Reaction of Formaldehyde and of Methanol with Xanthine Oxidase

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. In the signal‐giving species a single non‐exchangeable proton interacts with Mo(V) in the enzyme active site. Experiments with C 2 H 3 OH and with 2 HC 2 HO 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 at the active centre before taking part in the reaction.