JOURNAL OF BACrERIOLOGY, Nov. 1994, p. 6509-6517 Vol. 176, No. 21 0021-9193/94/$04.00+0 Copyright X 1994, American Society for Microbiology Sulfide Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus: a New Multifunctional Enzyme Involved in the Reduction of Elemental Sulfur KESEN MA AND MICHAEL W. W. ADAMS* Department ofBiochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602 Received 18 May 1994/Accepted 17 August 1994 Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 1000C by the fermentation of carbohydrates yielding acetate, C02, and H2 as the primary products. If elemental sulfur (SO) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). From the cytoplasm of this organism, we have now purified an enzyme, sulfide dehydrogenase (SuDH), which catalyzes the reduction ofpolysulfide to H2S with NADPH as the electron donor. SuDH is a heterodimer with subunits of52,000 and 29,000 Da. SuDH contains fiavin and approximately 11 iron and 6 acid-labile sulfide atoms per mol, but no other metals were detected. Analysis of the enzyme by electron paramagnetic resonance spectroscopy indicated the presence of four iron-sulfur centers, one of which was specifically reduced by NADPH. SuDH has a half-life at 950C of about 12 h and shows a 50o increase in activity after 12 h at 82C. The pure enzyme has a specific activity of 7 Fmol of H2S produced min-'m * mg of protein-' at 800C with polysulfide (1.2 mM) and NADPH (0.4 mM) as substrates. The apparent Km values were 1.25 mM and 11 iuM, respectively. NADH was not utilized as an electron donor for polysulfide reduction. P. furiosus rubredoxin (Km = 1.6 tuM) also functioned as an electron acceptor for SuDH, and SuDH catalyzed the reduction of NADP with reduced P. furiosus ferredoxin (Km = 0.7 FLM) as an electron donor. The multiple activities of SuDH and its proposed role in the metabolism of So and polysulfide are discussed. The ability of microorganisms to reduce elemental sulfur nism of H2S production from S0 has been investigated in only (SO) to H2S was discovered only recently (35) and is still a one autotrophic hyperthermophile, Pyrodictium brockii, an limited phenomenon in the microbial world (23, 40). The obligate S-reducing species which grows optimally at 1050C. notable exceptions are the hyperthermophiles, a recently dis- This organism was shown to have a primitive membrane-bound covered group of microorganisms that have the remarkable electron transport chain for coupling H2 oxidation and S0 property of growing at temperatures of 90'C and above (2, 3, reduction (36). The chain contained hydrogenase, a cyto- 45, 46). Hyperthermophiles have been isolated from a variety chrome, and a novel quinone, but the So-reducing entity was of geothermally heated environments, and almost all are not purified. In fact, there have been few reports on S5 classified as Archaea (formerly Archaebacteria) (33). The ma- reduction by mesophilic organisms. For example, Zophel and jority are strict anaerobes, and most are obligately dependent coworkers screened several different So-reducing bacteria for upon the reduction of elemental sulfur (S) to H2S for optimal sulfur oxidoreductase activity (50) and the enzyme responsible growth. Molecular H2 or organic compounds serve as electron in "Spirillum" strain 5175 (44) was postulated to be an donors for the apparent respiration of So. Some of the iron-sulfur protein possibly associated with a c-type cyto- heterotrophic species are able to grow in the absence of S by chrome (51). However, only one S0-reducing enzyme has been fermentation-type metabolisms that result in the production of purified and characterized from a mesophile, that from H2. In such cases, the addition of So to the growth medium Wolinella succinogenes (20, 21, 43), an organism which grows leads to H2S production and growth stimulation. Since the with formate as the electron donor and S0 as the electron hyperthermophiles are the most slowly evolving of known life acceptor (27). From sequencing analysis, it was postulated that forms, it has been suggested that S0 respiration may represent its reductase is composed of three subunits and is a one of the earliest mechanisms of energy conservation from an polysulfide evolutionary perspective (40, 47). molybdopterin-containing iron-sulfur protein (21). The mechanisms by which hyperthermophilic organisms We are investigating the So-reducing activities of heterotro- reduce So to H2S and the natures of the enzymes involved are phic hyperthermophiles such as P. furiosus (18). This obligate far from clear. The situation is complicated by the fact that the anaerobe grows optimally at 100'C by the fermentation of abiotic reduction of S0 can also occur at the growth tempera- carbohydrates and peptides in which excess reductant, gener- tures of these organisms (9). Also, because of the low solubility ated mainly in the form of reduced ferredoxin (2, 5, 30), is of S in aqueous media, golysulfide is thought to be the true disposed of either as H2, or if S is added to the growth substrate for microbial S reduction (9, 40, 41). The mecha- medium, as H2S (18). Both simple and complex sulfidic compounds serve as substrates for H2S production (9, 29, 49). Although S0 reduction was originally thought to be a mecha- * Corresponding author. Mailing address: Department of Biochem- nism of detoxifying inhibitory H2 (18), a more recent study istry, Life Sciences Bldg., University of Georgia, Athens, GA 30602. showed that S0 reduction plays a role in energy conservation Phone: (706) 542-2060. Fax: (706) 542-0229. Electronic mail address: (42). However, it was also shown (26) that the sulfur reductase [email protected]. (sulfur:reduced ferredoxin oxidoreductase) activity of P. furio- 6509 6510 MA AND ADAMS J. BACTERIOL. sus was located in the cytoplasm and that the enzyme respon- oxidoreductase (POR) of P. furiosus (8). The reaction mixture sible was the Ni-containing hydrogenase that had been already (2 ml) contained 100 mM EPPS (pH 8.0), pyruvate (10 mM), purified (11). Thus, this bifunctional enzyme, which is now coenzyme A (CoASH) (0.2 mM), POR (80 pug), ferredoxin termed sulfhydrogenase, reduces both protons to H2 and So (12.5 jxM), NADP (0.3 mM), and SuDH (10 jig). The reaction (and polysulfide) to H2S (26), although the bioenergetics of S' was measured at 80'C by the appearance of NADPH as reduction is completely unknown. described above. The results obtained were independent of Although heterotrophic hyperthermophiles such as P. fuio- whether the reaction was initiated by the addition of SuDH, sus have been proposed to contain an unusual "pyrosaccharo- ferredoxin, NADP, or POR as the final component. The lytic" pathway for carbohydrate fermentation that is indepen- reduction of polysulfide catalyzed by SuDH with reduced dent of nicotinamide nucleotides (30, 39), P. futiosus contains ferredoxin as the electron donor was measured by the produc- high concentrations of an NAD(P)-dependent glutamate de- tion of sulfide. The reaction was carried out in 8-ml sealed vials hydrogenase (16, 32, 38). In addition, an NADP-specific under Ar and shaken at 150 rpm at 80'C. The reaction mixture alcohol dehydrogenase has been purified from the related (2 ml) contained 100 mM EPPS (pH 8.0), pyruvate (10 mM), species Thermococcus litoralis (25). Clearly, these organisms CoASH (2.0 mM), POR (150 pg), ferredoxin (25 jiM), poly- generate significant amounts of NAD(P)H during oxidative sulfide (1.5 mM), and SuDH (40 jig). At 20-min intervals metabolism. Thus, in addition to sulfhydrogenase, whether typically over 2 h, aliquots of the reaction were removed with they contain an enzyme that couples the oxidation of a syringe and sulfide levels were determined by methylene blue NAD(P)H to So reduction and whether this enzyme is a formation (26). Glutamate dehydrogenase activity of P. furio- membrane-bound or cytoplasmic enzyme are not known. In sus was determined as described previously (38). One unit of the following, we describe the purification and properties of activity is defined as the 1 jimol of glutamate oxidized per min such an enzyme from the cytoplasm of P. furiosus, which we at 80°C. term sulfide dehydrogenase (SuDH). Enzyme purification. SuDH was purified from 400 g (wet weight) of cells under strictly anaerobic conditions (11) at MATERIALS AND METHODS 23°C. Frozen cells were thawed in 1.5 liters of buffer A (50 mM Tris-HCl [pH 8.0] containing 10% [vol/vol] glycerol, 2 mM Growth of organism. P. furiosus (DSM 3638) was routinely dithiothreitol [DTT], and 2 mM sodium dithionite) containing grown at 850C in a 500-liter fermentor with maltose as the lysozyme (1 mg/ml) and DNase I (10 ,ug/ml) and were lysed by carbon source as described previously (11). incubation at 35°C for 2 h. A cell extract was obtained by Enzyme assays. SuDH activity was determined at 80'C by centrifugation at 50,000 X g for 80 min. The supernatant (1.3 the polysulfide-dependent oxidation of NADPH measured at liters) was loaded onto a column (8 by 21 cm) of DEAE- 340 nm (molar absorbance of 6,200 M1 cm-'). The reaction Sepharose Fast Flow (Pharmacia LKB, Piscataway, N.J.) equil- mixture (2.0 ml) containing 100 mM EPPS [N-(2-hydroxy- ibrated with buffer A. The column was eluted with a linear ethyl)-piperazine-N'-(3-propanesulfonic acid)] buffer (pH 8.0), gradient (9.0 liters) from 0 to 0.5 M NaCl in buffer A, and NADPH (0.3 mM), and polysulfide (1.5 mM).