A Membrane-bound Multienzyme, Hydrogen-oxidizing, and Sulfur-reducing Complex from the Hyperthermophilic Bacterium Aquifex aeolicus

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 ...

Rhodanese Functions as Sulfur Supplier for Key Enzymes in Sulfur Energy Metabolism

“…Pieces of this intricate puzzle of sulfur metabolism are now better defined by the association of complementary approaches (proteomics and enzymatic studies) with the main players, SR, Sqr, SOR, well characterized (as described in Fig. 8 legend) (9,11,12). A role of SOR in the sulfur energy pathway is strongly supported by its membrane location also observed in Acidianus tengchongensis (37), which would allow transfer of its products to membrane energy enzymes (13,14).…”

“…A cytoplasmic sulfur oxygenase reductase (SOR), which catalyzes, in the presence of oxygen, the simultaneous oxidation and reduction of elemental sulfur into oxygen sulfur species and H 2 S, is also present in A. aeolicus (12). In addition, the presence of cytoplasmic sulfur globules of as yet unknown function has been observed in whole cells of A. aeolicus grown on S 0 (9). The simultaneous presence of all these sulfur enzymes, sulfur compounds and sulfur-containing struc-tures, is puzzling, and the interconnection between all these sulfur enzymes and pathways is our current main focus.…”

“…aeolicus VF5 was grown at 85°C in 2-liter bottles containing 400 ml of modified SME medium (29) at pH 6.8 in the presence of flowers of sulfur (7.5 g⅐liter Ϫ1 ) (which will be referred to as "S 0 " in this paper) and under 1.4 bar of H 2 /CO 2 (80:20) as described previously (9). Cells were harvested in the late exponential growth phase and stored at Ϫ80°C.…”

“…Sulfur Reductase Activity-Sulfur reductase activity was determined by quantifying the H 2 S formed by the reduction of S 0 with H 2 , as described previously (9), but performed in 50 mM Hepes, pH 7.6. To study the influence of SbdP on SR activity, SbdP (1,5,10,15, and 20 g⅐ml Ϫ1 , final concentrations) was added to the assay before the incubation at 85°C.…”

“…It also represents the earliest branching order in the bacterial domain (8). A. aeolicus is able to grow chemolithoautotrophically and obtains energy for growth in the presence of H 2 , O 2 , and CO 2 , but it also requires the presence of a sulfur compound such as S 0 , sodium sulfide (Na 2 S), or sodium thiosulfate (NaS 2 O 3 ) (9). After several years of study of the enzymes involved in the respiratory pathways of A. aeolicus growing in presence of S 0 , we have a better overall picture of its complex energy metabolism.…”