title: Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study.
abstract: Peroxide-activated Auricularia auricula-judae dye-decolorizing peroxidase (DyP) forms a mixed Trp377 and Tyr337 radical, the former being responsible for oxidation of the typical DyP substrates (Linde et al. Biochem. J., 2015, 466, 253-262); however, a pure tryptophanyl radical EPR signal is detected at pH 7 (where the enzyme is inactive), in contrast with the mixed signal observed at pH for optimum activity, pH 3. On the contrary, the presence of a second tyrosine radical (at Tyr147) is deduced by a multifrequency EPR study of a variety of simple and double-directed variants (including substitution of the above and other tryptophan and tyrosine residues) at different freezing times after their activation by H2O2 (at pH 3). This points out that subsidiary long-range electron-transfer pathways enter into operation when the main pathway(s) is removed by directed mutagenesis, with catalytic efficiencies progressively decreasing. Finally, self-reduction of the Trp377 neutral radical is observed when reaction time (before freezing) is increased in the absence of reducing substrates (from 10 to 60 s). Interestingly, the tryptophanyl radical is stable in the Y147S/Y337S variant, indicating that these two tyrosine residues are involved in the self-reduction reaction.
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena , I-53100 Siena, Italy.
authors.forename: Maria Camilla
authors.initials: MC
authors.lastname: Baratto
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena , I-53100 Siena, Italy.
authors.forename: Adalgisa
authors.initials: A
authors.lastname: Sinicropi
authors.affiliation: Centro de Investigaciones Biológicas, CSIC , Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Linde
authors.affiliation: Centro de Investigaciones Biológicas, CSIC , Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Sáez-Jiménez
authors.affiliation: Department of Chemistry, "Ugo Schiff" and INSTM RU, University of Florence , 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Sorace
authors.affiliation: Centro de Investigaciones Biológicas, CSIC , Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Ruiz-Duenas
authors.affiliation: Centro de Investigaciones Biológicas, CSIC , Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martinez
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena , I-53100 Siena, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Basosi
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena , I-53100 Siena, Italy.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Pogni
mesh_terms: D001487:Basidiomycota / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D011789:Quantum Theory
chemical_list: D004396:Coloring Agents; D010544:Peroxidases