Oxygen-17 splitting of the very rapid molybdenum(V) e.p.r. signal from xanthine oxidase. Rate of exchange with water of the coupled oxygen atom.

Studies have been carried out of effects of 17O substitution on a Mo(V) e.p.r. signal from xanthine oxidase, known as Very Rapid. This transient signal is believed to represent an intermediate in enzymic turnover. When Very Rapid was developed from enzyme equilibrate with 17O-enriched water, strong coupling of Mo(V) to a single oxygen atom was observed, with A(17O)1,2,3 1.34, 1.40, 1.36 mT. The isotropic character of the splittings is interpreted as favouring a structure of the type Mo--O--C. The rate of exchange with water of the oxygen atom detected in the signal was studied. In oxidized enzyme, which contains a terminal oxygen ligand, the exchange rate constant was 2--4 h-1 (pH 5.9--7.8 and about 20 degrees C). However, if the exchange was allowed to take place whilst the enzyme was turning over a substrate, then the process occurred within a few seconds. The present and previous results are interpreted as favouring an enzymic mechanism in which a terminal oxygen ligand reacts, as a nucleophile, with a substrate carbonium ion. To complete the reaction, product liberation, by hydrolysis of the enzyme-bound species, occurs in such a way as to cleave the Mo--O bond, thus explaining the fast oxygen exchange in the presence of the substrate.