H-2-FORMING METHYLENETETRAHYDROMETHANOPTERIN DEHYDROGENASE, A NOVEL TYPE OF HYDROGENASE WITHOUT IRON-SULFUR CLUSTERS IN METHANOGENIC ARCHAEA

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2=H4MPT) to methenyltetrahydromethanopterin (CH=H4MPT+) and H-2 and was therefore named H-2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H-2 or CH2=H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (V(max)) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between H-3(2) and H-1+ ; (d) the enzyme catalyzed the reduction of CH=H4MPT+ with H-3(2) generating [methylene-H-3]CH2=H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H-2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.