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|Title:||A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii - A combined multifrequency EPR and density functional theory study|
|Authors:||Pogni, Rebecca; Baratto, M. Camila; Teutloff, Christian; Giansanti, Stefania; Ruiz-Dueñas, F. J.; Choinowski, Thomas; Piontek, Klaus; Martínez, Ángel T.; Lendzian, Friedhelm; Basosi, Riccardo|
|Publisher:||American Society for Biochemistry and Molecular Biology|
|Citation:||Journal of Biological Chemistry 281(14): 9517-9526(2006)|
|Abstract:||Versatile peroxidases are heme enzymes that combine catalytic properties of lignin peroxidases and manganese peroxidases, being able to oxidize Mn2+ as well as phenolic and non-phenolic aromatic compounds in the absence of mediators. The catalytic process (initiated by hydrogen peroxide) is the same as in classical peroxidases, with the involvement of 2 oxidizing equivalents and the formation of the so-called Compound I. This latter state contains an oxoferryl center and an organic cation radical that can be located on either the porphyrin ring or a protein residue. In this study, a radical intermediate in the reaction of versatile peroxidase from the ligninolytic fungus Pleurotus eryngii with H2O2 has been characterized by multifrequency (9.4 and 94 GHz) EPR and assigned to a tryptophan residue. Comparison of experimental data and density functional theory theoretical results strongly suggests the assignment to a tryptophan neutral radical, excluding the assignment to a tryptophan cation radical or a histidine radical. Based on the experimentally determined side chain orientation and comparison with a high resolution crystal structure, the tryptophan neutral radical can be assigned to Trp164 as the site involved in long-range electron transfer for aromatic substrate oxidation.|
Different heme peroxidases are considered to be involved in the lignin biodegradation process, a key step for carbon recycling in terrestrial ecosystems. These are lignin peroxidase (LiP)4 and manganese peroxidase (MnP), first described in Phanerochaete chrysosporium (1-3), and the versatile peroxidase (VP), more recently described in fungi from the genera Pleurotus (4-6) and Bjerkandera (7, 8). VP is characterized by combining catalytic properties of the other two ligninolytic peroxidases, MnP and LiP. This enzyme is able to oxidize Mn2+ to Mn3+ and also exhibits manganese-independent activity toward veratryl alcohol and p-dimethoxybenzene. Furthermore, it oxidizes hydroquinones and substituted phenols that are not efficiently oxidized by LiP or MnP in the absence of veratryl alcohol and Mn2+, respectively. VP is even able to degrade directly high redox potential dyes, which can be eventually oxidized by LiP only in the presence of veratryl alcohol (9, 10). Two genes encoding VP isoenzymes VPL and VPS1, expressed in liquid- and solid-state fermentation cultures, respectively, have been cloned from Pleurotus eryngii (11, 12). The deduced amino acid sequences for both isoenzymes were used to build molecular models by homology modeling, taking advantage of sequence identity to P. chrysosporium LiP and MnP and Coprinopsis cinerea (synonym Coprinus cinereus) peroxidase (13). Very recently, the crystal structure of recombinant P. eryngii VP expressed in Escherichia coli and activated in vitro (14) has been determined at 1.33-Å resolution (Protein Data Bank code 2BOQ).
Two genes encoding VP isoenzymes VPL and VPS1, expressed in liquid- and solid-state fermentation cultures, respectively, have been cloned from Pleurotus eryngii (11, 12). The deduced amino acid sequences for both isoenzymes were used to build molecular models by homology modeling, taking advantage of sequence identity to P. chrysosporium LiP and MnP and Coprinopsis cinerea (synonym Coprinus cinereus) peroxidase (13). Very recently, the crystal structure of recombinant P. eryngii VP expressed in Escherichia coli and activated in vitro (14) has been determined at 1.33-Å resolution (Protein Data Bank code 2BOQ). Catalytically, VP would follow the classical heme peroxidase cycle, in which hydrogen peroxide is the final electron acceptor, acting as a 2-electron oxidizing substrate for the resting enzyme, which results in the formation of Compound I (15-17). Compound I is reduced back by the substrate in a two-step reaction that involves the formation of Compound II (a 1-electron oxidized form) and then the closure of the catalytic cycle to the resting state. It is known that H2O2 attains the distal side of the heme through the access channel present in all peroxidases. However, identification of sites involved in substrate oxidation has been successful only on a few occasions (18-20). In ligninolytic peroxidases, only the manganese interaction site in MnP, situated near the internal propionate of the heme, has been confirmed by site-directed mutagenesis and x-ray diffraction of MnP·Mn2+ complexes (21-23). A putative manganese-binding site similar to that of MnP has been identified in P. eryngii VP isoenzymes (12, 24). Substitution of one of the residues from the putative manganese interaction site in VPL yields an enzyme with its Mn2+ oxidation ability strongly impaired (25). Such a binding site is not present in LiP according to its lack of activity with this divalent cation.
In the case of LiP, there is no x-ray structure with any of its substrates available, and structure-function studies have been based on techniques that led to the indirect characterization of the substrate interaction sites. The veratryl alcohol molecule was initially modeled at the heme access channel (26), and the residues hypothetically involved are conserved also in the isoenzyme VPS1 of P. eryngii, which has a main heme access channel remarkably similar to that of LiP (24). Apart from the possibility of direct electron transfer to the heme from some substrates, long-range electron transfer (LRET) should be considered to justify the capacity of VP and LiP to oxidize high molecular size substrates that do not fit into the heme access channel. The first suggested LRET pathway involved the LiP distal histidine and started at His82 (27). This pathway does not exist in MnP and in the isoenzyme VPL of P. eryngii, but it has been identified in VPS1. Two additional pathways have been proposed in LiP. One starts at the second exposed histidine (LiP His239) to the proximal histidine and is absent in MnP and VPS1 and present in VPL (His232). The second pathway starts from an exposed tryptophan (Trp171) present in LiP and proceeds to the porphyrin ring. Its involvement in veratryl alcohol and non-phenolic lignin model compound oxidation has been confirmed in LiP, and Trp171 has been revealed to be a redox-active residue (28-32). Multisequence alignment revealed that the above-mentioned tryptophan residue is conserved in all LiP sequences as well as in Pleurotus VP and is absent in the typical MnP (15). The involvement of a surface tryptophan as a catalytic site in LiP was also inferred in a study on the S168W variant of MnP (33). Moreover, a putative electron transfer pathway from the exposed tryptophan residue was identified in P. eryngii isoenzymes VPL (Trp164) and VPS1 (Trp170) (12, 13, 24). It is interesting to point out that VPs, as all other ligninolytic enzymes described until now, have no Tyr residues in their sequences (15). In this work, we report the results from multifrequency (9.4 GHz, X-band; and 94 GHz, W-band) EPR studies on a freeze-quenched radical intermediate in the reaction of P. eryngii VP with H2O2. Comparison of the EPR and pulse electron nuclear double resonance (ENDOR) experimental data with theoretical results from density functional theory (DFT), in particular for the hyperfine (hf) tensor values, excludes assignment to a tryptophan cation radical or a histidine radical, but strongly suggests assignment to a tryptophan neutral radical. It has been shown in earlier studies on tryptophan radicals in ribonucleotide reductase variants that evaluation of the hf-tensors of the side chain methylene protons enables determination of the side chain geometry, which can be used for site-specific assignment (34, 35). Based on the side chain orientation deduced from the experimental hyperfine data of these protons, the observed tryptophan neutral radical can be assigned to Trp164, which is proposed as the surface site involved in LRET in VPL of P. eryngii.
|Description:||10 páginas, 9 figuras, 2 tablas -- PAGS nros. 9517-9526|
|Publisher version (URL):||http://dx.doi.org/10.1074/jbc.M510424200|
|Appears in Collections:||(CIB) Artículos|