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Abstract

Formylmethanofuran dehydrogenases, which are found in methanogenic Archaea, are molybdenum or tungsten iron-sulfur proteins containing a pterin cofactor. We report here on differences in substrate specificity, EPR properties and susceptibility towards cyanide inactivation of the enzymes from Methanosarcina barkeri, Methanobacterium thermoautotrophicum and Methanobacterium wolfei. The molybdenum enzyme from M. barkeri (relative activity with N-formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent Vmax, the dehydrogenation of N-furfurylformamide (11%), N-methylformamide (0.2%), formamide (0.1%) and formate (1%). The molybdenum enzyme from M. Wolfei could only use N-furfurylformamide (1%) and formate (3%) as pseudosubstrates. The molybdenum enzyme from M. thermoautotrophicum and the tungsten enzymes from M. thermoautotrophicum and M. wolfei were specific for N-formylmethanofuran. The molybdenum formylmethanofuran dehydrogenases exhibited at 77 K two rhombic EPR signals, designated FMDred and FMDox, both derived from Mo as shown by isotopic substitution with 97Mo. The FMDred signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMDox signal was displayed by an inactive form and was not quenched by O2. The tungsten isoenzymes were EPR silent. The molybdenum formylmethanofuran dehydrogenases were found to be inactivated by cyanide whereas the tungsten isoenzymes, under the same conditions, were not inactivated. Inactivation was associated with a characteristic change in the molybdenum-derived EPR signal. Reactivation was possible in the presence of sulfide.