Rapid reaction studies on the oxygenation reactions of catechol dioxygenase.

The reaction of oxygen with catechol 1,2-dioxygenase from Pseudomonas arvilla ATCC 23974 in complex with catechol, 4-methylcatechol, and 4-fluorocatechol has been studied using single turnover stopped flow spectrophotometry. Two sequential enzyme intermediates have been resolved and their visible spectra characterized by computer-assisted methods. These intermediates are spectrally similar to those observed in a similar study with protocatechuate dioxygenase (Bull, C., Ballou, D. P., and Otsuka, S. J. Biol. Chem. 256, 12681-12686 (1981), although the first intermediate seen with the latter enzyme was not observed in this study. The rate of formation of intermediate I is oxygen-dependent and also accelerated by electron-donating substituents on the C-4 of the substrate. This is consistent with the proposed substrate reduction of dioxygen to form a hydroperoxide. Intermediate I is thus suggested to be a 6-hydroperoxycyclohexa-3,5-diene-1-one. The decay of intermediate I is also accelerated by electron donors and is consistent with the rearrangement of intermediate hydroperoxide via an acyl migration mechanism. It is inconsistent with mechanisms involving nucleophilic attack at the carbonyl carbon. Intermediate II is proposed to be an enzyme-product complex based on the resemblance of its visible spectra to those of the benzoate complex of catechol 1,2-dioxygenase and enzyme-product complexes of protocatechuate dioxygenase. Careful 18O2-labeling experiments have shown that no label is lost to the solvent, implying that no free hydroxide forms during catalysis.