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 woljei.The molybdenum enzyme from M. barkeri (relative activity with N-formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent V,,,,,, 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 FMD,,, both derived from Mo as shown by isotopic substitution with 9 7 M~. The FMD,, signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMD,, signal was displayed by an inactive form and was not quenched by 0,. 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. Abbreviations. FMD,,,, EPR signal attributed to active formylmethanofuran dehydrogenase ; FMD,,, EPR signal attributed to inactive formylmethanofuran dehydrogenase ; R, Land6 splittina + co2 t P IE" = -497 mV (Thauer, 1990) factor; gx, gy-and g,, Land6 splitting factors in the x , y and directions for anisotropic systems; 1 U = 1 Fmol formylmethanofuran dehydrogenatedmin.The physiological electron accePtor/donor is not knownMethylviologen (E"' = -446 mV) can be used as artificial

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Formylmethanofurandehydrogenasc from Me//ratzobrrrfePizttt? //tenttoorr/utruphicro,l was purified to apparent homogeneity and found to contain per mol (apparent molecular mass I IO kDa) 0.6 mol molybdenum. 4 mol non-heme iron. 4 mol acid-labile sulfur, and in addition, 0.7 mol of a pterin-containing co-factor (apparent molecular mass 800 Da) which has been characterized. The pterin material was extracted after alkylation by iodoacetamide and the extract subjected to HPLC on Lichrospher 100 RP-18. Three pterin compounds were resolved. On the basis of their UV/visible spectra and of the products formed after cleavage by nucleotide pyrophosphatase and alkaline phosphatase they were identified as the [di(carboxamidomethyl)]-derivatives of molybdopterin guanine dinucleotide (MGD). of molybdopterin adenine dinucleotide (MAD). and of molybdopterin hypoxanthine dinucleotide (MHD). The three pterin dinucleotides were present in the proportions 1:0.4:O.l.

Cell extracts of Methanobacterium thermoautotrophicum catalyzed the reduction of methyl viologen (apparent K,,, = 0.1 mM) with formylmethanofuran (apparent K,,, = 10 PM) at a specific rate of 4 ymol mini mg protein-i.Coenzyme FaZO (apparent K,,,=25 PM) rather than NAD, NADP, FAD, or FMN was used as physiological electron acceptor. With coenzyme FbZO the specific activity was 0.4 prnol mm -I mg-i. More than 60% of the formylmethanofuran dehydrogenase activity was associated with the pellet fraction, which was obtained by centrifugation at 160000 xg. An enzyme system with very similar properties was also found to be present in cell extracts of Methanosarcina barkeri grown on methanol.In both organisms the formylmethanofuran dehydrogenase activity was rapidly inactivated by cyanide.