Sulfide Dehydrogenase from the Hyperthermophilic Archaeon

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). A stock poly- 90-ml fractions were collected. SuDH activity started to elute sulfide solution (0.5 M) was prepared by the reaction of 12 g of as 0.2 M NaCl was applied to the column. Those fractions Na2S with 1.6 g of elemental sulfur in 100 ml of anoxic water containing SuDH activity were combined (810 ml), concen- (19). Polysulfide was measured by cold cyanolysis (48). One trated by ultrafiltration (type PM-30 membrane; Amicon, unit of SuDH activity catalyzed the oxidation of 1 ,umol of Beverly, Mass.) and washed with buffer B. Buffer B was the NADPH per min. Where indicated, colloidal sulfur (0.05 % same as buffer A except sodium dithionite, which prevented [wt/vol]; Fluka, Ronkonkoma, N.Y.) and elemental sulfur SuDH from binding to Blue Sepharose, was omitted. The powder (5% [wt/vol]; J. T. Baker, Marietta, Ga.) were used as concentrated sample (150 ml) was applied to a column (5 by 12 the substrate in place of polysulfide. For reasons of higher cm) of Blue Sepharose (Pharmacia LKB) equilibrated with sensitivity and convenience, SuDH was routinely detected buffer B. The column was eluted with a linear gradient (1.4 during purification by its NADH:benzyl viologen oxidoreduc- liters) from 0 to 2.0 M NaCl in buffer B, and 50-ml fractions tase (NBVO) activity, namely, the NADH-dependent reduc- were collected. SuDH activity started to elute as 1.6 M NaCl tion of benzyl viologen at 80°C. The assay mixture (2.0 ml) was applied. Those fractions containing SuDH activity were contained 50 mM CAPS [3-(cyclohexylamino)-1-propanesulfo- combined (600 ml), concentrated to 30 ml by ultrafiltration nic acid] buffer (pH 10.3), NADH (0.3 mM), and benzyl (PM-30 membrane), and applied to a column (6 by 60 cm) of viologen (1 mM). A molarA580 of 7,800 M'- cm-1 was used to Superdex 200 (Pharmacia LKB) equilibrated with buffer B measure the reduction of benzyl viologen. One unit of SuDH containing 50 mM KCl. Fractions of 25 ml were collected. activity catalyzed the reduction of 2 ,umol of benzyl viologen Those containing SuDH activity were combined (50 ml) and per min. applied to a column (2.6 by 15 cm) of Q-Sepharose (high For the following electron acceptors used to measure performance; Pharmacia LKB) equilibrated with buffer B. The SuDH-catalyzed NADPH oxidation, the reaction was carried column was eluted with a gradient (0.5 liter) from 0 to 1.0 M out at 50°C (the molar absorbance at the indicated wavelength KCl in buffer B, and 20-ml fractions were collected. SuDH is given in parentheses): methyl viologen (9,700 M` cm- at activity started to elute as 0.24 M KCl was applied. Those 580 nm), 2,6-dichlorophenol indophenol (19,100 M1 cm-' at fractions judged homogeneous by sodium dodecyl sulfate 600 nm), potassium ferricyanide (1,020 M1 cm-1 at 420 nm), (SDS)-polyacrylamide gel electrophoresis were combined (80 cytochrome c (from horse heart, 19,520 M 1 cm-1 at 550 nm), ml), concentrated by ultrafiltration (PM-30 membrane), and methylene blue (30,500 M-' cm-' at 670 nm), and flavin stored as pellets in liquid N2. mononucleotide and flavin adenine dinucleotide (11,300 M1 Other methods. Gel filtration for molecular weight estima- cm'-I at 450 nm). The ferredoxin:NADP oxidoreductase assays tion was performed with a column (1.6 by 60 cm) of Superdex were performed at 80°C by monitoring the reduction of NADP 200 (Pharmacia LKB) equilibrated with 50 mM Tris-HCl at 365 nm (molar absorbance of 3,400 M-1 cm-1). For the buffer (pH 8.0) containing KCl (200 mM). The column was determination of ferredoxin:NADP oxidoreductase activity, calibrated with the following standard proteins (with Mr values reduced ferredoxin was generated by the pyruvate ferredoxin in parentheses): ferritin (450,000), catalase (240,000), lactate VOL. 176, 1994 P. FURIOSUS SULFIDE DEHYDROGENASE 6511 dehydrogenase (140,000), yeast alcohol dehydrogenase (150,000), TABLE 1. Sulfur reduction activities of pure SuDH bovine serum albumin (67,000), and ovalbumin (45,000). Poly- and cell extracts of P. furiosus acrylamide gel electrophoresis in the presence of SDS was with 12.5% the Activity Activity ratio (102) performed (wt/vol) polyacrylamide gels by Substrate (U/mg)a (cell method of Laemmli (22). Molecular weight markers were from Cell extract SuDH extract/SuDH) Sigma (St. Louis, Mo.). Protein concentrations were routinely Polysulfide 0.083 7.0 1.2 estimated by the Bradford method (10) with bovine serum Colloid sulfur 0.041 2.9 1.4 albumin as the standard. The protein content of samples of Sulfur powder 0.006 0.31 1.9 pure SuDH was also determined by the quantitative recovery of amino acids from compositional analyses (see below). The a The assay mixture (2.0 ml) contained either cell extract (1.0 mg) or pure SuDH (20 tLg) in 100 mM EPPS buffer (pH 8.0), using NADPH (0.4 mM) as the amounts of protein in all samples were 86% ± 5% of those reductant. The sulfur source was either polysulfide (1.25 mM), colloidal sulfur measured by the colorimetric protein assay (from three sepa- (0.05% [wt/vol]), or powdered sulfur (5% [wt/vol]). One unit of activity is the rate determinations). All analytical values for the pure protein oxidation of 1 pumol of NADPH per min at 80'C. that were based on the Bradford method have therefore been corrected by a factor of 0.86. The iron content and acid-labile sulfide content of SuDH of the SuDH activity (1.54 U/mg) and 83% ± 4% of the GDH were measured by using o-phenanthroline (24) and by meth- activity (0.97 U/mg). The residual activities in the resuspended ylene blue formation (13), respectively. A complete metal membrane fraction (6% ± 2% and 17% ± 3%, respectively) analysis (40 elements) was carried out by plasma emission corresponded to specific activities of 0.55 and 0.56 U/mg, spectroscopy, using a Jarrel Ash Plasma Comp 750 instrument respectively. The distribution and quantitative recovery of the in the Department of Ecology of the University of Georgia. two enzyme activities in the supernatant and membrane frac- For amino-terminal sequence and amino acid composition tions tend to rule out even a loose association of SuDH with analyses, the subunits of SuDH were separated by SDS- the cell membrane. In addition, more than 90% of activity of polyacrylamide gel electrophoresis and electroblotted onto the NADPH-dependent reduction of colloidal sulfur and of polyvinylidene difluoride protein-sequencing membranes, us- powdered elemental sulfur were also in the supernatant frac- ing a Bio-Rad electroblotting system. Electroblotting was tions from both the high- and low-speed centrifugations, carried out in buffer (pH 8.3) containing 25 mM Tris-HCl, 192 showing that these activities are also not membrane associated. mM glycine, and methanol (15% [vol/vol]) for 1 h at 100 V. In fact, the cell extract and the pure enzyme (see below) The amino-terminal sequences were determined with an Ap- showed the same specific activity ratios with these three plied Biosystems model 477 sequencer. Searches in the Na- substrates (Table 1), demonstrating that SuDH is the only tional Biomedical Research Foundation and Swiss protein enzyme in P. furiosus capable of catalyzing these reactions and databases for sequence similarities with the N-terminal se- that they occur in the cytoplasm rather than in the cell quences were carried out with the program FASTA (Univer- membrane. sity of Wisconsin Genetics Computer Group). Amino acid Purification of SuDH. During small-scale procedures to compositional analyses were carried out on an Applied Bio- purify SuDH using the NADPH-dependent reduction of poly- systems model 4240A analyzer after the hydrolysis of SuDH sulfide to measure its activity, it was found that the pure and of the individual subunits under Ar at 1650C for 1 h in the enzyme also catalyzed the reduction of benzyl viologen with presence of 6 M HCl, phenol (1% [wt/vol]), and thioglycolic NADH as the electron donor. Because of the increased acid (8% [wt/vol]). Serine and threonine were corrected for sensitivity and ease of this NBVO assay, it was used to monitor destruction. SuDH apoprotein was prepared and reduced with SuDH during the routine large-scale purification procedure. DTT as previously described (5). Tryptophan was determined The results of a typical purification of SuDH as measured by from the A280 value of the apoprotein after correction for NBVO activity are given in Table 2. Three peaks of NBVO tyrosine (17). The cysteine content of the reduced apoprotein activity were separated after chromatography on the Blue was estimated by the reaction with 5,5'-dithiobis(2-nitroben- Sepharose column (data not shown), but only the peak corre- zoic acid) (37). sponding to SuDH is shown in Table 2. This peak represented Electron paramagnetic resonance (EPR) spectra were re- approximately 40% of the total NBVO activity in the cell corded on an IBM-Bruker ER200D spectrometer interfaced to extract. The total NBVO activity recovered from the Blue an IBM 9001 microcomputer and equipped with an Oxford Sepharose column (Table 2) was slightly greater than that Instruments ESR-9 flow cryostat. Spin quantitations were measured in the cell extract. This is probably due to the determined by double integration of spectra recorded at 8 K presence of P. furiosus hydrogenase in the extract, which with 10-,uW microwave power. These spectra were compared with spectra of Cu (1 mM)-EDTA (10 mM) recorded under the same conditions. Ferredoxin (5) and POR (8) from P. furiosus were purified as described previously. TABLE 2. Purification of SuDH from P. furiosus Amt act Purification StepStep (mg) Activity' Sp Recovery(%) RESULTS of protein (U) (U/mg) (fold) Cellular location of SuDH. More than 90% of the SuDH Cell extract 38,267 28,667 0.75 100 1 activity, as measured by NADPH-dependent polysulfide reduc- DEAE-Sepharose 7,400 26,200 3.55 92 5 Blue Sepharose 145 15,720b 108 55 144 tion, was found in the supernatant fraction after low-speed Superdex 200 62 8,667 140 30 187 centrifugation of a cell extract of P. furiosus (50,000 X g for 80 Q-Sepharose 46 7,333 160 26 213 min, the conditions used for further SuDH purification). That the enzyme is located in the cytoplasm was confirmed by using "Activity was determined by the NADH-dependent reduction of benzyl viologen (NBVO activity) at 80'C, where 1 U is the reduction of 2 pumol of benzyl glutamate dehydrogenase (Mr, 280,000) as a marker cytoplas- viologen per min. mic enzyme (38). After high-speed centrifugation (110,000 X g bThis represents approximately 40% of the total NBVO activity recovered. for 2 h) of the cell extract, the supernatant retained 94% ± 4% See text for details. 6512 MA AND ADAMS J. BAC-MRIOL.

kD A B 0.8 66q_

45 0c 36r 0 29 (aa .o 24 _ .0e') 20W^ front

FIG. 1. SDS-polyacrylamide electrophoresis of purified SuDH from P. furiosus. Lane A contained standard proteins (6 rig) with the indicated molecular masses. Lane B contained purified polysulfide dehydrogenase (1 pug). Wavelength (nm) FIG. 2. UV-visible absorption spectra of sulfide dehydrogenase. rapidly oxidizes reduced benzyl viologen (11). Although pure The solid line is the spectrum of the enzyme (1.6 mg/ml) as isolated in SuDH was not inactivated by short-term exposure to oxygen 50 mM Tris-HCl (pH 8.0) containing 1 mM DIT, and the broken line (see below), the purification procedure was carried out under is the spectrum after the addition of sodium dithionite (3 mM). anaerobic conditions to prevent any aerobic degradation of its iron-sulfur clusters (see below). The rationale stems from our prior experience with other iron-sulfur proteins that once Catalytic properties of SuDH. Although pure SuDH cata- purified are oxygen stable (15). By the described procedure, lyzed the NADPH-dependent reduction of both polysulfide SuDH was purified over 200-fold. The final recovery of activity and benzyl viologen, the two activities differed in their re- from the first DEAE-Sepharose column (after separating sponse to pH, with optimal values of approximately 8.0 and SuDH from the other two peaks of NBVO activity) was about 10.3, respectively (Fig. 3). This was surprising in view of the 26%. fact that polysulfide is more stable at more alkaline pH values Molecular properties of SuDH. The molecular mass of (40). However, the activity with polysulfide, the putative phys- SuDH was estimated by gel filtration to be 90,000 ± 5,000 Da. iological substrate, is in accordance with physiological pH, in After polyacrylamide gel electrophoresis in the presence of contrast to the reduction of the artificial mediator, benzyl SDS, purified SuDH gave rise to two protein bands with viologen. Kinetic constants for these two activities are summa- molecular masses of 52,000 (a) and 29,000 (1) Da (Fig. 1). rized in Table 3. Additional analyses using NADP (0.06 to These results suggest that this enzyme is a heterodimer, and on 0.132 mM) and benzyl viologen (0.1 to 1.0 mM) in which the the basis of the latter analysis, a value of 81,000 Da for the concentration of one substrate was varied (over the indicated holoenzyme was used in all calculations. The amino acid range) and the concentration of the other was constant gave compositions of the two subunits were determined individually, rise to parallel Lineweaver-Burk plots (data not shown), and the sum was in reasonable agreement with the amino acid indicating a ping-pong type of catalytic mechanism for dye composition determined for the native enzyme (data not shown). Of note was the presence of 12 cysteinyl residues per mol of holoenzyme and an absence of methionine. The N- terminal amino acid sequences of the a and 13 subunits were RLIKDRVPTPERXVGY- and LRKERLAPGINLFEIESP RI-. No similarity was found with any known protein from the available protein databases. The UV-visible absorption spectra of the oxidized (as purified) form exhibited peaks near 390 and .2 450 nm, and these decreased in intensity upon the addition of sodium dithionite, indicating the presence of both iron-sulfur and flavin chromophores (Fig. 2). Assuming that only the flavin .U s contributes to the absorption at 450 nm upon reduction, the C. change in absorbance indicates that the holoenzyme contains C',Q1 C', 1.6 flavin residues per mol. By plasma emission analysis, the enzyme contained 3.2 ± 0.5 g-atoms of P per mol, suggesting the presence of two flavin adenine dinucleotide molecules per mole (rather than flavin mononucleotide). The presence of 7 8 9 10 11 12 iron-sulfur clusters was confirmed by calorimetric analyses. pH Pure SuDH contained 10.6 + 2.1 g-atoms of Fe per mol, as determined by colorimetric analysis. The measured amounts of FIG. 3. Effects of pH on the NADPH-dependent reduction of ± were polysulfide (closed symbols) and benzyl viologen (open symbols) by acid-labile sulfide (6.3 2.1 g-atoms per mol) lower than SuDH at 80'C. The assay mixtures (2.0 ml) contained either NADPH the iron values, which presumably reflects the inherent diffi- (0.3 mM), polysulfide (1.5 mM), and SuDH (20 ,g) (solid symbols) or culties in the assay (14) (see below). Analysis of the enzyme by NADPH (0.3 mM), benzyl viologen (1.0 mM), and SuDH (1.4 Rg) plasma emission spectroscopy showed that iron (approximately (open symbols). The buffers used were 100 mM EPPS (pH 7.3 to 8.8), 11 g-atoms/mol) was the only metal present in significant 50 mM glycine-NaOH (pH 9.1 to 9.5), and 50 mM CAPS (pH 10.3 to amounts (>0.1 g-atom/mol). 11.1). The pH values indicated were measured at 230C. VOL. 176, 1994 P. FURIOSUS SULFIDE DEHYDROGENASE 6513

TABLE 3. Kinetics parameters for NAD(P)H-dependent reductions TABLE 4. Substrate specificity of SuDHa catalyzed by SuDHa Substrate(mM) Reduction potential Activity Substrate (mM) (%)C Substrate Cosubstrate pH Apparent Apparent (mV)b (MM) pbKm (jiM) Vmax (U/mg)' Methyl viologen (1.0) -440 23 NADPH BVd (1.0) 9.5 11 263 Benzyl viologen (1.0) -350 100 NADH BV (1.0) 9.5 71 182 FADd (0.12) -220 27 Polysulfide NADPH (0.3) 8.0 1,250 14 FMNY (0.15) -190 4 Benzyl viologen NADPH (0.1) 9.5 125 278 Methylene blue (0.06) +11 43 Reduced ferredoxine NADP (0.3) 8.0 0.7 8 2,6-Dichlorophenol +220 68 Rubredoxin NADPH (0.2) 8.0 1.6 1 indophenol (0.08) Oxygen NADPH (0.24) 10.2 240f 166 Cytochrome c (0.05) +250 25 Ferricyanide (0.25) +360 133 a The polysulfide-, benzyl viologen-, and oxygen-reducing activities of SuDH were measured at the indicated pH at 80'C by NAD(P)H oxidation. Apparent a Activities were measured by the NADPH-dependent reduction of the Km and Vm. values were calculated from Lineweaver-Burk plots, using a indicated substrate at 50'C in 50 mM EPPS buffer (pH 8.0), using 0.3 mM constant concentration of the cosubstrate as indicated. NADPH. b Assay pH using either 50 mM EPPS (pH 8.0) or 50 mM CAPS (pH 9.5 or b Value at 25TC. 10.2). c 100% activity is 28 pumol of NADPH oxidized per min. Units are the oxidation of 1 pimol of NAD(P)H oxidized per min. dFAD, flavin adenine dinucleotide. d BV, benzyl viologen. e FMN, flavin mononucleotide. e Ferredoxin was reduced by using POR. See Materials and Methods. f Equivalent to air at a pressure of 1.4 atm at 80°C.

POR reaction, was an excellent substrate for the reduction of reduction. Information on the mechanism of the NADPH- NADP catalyzed by SuDH, with a Km value of less than 1 jxM dependent reduction of polysulfide could not be obtained, (Table 3). Therefore, although SuDH was identified by its since kinetic analyses, while yielding the apparent Km value for ability to reduce polysulfide with NADPH as an electron donor polysulfide (Table 3), were limited by the accurate determina- and is the only enzyme in P. furiosus with this catalytic ability, tion of catalytic activities (measured by NADPH oxidation) at SuDH also functions as a reduced ferredoxin:NADP oxi- NADPH concentrations below its apparent Km value (estimat- doreductase with a very high affinity for reduced ferredoxin. ed at -10 ,uM). Moreover, SuDH appeared to be specific for Since SuDH accepted electrons from reduced ferredoxin NADPH as the electron donor for polysulfide reduction: there and used them to reduce NADP, it was of interest to determine was no reaction when NADH replaced NADPH (as monitored if SuDH also catalyzed sulfide production from polysulfide at by the oxidation of NADH). In contrast, NADH was readily 80°C with reduced ferredoxin (generated by POR) as the utilized by SuDH in the reduction of benzyl viologen, although electron donor. In such an assay system, sulfide was produced the enzyme preferred NADPH over NADH with a 10-fold- at a fairly constant rate for more than 2 h, and there was no higher VmaJKm ratio (Table 3). These results, taken together reaction if SuDH was omitted. However, for unknown reasons, with the differences in the pH dependence of polysulfide the specific activity varied from assay to assay even with the reduction and benzyl viologen reduction, suggest that SuDH same reagents: the values obtained ranged from 0.3 to 1.5 might well catalyze these NADPH-dependent reactions by ,umol of H2S produced * min-' * mg of pure SuDH protein-' different mechanisms. at 80°C. These rates are 4 to 20% of the rate of polysulfide As shown in Table 3, pure SuDH catalyzed the NADPH- reduction catalyzed by SuDH with NADPH as the sole elec- dependent reduction of the rubredoxin from P. furiosus (7) and tron donor. It should be noted that to measure sulfide produc- had a very high affinity for this redox protein. In addition, tion, which is a very insensitive assay compared with NADPH SuDH also functioned as an NADPH oxidase (Table 3). Since oxidation, the reactions had to be carried out over a prolonged P. furiosus is a strictly anaerobic organism, the oxidase activity period. A systematic analysis of the thermal lability of the is assumed to be nonphysiological, as suggested by the ex- different components, such as CoASH and POR, under assay tremely high Km value. The broad specificity of SuDH was also conditions (in the presence of various sulfide and polysulfide shown by its ability to reduce various nonphysiological sub- concentrations) and of varying their initial concentrations in strates with a wide range of reduction potentials (Table 4), the reaction mixture, was not performed. Nevertheless, al- although the highest activity was observed with benzyl violo- though the assay is clearly not optimized, we conclude that gen. SuDH was unable to catalyze the reduction of NADP or polysulfide reduction is catalyzed by SuDH with reduced the oxidation of NADPH in the presence of fumarate (10 ferredoxin as an electron donor. However, the rate of sulfide mM), succinate (10 mM), nitrate (2 mM), nitrite (2 mM), production, at least in vitro, is much lower than when SuDH sulfate (2 mM), or sulfite (2 mM), and SuDH did not exhibit uses NADPH to provide reductant for polysulfide reduction. hydrogenase activity, as measured by H2 production from Interestingly, SuDH did not catalyze sulfide production from dithionite-reduced methyl viologen or NADPH and by the polysulfide if ferredoxin was initially reduced by sodium dithio- H2-dependent reduction of NADP or of oxidized benzyl or nite (10 mM) rather than by the POR system. methyl viologen. Stability of SuDH. Pure SuDH was quite stable at high Although SuDH showed high activity in the NADPH- temperatures. The time required for a 50% loss of activity at dependent reduction of benzyl viologen (360 U/mg, 80°C, pH 95°C was about 12 h (Fig. 4). Interestingly, this enzyme 8.0, with 1 mM benzyl viologen), the ferredoxin from P. exhibited an increase in catalytic activity by about 50% after furiosus (5) was a very poor substrate and exhibited barely incubation for several hours at 82°C (Fig. 4). The effect of detectable electron carrier activity (<0.1 U/mg with 50 ,uM temperature on the NADPH-dependent reduction of polysul- ferredoxin under the same conditions). This is in spite of the fide catalyzed by SuDH activity is also shown in Fig. 4. fact that benzyl viologen and ferredoxin have comparable Significant activity was observed at ambient temperature, reduction potentials (-350 and -360 mV at 25°C, respec- although the optimum was above 90°C. From the correspond- tively) (34). However, reduced ferredoxin, generated by the ing Arrhenius plot, which showed no obvious transition points 6514 MA AND ADAMS J. BACTERIOL.

200

0 0-"ZI 150 .5 0 l00' C.) U1)._ ci) CL cn 50

0.0 4.0 8.0 12.0 20 40 60 80 100 Time (hr) Temperature (IC) FIG. 4. Effects of temperature on the stability and activity of SuDH. (A) The pure enzyme (0.11 mg/ml in 50 mM Tris-HCl [pH 7.8] containing 2.0 mM DTr and 0.5 mM sodium dithionite) was incubated in serum-stoppered glass vials at 82 (open symbols) or 950C (solid symbols). Samples were removed with a syringe at intervals and assayed by the NADPH-dependent reduction of benzyl viologen at 800C. (B) The enzyme was assayed by the NADPH-dependent reduction of polysulfide at the indicated temperature. The assay mixture (2.0 ml) contained SuDH (20 ,ug), NADPH (0.3 mM), and polysulfide (2.5 mM) in 100 mM EPPS (pH 8.0). over the range 50 to 90'C, an activation energy of 81 kJ/mol observed near g = 2.04 and 1.91, indicating the presence of a was calculated. As suggested by its O2-reducing activity and in small amount of a second paramagnetic species (see below). contrast to several other oxidoreductase-type enzymes that The line shape and temperature dependence of the g = 2.01 have been purified from P. furiosus (2, 3), pure SuDH was signal are typical of the EPR properties of an oxidized stable upon prolonged exposure to 02 (air). There was no loss [3Fe-4S]1+ cluster (6). The EPR absorption of the oxidized of activity when the enzyme (as purified in the presence of 2 enzyme represented 0.54 to 0.72 spins per mol of holoenzyme. mM DTT) was left exposed to air for 16 h. Although 3Fe clusters can be generated by the oxidative EPR properties of SuDH. SuDH oxidized with excess thi- degradation of [4Fe-4S]1" centers (see below), this appears onine (reduction potential of +60 mV) at 230C exhibited a not to be the case with SuDH. This enzyme was purified under broad axial-type EPR signal centered atg = 2.01 which was not anaerobic conditions, and the enzyme lost no activity after observed above 40 K (Fig. 5, line a). Additional features were oxidation with thionine. Together with the significant concentration of this cluster in SuDH, these results suggest that the 3Fe center is part of the active enzyme. Upon reduction of oxidized SuDH with excess NADPH, the EPR signal from the 3Fe center was replaced with a spectrum (recorded at 12 K and 1-mW power) which indicated the a presence of at least two paramagnetic centers (Fig. 5, line c). This spectrum was observed at 40 K but not at 70 K. No signals were apparent at lower magnetic fields (<200 mT). The observed spectrum was not further resolved after variations in b temperature (4.2 to 40 K) or microwave power (0.01 to 100 mW) but appears to be a compilation of two rhombic type- spectra with an average g of -1.9. Such an interpretation is consistent with the determined spin concentration of 1.75 ± 0.13 spins per mol. Thus, NADPH appears to reduce the 3Fe center to an EPR-silent state, which further supports the notion that this center is not a purification artifact, as well as inducing EPR signals from two additional redox centers. It should be noted that this is a very unusual reaction, since it -l involves the reduction of the enzyme by three electrons, yet 2.108 1.984 1.874 NADPH is a two-electron carrier. The oxidized enzyme was g-value treated with excess NADPH, but it is not clear where the fourth electron goes (from the oxidation of two molecules of FIG. 5. EPR spectra of SuDH. SuDH (12.5 mg/ml) was prepared NADPH). The flavin moiety in SuDH might play a role, but anaerobically by ultrafiltration in 50 mM Tris-HCl, pH 8.0. SuDH after there was no evidence of a radical-type EPR signal from a oxidation by excess thionine (1.0 mM) (line a), after reduction by semiquinone species (Fig. 5, line c). This phenomenon is sodium dithionite (10 mM) (line b), and after reduction by NADPH under (2.0 mM) (line c) are shown. The spectrometer conditions were as currently study using varible-temperature potentiomet- follows: receiver gain, 1 x 105 (a) and 2 x 105 (b and c); microwave ric analyses. power, 1 mW; microwave frequency, 9.442 GHz; modulation fre- Reduction of oxidized SuDH with excess sodium dithionite quency, 100 kHz; modulation amplitude, 1 mT; time constant, 40.96 also gave rise to a complex EPR spectrum (Fig. 5, line b). This ms; and temperature, 12 K. spectrum appeared to consist of one of the paramagnetic VOL. 176, 1994 P. FURIOSUS SULFIDE DEHYDROGENASE 6515 species seen from the NADPH-reduced enzyme (represented (or PS) by features at g = 2.04 and 1.80), together with a second species not seen in the spectrum from the latter form of the enzyme. The former species appears to have a relatively high H2S reduction potential, since its EPR resonances are apparent in FERREDOXIN j e , * the thionine-oxidized enzyme (Fig. 5, line a). Like the H2 NADPH-reduced form, the EPR absorption of the dithionite- e reduced enzyme also represented approximately 1.7 spins per mol, and was observed at 40 K but not at 70 K. These data Metabolism 2H* suggest that reduced SuDH contains three paramagnetic cen- e ters with ground states of S (spin state) = 1/2, one reduced by I either sodium dithionite or NADPH, while the other two are SULFIDE |(NAD(P)H e each reduced only by one or the other reductant. The temper- NAD(P)H - DEHYDROGENASE FeS + FAD H2S ature dependence of the observed resonances are more consistent with the EPR properties of reduced [4Fe-4S]1" rather than [2Fe-2S]1" centers, although one or more of the latter cannot be excluded (12). Thus, from the EPR data of both the S' (or PS) oxidized and reduced states, we conclude that the enzyme FIG. 6. Proposed model for S0 and polysulfide (PS) reduction by contains a 3Fe center plus three additional FeS centers. The SuDH and sulfhydrogenase in P. furiosus. The cofactor contents of the latter could be all of the 4Fe type, all of the 2Fe type, or a two enzymes are indicated. mixture of both. The determined Fe content (10.6 ± 2.1 g-atoms/mol) does not rule out any of these possibilities, although three 4Fe and a 3Fe cluster (corresponding to 15 Fe yield (in grams [dry wt] per mole of maltose utilized) of P. atoms per mol) is perhaps the least likely. Obviously, addi- furiosus grown in the presence of So was almost twice that of tional spectroscopic analyses are required to clearly establish cells grown in its absence, suggesting that S0 reduction plays a the cluster content of SuDH. role in energy conservation (42). Moreover, on the basis of the unusual ferredoxin-dependent "pyrosaccharolytic" pathway DISCUSSION that P. furiosus is proposed to use for carbohydrate fermentation, it was calculated that So reduction is equivalent to an ATP Three enzymes that are capable of reducing So and polysul- yield of 0.5 mol of ATP per mol of S0 reduced (42). It was fide to H2S, SuDH and sulfhydrogenase from P. furiosus and therefore of some surprise to find that the sulfhydrogenase polysulfide reductase from the mesophile W succinogenes, are (sulfur:reduced ferredoxin oxidoreductase) activity of P. furio- now known. However, other than the ability to generate H2S, sus was located in the cytoplasm (26). In further contrast to the these enzymes have few properties in common. The mem- polysulfide reductase of W succinogenes, sulfhydrogenase is a brane-bound polysulfide reductase of W succinogenes was nickel-containing iron-sulfur protein which also functions as a solubilized by detergent treatment and consisted mainly of one hydrogenase (11). type of subunit (85 kDa) which contained iron and inorganic We show here P. furiosus contains a second S0-reducing sulfide but not flavin or b- or c-type hemes (20, 43). With enzyme, SuDH. This enzyme uses NADPH as a source of antibodies to the polysulfide reductase subunit, the gene for reductant for So reduction, but it too is a cytoplasmic enzyme. the enzyme (psrA) was recently cloned, together with two This obviously further complicates any attempt at rationalizing adjacent genes now termedpsrB andpsrC (21). From sequenc- the apparent coupling of So reduction to energy conservation ing analysis, it was postulated that the enzyme is composed of in this organism, at least by any conventional mechanism. three subunits and is a molybdopterin-containing protein, SuDH differs from both P. furiosus sulfhydrogenase and the wherein PsrA contains the molybopterin site, PsrB contains polysulfide reductase of W succinogenes in that it is a flavopro- ferredoxin-type clusters, and PsrC is a membrane anchor. tein and contains no metals other than iron. Data from Since W. succinogenes grows with formate and So as the sole spectroscopic and metal analyses suggest that the latter is carbon and energy sources (27), polysulfide reductase func- present as four paramagnetic centers, one of the 3Fe type and tions as the terminal enzyme of a membrane-bound electron three of the 2Fe or 4Fe type. In contrast to sulfhydrogenase, transport system and is thought to couple So reduction to SuDH did not evolve or oxidize H2. energy conservation by the pumping of protons. Indeed, re- The question therefore arises as to the physiological roles of constitution studies have indicated that a membrane-bound SuDH and sulfhydrogenase in P. furiosus. In the proposed formate dehydrogenase and polysulfide interact directly, with- fermentative pathway of this organism, the three oxidation out the participation of intermediate electron carriers such as steps in the conversion of glucose to acetate are each catalyzed quinones (43). by ferredoxin-linked oxidoreductases (30, 39). In addition, P. Like W succinogenes, the reduction of S0 by hyperthermo- furiosus contains several ferredoxin-linked oxidoreductases in- philic organisms was assumed to involve a membrane-bound volved in amino acid oxidation. These enzymes include two electron transport system wherein So via a So-reducing enzyme CoASH-dependent enzymes which oxidize indole pyruvate and functioned as the terminal electron acceptor. Thus, in autotro- 2-oxoglutarate (3, 28) and a CoASH-independent enzyme that phic hyperthermophiles, such a system would accept reductant oxidizes formaldehyde (31). Hence, the oxidative metabolism generated from H2 (36), whereas in heterotrophs, electrons of this organism would appear to generate significant amounts would be produced from the oxidative degradation of peptides of reduced ferredoxin, which could be directly oxidized by or carbohydrates (2, 3). On the other hand, some of these sulfhydrogenase leading to S0 reduction (Fig. 6). However, P. organisms, such as P. furiosus, are able to grow in the absence furiosus also contains an NAD(P)H-dependent glutamate de- of S0 by fermentation and produce H2. In these cases, So hydrogenase (16, 32, 38). Moreover, this represents about 20% reduction was originally thought to be a mechanism of detox- of the total cytoplasmic protein, and kinetic analyses indicate ifying inhibitory H2 (18). However, we have shown that the cell that it functions in glutamate oxidation and thus the produc- 6516 MA AND ADAMS J. BAC-1ERIOL.

tion of NAD(P)H (38). During peptide degradation by P. This research was supported by grants from the Office of Naval furiosus, significant amounts of glutamate would be generated Research (N00014-90-J-1894) and the Department of Energy (FG09- both by direct peptide hydrolysis and the transamination of 88ER13901). various amino acids (4). Thus, glutamate dehydrogenase would produce corresponding amounts of NAD(P)H, which could be REFERENCES directly oxidized by SuDH leading to S reduction (Fig. 6). 1. Adams, M. W. W. 1992. Novel iron sulfur clusters in metalloen- to zymes and redox proteins from extremely thermophilic bacteria. That SuDH makes a significant contribution H2S production Adv. Inorg. Chem. 38:341-396. in vivo is suggested by its activity relative to that of sulfhydro- 2. Adams, M. W. W. 1993. Enzymes and proteins from organisms that genase. For example, in cell extracts of P. furiosus, the poly- grow near and above 100TC. Annu. Rev. Microbiol. 47:627-658. sulfide-reducing activity of SuDH (0.083 U/mg) is about 50% 3. Adams, M. W. W. Biochemical diversity among sulfur-dependent that of sulfhydrogenase (0.2 pumol of H2S produced per min hyperthermophilic microorganisms. FEMS Microbiol. Rev., in per mg) (26). press. A complicating factor in assigning specific physiological 4. Andreotti, G., M. V. Cubellis, G. Nitti, G. Sannia, X. Mai, G. roles to SuDH and sulfhydrogenase in P. furiosus is the finding Marino, and M. W. W. Adams. 1994. Characterization of aromatic that SuDH also functions as a reduced ferredoxin:NADP aminotransferases in the hyperthermophilic archaeon Thennococ- in this cus litoralis. Eur. J. Biochem. 220:543-549. oxidoreductase. As shown Fig. 6, activity potentially 5. Aono, S., F. 0. Bryant, and M. W. W. Adams. 1989. A novel and couples ferredoxin oxidation to S reduction via SuDH. Our in remarkably thermostable ferredoxin from the hyperthermophilic vivo assays did not give definitive results on whether SuDH archaebacterium Pyrococcus furiosus. J. Bacteriol. 171:3433-3439. could use reduced ferredoxin as an efficient electron donor for 6. Beinert, H., and A. J. Thomson. 1983. Three-iron clusters in So reduction in the absence of NADP(H). Nevertheless, it iron-sulfur proteins. Arch. Biochem. Biophys. 222:333-361. appears that both reduced ferredoxin and NADPH could be 7. Blake, P. R., J.-B. Park, F. 0. Bryant, S. Aono, J. K. Magnuson, E. utilized by SuDH. This may be significant when carbohydrates Eccleston, J. B. Howard, M. F. Summers, and M. W. W. Adams. rather than peptides are the primary carbon and energy source, 1991. Determinants of protein hyperthermostability: purification since reductant generated by carbohydrate oxidation appears and amino acid sequence of rubredoxin from hyperthermophilic the ferredox- archaebacterium Pyrococcus furiosus and secondary structure of to exclusively reduce ferredoxin. Hence, reduced the zinc adduct by NMR. Biochemistry 30:10885-10895. in:NADP oxidoreductase activity of SuDH allows both it and 8. Blamey, J. M., and M. W. W. Adams. 1993. Purification and sulfhydrogenase to couple the reoxidation of reduced ferre- characterization of pyruvate ferredoxin oxidoreductase from the doxin to So reduction. hyperthermophilic archaeon, Pyrococcus furiosus. Biochim. Bio- In addition to NAD(P) and ferredoxin, SuDH utilized a phys. Acta 1161:19-27. variety of electron carriers including 02 and rubredoxin (Ta- 9. Blumentals, L. I., M. Itoh, G. J. Olson, and R. M. Kelly. 1990. Role bles 3 and 4). The 02-reducing activity of this enzyme is of polysulfides in reduction of elemental sulfur by the hyperther- surprising, since P. furiosus is an obligate anaerobe. Moreover, mophilic archaebacterium Pyrococcus furiosus. Appl. Environ. it is unlikely that SuDH functions as a scavenger of 02 when Microbiol. 56:1255-1262. to because the 10. Bradford, M. M. 1976. A rapid and sensitive method for the the organism is accidentally exposed air, quantitation of microgram quantities of protein utilizing the apparent Km value for 02 was 0.24 mM, which is equivalent to principle of protein-dye binding. Anal. Biochem. 72:248-254. air at 1.4 atm (1 atm = 101.29 kPa) (at 80°C). Interestingly, a 11. Bryant, F. O., and M. W. W. Adams. 1989. Characterization of rubredoxin:oxygen oxidoreductase was recently purified from hydrogenase from the hyperthermophilic archaebacterium, Pyro- the anaerobic bacterium Desulfovibrio gigas (14). It was pro- coccus furiosus. J. Biol. Chem. 264:5070-5079. posed to function in a membrane-bound electron transfer 12. Cammack, R. 1992. Iron-sulfur clusters in enzymes: themes and chain which leads to energy conservation upon exposure of the variations. Adv. Inorg. Chem. 38:281-322. organism to 02 (although the affinity of the enzyme for 02 was 13. Chen, J.-S., and L. E. Mortenson. 1977. Inhibition of methylene not reported). The absence of an analogous electron transport blue formation during determination of acid-labile sulfide of iron-sulfur protein samples containing dithionite. Anal. Biochem. system in P. furiosus suggests that the 02-reducing activity of 79:157-165. SuDH does not have a similar role. Nevertheless, the ability of 14. Chen, L., M.-Y. Liu, J. LeGall, P. Fareleira, H. Santos, and A. V. SuDH to reduce both 02 and So may be of significance in the Xavier. 1993. Rubredoxin oxidase, a new flavo-hemo-protein, is potential evolution of 02-reducing enzymes from those that the site of oxygen reduction to water by the "strict anaerobe" reduce So (40). Desulfovibrio gigas. Biochem. Biophys. Res. Commun. 193:100- Finally, it is also difficult to ascribe any physiological impor- 105. tance to the rubredoxin-reducing activity of SuDH. Even 15. Conover, R. C., A. T. Kowal, W. Fu, J.-B. Park, S. Aono, M. W. W. though the enzyme had a high affinity for oxidized rubredoxin Adams, and M. K. Johnson. 1990. Spectroscopic characterization (Table 3), the role or even necessity of this high-potential of the novel iron-sulfur cluster in Pyrococcusfuriosus ferredoxin. J. in P. Biol. Chem. 265:8533-8541. redox protein (reduction potential of -0 mV, 25°C) (7) 16. Consalvi, V., R. Chiaraluce, L Politi, R. Vaccaro, M. De Rosa, and furiosus, or indeed, in any of a variety of mesophilic anaerobes, R. Scandurra. 1991. Extremely thermostable glutamate dehydro- is not known (1). However, it is interesting that P. furiosus genase from the hyperthermophilic archaebacterium Pyrococcus rubredoxin was reported to have a stimulatory effect on the furiosus. Eur. J. Biochem. 202:1189-1196. So-reducing activity of sulfhydrogenase, even though its H2 17. Edelhoch, H. 1967. Spectroscopic determination of tryptophan and evolution activity was unperturbed (26). One is tempted to tyrosine in protein. Biochemistry 6:1948-1954. conclude that rubredoxin provides some form of link between 18. Fiala, G., and K. O. Stetter. 1986. Pyrococcus furiosus sp. nov. the two So-reducing enzymes of P. furiosus, although further represents a novel genus of marine heterotrophic archaebacteria experimentation is obviously required to substantiate such an growing optimally at 100'C. Arch. Microbiol. 145:56-61. 19. Ikeda, S. H., T. Satake, T. Hisano, and T. Terazawa. 1972. effect. Potentiometric argimetric method for the successive titration of sulphide and dissolved sulphur in polysulphide solutions. Talanta ACKNOWLEDGMENTS 19:1650-1654. 20. Klimmek, O., A. Krbger, R. Steudel, and G. Holdt. 1991. Growth We thank Zhi Hao Zhou for growing P. furiosus and for preparing of Wolinella succinogenes with polysulfide as terminal acceptor of cell extracts. phosphorylative electron transport. Arch. Microbiol. 155:177-182. VOL. 176, 1994 P. FURIOSUS SULFIDE DEHYDROGENASE 6517

21. Kraft, T., M. Bokranz, 0. Klimmek, L. Schroder, F. Fahrenholz, E. cus furiosus. J. Biol. Chem. 266:19351-19356. Kojro, and A. Kroger. 1992. Cloning and nucleotide sequence of 35. Pfennig, N., and H. Biebl. 1976. Desulfuromonas acetoxidans gen. the psr A gene of Wolinella succinogenes polysulfide reductase. nov. and sp. nov., a new anaerobic, sulfur-reducing, acetate- Eur. J. Biochem. 206:503-510. oxidizing bacterium. Arch. Microbiol. 110:3-12. 22. Laemmli, U. K. 1970. Cleavage of structural proteins during the 36. Pihl, T. D., L. K. Black, B. A. Schulman, and R. J. Maier. 1992. assembly of the head of bacteriophage T4. Nature (London) Hydrogen-oxidizing electron transport components in the hyper- 227:680-685. thermophilic archaebacterium Pyrodictium brockii. J. Bacteriol. 23. Le Faou, A., B. S. Rajagopal, L. Daniels, and G. Fauque. 1990. 174:137-143. Thiosulfate, polythionates and elemental sulfur assimilation and 37. Riddles, P. W., R. L. Blakeley, and B. Zerner. 1983. Reassessment reduction in the bacterial world. FEMS Microbiol. Rev. 75:351- of Ellman's reagent. Methods Enzymol. 91:49-60. 382. 38. Robb, F. T., J.-B. Park, and M. W. W. Adams. 1992. Character- 24. Lovenberg, W., B. B. Buchanan, and J. C. Rabinowitz. 1963. ization of an extremely thermostable glutamate dehydrogenase: a Studies on the chemical nature of ferredoxin. J. Biol. Chem. key enzyme in the primary metabolism of the hyperthermophilic 238:3899-3913. archaebacterium, Pyrococcus furiosus. Biochim. Biophys. Acta 25. Ma, K., F. T. Robb, and M. W. W. Adams. 1994. Purification and 1120:267-272. characterization of NADP-specific alcohol dehydrogenase and 39. Schafer, T., and P. Schonheit. 1992. Maltose fermentation to NADP-specific glutamate dehydrogenase from the hyperthermo- acetate, CO2 and H2 in the anaerobic hyperthermophilic archaeon philic archaeon Thermococcus litoralis. Appl. Environ. Microbiol. Pyrococcus furiosus: evidence for the operation of a novel sugar 60:562-568. fermentation pathway. Arch. Microbiol. 158:188-202. 26. Ma, K., R. N. Schicho, R. M. Kelly, and M. W. W. Adams. 1993. 40. Schauder, R., and A. Krdger. 1993. Bacterial sulfur respiration. Hydrogenase of the hyperthermophile Pyrococcus furiosus is an Arch. Microbiol. 159:491-497. elemental sulfur reductase or sulfhydrogenase: evidence for a 41. Schauder, R., and E. Muller. 1993. Polysulfide as a possible sulfur-reducing hydrogenase ancestor. Proc. Natl. Acad. Sci. USA substrate for sulfur-reducing bacteria. Arch. Microbiol. 160:377- 90:5341-5344. 382. 27. Macy, J. M., I. Schroder, R K. Thauer, and A. Kroger. 1986. 42. Schicho, R. N., K. Ma, M. W. W. Adams, and R. M. Kelly. 1993. Growth of Wolinella succinogenes on H2S plus fumarate and on Bioenergetics of sulfur reduction in the hyperthermophilic ar- formate plus sulfur as energy sources. Arch. Microbiol. 144:147- chaeon, Pyrococcus furiosus. J. Bacteriol. 175:1823-1830. 150. 43. Schroder, I., A. Kroeger, and J. M. Macy. 1988. Isolation of the 28. Mai, X., and M. W. W. Adams. 1994. Indolepyruvate ferredoxin sulphur reductase and reconstitution of the sulphur respiration of oxidoreductase from the hyperthermophilic archaeon, Pyrococcus Wolinella succinogenes. Arch. Microbiol. 149:572-579. fuiosus: a new enzyme involved in peptide fermentation. J. Biol. 44. Schumacher, W., P. M. H. Kroneck, and N. Pfennig. 1992. Chem. 269.16726-16732. Comparative systematic study on "Spirillum" 5175, Campylobacter 29. Malik, B., H. L. Wald, I. I. Blumentals, and R M. Kelly. 1989. and Wolinella species. Arch. Microbiol. 158:287-293. Growth and gas production for hyperthermophilic archaebacte- 45. Stetter, K. 0. 1986. Diversity of extremely thermophilic archae- rium, Pyrococcus furiosus. Biotech. Bioeng. 34:1050-1057. bacteria, p. 39-74. In T. D. Brock (ed.), The thermophiles: 30. Mukund, S., and M. W. W. Adams. 1991. The novel tungsten-iron- general, molecular and Applied microbiology. John Wiley, New sulfur protein of the hyperthermophilic archaebacterium, Pyrococ- York. cus furiosus, is an aldehyde ferredoxin oxidoreductase: evidence 46. Stetter, K. O., G. Fiala, G. Huber, R. Huber, and G. Segerer. 1990. for its participation in a unique glycolytic pathway. J. Biol. Chem. Hyperthermophilic microorganisms. FEMS Microbiol. Rev. 75: 266:14208-14216. 117-124. 31. Mukund, S., and M. W. W. Adams. 1993. Characterization of a 47. Stetter, K. O., and G. Gaag. 1983. Reduction of molecular sulphur novel tungsten-containing formaldehyde ferredoxin oxidoreduc- by methanogenic bacteria. Nature (London) 305:309-311. tase from the extremely thermophilic archaeon, Thermococcus 48. Then, T., and H. G. Truper. 1983. Sulfide oxidation in Ectothio- litoralis. A role for tungsten in peptide catabolism. J. Biol. Chem. rhodospira abdelmalekii. Evidence for the catalytic role of cyto- 268:13592-13600. chrome c-551. Arch. Microbiol. 135:254-258. 32. Ohshima, T., and N. Nishida. 1993. Purification and properties of 49. Tilstra, L., G. Eng, G. J. Olson, and F. W. Wang. 1992. Reduction extremely thermostable glutamate dehydrogenases from two hy- of sulphur from polysulphidic model compounds by the hyperther- perthermophilic archaebacteria, Pyrococcus woesei and Pyrococcus mophilic archaebacterium Pyrococcus furiosus. Fuel 71:779-783. furiosus. Biosci. Biotechnol. Biochem. 57:945-951. 50. Zophel, A., M. C. Kennedy, H. Beinert, and P. M. H. Kroneck. 33. Olsen, G. J., C. R Woese, and R Overbeek. 1994. The winds of 1988. Investigations on microbial sulfur respiration. 1. Activation (evolutionary) change: breathing new life into microbiology. J. and reduction of elemental sulfur in several strains of eubacteria. Bacteriol. 176:1-6. Arch. Microbiol. 150:72-77. 34. Park, J.-B., C. Fan, B. M. Hoffman, and M. W. W. Adams. 1991. 51. Zophel, A., M. C. Kennedy, H. Beinert, and P. M. H. Kroneck. Potentiometric and electron nuclear double resonance properties 1991. Investigations on microbial sulfur respiration-isolation, of the two spin forms of the [4Fe-4S]" cluster in the novel purification, and characterization of cellular components from ferredoxin from the hyperthermophilic archaebacterium, Pyrococ- Spirillum 5175. Eur. J. Biochem. 195:849-856.