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.
Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes the H2O2 dependent one- and two-electron oxidations of substrates. The catalytic cycle involves the oxidation of ferric-LiP by H2O2 by two electrons to compound I, which is an oxoferryl heme and a free radical. It has been speculated that the unpaired electron is in a pi delocalized porphyrin radical. However, no direct evidence for the presence of the free radical has been reported. We present electron paramagnetic resonance (EPR) detection and characterization of compound I of LiP. The LiP compound I EPR signal is different than those reported previously for compound I of horseradish peroxidase and chloroperoxidase. However, the EPR signal of compound I of LiP (axial g tensor extending from gperpendicular = 3.42 to gparallel approximately 2) is very similar to the EPR signals of compound I of ascorbate peroxidase and catalase from Micrococcus lysodeikticus, in which the radical has been identified as a porphyrin pi-cation radical. On the basis of the analysis of our data and comparison with the earlier published results for compounds I of other peroxidases, we interpret the LiP compound I signal by a model for exchange coupling between an S = 1 oxyferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S = 1/2) [Schulz, C.E., et al. (1979) FEBS Lett. 103, 102-105]. The exchange coupling is characterized by ferromagnetic rather than an antiferromagnetic interaction between the two species. The ferric-Lip EPR signal suggests that the iron in the heme is in near perfect orthogonal symmetry and provides additional evidence of the ferromagnetic interaction between the oxoferryl iron center and the porphyrin pi-cation radical.
Lignin peroxidases (LiP) catalyze the H2O2-dependent two-electron oxidation of veratryl alcohol (VA) to veratryl aldehyde. We present here, electron spin resonance (ESR) evidence for the formation of the one-electron oxidized intermediate, the veratryl alcohol cation radical (VA.+). The ESR spectrum of VA.+ was first obtained in a fast-flow system with Ce(IV) as an oxidant and 10% HNO3 to stabilize the radical. This ESR signal was deconvoluted, and the hyperfine splitting constants were determined. The identity of the radical was confirmed by computer simulation of the ESR spectrum and calculation of spin and charge densities on the radical. An identical radical signal was observed with LiP, also in a fast-flow incubation containing 10 microM LiP, 2 mM VA, and 500 microM H2O2 at pH 3.5. The Fourier transforms of the ESR signals further confirmed that the spectra obtained with both Ce(IV) and LiP were due to the same radical species. The VA.+ had a distinct visible spectrum in 98% H2SO4 with an absorbance maximum at 529 nm. The extinction coefficient of the VA.+ spectral band at 529 nm was calculated to be 11,000 M-1 cm-1. The VA.+ was found to be a strong acid, as are other cation radicals, with the pKa at -1.0 pH. This value was determined by quantitating both the concentration of VA.+ by visual and ESR spectrometry and the g-value of the ESR signal at various pH values.
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