Inhibition of milk xanthine oxidase by fluorodinitrobenzene

Milk xanthine oxidase reacted with fluorodinitrobenzene resulting in the modification of two lysine residues with a 6-fold decrease in catalytic activity. Continued reaction with fluorodinitrobenzene up to a total of 11 dinitrophenyl residues/equivalent of enzyme-bound FAD resulted in no further decrease in activity. Stopped flow studies revealed that the modification perturbed the reduction of the enzyme by xanthine; this was 6-fold lower with modified than with native enzyme. The reaction of the reduced modified enzyme with oxygen was qualitatively and quantitatively the same as with native enzyme. One nitro group of each dinitrophenyl lysine residue is slowly reduced by xanthine; reduction of both nitro groups is achieved by dithionite. The two dinitrophenyl lysine reduces can be distinguished on the basis of their kinetics of reduction. One appears to be located on the protein surface and is reduced in an intermolecular reaction, while the other appears to be located in a pocket of the enzyme and is reduced in a slow intramolecular reaction.