title: Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY.
abstract: Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn(2+), and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.
authors.affiliation: From the 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: the Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, I-53100 Siena, Italy.
authors.forename: Maria Camilla
authors.initials: MC
authors.lastname: Baratto
authors.affiliation: the Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, I-53100 Siena, Italy.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Pogni
authors.affiliation: the Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P. O. Box 1052, E-41080 Seville, Spain, and.
authors.forename: Jorge
authors.initials: J
authors.lastname: Rencoret
authors.affiliation: the Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P. O. Box 1052, E-41080 Seville, Spain, and.
authors.forename: Ana
authors.initials: A
authors.lastname: Gutiérrez
authors.affiliation: the NMR Facility, SGIKER, Universidad del País Vasco, UPV/EHU Donostia, 48940 Leioa, Bizkaia Spain.
authors.forename: José Ignacio
authors.initials: JI
authors.lastname: Santos
authors.affiliation: From the Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain, ATMartinez@cib.csic.es.
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
authors.affiliation: From the Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain, fjruiz@cib.csic.es.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Ruiz-Dueñas
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000502:physiology; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008031:Lignin / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D008031:Lignin; D006861:Hydrogen Peroxide; D009195:Peroxidase
keywords: HSQC NMR; electron paramagnetic resonance (EPR); enzyme kinetics; lignin degradation; lignosulfonate; nuclear magnetic resonance (NMR); peroxidase; transient-state kinetics; tryptophanyl radical; versatile peroxidase