Peroxidase-Catalyzed Oxidation of Pentachlorophenol

Abstract

Pentachlorophenol (PCP) was shown to function as a reducing substrate for horseradish peroxidase (HRP) and to stimulate the HRP-catalyzed reduction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to 5-phenyl-4-penten-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 mixtures. The major oxidation product was tetrachloro-1,4-benzoquinone which was identified on the basis of electronic absorption spectroscopy, mass spectrometry, and cochromatography with authentic standard. HRP-catalyzed oxidation of PCP yielded relatively stable, ESR-detectable pentachlorophenoxyl radical intermediates whose ESR spectra consisted of a symmetrical single line without hyperfine structure. Substitution of natural abundance isotopically-labeled PCP with 13C-labeled PCP resulted in broadening of the ESR signal line width from 6.1 G to 13.5 G. ESR spin trapping studies, with alpha-(1-oxy-4-pyridyl)-N tert-butylnitrone (4-POBN) as the spin trap demonstrated identical spectra using natural abundance isotopically-labeled PCP versus 13C-labeled PCP, suggesting oxyl addition, rather than carbon-centered radical addition to 4-POBN. The computer simulation of the observed spectra is consistent with two distinct 4-POBN adducts, with relative abundances of approximately 3:1, and hyperfine coupling constants of alpha N = (14.61 G)/alpha H = 1.83 G and alpha N = (14.76 G)/alpha H = 5.21 G, respectively. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of PCP are presented that are consistent with these results.

Nucleobase-Dependent Reactivity of a Quinone Metabolite of Pentachlorophenol

“…The generation of Cl 4 BQ in the absence or presence of DMSO correlates with the HRP concentration but saturates at 50 µM HRP for the high DMSO conditions compared to 25 µM in the absence of DMSO ( Figure S6 in the SI). Furthermore, in these control experiments, production of Cl 4 BQ was observed to be time dependent, consistent with a previous report that did not involve DMSO (10). The presence of DMSO did not appear to influence the relative rate of Cl 4 -BQ formation.…”

“…All chemicals were used as received. The HRP concentration was determined by measuring the absorbance at 402 nm (E 402 ) 9.5 × 10 4 M -1 cm -1 ) as described previously (10). Caution: The work described inVolVes the handling of hazardous agents and was therefore conducted in accordance with the NIH guidelines for the laboratory use of chemical carcinogens (18).…”

The Catalytic Product of Pentachlorophenol 4-Monooxygenase is Tetrachlorohydroquinone rather than Tetrachlorobenzoquinone

“…It was likely that TCBQ was produced by other enzymes rather than PcpB and/or by non-enzymatic oxidation of TCHQ, because TCBQ would accumulate to higher concentrations, inhibit the rate-limiting enzyme PcpB, and decrease the PCP degradation rate if it were produced by PcpB. Taking into consideration that PCP could be converted to TCBQ in vivo by peroxidases in fungi [ 30 ] and in vitro by horseradish peroxidase [ 31 , 32 ], we suspected that TCBQ was also produced from PCP by at least one peroxidase in S. chlorophenolicum strain ATCC 39723. We thus measured the peroxidase activity of the cell culture of strain ATCC 39723 and the supernatant of the bacterial cell lysate (Fig.…”

Ochratoxin A: Molecular Interactions, Mechanisms of Toxicity and Prevention at the Molecular Level

“…Another theory considers the activation by peroxidases as an important step to produce the phenoxyl radical from OTA [ 160 , 161 ]. Then, in the presence of glutathione, the phenoxyl radical may be converted to OTA again, but at the expense of the formation of a superoxide anion radical (O 2 • ¯) [ 162 , 163 ].…”