The reaction of reduced xanthine dehydrogenase with molecular oxygen - Reaction kinetics and measurement of superoxide radical

Xanthine dehydrogenase (XDH) from bovine milk contains significant activity in xanthine/oxygen turnover assays, The oxidative half-reaction of XDH with molecular oxygen has been studied in detail, at 25 degrees C, pH 7.5, to determine the basis of the preference of XDH for NAD over oxygen as oxidizing substrate. Spectral changes of XDH accompanying oxidation were followed by stopped-flow spectrophotometry, The amount of superoxide radicals formed during oxidation was investigated to assess the ability of XDH to catalyze production of oxygen radicals. Reduced XDH reacts with oxygen in at least 4 bi-molecular steps, with 1.7-1.9 mol of superoxide per mol of XDH formed from the last 2 electrons oxidized. A model is discussed in which the flavin hydroquinone transfers electrons to oxygen to produce hydrogen peroxide at a rate constant of at least 72,000 M(-1) s(-1) whereas flavin semiquinone reduces oxygen to form superoxide as slow as 16 M(-1) s(-1). Steady-state kinetics of xanthine/oxygen and NADH/oxygen turnover of XDH were determined to have h(cat) values of 2.1 +/- 0.1 and 2.5 +/- 0.9 s(-1), respectively, at 25 degrees C, pH 7.5, XDH is therefore capable of catalyzing the formation of reduced oxygen species at one-third the rate of xanthine/NAD turnover, 6.3 s(-1) (Hunt, J., and Massey, V. (1992) J. Biol. Chem. 267, 21479-21485), in the absence of NAD. As XDH contains a significant and intrinsic xanthine oxidase activity, estimates of relative amounts of XO and XDH based solely upon turnover assays must be made with caution, Initial-rate assays containing varying amounts of xanthine, NAD, and oxygen indicate that at 100% oxygen saturation, NADH formation is only inhibited at concentrations of xanthine and NAD below K-m for each substrate.