title: Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study.
abstract: Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H₂O₂-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (k(cat) > 200 s⁻¹) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 k(cat) ~20 s⁻¹) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.
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: †Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100, Siena, Italy.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Pogni
authors.affiliation: ‡Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, E-08034 Barcelona, Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: Cañellas
authors.affiliation: ‡Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, E-08034 Barcelona, Spain.
authors.forename: Fátima
authors.initials: F
authors.lastname: Lucas
authors.affiliation: ‡Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, E-08034 Barcelona, Spain.
authors.forename: Victor
authors.initials: V
authors.lastname: Guallar
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: Verónica
authors.initials: V
authors.lastname: Sáez-Jiménez
authors.affiliation: *Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Coscolín
authors.affiliation: *Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Romero
authors.affiliation: *Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Medrano
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-Dueñas
authors.affiliation: *Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
mesh_terms: D019943:Amino Acid Substitution; D001487:Basidiomycota / Q000201:enzymology*; D001665:Binding Sites; D055162:Biocatalysis; D004396:Coloring Agents / Q000737:chemistry* / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D005609:Free Radicals; D005656:Fungal Proteins; D006420:Hemeproteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D014364:Tryptophan; D010544:Peroxidases