A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei

Abstract

We have recently reported that the thermophilic archaeon Methanohucterium woCfei contains two formylmethanofuran dehydrogenases, I and 11. E'ormylmethanofuran dehydrogenase 11, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-ironsulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is amolybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per moll4h-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten ( < 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 Ujmg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent K , for the electron acccptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the CI and fl subunits were identical up to residue 23, indicating that the two proteins are isoenzymes.The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.Formylmethanofuran dehydrogenase (Borner et al., 1989) catalyzes the reversible reduction of C 0 2 plus methanofuran (MFR) to N-formylmethanofuran (CHO-MFR). This reaction is the first step in methane formation from COz and H2 in all methanogenic archaea (DiMarco et al., 1990; Schworer and Thauer, 1991). C02+MFK+2[H] + CHO-MF:K+H20 E"'= -497 mVThe enzyme has been isolated and characterized from Methanosarcina burkeri and from Methanobacferium thermoaututruphicum (Borner et al., 1991). The dehydrogenase from these archaea (Woese et al., 1990) contains molybdenum, a molybdopterin dinucleotide, and iron-sulfur clusters. The molybdenum enzymes from M . huvkeri and from M . thermuautotruphicum differ in that the former, as isolated, is composed of six subunits of apparent molecular mass 65 kDa, 50 kDa, 37 kDa, 34 kDa, 29 kDa, and 17 kDa and the latter of only two subunits of apparent molecular mass 60 kDa and 45 kDa. They also differ significantly in substrate specificity: formylmethanofuran dehydrogenase from M. barker; is able to catalyze the oxidation of N-furfurylformamide, of formamide, of N-methylformamide, and of formate (unpublished data), whereas the enzyme from M . thermouututrophicum can only mediate the oxidation of formylmethanofuran (Breitung et al., 1990). We have recently found that Mcthnnobacterium wulfei, which is a thermophile growing at 65 "C, contains two active formylmethanofuran dehydrogenases 1 and 11, which can be separated by anion-exchange chromatography on Mono Q and by hydrophobic chromatography on Phenyl-Superose. Formylmethanofuran dehyd...

Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

“…This indicates that molybdenum might exert an antagonistic effect on the tungsten-containing enzyme, similar to that described for carbonic acid reductase of C. thermoaceticum (43). In M. wolfei and M. thermoautotrophicum, two isoenzymes of formylmethanofuran dehydrogenase are synthesized in the presence of molybdate and tungstate, one containing molybdenum and the other containing tungsten (6,37). Growth of P. acetylenicus after addition of only molybdate to the medium (Fig.…”

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

“…Tungsten is not a very common element in active enzymes; it has only been found in some formylmethanofuran dehydrogenases of thermophilic methanogens (2,27), in a few formate dehydrogenases from acetogenic Clostridium strains (36), in aldehyde oxidoreductases from the same strains (34) and from hyperthermophilic archaea (19,20), and in a glyceraldehyde-3-phosphate ferredoxin oxidoreductase from P. furiosus (21). All of these enzymes have a molybdopterin cofactor (16,23).…”

Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine‐dinucleotide‐type of molybdenum cofactor in Hydrogenophaga pseudoflava

“…nase [3,4], carboxylic acid reductase [5,6] and aldehyde dehydrogenase [7,8]. Most mesophilic, MoϪdependent organisms, when grown in the presence of tungstate, produce either inactive enzymes lacking any metal or W-substituted enzymes that have little or no catalytic activity [2].…”