Complex-formation between reduced xanthine oxidase and purine substrates demonstrated by electron paramagnetic resonance

Abstract

The origin of the Rapid molybdenum electron-paramagnetic-resonance signals, which are obtained on reducing xanthine oxidase with purine or with xanthine, and whose parameters were measured by Bray & Vänngård (1969), was studied. It is concluded that these signals represent complexes of reduced enzyme with substrate molecules. Xanthine forms one complex at high concentrations and a different one at low concentrations. Purine forms a complex indistinguishable from the low-concentration xanthine complex. There are indications that some other substrates also form complexes, but uric acid, a reaction product, does not appear to do so. The possible significance of the complexes in the catalytic cycle of the enzyme is discussed and it is suggested that they represent substrate molecules bound at the reduced active site, waiting their turn to react there, when the enzyme has been reoxidized. Support for this role for the complexes was deduced from experiments in which frozen samples of enzyme-xanthine mixtures, prepared by the rapid-freezing method, were warmed until the signals began to change. Under these conditions an increase in amplitude of the Very Rapid signal took place. Data bearing on the origin of the Slow molybdenum signal are also discussed. This signal disappears only slowly in the presence of oxygen, and its appearance rate is unaffected by change in the concentration of dithionite. It is concluded that, like other signals from the enzyme, it is due to Mo(v) but that a slow change of ligand takes place before it is seen. The Slow species, like the Rapid, seems capable of forming complexes with purines.

Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme

“…For the bovine enzyme, reduction with purine gave the Rapid signal from the functional enzyme, as expected ( Figure 2A, trace b), while reduction with Na # S # O % gave the Slow signal from the desulpho-enzyme ( Figure 2A, trace d ; the line shape of this signal is slightly different from the normal one [27] due to the presence of purine [28]). The recombinant enzyme also gave a signal that, though slightly different from that for the bovine enzyme, is clearly of the Slow form (Figure 2A, trace c).…”

Kinetic and Spectroscopic Studies of the Molybdenum-Copper CO Dehydrogenase from Oligotropha carboxidovorans

“…In the "Rapid type 2" signal of this enzyme, the Mo-OH splitting grows to 13 G, yielding a 1:2:1 triplet due to two approximately equally strongly coupled protons. As demonstrated in the classic work of Bray and coworkers (33,34), the Mo-OH and Mo-SH protons of the "rapid" signals of xanthine oxidase are solvent-exchangeable, and substitution into D 2 O collapses the hyperfine structure to give a single line for each principal g-value of the signals. 3 The complexity of the EPR signal seen here with CO dehydrogenase notwithstanding, its linewidths (5-6 G) are comparable to those seen for the signals observed with xanthine oxidase and loss of proton splitting even as weak as 3 G would have led to a demonstrable narrowing of the features upon substitution into D 2 O.…”

Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases

“…g= 1.973 1( a) (cf. Pick & Bray, 1969) had no effect on the activity of the enzyme, showing that no major irreversible change in the structure of the protein is caused by the denaturing agent under the conditions used.…”