[NMR paper] Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A Transient-state Kinetics, Directed Mutagenesis, EPR and NMR Study.

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Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A Transient-state Kinetics, Directed Mutagenesis, EPR and NMR Study.

Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A Transient-state Kinetics, Directed Mutagenesis, EPR and NMR Study.

J Biol Chem. 2015 Aug 3;

Authors: Saez-Jimenez V, Baratto MC, Pogni R, Rencoret J, Gutierrez A, Santos JI, MartInez AT, Ruiz-Duenas FJ

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, since it was reduced during lignosulfonate oxidation resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation 355 nm/emission 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in 1D and 2D 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 neighbor to 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.


PMID: 26240145 [PubMed - as supplied by publisher]



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