The molybdenum centre of native xanthine oxidase. Evidence for proton transfer from substrates to the centre and for existence of an anion-binding site

Abstract

The observation by Bray and Knowles of direct transfer, during the catalytic reaction, of H atoms from substrate molecules to the enzyme xanthine oxidase was reinvestigated. The experimental phenomenon and its basic interpretation were confirmed and extended. In the reduced functional enzyme, Mo(V) interacts with 2 enzyme-bound protons, which are exchangeable with solvent protons. One of these is coupled to the metal with .**GRAPHIC**. 1.4mT and the other with .**GRAPHIC**. 0.3mT. The molecule also contains a site for the binding of anions, presumably as ligands of M. This is shown by effects of nitrate ions on the EPR spectra. The spectra of the nitrate and 1-methylxanthine complexes of the reduced enzyme are very similar to one another, and are designated Rapid type-1 spectra. In the Michaelis complex, the substrate molecule probably occupies the anion site, being bound to Mo via the N in its 9-position. During the turnover process, H from the substrate C-8 position, after transfer to the enzyme, appears as the proton more strongly coupled to Mo. This proton then exchanges with solvent deuterium with a rate constant of 27 s-1, at pH 8.2 and 12.degree. C. Substrate molecules occupying the anion site do not interfere with observation of the transfer and exchange processes.