The reductive half-reaction of xanthine oxidase. Identification of spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine.
The reaction of xanthine oxidase with 2-hydroxy-6-methylpurine (also called 2-oxo-6-methylpurine) has been studied under both anaerobic and aerobic conditions. Reaction of enzyme with substoichiometric concentrations of hydroxymethylpurine in aerobic 0.1 M 3-(cyclohexylamino)propanesulfonic acid, 0.1 N KCl, 0.3 mM EDTA, pH 10.0, exhibits two reaction intermediates detectable by UV-visible spectrophotometry. The rate constants for formation of the first intermediate, conversion of the first to the second, and the decay of the second to give oxidized enzyme are 18, 1.2, and 0.13 s-1, respectively. The difference spectra of these two intermediates relative to oxidized enzyme are characterized by absorbance maxima at 470 and 540 nm, respectively, with extinction changes (relative to oxidized enzyme) of approximately 410 M-1 cm-1. The 0.13 s-1 decay of the second intermediate agrees well with kcat of 0.11 s-1 determined under the same conditions. Based on a comparison of the kinetics of the reaction as monitored by UV-visible absorption and electron paramagnetic resonance spectrometry, it is concluded that these spectral intermediates arise from the molybdenum center of the enzyme in the MoIV and MoV valence states, respectively, the latter corresponding to the species exhibiting the "very rapid" MoV EPR signal known to be formed in the course of the reaction. This conclusion is supported by the results of experiments using cytochrome c reduction to follow the formation of superoxide production in the course of the aerobic reaction of xanthine oxidase with substoichiometric hydroxymethylpurine, which demonstrate unequivocally that the species exhibiting the very rapid EPR signal is formed by one-electron oxidation of a MoIV species rather than direct one-electron reduction of MoVI by substrate. No evidence is found for the formation of any of the MoV EPR signals designated "rapid" in the present studies, and it is concluded that this species is not a bona fide catalytic intermediate in the reductive half-reaction of xanthine oxidase.
McWhirter
R B
RB
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Hille
R
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D000332:Aerobiosis; D000693:Anaerobiosis; D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008892:Milk / Q000201:enzymology; D010084:Oxidation-Reduction; D011687:Purines / Q000378:metabolism*; D013053:Spectrophotometry; D014969:Xanthine Oxidase / Q000378:metabolism*
D011687:Purines; D005182:Flavin-Adenine Dinucleotide; C071987:2-hydroxy-6-methylpurine; D014969:Xanthine Oxidase