REDUCTIVE HALF-REACTION OF XANTHINE-OXIDASE - MECHANISTIC ROLE OF THE SPECIES GIVING RISE TO THE RAPID TYPE-1 MOLYBDENUM(V) ELECTRON-PARAMAGNETIC RESONANCE SIGNAL

The reaction of xanthine oxidase with xanthine, 1-methylxanthine, and 2-hydroxy-6-methylpurine has been reinvestigated with the aim of elucidating the mechanistic role of the species giving rise to the ''rapid'' Mo(V) electron paramagnetic resonance (EPR) signal. It is found that addition of 2.0 mM 1-methylxanthine or 2-hydroxy-6-methylpurine to partially reduced enzyme generates substantial amounts of the Type 1 form of the ''rapid'' EPR signal, characterized by superhyperfine coupling to one strongly interacting (a(av) = 13 G) and one weakly interacting (a(av) = 3 G) proton. The ''rapid'' signals observed with both substrates are identical to those observed in the course of the anaerobic reaction of enzyme with a stoichiometric excess of substrate. With 2-hydroxy-6-methylpurine at pH 10, a burst phase in the formation of the species giving rise to the ''rapid Type 1'' signal is observed that is fast relative to the rate of formation of the species giving rise to the ''very rapid'' EPR signal. At pH 8.5, partial reduction of enzyme prior to reaction with xanthine, 1-methylxanthine, or 2-hydroxy-6-methylpurine reverses the relative amounts of ''rapid'' and ''very rapid'' EPR signal observed at the shortest reaction times. The substantial amounts of ''rapid Type 1'' signal formed by addition of substrates to partially reduced enzyme or by reaction of oxidized enzyme with a stoichiometric excess of substrate contrasts with previous work, which has shown that under single-turnover conditions none of the substrates investigated generates an appreciable amount of ''rapid'' EPR signal. It is concluded that the species giving rise to the ''rapid Type 1'' EPR signal is not a bona ride catalytic intermediate, as has long been thought, but rather arises from the dead-end complex of substrate with enzyme possessing Mo(V) rather than the Mo(VI) required for the initiation of the reductive half-reaction. Using 2-hydroxy-6-methylpurine labeled with H-2 at the C-8 position, additional evidence is found that the C-8-H proton of bound substrate in the species giving rise to the ''rapid Type 1'' EPR signal is not detectably coupled magnetically.