Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5341-5344, June 1993 Biochemistry Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: Evidence for a sulfur-reducing hydrogenase ancestor (hydrogen activation/polysulfide reduction/geothermal biolog/evolution) KESEN MA*, RICHARD N. SCHICHOt, ROBERT M. KELLYt, AND MICHAEL W. W. ADAMS*§ *Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602; tDepartment of Chemical Engineering, The Johns Hopkins University, Baltimore, MD 21218; and kDepartment of Chemical Engineering, North Carolina State University, Raleigh, NC 27695-7905 Communicated by Helmut Beinert, February 22, 1993 (receivedfor review December 23, 1992) ABSTRACT Microorganisms growing near and above tion in mesophilic organisms. The activity was also shown to 100C have recently been discovered near shaflow and deep sea be membrane-associated in Wollinella succinogenes (9) and hydrothermal vents. Most are obligately dependent upon the Spirillum 5175 (10, 11), whereas it was associated with a reduction of elemental sulfur (S°) to hydrogen sulfide (H2S) for periplasmic protein in some Desulfovibrio sp. (12) and with a optimal growth, even though SO reduction readily occurs soluble protein in Chlorobium thiosulfatophilum (13). Only abioticafly at their growth temperatures. The sulfur reductase one sulfur reductase has been partially purified: that from W. activity of the anaerobic archaeon Pyrococcusfuriosus, which succinogenes, an organism that can obtain energy by S0 grows optimally at 100°C by a metabolism that produces H2S respiration (10). This membrane-bound enzyme contained an if S0 is present, was found in the cytoplasm. It was purified Fe-S cluster but no heme iron and appeared to use either SO anaerobically and was shown to be identical to the hydrogenase or polysulfide as the electron acceptor (14). that had been previously purified from this organism. Both S0 We report here on the SO-reducing activity of the hyper- and polysulfide served as substrates for H2S production, and thermophile P. furiosus, which grows optimally at 100°C (6). the SO reduction activity but not the H2-oxidation activity was This organism reduces either S0 or polysulfide to H2S during enhanced by the redox protein rubredoxin. The H2-oxidizing growth and can do so remote from the insoluble sulfur and SO-reduction activities of the enzyme also showed different particles present in the medium (15). However, the abiotic responses to pH, temperature, and inhibitors. This bifunc- reduction of S to H2S also occurs at the growth temperature tional "sulfhydrogenase" enzyme can, therefore, dispose ofthe of P. furiosus (15), and so it was far from clear whether any excess reductant generated during fermentation using either enzyme is actually involved in catalyzing S0 reduction. In protons or polysulfides as the electron acceptor. In addition, addition, assuming the reaction is enzymatic, two mecha- purified hydrogenases from both hyperthermophilic and me- nisms have been suggested by which the excess reductant sophilic representatives of the archaeal and bacterial domains produced during fermentation might be coupled to SO reduc- were shown to reduce SO to H2S. It is suggested that the function tion rather than HI reduction (H2 production) (4). These did of some form of ancestral hydrogenase was SO reduction rather not involve membrane electron transport, which might be than, or in addition to, the reduction of protons. anticipated if S0 reduction is an energy-conserving reaction (7). However, we show here that P.furiosus does have sulfur The ability of microorganisms to reduce elemental sulfur (S0) reductase activity, defined as the release of H2S from S, but is a very recent discovery (1) and is still limited in the it is located in the cytoplasmic fraction. Moreover, it is microbial world (2). The notable exceptions are the hyper- associated with and seems to be identical to the hydrogenase thermophilic archaea (formerly archaebacteria) that thrive at previously purified from this organism (16). temperatures near and even above 100°C in shallow and deep sea hydrothermal vents (3-5). All of these organisms have MATERIALS AND METHODS been shown to be dependent, to a greater or lesser extent, Growth ofBacterium. P.furiosus (DSM 3638) was routinely upon S0 reduction for optimal growth. The majority are grown at 85°C in a 500-liter fermenter, as described (16). obligately dependent upon the reduction of S0 to H2S and use Enzyme Assays. Sulfur reductase was assayed at 80°C in either H2 or organic compounds as electron donors. On the 8-ml sealed vials with H2 as the gas phase. The 2-ml assay other hand, some of these organisms can grow by fermenta- mixture routinely contained 100 mM N-(2-hydroxyethyl)- tive-type metabolisms and produce H2. In these cases addi- piperazine-N'-3-propanesulfonic acid (EPPS)/NaOH, pH tion of SO to the growth medium leads to the production of 8.4, 0.1 g of sublimed sulfur (Baker), and 0.8 mM sodium H2S and usually stimulates growth. It has been suggested that dithionite. The vials were shaken at 160 rpm in a water bath, sulfur reduction is a "detoxification" mechanism to lower the and cell-free extract or purified fractions were added to amount of H2 produced because H2 inhibits growth (6). initiate the reaction. Where indicated, P. furiosus hydrogen- However, we have recently shown that in at least one ase (5 ug) was added immediately before the addition of the fermentative hyperthermophile, Pyrococcus furiosus (6), S0 extract. At intervals, aliquots of the assay mixture were reduction appears to be an energy-conserving reaction (7). removed and assayed for hydrogen sulfide (H2S) by methyl- Little is known about the mechanism of S0 reduction in ene blue formation (17). Because the H2S is distributed hyperthermophilic organisms. Pihi et al. (8) reported that the between the gas and liquid phases, the total amount of H2S activity was membrane-bound in the autotrophic, S°- produced in this assay was calculated from a standard curve respiring Pyrodictium brockii, which grows optimally at prepared by using known amounts of sodium sulfide (Na2S) 105°C. Moreover, there have been few reports on SO reduc- under the same conditions. One unit of sulfur reductase activity is defined as the production of 1 ,umol ofH2S per min, The publication costs of this article were defrayed in part by page charge calculated from the amount of H2S produced over a 3-hr payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 5341 Downloaded by guest on October 2, 2021 5342 Biochemistry: Ma et al. Proc. Natl. Acad. Sci. USA 90 (1993) period. Hydrogenase activity was determined by H2 evolu- that in the sulfur reductase assay used, the production ofH2S tion of H2 oxidation as described (15). One unit of activity is from SO was measured by using H2 as the electron donor. defined as 1 ,umol of H2 evolved or consumed per min. Under all conditions tested and with either cell-free extracts Polysulfide Preparation and Determination. A 0.5 M or purified fractions, H2S could not be detected unless a polysulfide solution was prepared by reaction of 12 g ofNa2S protein-containing fraction was added to the assay mixture. with 1.6 g of elemental sulfur in 100 ml of anoxic water (18). In the initial attempts to purify the sulfur reductase activity Polysulfide was measured by cold cyanolysis (19). from cell-free extracts, purified P. furiosus hydrogenase was Purification of Sulfur Reductase. All steps were performed added to the assay medium to provide a means of activating under anaerobic conditions at room temperature. All buffers H2 as a source of reductant. However, control experiments were repeatedly degassed and flushed with Ar and were showed that addition of hydrogenase, although stimulatory, maintained under a positive pressure of Ar. They all con- was not obligatory for H2S production, and it was therefore tained dithiothreitol (1 mM) to protect against trace 02 omitted for routine assays. The sulfur reductase activity was contamination. All columns were controlled by a fast protein purified -30-fold compared with the cell-free extract with an liquid chromatography system (Pharmacia LKB). P.furiosus overall yield of 10% (Table 1). Analysis of the purified cells (12 g, wet weight) were suspended in 50 mM Tris-HCl, enzyme by SDS/gel electrophoresis revealed why the addi- pH 8.1 (1 g of cells per 4-ml buffer) containing lysozyme (1 tion of hydrogenase was not required for H2 activation: the mg/ml) and DNase (0.1 mg/ml). The cells were lysed during purified sulfur reductase showed the same pattern ofsubunits incubation ofthe cell suspension at 35°C for 2 hr with stirring. as that exhibited by the hydrogenase previously purified from A cell-free extract was obtained by centrifugation at 50,000 x this organism (16). Furthermore, that the enzymes respon- g for 1 hr. The cell-free extract was applied to a column (2.8 sible for SO reduction and H2 evolution inP.furiosus were one x 13 cm) of Q Sepharose Fast Flow (Pharmacia LKB) that and the same was confirmed by the co-elution of these two was equilibrated with 50 mM Tris HCl buffer, pH 8.1, at 2.5 activities throughout the purification procedure. In addition, ml per min. The absorbed proteins were eluted with a 500-ml pure samples of P. furiosus hydrogenase previously purified lineargradient from 0 to 0.7 M NaCl in the same buffer. Sulfur in our laboratory all exhibited sulfur reductase activity. The reductase activity was located in the fractions eluting at enzymatic nature of the H2S-producing activity of what we 0.35-0.5 M NaCl.