Comparison of the molybdenum centres of native and desulpho xanthine oxidase. The nature of the cyanide-labile sulphur atom and the nature of the proton-accepting group

Abstract

The non-functional form of xanthine oxidase known as the desulpho enzyme was compared with the functional enzyme in various ways, to obtain information on the structure of the molybdenum centre and the mechanism of the catalytic reaction. The desulpho enzyme, like the functional one, possesses a site for the binding of anions, presumably as ligands of molybdenum. Evidence is presented that in the Mo(V) e.p.r. signal from the desulpho-enzyme, as in that from the functional enzyme, a weakly coupled proton, in addition to a strongly coupled proton, interacts with the metal. Measurements were carried out by e.p.r. on the rate at which the proton strongly coupled to molybdenum exchanged, on diluting enzyme samples with 2H2O. For the desulpho enzyme the exchange rate constant was 0.40s-1, at pH 8.2 and 12 degrees C, and for the functional enzyme it was 85 s-1. It is shown that the great majority of reported differences between the enzyme forms are consistent with functional enzyme containing an (Enzyme)-Mo=S grouping, replaced in the desulpho form by (Enzyme)-Mo=O. Protonation of these groups, with pK values of about 8 and 10 respectively, would give (Enzyme)-Mo-SH and (Enzyme)-Mo-OH, these being the forms observed by e.p.r. The accepting group in the functional enzyme, for the proton transferred from the substrate while molybdenum is reduced in the catalytic reaction [Gutteridge, Tanner & Bray (1978) Biochem J. 175 869-878], is thus taken to be Mo=S.

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

“…5) and the EPR parameters found are presented in Tables 2 and 3. The signals obtained are closely related to those observed and described for bovine milk XO and for avian liver XD [39][40][41][42][43][44][45]. Both active and desulfo enzymes are reduced simultaneously by dithionite and, accordingly, the Mo(V) Fig.…”

Multiple States of the Molybdenum Centre of Dimethylsulphoxide Reductase from Rhodobacter Capsulatus Revealed by EPR Spectroscopy

“…It bears a striking similarity (Table 1) to a signal (Fig. 4a) from the desulpho form of xanthine oxidase, known as the Slow signal, and in particular to the variant of this obtained in the presence of nitrate ions (Gutteridge et al, 1978). The Low-g type-2 signal ( Fig.…”

“…The rapid freezing procedure of Bray (1961) was used as modified in later work (Gutteridge et al, 1978). Samples were frozen 10 ms or 80 ms after mixing the enzyme in one syringe, with a mixture of M e 3 0 and MV'+ (prepared by reduction with substiochiometric Na,S,O,) in another.…”

“…The simulation of the Slow (nitrate) signal from desulpho xanthine oxidase (Gutteridge et al, 1978) is presented in (a) for comparison. Spectra were generated using procedures described in Materials and Methods.…”

The mechanism of action of xanthine oxidase. The relationship between the rapid and very rapid molybdenum electron-paramagnetic-resonance signals

“…A chemical interpretation of Scheme 2, is shown in Scheme 3, taking account of (Enzyme)-Mo=S as the proton-accepting group in the enzyme (Gutteridge et al, 1978b). Attack on the substrate takes place in the Michaelis complex (I), in which the substrate is co-ordinated, via its N-9 position, to the anionbinding site in the enzyme (Gutteridge et al, 1978a;Bergmann & Levene, 1976).…”