the journal of vol. 264, no. 9. of 25. pp. 5070-5079,1969 ... · all are archaebacteria (3) ... the...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1989 by The American Society €or Biochemistry and Molecular Biology, Inc. Vol. 264, No. 9. Issue of March 25. pp. 5070-5079,1969

Printed in U.S.A.

Characterization of Hydrogenase from the Hyperthermophilic Archaebacterium, Pyrococcus furiosus"

(Received for publication, August 29,1988)

Frank 0. Bryant and Michael W. W. Adams From the Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602

The archaebacterium, Pyrococcus furiosu~, grows optimally at 100 "C by a fermentative type metabolism in which H2 and COz are the only detectable products. The organism also reduces elemental sulfur (So) to &S. Cells grown in the absence of So contain a single hydrogenase, located in the cytoplasm, which has been purified 360-fold to apparent homogeneity. The yield of HZ evolution activity from reduced methyl viologen at 80 "C was 40%. The hydrogenase has a M, value of 186,000 f 15,000 and is composed of three subunits of M. 46,000 (a), 27,000 (B), and 24,000 (7). The enzyme contains 31 % 3 g atoms of iron, 24 2 4 g atoms of acid-labile sulfide, and 0.98 f 0.06 g atoms of nickel/ 186,000 g of protein. The Ha-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 16 I(, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by Oa and is remarkably thermostable. Most of its Hs evolution activity is retained after a l-h incubation at 100 "C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of HZ evolution is observed between 46 and 90 OC. The hydrogenase also catalyzes Hz oxidation with methyl viologen or metb- ylene blue as the electron acceptor. The temperature optimum for both Hz oxidation and Hz evolution is >96 "C. Arrhenius plots show two transition points at -60 and -80 "C independent of the mode of assay. That occurring at 80 "C is associated with a dramatic increase in Hz production activity. The enzyme preferentially catalyzes HZ production at all temperatures examined and appears to represent a new type of "evolution'' hydrogenase.

Very recently some remarkable microorganisms have been discovered that grow optimally at temperatures above 80 "C (1). Largely through the efforts of Stetter and colleagues, some 20 species of these extremely thermophilic bacteria have been isolated, mainly from shallow submarine and deep sea geothermal environments (see Ref. 2). All are archaebacteria (3) and as such are very distinct from thermophilic eubacteria which also have lower optimum growth temperatures (4). Most of the extreme thermophiles are strict anaerobes and depend on the reduction of elemental sulfur (So) for growth. Included in this group are the so-called "hyperthermophiles"

* This research was supported by grants from the University of Georgia Research Foundation and Department of Energy Grant FG09-88ER13901 (to M. W. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

that grow optimally around 100 "C. So far these are represented by three distinct genera, Pyrodictium (5), Pyrococcus (6), and Pyrobaculum (7). Pyrodictium brockii (Topt 105 'C) is an obligate autotroph which obtains energy by reducing So to HzS with HZ (5) while Pyrobaculum islandicum (Topt 100 OC) is a facultative heterotroph that uses either organic substrates or HZ to reduce So (7). In contrast, Pyrococcus furiosus (To,, 100 "C) grows by a fermentative-type metabolism rather than by So respiration (6). It is a strict heterotroph that utilizes both simple and complex carbohydrates where only Hz and COZ are the detectable products. The organism reduces So to H2S apparently as a form of detoxification since Hz inhibits growth.

The discovery of bacteria growing optimally around 100 "C has generated considerable interest in both academic and industrial communities, e.g. Refs. 2, 4, 8-10. In addition to providing basic information on the mechanisms by which various biomoIecules are stabilized at high temperatures, one can anticipate the development of processes that take advan- tage of their thermostable enzymes and novel metabolic reactions. Both the organisms and their enzymes have the potential to bridge the gap between biochemical catalysis and many industrial chemical conversions. However, our knowl- edge of the metabolism of the hyperthermophilic bacteria is limited to the papers describing their isolation, and no enzyme has been purified from them. A characteristic of these bacteria appears to be their ability to oxidize or evolve Hz, reactions of considerable biotechnological relevance (see Ref. 11). We have therefore focused on the metabolism of Hz by P. furiosus and report here on the purification and properties of its hydrogenase.

Hydrogenase has the unique ability to reversibly activate the simplest of molecules, Hz, according to Equation I:

HZ H 2H+ + 2e- (1)

The enzyme is widely distributed in bacteria and its presence enables an organism to either use Hz as a source of energy and reductant, or use protons as a terminal acceptor and evolve Hz (12-15). Hydrogenases have been purified from a variety of bacteria, but they are a very heterogeneous group of enzymes varying in molecular composition, catalytic activity, sensitivity to Oz, cofactor content, and electron carrier specificity. However, they have one common feature: all are iron-sulfur proteins and the majority also contain nickel. The NiFe-hydrogenases have been isolated from both aerobic and anaerobic bacteria and in spite of an array of molecular properties, the minimum metal requirement for catalytic activity appears to be a Ni center and a 4Fe cluster. There is general agreement that a Ni-S cluster (16, 17) is involved in the reaction with Hz (18-20), but controversy exists over the redox state of the nickel and the role of the 4Fe-cluster (18- 22). In contrast to the NiFe hydrogenases, those that contain only Fe are quite similar (see Ref. 23). They have been isolated

5070

P. furiosus Hydrogenase 5071

only from anaerobic bacteria and are the most active hydrogenases known, often by an order of magnitude. Catalysis of H2 oxidation and Hz evolution is thought to reside with a novel type of iron-sulfur cluster (24-27). To date, the hydrogenases of only the two following thermophilic organisms have been well characterized from the methanogen, Methanobac- terium thermoautotrophicum ( Topt 60 “C; 28-30), and from the saccharolytic anaerobe, Acetobacterium flauidum (To,, 58 “C, 31). These contain NiFe- and Fe-hydrogenases, respectively, whose properties, including thermostability, are very similar to their mesophilic counterparts.

The characterization of hydrogenase from an hyperthermophilic bacterium is therefore of considerable interest from both scientific and technological standpoints. We show here that the enzyme from P. furiosus is remarkably thermostable and represents a new type of hydrogenase. In contrast to all other hydrogenases, it preferentially catalyzes Hz production. Furthermore, it shares certain characteristics peculiar to the hydrogenases of some aerobic eubacteria, of some anaerobic eubacteria, and of other archaebacteria, consistent with the notion of the hyperthermophiles representing an ancient and primitive phenotype.

MATERIALS AND METHODS

Growth of Bacterium P. furiosus (DSM 3638) was routinely grown at 85-88 ‘C as closed

static cultures in a medium containing maltose (5 g/liter), NHFI (1.25 g/liter), elemental sulfur (So, 5 g/liter), NazS (0.5 g/liter), synthetic sea water (SME, 5), a vitamin mixture (32), FeCb (25 p ~ ! , NazWO, (10 p ~ ) , and yeast extract (1.0 g/liter). Growth was mom- tored by direct cell count and by the increase in turbidity at 600 nm. For large scale cultures, sulfide was replaced by titanium (111) citrate, and So was omitted which necessitated sparging with Ar (see “Re- sults”). Two 20-liter cultures served as an innoculum for growth in a 400-liter fermentor where the culture was maintained at 88 “C, bub- bled with Ar (7.5 liters/min), and stirred (50 rpm). Cells were har- vested after approximately 20 h (ODm -0.5) with a Sharples centri- fuge at 100 liters/h. They were immediately frozen in liquid NZ and stored at -80 “C.

Methods Hydrogenase activity was routinely determined by Hz evolution

from reduced methyl viologen (1 mM) as described previously (33), except the buffer was 50 mM EPPS,’ pH 8.4, and the temperature was 80 “C. The Hz produced was measured using a gas chromatograph (model 3300, Varian Associates) which was calibrated with known amounts of HZ withdrawn from vials maintained at the assay temperature. One unit of hydrogenase activity catalyzes the production of 1 pmol of Hz/min. Hz oxidation activity was determined by the Hz- dependent reduction of methylene blue or methyl viologen (0.04 and 1 mM, respectively) in 50 mM EPPS buffer, pH 8.4, at 80 “C in serum- stoppered cuvettes (33). Absorbance changes were measured on a DMS 200 spectrophotometer (Varian Associates) equipped with ther- mostatted cuvette holders and a thermo-insulated cell compartment. Results are expressed as pmol of Hz oxidized/min. Protein was estimated by the Lowry method using bovine serum albumin as the standard (34).

Electrophoresis under nondenaturing conditions (35) and in the presence of SDS (36) were performed as described in the references. Hydrogenase activity was located on the gels by the Hz-dependent reduction of methyl viologen at 70 “C, and the activity bands were permanently stained using 2,3,5-triphenyl tetrazolium chloride (37). The molecular weight of the native hydrogenase was determined by

The abbreviations used are: EPPS, N-(2-hydroxyethyl)piperzine- N”3-propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazinesulfonic acid; CAPS, 3- (cyclohexy1amino)-1-propanesulfonic acid; MOPS, 3-(N-morpho- 1ino)propanesulfonic acid; CHES, 2-(N-cyclohexylamino) ethanesulfonic acid; FPLC, fast protein liquid chromatography; SDS, sodium dodecyl sulfate; Tricine, N-(2-hydroxyl-l,l-bis (hydroxymethy1)ethyl-glycine; W, watt.

gel filtration on columns of Superose 6 and Superose 12 operated by a Pharmacia LKB Biotechnology Inc. FPLC System. In both cases, two HR 10/30 columns were connected and calibrated with proteins of known molecular weight using 50 mM Tris-HC1 buffer, pH 8.0, containing 200 mM NaCI, as the eluent. The iron and acid-labile sulfide contents of the hydrogenase were measured using o-phenan- throline (38) and by methylene blue formation (39), respectively. Ferredoxin (8Fe, 8Sz-) from Clostridium pusteurianum (40) was used as a reference protein in both assays. Nickel was estimated by plasma emission spectroscopy using a Jarrel Ash Plasma Comp 750 instru- ment. The amino acid composition of the hydrogenase was determined by Dr. James B. Howard of the University of Minnesota (41). The purification and properties of P. furiosus ferredoxin will be described elsewhere? Cofactor F,m of Methanobacterium thermoautotrophicurn (strain AH, see Ref. 29) was a gift of Dr. R. Scott of the Department of Chemistry.

Reduced hydrogenase samples for EPR spectroscopy were prepared under HZ in 50 mM Tris-HC1 buffer, pH 8.0, containing 1 mM sodium dithionite. The enzyme was oxidized by the addition of anaerobic thionine (4 mM in 50 mM Tris-HC1 buffer, pH 8.0) until a slight blue color of the oxidized dye was visible. Samples were then rapidly frozen in a heptane/liquid N P mixture. EPR spectra were recorded on an IBM-Bruker ER 200D spectrometer interfaced to an IBM 9001 mi- crocomputer and equipped with an Oxford Instruments ESR-9 flow cryostat. Spin quantitations of the EPR signals from the reduced

corded at 70 K (100 pW and 1 mW) and 10 K (20 pW) with the hydrogenase were determined by double integration of spectra re-

indicated microwave power. These were compared with spectra of Cu (1 mM)/EDTA (100 mM) recorded under identical conditions.

Purification of Hydrogenase

All steps were performed under anaerobic conditions at room temperature. All buffers were repeatedly degassed and flushed with Ar, prepurified with heated BASF catalyst (Kontes, NJ) to remove traces of Oz, and were maintained under a positive pressure of Ar. They all contained 2 mM sodium dithionite (Sigma), to protect against trace 02 contamination, and ethanol (2.5%, v/v), to minimize bacte- rial contamination. The latter had no effect on hydrogenase activity (see “Results”).

Preparation of Cell Extract-Frozen cells (450 g) were thawed under Ar with repeated degassing and flushing and then suspended in 50 mM Tris-HC1 buffer, pH 8.0, containing lysozyme (1 mg/ml) and DNase (0.1 mg/ml). The cells were lysed, as determined by micro- scopic observation, during incubation of the cell suspension at 30 “C for 1 h with stirring. A cell-free extract was obtained by centrifugation at 50,000 X g for 40 min in a L8-70 ultracentrifuge (Beckman Instruments).

Q Sephurose Chromatography-The cell-free extract was applied directly to a column (5 X 45 cm) of Q Sepharose (Pharmacia LKB Biotechnology, Inc.) previously equilibrated with 50 mM Tris-HC1 buffer, pH 7.8, at 8 ml/min. This and all subsequent columns were controlled using an FPLC system. The column was washed with 1.5 liters of the equilibration buffer, and the absorbed proteins were eluted in 90-ml fractions with a 4.2-liter linear gradient from 0 to 0.6 M NaCl at 6 ml/min in the same buffer. Hydrogenase activity started to elute when 0.33 M NaCl was applied to the column.

DEAE-Sephmel Chromatography-Fractions from the Q Sepha- rose column with a specific activity in the HZ evolution assay greater than 4 units/mg were applied to a column (2.5 X 30 cm) of DEAE- Sephacel (Pharmacia LKB Biotechnology Inc.), pre-equilibrated with 50 mM Tris-HC1 buffer, pH 8.3, a t 3 ml/min. The FPLC system was used to dilute the fractions with an equivalent volume of equilibration buffer as they were applied. After washing the column with the same buffer (300 ml), the protein was eluted in 40-ml fractions with sequential linear gradients from 0 to 0.1 M NaCl (50 ml) and 0.1-0.4 M NaCl (1.25 liters) at 2.5 ml/min. Hydrogenase activity started to elute as 0.26 M NaCl was applied.

Hydroxyupatite Chromatography-Fractions from the previous column with a specific activity above 60 units/mg were applied directly to a column (1.5 X 35 cm) of hydroxyapatite (High Resolution, Behring Diagnostics) previously equilibrated with 50 mM Tris-HCl buffer, pH 8.0, a t 1.5 ml/min. The column was washed with 90 ml of the same buffer and the protein was eluted in 5-ml fractions with a 600-ml linear gradient from 0 to 0.20 M potassium phosphate in the

S. Aono, F. 0. Bryant, and M. W. W. Adams, manuscript in preparation.

5072 P. furiosus Hydrogenase

same buffer, pH 8.0, at 0.8 mI/min. The hydrogenase eluted as 0.08 M phosphate was applied. Fractions with specific activity above 120 units/mg were then concentrated to -1.0 ml using an HR 5 / 5 Mono Q column (Pharmacia LKB Biotechnology Inc.). The samples were applied to the Mono Q column, pre-equilibrated with 50 mM Tris- HCI buffer, pH 8.0, at 1.0 ml/min, using the FPLC system to effect dilution with an equivalent volume of the same buffer. The protein was eluted at 0.3 ml/min in 0.5-ml fractions with a linear gradient (3 ml) from 0 to 1.0 M NaCl in the same buffer.

Superose 6 Chromatography-The concentrated hydrogenase was applied to a column (HR 16/50, Pharmacia LKB Biotechnology Inc.) of Preparative Superose 6 pre-equilibrated with 50 mM Tris-HCI buffer, pH 8.0, containing 0.2 M NaCI. The column was run at 0.8 ml/min and samples of 5 ml were collected. Those judged pure by native and SDS-gel electrophoresis (see "Results") were combined, concentrated by Mono Q chromatography to -5 mg/ml, and were stored either at room temperature or as pellets in liquid N2.

RESULTS

Growth Studies-Yields of P. furiosus cells grown in closed static cultures using a complex medium containing So (6) were lower than those reported by Stetter and co-workers (final cell titer -1 X 10' ceHs/min uersus 3 X IO8 ceUs/ml given in Ref. 6). However, the concentration of So used by them (30 g/liter) was inhibitory under our conditions since the cell yields increased over %fold when the concentration of So was lowered to 5 g/liter. They also obtained reasonable growth (final cell yields of -5 x IO7 cells/ml) using maltose as the carbon source and casamino acids as the nitrogen source, in place of yeast extract and peptone. We found that the growth yields on maltose increased 4-fold if ammonia (NHrCl) was used as the nitrogen source and a vitamin mixture (32) was also added. A low concentration of yeast extract was also required for optimal growth on maltose. The yeast extract could not be replaced by increasing the vitamin mixture (32) or the mineral solution (5) . Attempts to increase the cell yields by including salts of vanadium, selenium, cesium, flu- orine, lead, and rubidium (c.f composition of natural sea water) or by adding additional nickel (all up to 10 FM final concentration) were unsuccessful? Although high concentrations of the mineral solution (210 times that given in Ref. 5 ) inhibited growth (final concentration -2 x lo7 cells/ml), the highest cell yields of P. furiosus were obtained when tungsten and additional iron were added (to final concentrations of 10 and 25 PM, respectively). Growth in this medium was poor in the absence of added So presumably due to inhibition by the Hz produced during fermentation (6). In support of this, the inhibition was completely relieved by sparging with Ar. A sulfur-free medium (lacking So and sulfide) was obtained by replacing sulfide, used as the initial reductant, with either the citrate (42) or nitrilotriacetate (43) salt of titanium (111). Cell yields from a 400-liter fermentor using So-free media (to prevent the production of corrosive HZS) with either sulfide or titanium as the reductant were 450-1100 g (wet weight). The cause of this variation is not known although low agita- tion rates (530 rpm) seem to favor higher cell yields. These yields are significantly higher than that originally obtained (70 g/300 liters, 6). Static cultures of P. furiosus grown both with and without So also served as stock cultures. They were stored at 23 "C and remained viable for at least 6 months. Cells maintained at 4 "C lost viability within a few days unless So was present.

Purification of Hydrogenase-P. furiosus cells were very sensitive to lysis by lysozyme, and this method was used to prepare cell-free extracts. The Hz evolution activity of such extracts was scarcely detectable at 30 "C but was readily measured at 80 "C, increasing -23-fold between these two

J. B. Park, S. Aono, and M. W. W. Adams, unpublished data.

temperatures. The responsible hydrogenase(s) was soluble since more than 90% of the activity remained in the yellowish- brown supernatant after centrifugation. The specific activity (at 80 "C) of the supernatant solution was found to vary from one batch of cells to another. The range was 0.25-2.8 units/ mg. The cause of this variation is currently under investigation, but it is not dependent on the presence or absence of So, titanium, or sulfide? Cell-free extracts prepared from the same batch of cells in 50 mM Tris-HC1 buffer, pH 8.0, either aerobically or anaerobically in the presence of sodium dithionite (2 mM), had the same Hz evolution activity. However, the Hz evolution activity was somewhat sensitive to inactivation by 02 since extracts prepared aerobically lost -50% of their HZ evolution activity after 6 days at both 4 and 23 "C, while those prepared anaerobically (with and without sodium dithionite) retained all activity over the same period. The stability of the hydrogenase also depended on the buffer (and pH) used even under anaerobic conditions. Substantial losses of Hz evolution activity were observed after a 2-day incubation of cell-free extracts prepared anaerobically in the following buffers (all 50 mM concentration): citrate/acetate, pH 4.5, MES, pH 6.0, triethanolamine, pH 7.5, HEPES, pH 7.5, Tricine, pH 8.2, ethanolamine, pH 9.2, glycine, pH 9.3, and CAPS, pH 10.1. In contrast, no activity was lost when Tris- HC1, pH 7.5, 8.0, and 8.5, potassium phosphate, pH 7.2, MOPS, pH 7.2, or EPPS, pH 8.4, were used. These buffers were therefore used for the purification procedure.

Electrophoresis of cell-free extracts under nondenaturing conditions (lo%, w/v, acrylamide) revealed two bands of hydrogenase activity (RF values of 0.20 and 0.34) suggesting the presence of two distinct hydrogenase species. Initial attempts to separate these by ion-exchange chromatography on a Mono Q column (HR 5 / 5 ) gave confounding results. Hy- drogenase activity (Hz evolution) was detectable in almost all fractions between 0.2 and 1.0 M C1- after gradient elution (0- 1.0 M C1-, pH 8.0) with two main peaks of activity at -0.28 and -0.43 M C1- and a minor peak at -0.59 M Cl-. Moreover, all active fractions gave rise to the same two hydrogenase activity bands after electrophoresis on nondenaturing gels. Gel filtration on separate calibrated columns of Superose 6 (using 50 mM Tris-HC1 buffer, pH 8.0, containing 0.2 M NaCl as the eluant) of the two main activity peaks from the Mono Q column both gave similar results: no definitive protein peaks and a spread of hydrogenase activity corresponding to M, values between 200,000 and several million. It was subsequently found that these anomalous results were obtained only when cell-free extracts had been previously stored over- night at 4 "C. Extracts maintained at 23 "C gave only a single peak of hydrogenase activity eluting at -0.28 M c1- after applying a gradient (0-1.0 M C1-, pH 8.0) to a Mono Q ion- exchange column, and gave one major peak of Hz evolution activity (M, -200,000) after gel filtration on a Superose 6 column. However, subsequent analyses of these active fractions still gave rise to two activity bands on nondenaturing gels (see below).

Prolonged incubation of cell-free extracts at 4 "C therefore appears to promote some form of aggregation or association of the hydrogenase(s) and this gives rise to the anomalous chromatographic behavior described above. The "aggregated" form could also be distinguished by its kinetic properties. The rate of Hz production at 80 "C was nonlinear and a lag of phase of 4-8 min was observed. In contrast, cell-free extracts maintained at 23 "C gave linear rates of Hz evolution over the 10-min assay period. Attempts to prevent and/or reverse this apparent aggregation phenomenon using urea (6 M), high salt (1.0 M NaCl), glycerol (up to lo%, v/v), polyethylene glycol

P. furiosus Hydrogenase 5073 TABLE I

Purification of hydrogenase from P. furiosus Step Volume Protein Activity Specific activity Purification Recovery

ml mg units unitslmg fOM % Cell extract 1000 9460 9900 1.04 1 100 Q-Sepharose, pH 8.3 351 926 6938 7.5 7.2 70 DEAE-Sephacel, pH 7.7 150 56 5342 95.4 92 54 Hydroxyapatite 86 22 4180 190 163 42 Superose 6 19 11 3960 360 350 40

(up to lo%, w/v), dithiothreitol (up to 10 mM), or ethanol (up to 20%, v/v) were unsuccessful. However, the HZ evolution activity was remarkably stable since none of these reagents caused significant inhibition.

The purification procedure was therefore carried out at 23 "C and active fractions were routinely maintained at this temperature. All steps were performed anaerobically and only Tris and phosphate buffers were used. The results of the purification procedure are summarized in Table I. The Hz evolution activity was purified 350-fold over the cell extract with a yield of 40%. About 11 mg of the purified enzyme with a specific activity of -360 units/mg were obtained from 450 g of cells (wet weight).

Molecular Composition of Hydrogenuse-The hydrogenase preparation purified by the above procedure gave rise to a single peak of protein coincident with a single peak of HP evolution activity after elution from columns of Mono Q (eluting at 0.28 M C1-) and Superose 6. It eluted from calibrated columns of both Superose 6 and Superose 12 with an apparent M, of 185,000 f 15,000. However, these enzyme samples still exhibited two protein bands with RF values of 0.20 and 0.34 after electrophoresis on nondenaturing gels (lo%, w/v acrylamide). The estimated stain intensities were 75% (upper band) and 25% (lower band). Both of these protein bands stained for hydrogenase activity, and they corresponded to the two activity bands observed when cell-free extracts were subjected to electrophoresis and stained for hydrogenase activity. Furthermore, the two protein species from the pure hydrogenase had identical subunit compositions as judged by SDS-gel electrophoresis (see below). These two active protein bands therefore appear to be electrophoretically distinguish- able components of a single hydrogenase rather than two distinct enzymes.

The pure hydrogenase also exhibited anomalous behavior upon SDS-gel electrophoresis. The results depended on the pretreatment temperature and the gel pore size. For example, after treating the hydrogenase with SDS at 90 or 105 "C for 10 min, electrophoresis in the presence of SDS (using lo%, w/v, acrylamide) revealed one major and very sharp protein band corresponding to a M, value of approximately 46,000, and two minor and rather diffuse protein bands of M, = 27,000 and 24,000. However, as shown in Fig. lA (lanes 3 and 4) , on gels containing 215% (w/v) acrylamide, (a) the two lower molecular bands were much sharper and of intensity comparable to the M, = 46,000 species, and (b) an additional and rather diffuse protein band corresponding to M, = 41,000 was observed. When the M, = 41,000 and 46,000 bands were excised from unstained gels (15%) and rerun on gels containing 10% (w/v) acrylamide, only the M, = 46,000 band was apparent. This suggests that the M, = 41,000 species may be an artifact, possibly a dimer of two lower molecular weight bands which are not readily observed on 10% gels. In addition, a rather diffuse band which migrated ahead of the dye front was also observed from the hydrogenase preparation independent of the acrylamide concentration used (Fig. lA). This

1 2 3 4 2 3 4 FIG. 1. SDS-polyacrylamide gel electrophoresis of P. turio-

SUB hydrogenase. Hydrogenase samples (1.1 mg/ml) were incubated with an equal volume of SDS (I%, w/v) for 10 min at 80 'C ( l a n e 2), 90 "C ( l a n e 3) , and 105 "C ( l a n e 4) prior to electrophoresis on 15% (w/v) acrylamide gels containing SDS (O.l%, w/v). Approximately 25 pg of protein were applied to each lane. After electrophoresis, gel A was stained for protein andgel B was stained for hydrogenase activity as described under "Materials and Methods." Lane 1 contains the following proteins with the indicated subunit molecular weight (top to bottom): phosphorylase a (92,5001, ovalbumin (45,000), chymo- trypsinogen A (25,000), lysozyme (14,000), and cytochrome c (12,000).

low molecular weight species is ubiquitous in P. furwsus since it is evident upon SDS-gel electrophoresis of cell-free extracts or any (partially) purified component examined so far. At- tempts to isolate this low molecular weight moiety are under- way since an intriguing possibility is that it is related to the high thermostability of proteins from this organism.

When the hydrogenase was incubated with SDS at only 80 "C for 10 min, yet another protein band was observed after electrophoresis and this corresponded to M, = 92,500 (Fig. lA, lane 2). This band retained hydrogenase activity as determined by activity staining, in contrast to any of the lower molecular weight bands (Fig. lB, lane 2). The M, = 92,500 species appears to be the precursor of the lower molecular weight bands since it was the predominant protein band when the hydrogenase was pretreated with SDS at 60 "C. Since the M, = 41,000 species may be an artifact of electrophoresis, the hydrogenase appears to be comprised of three different subunits with M, values of 46,000 (a), 27,000 (@), and 24,000 (y), giving a minimum M, of 97,000 (a@y). It is therefore tempting to suggest that the M, = 92,500 species is a trimer (a@y), while the holoenzyme (M, - 185,000) is hexameric (a&yz). However, since the former is catalytically active, it is unlikely to be unfolded and would migrate faster than its true molecular weight. Further investigation is therefore required to determine the nature of the M, = 92,500 species. The two active protein bands observed from the native enzyme on nondenaturing gels were found to have the same subunit compositions since they gave rise to identical patterns of protein bands after excision and SDS-gel electrophoresis. It therefore appears that under electrophoretic conditions, the


holoenzyme (M, - 185,000) either partially associates to a higher molecular weight form, or partially dissociates (perhaps to the active M, = 92,500 species), and these give rise to the two active protein bands observed on nondenaturing gels.

The pure hydrogenase samples were yellowish-green in color and showed increased absorption with decreasing wave- length in the visible region, but there were no defined peaks. The extinction coefficient at 420 nm was 78 mM- cm". The absorption at 420 nm increased approximately 30% upon air oxidation suggesting the presence of iron-sulfur chromo- phores. Analysis of three different hydrogenase samples using colorimetric assays revealed the presence of 31 f 3 g atoms of iron and 24 * 4 g atoms of acid-labile sulfide/183,000 g of protein. In addition, two preparations of the hydrogenase were analyzed for nickel by plasma emission spectroscopy and were found to contain 0.98 f 0.05 g atoms/mol. Since this corresponds to only -0.5 atoms of nickel/minimum M, of 97,000 (aj3y), either (a) a significant fraction of the nickel is lost during purification, (b) the nickel is a structural component not involved in catalysis, or (c) the nickel is adventi- tiously bound. At the present time we cannot distinguish between these possibilities (see below). The amino acid composition of the hydrogenase is given in Table 11. The protein contains approximately the same number of cysteine residues as it does iron atoms suggesting that most, if not all, of these residues are involved in coordinating iron-sulfur clusters, assuming the latter are of the conventional type (see below). The protein concentration of the hydrogenase determined by amino acid analysis agreed (+8%) with that measured by the Lowry colorimetric assay.

Stability of Hydrogenase-The hydrogenase was much more sensitive to inactivation by Oz in a pure state compared with cell-free extracts. When the pure enzyme (1 mg/ml in 50 mM Tris-HC1 buffer, pH 8.0, containing 2 mM sodium dithionite) was briefly shaken in air to oxidize the sodium dithionite and then left exposed for 6 h, 53% of the Hz evolution activity remained when the sample was subsequently degassed and flushed with Ar and assayed under standard conditions at 80 "C. However, the inhibition was reversible since >95% of the original activity was recovered if the air-treated sample was subsequently degassed and flushed with Ar and incubated with sodium dithionite (10 mM in 50 mM EPPS buffer, pH 8.4) for 30 min prior to performing the assay. Under anaerobic conditions the hydrogenase lost no activity after 2 months at

TABLE rr Amino acid composition of P. furiosus hydrogenase

Residue Residues/mol"

Asp/Asn 153 Thr 61 Ser 50 Glu/Gln 184 Pro 94 G ~ Y 141 Ala 112 Cysb 33 Val 121 Met 41 Ile 106 Leu 149 TYr 77 Phe 86 His 31 LYS 135 Arg 93 Trp ND'

Based on a calculated molecular weight of 187,572. Based on performic acid oxidation.

e ND, not determined.

23 "C. There was also no loss of activity upon freezing the enzyme in liquid NZ and thawing under Ar.

The hydrogenase was remarkably thermostable since at low concentrations in dilute anaerobic buffer it retained most of its HZ evolution activity after a 1-h incubation at 100 'C (Fig. 2). The times required for a 50% loss of activity ( t6OW) at 105, 100,90, and 80 "C were approximately 5 min, 2 h, 3, and 21 h, respectively. Curiously, over shorter periods (up to 1 h), the enzyme was slightly more stable at 100 "C than 90 "C (Fig. 2). The hydrogenase was much less stable under reducing conditions since, as shown in Fig. 2, the t60% value decreased to -30 min at 100 "C if sodium dithionite was also present. Rather surprisingly, the presence of P. furwsus ferredoxin, which is stable at 100 "C for 1 h,2 decreased the tsow value for the reduced hydrogenase to -10 min at 100 "C. The hydrogenase was even less thermostable under aerobic conditions: t 6 0 ~ at 100 "C was less than 5 min. All of these results were obtained by the direct transfer of the hydrogenase samples from the incubation vials to the assay medium at 80 "C. Almost identical results were obtained if the enzyme sample was first cooled to 23 or 4 "C for 5 min before assaying.

Kinetic Properties-The rate of hydrogenase-catalyzed Hz evolution with methyl viologen as the electron carrier increased 40-fold between 45 and 95 "C, as shown in Fig. 3a. A triphasic Arrhenius plot was obtained with transition temperatures of 60 and 80 'C (Fig. 4a). P. furwsus ferredoxin also served as an efficient electron carrier for Hz evolution by the hydrogenase, and we presume that it is the physiological electron donor. Compared with methyl viologen, an even more dramatic increase in the rate of Hz evolution with increasing temperature was observed with ferredoxin (Fig. 3b). There was a 350-fold increase between 45 and 90 "C. This also gave rise to a triphasic Arrhenius plot (Fig. 46) with transition temperatures of 65 and 80 "C. The calculated Esct values (in kcal/mol) at 55, 75, and 95 "C are 25.6, 6.8, and 23.6 for methyl viologen and 18.3, 35.1, and 57.2 (at 90 'C) for ferredoxin, respectively. The optimum temperature for Hz evolution from reduced methyl viologen was >95 "C compared with -90 "C for reduced ferredoxin. Double reciprocal plots of Hz evolution activity at 80 "C uersw electron carrier concentration were linear for both methyl viologen (concentration range 0.3-10.0 mM) and ferredoxin (5-250 phi). The apparent K,,, and V, values are 5.3 mM and 2900 clmol HP evolved/min/mg for methyl viologen and 79 ~ L M and 250 pmol HZ evolved/min/ mg for ferredoxin, respectively. In the absence of an electron

120 , I 100

80

60

40

20

0 0 20 4 0 6 0 80 100 120 1 4 0

Time (mln)

FIG. 2. Thermostability of P. furioeucr hydrogenase. The hydrogenase (0.15 mg/ml in anaerobic 50 mM EPPS, pH 8.4, final volume 300 p1) was incubated under Ar in sealed 8-ml vials in a Temp Blok Module Heater (American Scientific Products) at the indicated temperature. Samples (10 pl) were removed at various times and injected directly into assay vials (at 80 "C) to determine residual Hz evolution activity (see "Materials and Methods"). The activity is expressed as a percentage of that measured at time 0. +DT indicates that the incubation buffer contained sodium dithionite (1 mM).


1600 a) METHYL VIOLOGEN

i200 i 800 i 400

0 / 350 - b) FERREDOXIN

300 - 250 - 200

150 - 100

-

-

4 0 50 60 70 80 90 100

Temperature ("C)

FIG. 3. Effect of temperature on the catalytic activity of P. furiosus hydrogenase. The Hz evolution (a and b) and Hz oxidation (c) activity of the hydrogenase was determined at various temperatures using methyl viologen (1 mM), ferredoxin (0.02 mM), or methylene blue (0.04 mM) as the electron carrier. Assays were performed as described under "Materials and Methods". The results are expressed as Fmoles of Hz evolved (a and b) or consumed ( c ) per min/ mg.

carrier, the rate of Hz evolution from sodium dithionite (20 mM) at both 60 and 80 "C was -3% of that observed with methyl viologen (1 mM). This activity was unaffected by the presence of NADH (0.1-10 mM, see Ref. 44) showing that this nucleotide does not act as an electron donor to the hydrogenase.

A comment is appropriate on the problems of determining hydrogenase activity at high temperatures. Changes in pH over the temperature range investigated were minimized by using EPPS buffer (pKa = 8.0 at 20 "C). This shows a rela- tively small decrease in pK. with temperature (ApK,/"C = -0.007), in contrast to Tris-HC1 buffer (pK, = 8.3 at 20 "C, ApKJC = -0.031), the usual choice for hydrogenase assays. Thus, because of the instability of sodium dithionite (the reductant in the Hz evolution assay) at low pH (45), the ODm of the blue assay mixture of reduced methyl viologen in 50 mM Tris-HC1 buffer, pH 8.0 (at 23 "C) decreased by 90% at 90 "C (the actual pH was probably 6 or less) whereas the ODsw remained constant between 23 and 90 "C in 50 mM EPPS buffer, pH 8.4 (at 23 "C). When MES, pH 6.2, potassium phosphate, pH 7.2, MOPS, pH 7.2, triethanolamine, pH

7.2, HEPES, pH 7.8, ethanolamine, pH 9.2, CHES, pH 9.3, and CAPS, pH 10.3, all at concentrations of 100 mM, were each used to buffer a solution of sodium dithionite (10 mM) and methyl viologen (1 mM) using only HEPES, CHES, and CAPS there were no significant differences in the absorption at 600 nm after a 10-min incubation at 23 and 90 "C. In addition, the quality (purity) of the sodium dithionite used in the Hz evolution assay was critical. Material containing <70% Na2S204 (w/w, as determined by anaerobic titration with FMN: 45) maintained the blue color of reduced methyl viologen (in EPPS buffer, pH 8.4) for less than 2 min at 90 "C.

P. furiosus hydrogenase also catalyzed HZ oxidation with methylene blue as the electron acceptor. A double reciprocal plot of Hz oxidation activity at 80 "C versus methylene blue concentration was linear in the range 0.004-0.1 mM. The apparent K, value was 0.08 mM and V, was 261 pmol of HZ oxidized/min/mg. Methyl viologen also served as an electron acceptor for hydrogenase-catalyzed Hz oxidation. However, the double reciprocal plot was linear only in the range 0.05- 1.0 mM: concentrations of methyl viologen above 1.0 mM strongly inhibited the reaction. The calculated V,,, and K,,, values at 80 "C were 845 pmol of HZ oxidized/min/mg and 1.9 mM, respectively. Under standard assay conditions at 80 "C (using 1 mM methyl viologen or 0.04 mM methylene blue as the electron acceptor, see "Materials and Methods"), these two electron carriers supported similar rates of Hz oxidation (84-91 pmol of Hz oxidized/min/mg). However, these activities are only -25% of the Hz evolution activity of the hydrogenase at 80 "C (360 units/mg using 1 mM reduced methyl viologen). The effect of temperature on the rate of Hz oxidation using methylene blue as the electron acceptor is shown in Fig. 3c). The optimum was >95 "C. The corresponding Arrhenius plot (Fig. 4c) shows two transition points at 60 and 82 "C. The E,,, values a t 55, 75, and 95 "C were 26.8, 7.2, and 2.6 kcal/mol, respectively. The 95 "C value is about 10-fold less than that for Hz evolution with either methyl viologen or ferredoxin as the electron carrier. Neither NAD (0.1-5 mM, see Ref. 44) or coenzyme F4z0 (20-100 pM, see Ref. 29) were reduced by the hydrogenase in the presence of HZ at either 60 or 80 "C.

The thermodynamic data show that there are two temperature-dependent transition points during catalysis by the hydrogenase occurring at approximately 60 and 80 "C. Both are independent of the electron carrier used and the mode of assay. A plot of the ratio of Hz evolution activity/Hz oxidation activity versus temperature (Fig. 5) reveals that the higher of the two transition points appears to be associated with a dramatic change in the catalytic activities of the enzyme in favor of Hz evolution. The transition at -60 "C is not reflected by a change in the catalytic activity of the enzyme since the ratios remain more or less unchanged until above 80 "C. Of relevance is the fact that P. furiosus shows reasonable growth only above 80 "C with an optimum around 100 "C (6). This fermentative-type organism presumably has to evolve Hz to reoxidize reduced electron carriers, e.g. ferredoxin, a process that must occur for growth to continue. Hence the hydrogenase exhibits a remarkable catalytic shift in favor of Hz evolution at the growth temperature. Similarly, the rate of Hz production with P. furiosus ferredoxin as the electron donor becomes significant only at 80 "C and above (Fig. 3b). How- ever, the in vitro thermostability of the hydrogenase and of the ferredoxin at the growth temperatures is puzzling. Both proteins are independently very stable 280 "C, but the stability of the hydrogenase decreases significantly when it is reduced and when ferredoxin is present. Since both of these

5076 P. furiosus Hydrogenase 3.50 1 I

a) METHYL VIOLOGEN

2.50 - 2.25 -

2.00

1.75

- -

1.5

1 .o

0.5

0.0 I

2.0

1.8

1.6

1.4

1.2

1 .o

0 . 8 ~ ” ” ” ” ’ ~ 2.7 2.8 2.9 3.0 3.1 3.2

1000/K

FIG. 4. Arrhenius plots of Hz evolution and Hz oxidation catalyzed by P. furiosus hydrogenase. The data are taken from Fig. 3 and the electron carrier is as indicated. a and b represent H2 evolution activity while c depicts H, oxidation activity.

11 -

9 -

7 -

5 -

3 1 & , , , I 1 40 50 60 70 80 90 100

0

Temperature (“C)

FIG. 5. Effect of temperature on the catalytic preference of P. furiosus hydrogenase. The data are taken from Fig. 3 where MV/MeB and Fd/MeB are the ratios of specific activities in HZ evolution (with methyl viologen ( M V ) or ferredoxin (Fd) as the electron carrier)/Ha oxidation (with methylene blue as the electron acceptor) at the indicated temperature.

conditions probably exist in growing cells, other stabilizing factors must exist in vivo.

Electron Paramagnetic Resonance Properties-The hydrogenase as isolated in its reduced form exhibited a sharp rhombic EPR signal (gz = 2.03, g, = 1.93 and g, = 1.92) at 70 K that represented 0.96 f 0.07 spins/mol (Fig. 6a). This was the only signal evident upon lowering the temperature to 20 K (Fig. 6b), conditions under which EPR absorption from reduced [4Fe-4S] clusters is typically seen (46). The g values, line- shape, and temperature dependence of the observed EPR signal are consistent with the presence of a single, reduced [2Fe-2S] cluster. A much more complex spectrum was seen below 15 K (Fig. 6c) with additional features at g values of 1.88, 1.96, 1.99, and 2.05, together with a broad line around g = 2.14. The temperature dependence and complexity of the EPR spectrum at 10 K suggests the presence of (at least) two interacting iron-sulfur clusters with extremely rapid spin relaxation rates. Interestingly, EPR signals from the iron-sulfur clusters of P. furiosus ferredoxin also have quite remarkable relaxation properties.’ It remains to be determined, however, if this is a general feature of iron-sulfur proteins from extreme thermophiles. The complete EPR spectrum seen at 10 K quantitated to only 1.90 & 0.15 spins/mol indicating that a significant fraction of the iron in the reduced enzyme is not present as conventional iron-sulfur clusters. No EPR absorption was detected from the reduced enzyme at lower magnetic fields (c0.31 Tesla) between 4.5 K and 70 K. The EPR signals from the reduced hydrogenase disappeared upon anaerobic oxidation of the enzyme with excess thionine (E,,, = +11 mV) giving an EPR silent state. There was no evidence of the rhombic EPR signal from Ni I11 ( g z typically 2.30) seen from other Ni-hydrogenases.

DISCUSSION

Although hydrogenases are a heterogeneous group of enzymes, all are iron-sulfur proteins and the majority also

0.31 0.33 0.35 0.37 Magnetic Field (T)

FIG. 6. Electron paramagnetic resonance spectra of reduced P. furiosus hydrogenase. The enzyme (7.5 mg/ml) was prepared as described under “Materials and Methods.” The temperature, microwave power, and gain settings were Q, 70 K, 2 mW and 5 x los, b, 20 K, 2 mW and 5 X lo5, and c, 10 K, 50 mW and 4 X lo‘. Other conditions were: time constant, 0.164 s; sweep time, 870 s; modulation amplitude, 2.5 millitesla; microwave frequency, 9.41 GHz.


contain nickel (14, 15). In comparison with other hydrogenases, the P. furiosus enzyme has several unique properties, yet it also shares certain characteristics peculiar to the hydrogenases of some aerobic eubacteria, of some anaerobic eubacteria, and of other archaebacteria. For example, it is the only hydrogenase that preferentially catalyzes HZ evolution and is the most thermostable hydrogenase so far purified. On the other hand, (a ) its physiological role is to catalyze HZ production, ferredoxin appears to be its electron carrier i n vivo and it catalyzes Hz evolution at high rates i n uitro, all characteristics of the "Fe-only" hydrogenases of the anaerobic clostridia; (b) it gives rise to an EPR signal typical of a [2Fe- 2S] cluster but does not exhibit an EPR signal from nickel, characteristics of the NiFe-hydrogenases of the aerobic hydrogen bacteria; and (c ) its subunit structure and metal content are very similar to the hydrogenase of a methanogenic archaebacterium. We will briefly discuss each one of these points.

Hydrogenases are generally considered to be rather thermostable enzymes, and heat treatment steps have been used in their purification (12-14). However, almost all are rapidly inactivated upon incubation for several minutes at temperatures above 70 "C. The most stable are some of the enzymes from sulfate-reducing bacteria, e.g. Desulfouibrio desulfuricans (47) and D. gigas (see Ref. 48), and from photosynthetic bacteria, e.g. Thiocapsa roseopersicina (49) and Rhodospirillum rubrum (50), yet even these lose at least 50% of their activity after a 20-min incubation at 80-85 "C. For comparison, the time required for P. furiosus hydrogenase to lose 50% of its activity at 80 "C was -21 h. Indeed, the P. furiosus enzyme appears to be one of the most thermostable enzymes known at present, comparable to the most stable proteinase (t50% -80 min at 95 "C, Ref. 51) and a-amylase (tso% 1-2 h at 95 "C; see Refs. 8, 52). The mechanisms of the thermoinactivation of proteins at 100 "C were elegantly elucidated by Klibanov and co-workers (53) who showed that, in the case of lysozyme, deamidation of asn residues, hydrolysis of certain peptide bonds, destruction of cystine residues, and the formation of incorrect structures upon cooling were all involved. The latter mechanism appears not to be significant with P. furiosus hydrogenase, however, since similar results were obtained regardless of whether the enzyme solution was first cooled to 23 "C (or 4 "C) before assaying (at 80 "C).

The optimum temperatures for catalyzing Hz evolution and Hz oxidation by P. furiosus hydrogenase using artificial electron carriers were >95 "C. For comparison, the highest temperature previously reported for Hz production was 78-80 "C for the hydrogenase of T. roseopersicina (49). This gave a linear Arrhenius plot (30-80 "C) and a calculated E,, value of 16 kcal/mol. Comparable values of 6.8 kcal/mol (at 75 "C) and 23.6 kcal/mol (at 95 "C) were obtained for Hz production by P. furiosus hydrogenase. Schlegel and co-workers (54) reported a temperature optimum of ~ 9 0 "C for Hz oxidation with methylene blue as the electron acceptor by a partially purified hydrogenase from the thermophile, Bacillus schlegelii (To,, -70 "C), with an E.,, value of 4.7 kcal/mol (the stability of this enzyme at high temperatures was not reported). This compares with values of 7.2 kcal/mol (at 75 "C) and 2.6 kcal/ mol (at 95 "C) for the same reaction catalyzed by the P. furiosus enzyme. These low activation energies suggest that Hz oxidation catalyzed by these enzymes may be diffusion limited at high temperatures. The F420-reducing hydrogenase of the thermophilic methanogen, M. thermoautotrophicum ( Top, -60 "C), which has a temperature optimum for catalyzing HZ oxidation of -80 "C (55), and the hydrogenase of B. schlegelii (54), both show breaks in their Arrhenius plots for

Hz oxidation at -15 "C below the optimum growth temperature of the organism. Transition temperatures at 60 and 80 "C were observed with P. furiosus hydrogenase ( To,, for growth, -100 "C), independent of the mode of assay and the electron carrier used. That occurring at 80 "C was associated with a dramatic switch in the catalytic preference of the enzyme, a phenomenon not investigated with the hydrogenases of these other organisms.

The NiFe-hydrogenases are usually referred to as "uptake" hydrogenases, a term which reflects their physiological role and their ability to preferentially catalyze HZ oxidation in uitro. Indeed, many exhibit barely detectable rates of Hz production. In contrast, the "Fe-only" hydrogenases, SO far purified only from anaerobic bacteria where they function to produce Hz, are, with one exception (56), very active in catalyzing Hz evolution i n vitro (see Ref. 23). It is therefore surprising that the rate of Hz evolution catalyzed by the Ni- containing hydrogenase of P. furiosus is comparable to those exhibited by the Fe-hydrogenases, e.g. 2900 units/mg (at 80 "C) uersus 4000 units/mg for Clostridium pasteurianum hydrogenase I (57). Moreover, under the usual assay conditions, even the Fe-hydrogenases show much higher rates of Hz oxidation, e.g. with methylene blue as the electron acceptor, compared to rates of Hz evolution, i.e. with reduced methyl viologen as the electron donor (see Ref. 23). Activity ratios (Hz evolution/HZ oxidation) are always <<1, e.g. for C. pasteurianum hydrogenase I it is 0.2 (56). The hydrogenase of P. furiosus therefore appears to represent a new type of "evolution" hydrogenase that is thermodynamically primed to catalyze Hz production even at low temperatures (45 "C). This is especially apparent at the growth temperature of the organism where the in vitro activity ratio of the hydrogenase rises from 4 to about 12 (Fig. 5).

All Ni-hydrogenases have so far been purified aerobically and are typically inactive in catalyzing Hz oxidation without some form of reductive activation. Furthermore, the enzymes as isolated characteristically exhibit a novel rhombic EPR signal with g values of -2.3, 2.2, and 2.0 (see Refs. 14, 15). This signal, first identified by Lancaster (58), has been un- ambiguously assigned to nickel (probably as Ni 111) in several hydrogenases and is referred to as the Ni-A signal (E,,, range, -150 to -400 mV: see Ref. 15). In addition, two other rhombic EPR signals arising from nickel (Ni-B and Ni-C, both g,, >2) have been observed from various hydrogenases during reductive activation. The Ni-C EPR signal (E , "330 mV), possibly representing a Ni (I) species, is thought to be involved in the reaction with HP (21). Many Ni-hydrogenases as isolated also exhibit EPR signals from an oxidized 3Fe-cluster (E , range +25 to -50 mV; see Ref. 15). In contrast to almost all Ni-hydrogenases, EPR signals attributable to a Ni center or a 3Fe-cluster were not observed from P. furiosus hydrogenase in its oxidized state. It should be noted that this is the first Ni-hydrogenase that has been purified under anaerobic (reducing) conditions and, as isolated, it does not require reductive activation. Transient EPR signals from nickel, e.g. Ni-C, might be apparent only during a redox titration. How- ever, the soluble, aerobically prepared, Ni-hydrogenases from aerobic hydrogen bacteria, e.g. Alcaligenes eutrophus (59, 60) and Nocardia opaca (61), do not exhibit EPR signals from nickel. These are also the only hydrogenases to date that exhibit high temperature (>40 K) EPR signals typical of reduced [2Fe-2S] clusters (59). Moreover, like the P. furiosus enzyme, they are multisubunit enzymes that, in the case of N. opaca, show anomalous behavior upon gel electrophoresis (62). The catalytic role of nickel in N. opaca hydrogenase is unclear since, in addition to two tightly bound nickel atoms,


two loosely bound nickel ions are required to hold together the subunits of the enzyme (61,62). Since P. furwsus hydrogenase as isolated contains only -1 Ni atom/mol, and exhibits catalytic properties similar to an Fe-hydrogenase, nickel may play a structural rather than catalytic role in the P. furiosus enzyme. P. furiosus hydrogenase is the first hydrogenase to be iso-

lated from a non-methanogenic archaebacterium and has some properties in common with the hydrogenases of other archaebacteria, namely methanogens. These organisms contain two different types of hydrogenases, one reduces the methanogenic coenzyme factor 420 (Fdz0) while the other does not (29,48,63). It is with the former type that the P. furiosus enzyme has some similarities. For example, the F420-reducing hydrogenase of the thermophilic methanogen, M. thermoautotrophicum (strain AH, 31) has an almost identical subunit structure and metal content, it also exists as a higher molecular weight aggregate at 4 "C, and it also exhibits several activity bands after nondenaturing gel electrophoresis (64). However, in contrast to P. furiosus hydrogenase, the M. thermoautotrophicurn enzyme readily reduces coenzyme F420

with HZ and exhibits EPR signals characteristic of nickel but not of a reduced [2Fe-2S] cluster (64).

Thus, of all hydrogenases purified to date, that of P. furiosus has some properties in common with both the soluble hydrogenases of mesophilic, aerobic hydrogen bacteria, and with the F4zo-reducing hydrogenase of a methanogenic bacterium. The physiological role of hydrogenase is Hz oxidation in both the aerobic hydrogen bacteria and in the methanogens wherein NAD and F 4 2 0 is the natural electron carrier, respectively. Indeed, these hydrogenases will not evolve Hz from reduced ferredoxin. In contrast, P. furwsus hydrogenase functions in vivo to evolve Hz, and reduced ferredoxin appears to be the physiological electron donor, as it is for the Hz-evolving hydrogenases of the anaerobic, fermentative clostridia (see Ref. 13). Since the So-metabolizing, extremely thermophilic bacteria are considered an ancient and primitive phenotype, perhaps the most closely related of all life forms to a universal ancestor (see Ref. 3), it is interesting that the hydrogenase of P. furiosus has characteristics of the enzymes from other archaebacteria, and from both anaerobic and aerobic eubacteria.

Acknowledgments-We thank Dr. Juergen Wiegel for his assistance in culturing the bacterium and for many helpful discussions, Tracy Miller for excellent technical support, and Dr. James B. Howard for amino acid analyses.

1.

2.

3. 4.

5.

6. 7.

8.

9. 10.

11.

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the journal of vol. 264, no. 9. of 25. pp. 5070-5079,1969 ... · all are archaebacteria (3) ... the...

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