STABILIZATION OF THE VERATRYL ALCOHOL CATION-RADICAL BY LIGNIN PEROXIDASE

Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of vera tryl 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 f or the enzyme-generated cation radical did not agree with the decay co nstant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 27 0, 16745-16748] for the chemically generated radical. Moreover, we hav e found that the chemically generated VA(.+) formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxy gen-saturated solutions, with a decay constant of 1.2 x 10(3) s(-1), a nd with a second-order mechanism in argon-saturated solution. The firs t-order decay constant was pH-independent suggesting that the rate-lim iting step in the decay was deprotonation. When VA(.+) was generated b y oxidation with LiP the decay also occurred with a first-order mechan ism but was much slower, 1.85 s(-1), and was the same in both oxygen- and argon-saturated reaction mixtures, However, when the enzymatic rea ction 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 stabilizati on of VA(.+) may be due to the acidic microenvironment in the enzyme a ctive site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potentia l of VA(.+)/VA couple using cyclic voltammetery. Due to the instabilit y of VA(.+) in aqueous solution the reversible redox potential was mea sured in acetone, and was 1.36 V vs normal hydrogen electrode. Our dat a allow us to propose that enzymatically generated VA(.+) can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lign in or pollutant degradation.