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 & Knowles [Proc. R. Soc. London Ser. A (1968) 302, 351--353] of direct transfer, during the catalytic reaction, of hydrogen 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, molybdenum(V) interacts with two enzyme-bound protons, which are exchangeable with solvent protons. One of these is coupled to the metal with AHav. 1.4mT and the other with AHav. 0.3mT. The molecule also contains a site for the binding of anions, presumably as ligands of molybdenum. This is shown by effects of nitrate ions on the e.p.r. 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. It is concluded that, in the Michaelis complex, the substrate molecule occupies the anion site, probably being bound to molybdenum via the nitrogen in its 9-position. During the turnover process, hydrogen from the substrate C-8 position, after transfer to the enzyme, appears as the proton more strongly coupled to molybdenum. This proton then exchanges with solvent deuterium with a rate constant of 27s-1, at pH 8.2 and 12 degrees C. It has been confirmed that substrate molecules occupying the anion site do not interfere with observation of the transfer and exchange processes.

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

“…This would bring the aldehydic hydrogen closer to the sulfido group (MOH3) for transfer to occur. The transferred hydrogen from the substrate is observed as the proton strongly coupled to the molybdenum in the rapid type 1 EPR signal (32)(33)(34). The sulfhydryl hydrogen may indeed be closer than 2.5 A to the metal.…”

Structure and function of the xanthine-oxidase family of molybdenum enzymes

“…While this "very rapid" signal is quite anisotropic and exhibits no proton hyperfine coupling, the so-called"rapid" Mo v EPR signal, which also arises in the course of reaction with substrate, belongs to two different types, "rapid type 1" and "rapid type 2", depending on the nature of the proton hyperfine coupling observed [7,10]. The Mo v EPR signals of ,type 2", exhibited by xanthine oxidase upon reaction with xanthine [49] M6ssbauer spectroscopy also allows one to distinguish between both Fe/S centers in the reduced enzymes: one of the centers (probably Fe/S I) exhibits a rather normal quadrupole splitting AEQ of 2.4 mm/s and 2.69(2) mm/s for the ferrous site in xanthine oxidase (at 175 K) [54] and Mop (at 180 K) [55] respectively, while the other center exhibits an unusually large quadrupole splitting of 3.2 mm/s and 3.14(2) mm/s for xanthine oxidase and Mop respectively.…”

Mo(V) Electron Paramagnetic Resonance Signals from the Periplasmic Nitrate Reductase of Thiosphaera Pantotropha

“…The name 'High-g Split' is therefore adopted here for that nitrate reductase signal. The EPR parameten of a second set of signals were found to be remarkably like those of the Rapid type 1 signal from bovine milk xanthine oxidase (Gutteridge et a]., 1978) and these signals are therefore designated the 'Pseudo-Rapid signals' (Fig. 2).…”

“…2a) and for 100 rnin (Fig. 2e) each appear to be from single, defined chemical species and the EPR parameters are given in Table 1 along with those (Gutteridge et al, 1978) for the bovine milk xanthine oxidase Rapid type 1 Mo(V) signal (Fig. 2b).…”