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Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens

Simone Laska

Friedrich Lottspeich

Institute of Microbiology and Genetics, Darmstadt University of Technology, Schnittspahnstrasse 10, 64287 Darmstadt, Germany

Correspondence
Arnulf Kletzin
Kletzin{at}bio.tu-darmstadt.de

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U (mg protein)^-1] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U (mg protein)^-1 with methyl viologen and 833 U (mg protein)^-1 with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 µmol Fe and 2 µmol Ni (g protein)^-1; in addition, 2·5 µmol Mo (g protein)^-1 was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.

The EMBL accession numbers for the sequences reported in this paper are AJ320523 and AJ345004.

Present address: Max-Planck-Institute of Biophysics, Marie-Curie Strasse 15, 60439 Frankfurt, Germany.

Present address: Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, 80387 Martinsried, Germany.

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