PEROXIDASE-CATALYZED OXIDATION OF PENTACHLOROPHENOL

Pentachlorophenol (PCP) was shown to function as a reducing substrate for horseradish peroxidase (HRP) and to stimulate the HRP-catalyzed re duction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to 5-phenyl-4-p enten-1-ol. HRP catalyzed the hydroperoxide-dependent oxidation of PCP , using H2O2, PPHP, or ethyl hydroperoxide as substrates, as evidenced by UV spectroscopic and reverse phase HPLC analysis of reaction mixtu res. The major oxidation product was tetrachloro-1,4-benzoquinone whic h was identified on the basis of electronic absorption spectroscopy, m ass spectrometry, and cochromatography with authentic standard. HRP-ca talyzed oxidation of PCP yielded relatively stable, ESR-detectable pen tachlorophenoxyl radical intermediates whose ESR spectra consisted of a symmetrical single line without hyperfine structure. Substitution of natural abundance isotopically-labeled PCP with C-13-labeled PCP resu lted in broadening of the ESR signal line width from 6.1 G to 13.5 CT. ESR spin trapping studies, with alpha-(1-oxy-4-pyridyl)-N tert-butyln itrone (4-POBN) as the spin trap demonstrated identical spectra using natural abundance isotopically-labeled PCP versus C-13-labeled PCP, su ggesting oxyl addition, rather than carbon-centered radical addition t o 4-POBN. The computer simulation of the observed spectra is consisten t with two distinct 4-POBN adducts, with relative abundances of simila r to 3:1, and hyperfine coupling constants of alpha(N) = (14.61 G)/alp ha(H) = 1.83 G and alpha(N) = (14.76 G)/alpha(H) = 5.21 G, respectivel y. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of PCP are presented that are consistent with these results.