Studies on methanol dehydrogenase from Hyphomicrobium X. Isolation of an oxidized form of the enzyme.

1. Double-reciprocal plots of initial reaction rates of methanol dehydrogenase [alcohol--(acceptor) oxidoreductase, EC 1.1.99.8] in vitro show patterns of parallel lines. The results with various methanol, ammonia and phenazine methosulphate concentrations can be described by an equation valid for a Ping Pong kinetic mechanism with three reactants. 2. The overall maximum velocity was the same for several primary alcohols, C(2)-deuterated ethanols and different electron acceptors, but it was significantly lower for C(1)-deuterated substrates. 3. Oxidation of the isolated enzyme with electron acceptors required the presence of ammonia and a high pH. The inclusion of cyanide or hydroxylamine during the incubation was essential to prevent enzyme inactivation. The absorbance spectrum of an oxidized form of the enzyme was clearly different from that of the isolated enzyme and the free radical was no longer present. On addition of substrate, the original absorption spectrum and electron-spin-resonance signal reappeared and a concomitant substrate oxidation was found. This reaction could be carried out at pH 7 and ammonia was not required. 4. Based on the activity of the enzyme with one-electron acceptors, the presence of a free radical and the kinetic behaviour, an oxidation of the enzyme via one-electron steps is proposed.