Earlier studies on inactivation of xanthine oxidase induced by methanol and accompanied by development of a specific electron paramagnetic resonance signal (the Inhibited signal) have been extended. The same reaction takes place when enzyme is treated with formaldehyde. With this, time-courses for signal development and disappearance of enzymic activity cannot be distinguished. Under a variety of conditions, disappearance of the signal accompanied by partial restoration of activity could be achieved. Side reactions also take place in the presence of formaldehyde. Electron paramagnetic resonance parameters of the Inhibited signal are reported. I n the signal-giving species a single non-exchangeable proton interacts with Mo(V) in the enzyme active site. Experiments with C2H30H and with 2HC2H0 show that this proton is derived from the inactivating agent. It is proposed that a formyl residue, -CHO, becomes attached to a group in the active centre and that interaction of molybdenum with this residue stabilizes the five-valent state of the metal, thus preventing enzyme turnover. Methanol is presumed to be oxidized to formaldehyde a t the active centre before taking part in the reaction.Studies on the interaction of enzymes with specific irreversible inhibitors may yield information important in understanding their catalytic mechanisms. Specific inhibition of xanthine oxidase by methanol seems first to have been noted by Polonovski [l]. More detailed studies were carried out by Handler and co-workers (e.g. [2,3]), who found that inhibition develops progressively and only while the enzyme is turning over in the presence of substrates plus methanol. They proposed [4] that this somewhat unusual inhibition involves a molecule of methanol substituting for one of water in the coordination sphere of molybdenum in the active site of the enzyme. They further reported [4] tion [6]) that the doublets were replaced by single lines, establishing the interacting proton t o be derived from the methyl group of methanol. We now report further studies carried out with the aim of understanding the reaction between milk xanthine oxidase and methanol.

1. An electron-spin-resonance signal with g( parallel)2.08 and g( perpendicular)2.00 is observed by the rapid-freezing technique during the oxidation of substrates by molecular oxygen catalysed by xanthine oxidase at pH10. 2. The intensity of this signal is shown to depend on oxygen rather than on enzyme concentration, indicating that it is due to an oxygen free radical and not to the enzyme. 3. The same species is shown to be produced in the reaction at pH10 between hydrogen peroxide and periodate ions. Studies with this system have facilitated comparison of the properties of the oxygen radical with data in the literature on the products of pulse radiolysis of oxygenated water over a wide pH range. 4. It is concluded that the species observed is the superoxide ion, O(2) (-), and that the stability of this ion is greatly increased in alkaline solution. A mechanism explaining the alkaline stability is proposed. 5. The importance of O(2) (-) in the enzymic reaction is discussed.

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.

No abstract

No abstract