Xanthine oxidoreductase from bovine milk can be prepared in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH), depending on the number of protein cysteines versus cystines. Enzyme forms differ in respect to their oxidizing substrates; XDH prefers NAD to molecular oxygen, whereas XO only reacts significantly with oxygen. The preference for oxidizing substrate is partially explained by thermodynamics. Unlike XDH, the 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 distinguish between changes in thermodynamics and in substrate binding, samples of both 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 flavin midpoint potential similar to native XDH and that 8-CN-XO and 8-CN-XDH each have a flavin potential higher than XO. Both the low potential 1-deaza-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 ultrafiltration and isothermal titration calorimetry. The Kd for the binding of NAD to XDH was determined to be 280 +/- 145 microM by ultrafiltration and 160 +/- 40 microM by isothermal titration calorimetry. No evidence for the binding of NAD to XO by either method could be obtained. A low flavin midpoint potential is necessary but not sufficient for dehydrogenase activity.