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 (aav = 13 G) and one weakly interacting (aav = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hille
R
R
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Kim
J H
JH
Hemann
C
C
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008892:Milk / Q000201:enzymology; D008982:Molybdenum; D010084:Oxidation-Reduction; D011687:Purines / Q000378:metabolism; D013053:Spectrophotometry; D013997:Time Factors; D019820:Xanthine; D014969:Xanthine Oxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism
D011687:Purines; D014970:Xanthines; D005182:Flavin-Adenine Dinucleotide; D019820:Xanthine; C071987:2-hydroxy-6-methylpurine; C053085:1-methylxanthine; D008982:Molybdenum; D014969:Xanthine Oxidase