Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

No abstract

Aquifex aeolicus is a hyperthermophilic, chemolithoautotrophic, hydrogen-oxidizing, and microaerophilic bacterium growing at 85°C. We have shown that it can grow on an H 2 /S°medium and produce H 2 S from sulfur in the later exponential phase. The complex carrying the sulfur reducing activity (electron transport from H 2 to S°) has been purified and characterized. It is a membranebound multiprotein complex containing a [NiFe] hydrogenase and a sulfur reductase connected via quinones. The sulfur reductase is encoded by an operon annotated dms (dimethyl sulfoxide reductase) that we have renamed sre and is composed of three subunits. Sequence analysis showed that it belongs to the Me 2 SO reductase molybdoenzyme family and is similar to the sulfur/polysulfide/thiosulfate/tetrathionate reductases. The study of catalytic properties clearly demonstrated that it can reduce tetrathionate, sulfur, and polysulfide, but cannot reduce Me 2 SO and thiosulfate, and that NADPH increases the sulfur reducing activity. To date, this is the first characterization of a supercomplex from a bacterium that couples hydrogen oxidation and sulfur reduction. The distinctive feature in A. aeolicus is the cytoplasmic localization of the sulfur reduction, which is in accordance with the presence of sulfur globules in the cytoplasm. Association of this sulfur-reducing complex with a hydrogen-oxygen pathway complex (hydrogenase I, bc 1 complex) in the membrane suggests that subcomplexes involved in respiratory chains in this bacterium are part of supramolecular organization.The production of biomass in extreme light-independent environments is energized by chemolithoautotrophic oxidation and reduction of inorganic compounds like elemental sulfur (S°), 2 hydrogen, and nitrate (1, 2). Sulfur and sulfur compounds are the most abundant sources of both electron acceptors and electron donors in extremophilic environments (like volcanic environments) and are used by many microorganisms to support growth (3-6). Reduction and oxidation of sulfur compounds (sulfate, sulfite, thiosulfate, organic sulfoxide, elemental sulfur, polysulfides, and organic disulfide) are vital processes for many bacteria and essential steps in the global sulfur cycle. Because of the multiple oxidation states of sulfur, the biochemistry and chemistry of this cycle are complex and still not completely understood (7,8). This problem is exacerbated by the reactivities of the sulfur species at various oxidation states toward each other (7). The ability to reduce elemental sulfur is mostly found among hyperthermophilic micro-organisms (Archaea and Bacteria) (8), the majority of which depend on S°reduc-tion for optimal growth. They use either molecular hydrogen or organic compounds as electron donors. It has been suggested that S°respiration is one of the earliest mechanisms for energy conservation, because the hyperthermophiles are the forms of life evolving the most slowly (9).The processes by which micro-organisms reduce S°to H 2 S are still unclear for most of them, and only few ...