Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily

Thiols represent preferential targets of peroxynitrite in biological systems. In this work, we investigated the mechanisms and kinetics of the reaction of peroxynitrite with the dithiol dihydrolipoic acid (DHLA) and its oxidized form, lipoic acid (LA). Peroxynitrite reacted with DHLA being oxidation yields higher at alkaline pH. The stoichiometry for the reaction was two thiols oxidized per peroxynitrite. LA formation accounted for approximately 50% DHLA consumption at pH 7.4, probably reflecting secondary reactions between LA and peroxynitrite. Indeed, peroxynitrous acid reacted with LA with an apparent second-order rate constant (k(2app)) of 1400 M(-1) s(-1) at pH 7.4 and 37 degrees C. Nitrite and LA-thiosufinate were formed as reaction products. Surprisingly, the k(2app) for peroxynitrite-dependent DHLA oxidation was only 250 M(-1) s(-1) per thiol, at pH 7.4 and 37 degrees C. Testing various low-molecular-weight thiols, we found that an increase in the thiol pK (pK(SH)) value correlated with a decrease of k(2app) for the reaction with peroxynitrite at pH 7.4. The pK(SH) for DHLA is 10.7, in agreement with its modest reactivity with peroxynitrite.