THE REACTION OF REDUCED XANTHINE DEHYDROGENASE WITH MOLECULAR-OXYGEN - REACTION-KINETICS AND MEASUREMENT OF SUPEROXIDE RADICAL

Xanthine dehydrogenase (XDH) from bovine milk contains significant act ivity 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 ov er 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 as sess the ability of XDH to catalyze production of oxygen radicals. Red uced 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 trans fers electrons to oxygen to produce hydrogen peroxide at a rate consta nt of at least 72,000 M(-1) s(-1) whereas flavin semiquinone reduces o xygen to form superoxide as slow as 16 M(-1) s(-1). Steady-state kinet ics 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), respectiv ely, at 25 degrees C, pH 7.5, XDH is therefore capable of catalyzing t he formation of reduced oxygen species at one-third the rate of xanthi ne/NAD turnover, 6.3 s(-1) (Hunt, J., and Massey, V. (1992) J. Biol. C hem. 267, 21479-21485), in the absence of NAD. As XDH contains a signi ficant and intrinsic xanthine oxidase activity, estimates of relative amounts of XO and XDH based solely upon turnover assays must be made w ith caution, Initial-rate assays containing varying amounts of xanthin e, NAD, and oxygen indicate that at 100% oxygen saturation, NADH forma tion is only inhibited at concentrations of xanthine and NAD below K-m for each substrate.