine, indicating that Cicpx was not involved in the lignification process. Thus, further investigations are necessary to assign a specific function to this particular isoperoxidase.     

Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation

Plant peroxidases, as typified by horseradish peroxidase (HRP), primarily catalyze the one-electron oxidation of phenols and other low oxidation potential substrates. In contrast, the mammalian homologues such as lactoperoxidase (LPO) and myeloperoxidase primarily oxidize halides and pseudohalides to the corresponding hypohalides (e.g., Br(-) to HOBr, Cl(-) to HOCl). A further feature that distinguishes the mammalian from the plant and fungal enzymes is the presence of two or more covalent bonds between the heme and the protein only in the mammalian enzymes. The functional roles of these covalent links in mammalian peroxidases remain uncertain. We have previously reported that HRP can oxidize chloride and bromide ions, but during oxidation of these ions undergoes autocatalytic modification of its heme vinyl groups that virtually inactivates the enzyme. We report here that autocatalytic heme modification during halide oxidation is not unique to HRP but is a general feature of the oxidation of halide ions by fungal and plant peroxidases, as illustrated by studies with Arthromyces ramosus and soybean peroxidases. In contrast, LPO, a prototypical mammalian peroxidase, is protected from heme modification and its heme remains intact during the oxidation of halide ions. These results support the hypothesis that the covalent heme-protein links in the mammalian peroxidases protect the heme from modification during the oxidation of halide ions.     

Catalytic properties of tryptophanless recombinant horseradish peroxidase

Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.     

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.     

Reaction of haem containing proteins and enzymes with hydroperoxides: The radical view

The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe³⁺ (ferric) state reacts with hydroperoxides with a formation of the Fe⁴⁺=O (oxoferryl) haem state and a free radical primarily located on the π-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; αTyr42, αTrp14, βTrp15, βCys93, (αTyr24−αHis20), all in the α- and β-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases.     

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.     

Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase–peroxidases

Catalase–peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase–catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV–Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.