Protein Radicals in Fungal Versatile Peroxidase

Abstract

Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k 2app ) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k 2 ) was more affected than the dissociation constant (K D2 ). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k 3 ) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.Lignin degradation, a key step for carbon recycling in land ecosystems and a central issue for industrial use of lignocellulosic biomass (e.g. in paper pulp manufacture and bioethanol production), is initiated in nature by 1-electron oxidation of the benzenic rings of lignin by specialized high redox potential fungal peroxidases (1-5). The latter include lignin peroxidase (LiP; 6 EC 1.11.1.14), first described in Phanerochaete chrysosporium together with manganese peroxidase (EC 1.11.1.13), and versatile peroxidase (VP; EC 1.11.1.16), described in Pleurotus and Bjerkandera species as a hybrid enzyme combining the catalytic properties of LiP and manganese peroxidase (6, 7). Oxidation of high redox potential aromatics, including nonphenolic lignin models, by LiP (8, 9) and VP (10) takes place at an exposed tryptophan by long range electron transfer (LRET) to heme in the 2-electron (Compound I) and 1-electron (Compound II) activated forms of the enzyme. This basically differs from Mn 2ϩ oxidation by manganese peroxidase (11) and VP (12) and oxidation of different phenolic substrates and dyes by classical plant peroxidases (13) that is produced by direct electron transfer to the activated heme cofactor.The existence of a tryptophan radical in a peroxidase (in this case neighbor to heme) has been re...

Crystallographic, Kinetic, and Spectroscopic Study of the First Ligninolytic Peroxidase Presenting a Catalytic Tyrosine

“…This results in tyrosyl radical and oxo-ferryl heme formation, i.e. a compound ES-type intermediate (34), with the same behavior as the so-called Compound IB of the W164Y variant of P. eryngii VP (46). The theoretical dihedral angles for the tyrosyl radical estimated from the EPR data were nearly identical to those obtained for the Tyr-181 side chain in the T. cervina LiP* crystal structure, confirming radical location at this tyrosine residue.…”

Reaction Kinetics of Versatile Peroxidase for the Degradation of Lignin Compounds

“…O 2 •-denotes superoxide radical anions. Pathway 1-3 depicts the usual POX reaction cycle with the reversible steps described by Equation (a) and (b) (Ruiz-Dueñas et al, 2009b). The cycle will be initiated by H 2 O 2 (pathway 1).…”

“…Based on investigation results of transient state kinetics using a recombinant native VP (obtained by E. coli), both E I and E II reduction by VA oxidation exhibited reactions which can be described by Equation (6) and (7) (Ruiz-Dueñas et al, 2009b):…”

“…Moreover, it will be supposed that E II is directly reduced by adlerol (assumption is general accepted, e.g. for VA oxidation (Ruiz-Dueñas et al, 2009b)). However, the evidence for this assumption is still pending.…”

“…Despite a further review regarding applicability of the kinetic models described through Equation (13) or (19) as well. An approval is given by Ruiz-Dueñas et al (2009b) who investigated transient state kinetics of a recombinant native VP (obtained by E. coli) with the substrate VA (Equation (6)- (7)). E I formation via twoelectron oxidation of E 0 by H 2 O 2 "has long been understood" (Dunford, 2010).…”