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

Thermococcus litoralis is a hyperthermophilic archaeon that grows at temperatures 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 production, 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 oxidation, using artificial electron carriers, was around 80 degrees C. The operon encoding the T litoralis enzyme is composed of four genes forming one transcriptional unit, and transcription is not regulated by S-0. An un usual transcription-initiation site is located 139 bp upstream from the translational start point. Sequence analyses indicated the presence of new putative nucleotide-binding domains. Upstream from the hydrogenase operon, ORFs probably encoding a molybdopterin oxidoreductase enzyme have been identified. Based on sequence, biochemical and biophysical analyses, a model of the enzyme and the pathway of electron flow during catalysis is proposed.