Investigation of acid–base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

Abstract

The extradiol and intradiol catechol dioxygenase reaction mechanisms proceed via a common proximal hydroperoxide intermediate, which is processed via different Criegee 1,2-rearrangements. An R215W mutant of extradiol dioxygenase MhpB, able to produce a mixture of extradiol and intradiol cleavage products, was analysed at pH 5.2–8.6, and the yield of extradiol product was found to be highly pH-dependent, whereas the yield of intradiol product was pH-independent. The acid–base chemistry of a biomimetic reaction for extradiol oxidative catechol cleavage was also investigated, using 1,4,7-triazacyclononane, FeCl₂, and pyridine in methanol, in which pyridine is proposed to act as both a general base and (in protonated form) a general acid. Kinetic experiments using a range of meta- and para-substituted pyridines gave a Brønsted plot of log(v) vs. pK_a showing a bell-shaped plot. Oxidative catechol cleavage by a pyridine-monosubstituted β-cyclodextrin in the presence of TACN and FeCl₂ in methanol yielded only intradiol cleavage products. It is therefore proposed that bifunctional acid–base catalysis is required for iron (II)-dependent extradiol catechol cleavage, whereas the rate-determining step for intradiol catechol cleavage does not involve acid–base catalysis.