Xanthine oxidase-catalyzed reductive debromination of 6-(bromomethyl)-9H-purine with concomitant covalent modification of the FAD prosthetic group

Bovine milk xanthine oxidase was potently inhibited by 6-(bromomethyl)-9H-purine in a time-dependent process with O2 as the electron acceptor. If the enzyme were assayed with phenazene ethosulfate as an electron acceptor, 6-(bromomethyl)-9H-purine was not an inhibitor. The rate of formation of inhibited enzyme increased with increasing concentrations of 6-(halomethyl)-9H-purine, decreased with increasing concentrations of O2, and increased in the presence of xanthine. The inhibited enzyme regained activity nonactinically at pH 7 with a t1/2 of 31 h. The optical difference spectrum between native enzyme and inhibited enzyme suggested that the enzyme-bound FAD was modified. This conclusion was confirmed by demonstrating that activity was restored to the inhibited enzyme if the enzyme-bound flavin was removed by treatment with CaCl2 and the resulting apoenzyme was reconstituted with FAD. Aerobically, 6-(bromomethyl)-9H-purine was oxidized by the enzyme to a species having a UV spectrum consistent with hydroxylation of the purine ring to form a urate analogue. Anaerobically, the enzyme reduced 6-(bromomethyl)-9H-purine to 6-methylpurine with 1 mol of enzyme being completely inhibited after reduction of 23 mol of 6-(bromomethyl)-9H-purine. Thus, 6-(bromomethyl)-9H-purine was not only oxidized by xanthine oxidase but was also reduced by the enzyme in a reaction that partitioned between formation of 6-methylpurine and inhibition of the enzyme by modification of the enzyme-bound flavin. Similar results were found when 6-(chloromethyl)-9H-purine was the inhibitor.