Role of the flavin midpoint potential and NAD binding in determining NAD versus oxygen reactivity of xanthine oxidoreductase

Xanthine oxidoreductase from bovine milk can be prepared in two interconver tible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH), depend ing on the number of protein cysteines versus cystines, Enzyme forms differ in respect to their oxidizing substrates; XDH prefers NAD to molecular oxy gen, whereas XO only reacts significantly with oxygen. The preference for o xidizing substrate is partially explained by thermodynamics. Unlike XDH, th e midpoint potential of the FAD, the center at which oxygen and NAD react, is too high in XO to efficiently reduce NAD (Hunt, J,, Massey, V., Dunham, W.R,, and Sands, R.H. (1993) J, Biol, Chem, 268, 18685-18691), To distingui sh between changes in thermodynamics and in substrate binding, samples of b oth XO and XDH have been prepared in which the native FAD has been replaced with an FAD analog of different redox potential, 1-deaza-FAD or 8-CN-FAD. Reductive titrations indicate that both 1-deaza-XO and 1-deaza-XDH have a f lavin midpoint potential similar to native XDH and that 8-CN-XO and 8-CN-XD H each have a flavin potential higher than XO, Both the low potential. 1-de aza-XO and the high potential 8-CN-XDH contain essentially no xanthine/NAD activity. However, 1-deaza-XDH does exhibit xanthine/NAD activity, and 8-CN -XO has normal xanthine/oxygen activity. The binding of NAD to oxidized XO and XDH was investigated by ultrafiltrati on and isothermal titration calorimetry. The K-d for the binding of NAD to XDH was determined to be 280 +/- 145 mu M by ultrafiltration and 160 +/- 40 mu M by isothermal titration calorimetry. No evidence for the binding of N AD to XO by either method could be obtained. A low flavin midpoint potentia l is necessary but not sufficient for dehydrogenase activity.