Tryptophan-Based Radical in the Catalytic Mechanism of Versatile Peroxidase from Bjerkandera adusta ² Rebecca Pogni,* ,‡ M. Camilla Baratto, ‡ Stefania Giansanti, ‡ Christian Teutloff, § Jorge Verdin, | Brenda Valderrama, | Friedhelm Lendzian, § Wolfgang Lubitz, ⊥ Rafael Vazquez-Duhalt, | and Riccardo Basosi ‡ Department of Chemistry, UniVersity of Siena, Via Aldo Moro, 53100 Siena, Italy, the Max-Volmer Laboratory for Biophysical Chemistry, Technical UniVersity, Berlin, Germany, the Max-Planck-Institute for Bioinorganic Chemistry, Mu ¨lheim, Germany, and Instituto de Biotecnologia, UNAM, Apartado Postal 510-3, CuernaVaca, Morelos 62250, Mexico ReceiVed December 2, 2004; ReVised Manuscript ReceiVed January 18, 2005 ABSTRACT: 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 ∼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. The extracellular enzymatic system from white rot fungi, which is involved in lignin degradation, consists mainly of oxidative enzymes: laccase, lignin peroxidase (LiP), 1 and manganese peroxidase (MnP) (1). However, active lignin- degrading strains of Pleurotus eryngii were shown to produce a peroxidase different from P. chrysosporium peroxidases, which can both efficiently oxidize Mn II to Mn III and carry out Mn II -independent activity on aromatic substrates (2). A related novel manganese-lignin peroxidase hybrid enzyme, called versatile peroxidase (VP), was described for Bjerkan- dera sp. BOS55 and is able to oxidize various phenolic and nonphenolic substrates, such as 2,6-dimethoxyphenol, guaiacol, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and veratryl alcohol (VA), in the absence of Mn II (3). Similar VPs have been reported in Pleurotus eryngii (4- 6), Pleurotus pulmonarius (7), Pleurotus ostreatus (8), as well as in Bjerkandera adusta (3, 9-11). VPs from Bjer- kandera and Pleurotus species show comparable structural and catalytic parameters, and this allows us to infer the same catalytic intermediates for these enzymes. VP shows high identity with LiP (58-60%) and MnP (55%) both from Phanerochaete chrysosporium (5). The heterologous expression of VP in Aspergillus nidulans confirmed the ability of this hybrid enzyme to oxidize Mn II and different aromatic compounds in the absence of the mediator (6). This enzyme seems to have a long-range electron-transfer pathway similar to those postulated for LiP (12, 13). Putatively, VP catalyzes the electron transfer from an oxidizable substrate to a hydrogen peroxide molecule fol- lowing the classic peroxidase mechanism (14-17). First, in the presence of peroxide, a two-oxidizing equivalent inter- mediate, compound I, is produced. One-oxidizing equivalent is stored as a ferryl (Fe IV ) state with S ) 1 and the second, as a porphyrin π radical. In some cases, the second oxidizing equivalent is localized on a spatially removed paramagnetic species with S ) 1 / 2 , namely, a protein-based radical, as the tryptophan radical found in cytochrome c peroxidase (18). This intermediate is then sequentially reduced back by substrate molecules in a two-step reaction. ² This work was funded by PRIN 2002 (Italy), University of Siena, and CONACYT (Mexico). Financial support by Deutsche For- schungsgemeinschaft DFG (SPP 1051, Le812/3-1 to F.L. and Lu 315/ 16-2 to W.L.) and German DAAD (VIGONI program, to W.L. and R.B.) is gratefully acknowledged. * To whom correspondence should be addressed: Telephone: +39- 0577-23-4258. Fax: +39-0577-23-4239. E-mail: pogni@unisi.it. ‡ University of Siena. § Technical University. | UNAM. ⊥ Max-Planck-Institut. 1 Abbreviations: LiP, lignin peroxidase; MnP, manganese peroxidase; VP, versatile peroxidase; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline- 6-sulfonate); VA, veratryl alcohol; EPR, electron paramagnetic reso- nance; GPC, gel-permeation chromatography; RNR, ribonuclotide reductase; NBS, N-bromosuccinimide. 4267 Biochemistry 2005, 44, 4267-4274 10.1021/bi047474l CCC: $30.25 © 2005 American Chemical Society Published on Web 02/19/2005