Oxidation-reduction potentials of molybdenum, flavin and iron-sulphur centres in milk xanthine oxidase

Abstract

The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by EPR spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH 8.2, are: Fe/S center I, -343 .+-. 15 mV; Fe/S II, -303 .+-. 15 mV; FAD/FADH.; -351 .+-. 20 mV; FADH./FADH2, -236 .+-. 20 mV; Mo(VI)/Mo(V)(Rapid), -355 .+-. 20 mV; Mo(V)(Rapid)/Mo(IV), -355 .+-. 20 mV. Behavior of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V)(Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with 6 other buffers. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow Mo signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centers. The potentials associated with the Slow signal are: Mo(VI)/Mo(V)(Slow), -440 .+-. 25 mV; Mo(V)(Slow)/Mo(IV), -480 .+-. 25 mV. This signal exhibits very sluggish equilibration with the mediator system. The deviations from ideal behavior are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centers.