Cis-trans isomerization of a cyclopropyl radical trap catalyzed by extradiol catechol dioxygenases: Evidence for a semiquinone intermediate

Substrate analogues cis- and trans-2-(2,3-dihydroxyphenyl)cyclopropane-1-carboxylic acid were synthesized as probes for a semiquinone radical intermediate in the (2,3-dihydroxyphenyl)propionate 1,2-dioxygenase reaction. These analogues were found to be substrates for oxidative cleavage by extradiol dioxygenases from Escherichia coli and Alcaligenes eutrophus. The stereochemistry of the ring fission products was analyzed by conversion to cyclopropane-1,2-dicarboxylic acids using the ensuing hydrolase enzyme MhpC, followed by GCMS analysis. This analysis revealed 85-94% trans product and 6-15% cis products, implying that cis/trans isomerization of the cyclopropyl ring substituents had taken place during the enzymatic conversion. These results are consistent with a reversible opening of the cyclopropyl ring, and hence consistent with the intermediacy of a semiquinone radical intermediate in the extradiol catechol hioxygenase reaction.