Stabilization of the Veratryl Alcohol Cation Radical by Lignin Peroxidase

Abstract

Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA) to veratryl aldehyde, with the enzyme-bound veratryl alcohol cation radical (VA.+) as an intermediate [Khindaria et al. (1995) Biochemistry 34, 16860-16869]. The decay constant we observed for the enzyme generated cation radical did not agree with the decay constant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 270, 16745-16748] for the chemically generated radical. Moreover, we have found that the chemically generated VA.+ formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxygen-saturated solutions, with a decay constant of 1.2 x 10(3) s-1, and with a second-order mechanism in argon-saturated solution. The first-order decay constant was pH- independent suggesting that the rate-limiting step in the decay was deprotonation. When VA.+ was generated by oxidation with LiP the decay also occurred with a first-order mechanism but was much slower, 1.85 s-1, and was the same in both oxygen- and argon-saturated reaction mixtures. However, when the enzymatic reaction mixture was acid-quenched the decay constant of VA.+ was close to the one obtained in the Ce(IV) oxidation system, 9.7 x 10(2) s-1. This strongly suggested that the LiP-bound VA.+ was stabilized and decayed more slowly than free VA.+. We propose that the stabilization of VA.+ may be due to the acidic microenvironment in the enzyme active site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potential of VA.+/VA couple using cyclic voltammetery. Due to the instability of VA.+ in aqueous solution the reversible redox potential was measured in acetone, and was 1.36 V vs normal hydrogen electrode. Our data allow us to propose that enzymatically generated VA.+ can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lignin or pollutant degradation.

Anionic tobacco peroxidase is active at extremely low pH: veratryl alcohol oxidation with a pH optimum of 1.8

“…This could be either EIIVAd + (k u h) or E-VAd + (k u d), or if both complexes have similar dissociation rates, either could be partially rate-limiting. This is consistent with the recently shown existence of such a complex for lignin peroxidase Compound II [16,17].…”

Relevância de compostos de baixa massa molar produzidos por fungos e envolvidos na biodegradação da madeira

“…58,59 Por outro lado, Popp ) como, por exemplo, pelo ácido oxálico, como descrito em detalhes mais adiante.…”

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

“…Reduction of compound (I) by a single electron transfer, usually from an organic substrate (such as veratryl alcohol), yields a second spectroscopically detectable intermediate, compound (II), that lacks the porphyrin cation radical, but retains the ferryl haem. Compound (II) can also oxidize substrates by one electron, thereby completing the catalytic cycle of LiP [17][18][19][20]. Many electron donating substrates oxidized by LiP have redox potentials beyond the reach of plant peroxidases [21,22].…”