Quinoprotein (PPQ-containing) alcohol dehydrogenases

BiotechnologyApplied Science

Identification of Topaquinone, as Illustrated for Pig-Kidney Diamine Oxidase and Escherichia-Coli Amine Oxidase

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Studies on electron transfer from methanol dehydrogenase to cytochrome cL, both purified from Hyphomicrobium X

Ferricytochrome CL isolated from Hyphomicrobium X is an electron acceptor in assays for homologous methanol dehydrogenase (MDH), albeit a poor one compared with artificial dyes. The intermediates of MDH seen during the reaction are identical with those observed with Wurster's Blue as electron acceptor, indicating that the reaction cycles are similar. The assay showed a pH optimum of approx. 7.0 and scarcely any stimulation by NH4C1, this being in contrast with assays with artificial dyes, where strong activation by NH4C1 and much higher pH optima have been reported. From the results obtained with stopped-flow as well as steady-state kinetics, combined with the isotope effects found for C2H30H, it appeared that the dissimilarities-between the electron acceptors can be explained from different rate-limiting steps in the reaction cycles.- Ferricytochrome CL is an excellent oxidant of the reduced MDH forms at pH 7.0, but the substrate oxidation step is very slow and the activation by NH4Cl is very poor at this pH. At pH 9.0 the reverse situation exists: ferricytochrome CL is a poor oxidant of the reduced forms ofMDH at this pH. No C2H3OH isotope effect was observed under these conditions, indicating that substrate oxidation is not rate-limiting, so that activation by NH4C1 cannot be found. Since just the opposite holds for assays with artificial dyes, the poor electron-acceptor capability and the different pH optimum of ferricytochrome CL as well as the insignificant activating effect of NH4C1 (all compared with artificial assays) can be explained. Althoug