Reductive half-reaction of xanthine oxidase with xanthine. Observation of a spectral intermediate attributable to the molybdenum center in the reaction of enzyme with xanthine.
The reductive half-reaction of xanthine oxidase with substoichiometric concentrations of xanthine and 1-methylxanthine at pH 10 and 8.5 has been examined by UV-visible stopped-flow and rapid-quench electron paramagnetic resonance (EPR) kinetic experiments. A spectral intermediate is observed in stopped-flow experiments with xanthine which exhibits a difference absorbance maximum relative to oxidized enzyme at 480 nm and which decays at the same rate as the decay of the "very rapid" MoV EPR signal observed by freeze-quench EPR experiments both at 5 and 20 degrees C. The intermediate is observed in experiments using enzyme that has had its flavin removed, and most likely arises from the molybdenum center. With 40 microM xanthine oxidase and 10 microM xanthine, rate constants for the appearance and decay of this intermediate at pH 10 are 11 and 1.1 s-1, respectively; at pH 8.5 the corresponding values are 20 and 2.5 s-1. Based on the correlation of the stopped-flow kinetics with the appearance and decay of the MoV EPR signal designated very rapid as monitored in freeze-quench experiments, it is concluded that the spectral intermediate corresponds to the species exhibiting the very rapid EPR signal, the MoIV species that gives rise to it, or a combination of the two. None of the MoV EPR signals designated "rapid" is observed under single turnover conditions with either xanthine or 1-methylxanthine as substrate at pH 8.5, by contrast with the substantial amounts observed in both cases under conditions of excess substrate. These results call into question the prevailing view that the species giving rise to the rapid EPR signal lies downstream in the catalytic cycle from that exhibiting the very rapid signal.
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
J H
JH
Kim
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R
Hille
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D007700:Kinetics; D008433:Mathematics; D008892:Milk / Q000201:enzymology; D008982:Molybdenum / Q000378:metabolism*; D013053:Spectrophotometry; D013816:Thermodynamics; D013997:Time Factors; D019820:Xanthine; D014969:Xanthine Oxidase / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism*
D014970:Xanthines; D019820:Xanthine; C053085:1-methylxanthine; D008982:Molybdenum; D014969:Xanthine Oxidase