EVIDENCE FOR FORMATION OF THE VERATRYL ALCOHOL CATION-RADICAL BY LIGNIN PEROXIDASE

Lignin peroxidases (LiP) catalyze the H2O2-dependent two-electron oxid ation of veratryl alcohol (VA) to veratryl aldehyde. We present here, electron spin resonance (ESR) evidence for the formation of the one-el ectron oxidized intermediate, the veratryl alcohol cation radical (VA( .+)). The ESR spectrum of VA(.+) was first obtained in a fast-flow sys tem with Ce(IV) as an oxidant and 10% HNO3 to stabilize the radical. T his ESR signal was deconvoluted, and the hyperfine splitting constants were determined. The identity of the radical was confirmed by compute r simulation of the ESR spectrum and calculation of spin and charge de nsities on the radical. An identical radical signal was observed with Lip, also in a fast-flow incubation containing 10 mu M LiP, 2 mM VA, a nd 500 mu M H2O2 at pH 3.5. The Fourier transforms of the ESR signals further confirmed that the spectra obtained with both Ce(IV) and LiP w ere due to the same radical species. The VA(.+) had a distinct visible spectrum in 98% H2SO4 With an absorbance maximum at 529 nm. The extin ction 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 pK(a) at -1.0 pH. This value wa s determined by quantitating both the concentration of VA(.+) by visua l and ESR spectrometery and the g-value of the ESR signal at various p H values.