The reaction of oxygen with protocatechuate 3,4-dioxygenase from Pseudomonas putida. Characterization of a new oxygenated intermediate.

The reactions of protocatechuate dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase, EC 1.13.11.3) with substrates and oxygen have been studied at 4 degrees C using rapid kinetic techniques. In this study, two oxygenated intermediates were kinetically and spectrally characterized. The rate of oxygen addition to the enzyme-substrate complex was determined to be 5 X 10(5) M-1 s-1. This oxygenated complex rapidly converts (450 s-1) to another spectrally identifiable compound which then breaks down into free enzyme and the product, beta-carboxy-cis,cis-muconic acid. To carry out these experiments properly, it was necessary to use hyperbaric oxygen, thus obtaining approximately 6 mM final concentration of oxygen. The procedure is described in this work. It was also shown that the normally slow substrate, dihydroxyphenyl propionate, when reacted with the enzyme and oxygen, rapidly forms two sequential intermediates. However, the second intermediate was not spectrally the same as that observed with protocatechuate, but was more like that of enzyme-product complexes. Similar, although less extensive, studies using the protocatechuate dioxygenase from Pseudomonas aeruginosa showed that very analogous behavior occurred with both substrates as did with the Pseudomonas putida enzyme.