Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enzyme by sulphide and by selenide

Abstract

1. Turkey liver xanthine dehydrogenase engaged in catalysing the oxidation of xanthine by dichlorophenol-indophenol was progressively inactivated by methanol. This inactivation was reversible by NAD(+). 2. Reaction with arsenite and with cyanide, in each case first-order with respect to enzyme, resulted in characteristic alterations in the visible absorption spectrum of the enzyme. The rate of spectral change on reaction with either agent paralleled the rate of loss of enzyme activity. 3. Cyanide inactivation was accompanied by elimination from the enzyme of sulphur as thiocyanate. Partial restoration of activity was effected by incubation with sulphide or with selenide. The results suggest that turkey liver xanthine dehydrogenase, like milk xanthine oxidase (Massey & Edmonson, 1970), contains at the active centre a cyanolysable persulphide group essential to catalytic activity and that selenium may replace sulphur in this group to give an active enzyme. 4. Incubation of the native enzyme with sulphide or with selenide resulted in the rapid loss of half of the xanthine-oxidizing activity, apparently by disrupting the molybdenum and (Fe/S)II loci. This may indicate non-equivalence of the intramolecular electron-transfer systems.

Reversible interconversion between sulfo and desulfo xanthine oxidase in a system containing rhodanese, thiosulfate, and sulfhydryl reagent.

“…EC 1.2.3.2) (1), xanthine dehydrogenase (2,3), and aldehyde oxidase (4) can be converted into inactive forms by treatment with cyanide, which results in the release of an essential sulfur atom as thiocyanate. Recent advances in characterization of the desulfo enzyme show that.…”

On the origin of the cyanolysable sulphur in molybdenum iron/sulphur flavin hydroxylases

“…As outlined in this paper, an active North-Holland Publishing Company -Amsterdam centre cysteine residue could equally well be considered as the source of the cyanolysable sulphur. Such a hypothesis also provides one explanation for the finding [2] of one mole of dehydroalanine per mole of enzyme, whether cyanide-inactivated or untreated (cf. later).…”

“…In the first place, the 'desulphoenzyme' has the full compliment of acid-labile sulphur [ 1 ] . Secondly, the change in the visible absorption spectra of these enzymes on treatment with cyanide is much less than would be expected had these chromophores been disrupted [ 1,2,9] . On the basis of these findings, Massey and Edmonson [l] proposed that the sulphur atoms released following cyanide treatment originate from a persulphide group (R-S-S-)…”

“…Cyanide treatment of xanthine oxidase [l] , xanthine dehydrogenase [2] and of aldehyde oxidase [3] results in the removal of sulphur, as free thiocyanate, from the active centres of these enzymes. The sulphur atoms in question may also be lost spontaneously during purification or as a result of 'aging'.…”

“…Many, though not all, of the properties of each enzyme are affected as a result [l-7] . Treatment of the resulting 'desulphoenzyme' (terminology of Bray [8] ) with sulphide effects partial restoration of activity [ 1,2,4] . Under the reactivation conditions used, the incorporation of 35S2-has been demonstrated as has the release of 35SCN-on subsequent treatment with cyanide [ 1 J .…”