Biochemical and molecular characterization of the [NiFe] hydrogenase from the hyperthermophilic archaeon, Thermococcus litoralis

Thermococcus litoralis is a hyperthermophilic archaeon that grows at temper atures up to 98 degrees C by fermentative metabolism and reduces elemental sulfur (S-0) to H2S. A [NiFe] hydrogenase, responsible for H2S or H-2 produ ction, has been purified and characterized. The enzyme is composed of four subunits with molecular mass 46, 42, 34 and 32 kDa. Elemental analyses gave approximate values of 22 Fe, 22 S and 1 Ni per hydrogenase. EPR spectra at 70 and 5 K indicated the presence of four or five [4Fe-4S] and one [2Fe-2S ] type clusters. The optimal temperature for both Hz evolution and oxidatio n, using artificial electron carriers, was around 80 degrees C. The operon encoding the T litoralis enzyme is composed of four genes forming one trans criptional unit, and transcription is not regulated by S-0. An un usual tra nscription-initiation site is located 139 bp upstream from the translationa l start point. Sequence analyses indicated the presence of new putative nuc leotide-binding domains. Upstream from the hydrogenase operon, ORFs probabl y encoding a molybdopterin oxidoreductase enzyme have been identified. Base d on sequence, biochemical and biophysical analyses, a model of the enzyme and the pathway of electron flow during catalysis is proposed.