Abstract
The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (αβγδ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H2 production and the reduction of elemental sulfur to H2S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an Mr of 320,000 ± 20,000 and contains four different subunits with Mrs of 52,000 (α), 39,000 (β), 30,000 (γ), and 24,000 (δ). The heterotetramer contained Ni (0.9 ± 0.1 atom/mol), Fe (21 ± 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 ± 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H2 production with Km values near 70 μM and kcat/Km values near 350 min−1 mM−1. In contrast to hydrogenase I, hydrogenase II catalyzed the H2-dependent reduction of NAD (Km, 128 μM; kcat/Km, 770 min−1 mM−1). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H2 and NADPH served as electron donors for the reduction of elemental sulfur (S0) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H2 using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the β subunit and three in the δ subunit) and one [2Fe-2S] cluster (in the γ subunit), as well as two putative nucleotide-binding sites in the γ subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S0 concentrations since it has a higher affinity than hydrogenase I for both S0 and polysulfide.
Pyrococcus furiosus is an archaeon that grows optimally near 100°C by the fermentation of carbohydrates and peptides to produce acetate, CO2, and H2 (9). If elemental sulfur (S0) is present in the medium, it is reduced to H2S. It has previously been shown that the organism contains two enzymes that are able to catalyze the reduction of S0 to H2S, hydrogenase I (H-I) and sulfide dehydrogenase. The hydrogenase, also known as sulfhydrogenase because of its S0-reducing activity, is a Ni-containing, iron-sulfur flavoprotein that also serves to reduce H+ to H2 (16). Sulfide dehydrogenase is an iron-sulfur flavoprotein which also functions as a ferredoxin:NADP oxidoreductase (13). The two enzymes are thought to transfer electrons from reduced ferredoxin and NADPH, generated from the fermentation pathways, to the terminal electron acceptors, H+ and S0.
It was assumed that S0 reduction by P. furiosus was merely a means of removing the H2 produced during fermentation, as H2 inhibits growth (9). However, the presence of S0 in the growth medium both stimulates the growth rate and increases the cell yield by about 50%, suggesting that H2S production is an energy conservation process (19). Yet, H-I and sulfide dehydrogenase are cytoplasmic enzymes and a conventional respiratory mechanism with S0 as a terminal electron acceptor is not present in this organism. It is thought that S0 reduction may increase the ratio of oxidized to reduced electron carriers, thereby favoring the oxidative rather than the nonoxidative decarboxylation of the 2-keto acids produced by ferredoxin-linked oxidoreductases during fermentation (14). How this process is regulated, however, is not known.
In this paper, we show that the energy metabolism of S0 by P. furiosus may be even more complicated than was originally thought. A second enzyme, termed hydrogenase II (H-II), has been identified and purified. It is demonstrated that both it and H-I catalyze the reduction of S0 to H2S using NADPH as the electron donor. In fact, both enzymes preferentially reduce S0 rather than protons. The biochemical and molecular characterization of this new H2-evolving, S0-reducing enzyme is presented.
MATERIALS AND METHODS
Growth of P. furiosus.
P. furiosus (DSM 3638) was routinely grown at 90°C in a 600-liter fermentor with maltose as the carbon source as described previously (6).
Enzyme assays.
The activity of H-II was routinely measured during purification by the H2-dependent reduction of benzyl viologen at 80°C. The assay mixture (2 ml) contained 100 mM EPPS [N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid] (pH 8.0) and 1 mM benzyl viologen with H2 in the gas phase. The reaction was monitored at 580 nm, and a molar absorptivity of 7,800 M−1 cm−1 was used for reduced benzyl viologen. The S0 reduction activity of hydrogenase was determined at 80°C by measuring H2S production by methylene blue formation (16) or by NADPH oxidation at 365 nm using a molar absorbance of 3,200 M−1 cm−1. Reaction mixtures contained either S0 (5% [wt/vol]) under H2 (1 atm), S0 (5% [wt/vol]) and NADPH (0.4 mM) under Ar, or polysulfide (up to 3 mM) and NADPH (0.4 or 1 mM) under Ar. Rates of sulfide production measured by methylene blue formation were calculated over a time of 15 min. The H2 oxidation and H2 evolution activities of the enzyme were also measured at 80°C using methyl viologen, NAD(H), NADP(H), and P. furiosus ferredoxin as the electron carriers as previously described (16, 17). When ferredoxin was reduced using pyruvate as the electron donor via pyruvate ferredoxin oxidoreductase (POR) from P. furiosus, the 2-ml assay mixture contained 100 mM EPPS buffer (pH 8.0), pyruvate (10 mM), coenzyme A (4 mM), thiamine PP, (0.05 mM), ferredoxin (44 μM), POR (324 μg), and H-II (250 μg) or H-I (234 μg). Where indicated, the reaction mixtures also contained ferredoxin:NADP oxidoreductase (FNOR) from P. furiosus (100 μg) and NADP (1.6 mM). For the H2-dependent reduction of ferredoxin, the 2-ml assay mixture contained, under an atmosphere of H2, 75 mM EPPS buffer (pH 8.0), ferredoxin (0.11 mM), and H-I (234 μg) or H-II (250 μg). H-I (16), POR (3), FNOR (13), and ferredoxin (1) were purified from P. furiosus as described in the references. All activities for hydrogenase are expressed in units per minute, where 1 U catalyzes the oxidation of production of 1 μmol of H2 per min.
Enzyme purification.
P. furiosus H-II was purified at 23°C under anaerobic conditions. Frozen cells (100 g [wet weight]) were thawed in 400 ml of buffer A (50 mM Tris-HCl [pH 7.8] containing 2 mM dithiothreitol and 2 mM sodium dithionite) containing DNase I (10 μg/ml) and 0.2 mg of lysozyme/ml. The cells were lysed by incubation at 37°C for 2 h. A cell extract was obtained by centrifugation at 50,000 × g for 2 h. The supernatant (340 ml) was loaded onto a column (5 by 12 cm) of DEAE-Sepharose Fast Flow equilibrated with buffer A. The column was eluted with a linear gradient (1.2 liter) of 0 to 0.6 M NaCl in buffer A, and 50-ml fractions were collected. H-II started to elute as 0.4 M NaCl was applied to the column. The active fractions were combined (250 ml) and loaded onto a column (5 by 10 cm) of hydroxyapatite (Bio-Rad) equilibrated with buffer A. The flow rate was 4 ml/min, and 50-ml fractions were collected. The column was eluted with a 1.0-liter linear gradient (0 to 0.5 M potassium phosphate) in buffer A. The H-II activity started to elute as 0.25 M potassium phosphate was applied to the column. Fractions containing H-II activity were combined (230 ml), and 2 M (NH4)2SO4 was added to give a final concentration of 0.8 M. This was loaded onto a column of phenyl-Sepharose (3.5 by 10 cm) equilibrated with buffer A containing 0.8 M (NH4)2SO4. The column was eluted with a 320-ml linear gradient of 0.8 to 0 M (NH4)2SO4. The flow rate was 4 ml/min, and 30-ml fractions were collected. H-II activity started to elute from the column as 0.01 M (NH4)2SO4 was applied. Fractions containing H-II activity were combined and concentrated by ultrafiltration (Amicon ultrafilter; PM-30 membrane). The concentrated fractions (10 ml) were applied to a column of Superdex 200 (6 by 60 cm) equilibrated with buffer A containing 100 mM NaCl. The flow rate was 6 ml/min, and 30-ml fractions were collected. Those fractions containing pure H-II as judged by electrophoretic analysis were combined (120 ml), concentrated by ultrafiltration to 2 ml, and stored in liquid N2.
Other methods.
Protein concentrations were routinely estimated by the method of Bradford (4) with bovine serum albumin as the standard. The iron (12) and acid-labile sulfide (7) contents were measured as described elsewhere. A complete metal analysis (32 elements including nickel and iron) was performed by plasma emission spectroscopy using a Jarrel Ash Plasma Comp 750 instrument at the Riverbend Research Laboratories, University of Georgia. The flavin adenine dinucleotide (FAD) content was estimated by the change of absorption at 450 nm upon addition of sodium dithionite using a molecular absorptivity of 11,500 M−1 cm−1 (2). Flavin was analyzed using thin-layer chromatography and UV-visual light spectroscopy (22). The molecular weight of H-II was estimated by gel filtration (15), and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was carried out by the method of Laemmli (11). The N-terminal amino acid sequences were determined by using an Applied Biosystems model 477 sequencer (8). Electron paramagnetic resonance (EPR) spectra were recorded on an IBM-Bruker ER 300D spectrometer interfaced to an ESP 3220 data system and equipped with an Oxford Instrument ITC-4 flow cryostat. H-II in 50 mM Tris-HCl (pH 7.8) was oxidized by thionine (0.5 mM) under anaerobic conditions in an Amicon ultrafilter (PM-30 membrane), and the dye was removed by repeated washings with buffer. The oxidized enzyme (3.6 mg/ml) was reduced by adding 5 mM sodium dithionite, and after about 30 s at 80°C, the sample was frozen immediately in liquid nitrogen for analysis.
Nucleotide sequence accession number.
The DNA sequences of the genes encoding the four subunits (β-γ-δ-α) of H-II are available from GenBank under accession no. AF176650.
RESULTS
Purification of H-II.
More than 90% of H2-dependent S0-reducing activity of P. furiosus was in the supernatant after centrifugation (100,000 × g for 2 h) of a cell extract, indicating that the enzyme(s) responsible is located in the cytoplasmic fraction. During the routine purification of H-I (6, 16), the activity of which is measured by the H2-dependent reduction of benzyl viologen, a second distinct peak of hydrogenase activity eluted from the first ion-exchange (DEAE-Sepharose) column. The first peak eluted near 0.26 M NaCl (pH 8.0), contained approximately 90% of the total activity, and represented H-I. The second peak, containing the remaining 10%, eluted as 0.40 M NaCl was applied to the column. The hydrogenase responsible for the latter activity was purified further and, as discussed below, was shown to also catalyze the H2-dependent reduction of S0 to H2S. This second hydrogenase of P. furiosus is therefore referred to as H-II (or sulfhydrogenase II). The results of a typical purification are shown in Table 1. Compared to the cell extract, H-II was purified 24-fold with a yield of 4%, but since H-I is responsible for most of the activity in the extract, the true yield of H-II is closer to 40%.
TABLE 1.
Purification of H-II from P. furiosus
| Step | Protein (mg) | Activitya (U) | Sp act (U/mg) | Purification (fold) | Recovery (%) |
|---|---|---|---|---|---|
| Cell extract | 8,090 | 43,900 | 5.4 | 1 | 100 |
| DEAE-FF | 384 | 4,100 | 10.7 | 2 | 9.3 |
| Hydroxyapatite | 83 | 2,530 | 30.4 | 5.6 | 5.7 |
| Phenyl-Sepharose | 20 | 2,310 | 116 | 21.5 | 5.3 |
| Superdex-200 | 14 | 1,830 | 131 | 24.3 | 4.2 |
H2 oxidation activity at 80°C using benzyl viologen as the electron carrier.
Physical properties.
The purified enzyme gave rise to four protein bands after SDS-gel electrophoresis (Fig. 1) which corresponded to Mrs of 52,000, 39,000, 30,000, and 24,000 Da. H-I also contains four subunits with Mrs of 50,000, 43,000, 33,000, and 29,000 Da, which are of comparable size although clearly distinct from those of H-II (see below). The apparent Mr of the H-II holoenzyme was 320,000 ± 2,000, which suggests that the enzyme is a dimer of heterotetramers, (αβδγ)2. Plasma emission spectroscopy and chemical analyses showed that purified H-II contained (mole(s) per mole of αβγδ) Ni (0.9 ± 0.1), Fe (21 ± 1.6), and acid-labile sulfide (19 ± 2.1). Flavin was also present, and this was identified using thin-layer chromatography as FAD. Visible spectroscopy showed that the enzyme contained 0.83 ± 0.1 mol of FAD per mol of αβγδ. As shown in Fig. 2, the dithionite-reduced H-II exhibited at 50K an axial EPR spectrum characteristic of a single reduced [2Fe-2S] center. The spectrum became more complex and was considerably broadened as the temperature was decreased, consistent with the presence of multiple reduced [4Fe-4S] centers. The EPR absorption at 4K represented 2.7 ± 0.3 spin/mol of heterotetramer [αβγδ], indicating that the enzyme contained at least two 4Fe and one 2Fe cluster. The oxidized enzyme showed an isotropic-type EPR signal near g = 2.03 which is typical of an oxidized [3Fe-4S] cluster (data not shown). This corresponded to 0.2 ± 0.03 spins per heterotetramer [αβγδ] and presumably arose from oxidative damage of a [4Fe-4S] center. EPR resonances that might originate from Ni were not apparent from the reduced or oxidized enzyme.
FIG. 1.
SDS–12.5% polyacrylamide gel electrophoresis of H-II purified from P. furiosus. Lanes 1 and 4, molecular mass markers; lanes 2 and 3, 1 and 2 μg of H-II, respectively.
FIG. 2.
EPR spectra of reduced H-II. The spectra were recorded using the dithionite-reduced enzyme (3.6 mg/ml in 50 mM Tris-HCl [pH 7.8]) at the indicated temperature. The spectrometer settings were as follows: microwave power, 20 mW; microwave frequency, 9.596 GHz; modulation frequency, 100 kHz; modulation amptitude, 6.366 G; time constant, 81.92 ms; gain, 104.
Catalytic properties.
The activities of H-II under various assay conditions are shown in Table 2. Values for H-I from P. furiosus are given for comparison. H-II was about five times less active than H-I in the standard H2-dependent, benzyl viologen reduction assay. Hence, from the total activity in the cytoplasmic extract (Table 1), the cellular concentrations of the two enzymes are roughly comparable. Similarly, while H-II catalyzed the reduction of S0 to H2S using H2 as electron donor, the specific activity was about 30-fold lower than that of H-I. H-II was also about an order of magnitude less active than H-I in catalyzing both H2 oxidation and H2 evolution using methyl viologen as the electron carrier (Table 2).
TABLE 2.
Catalytic properties of H-I and H-II from P. furiosus
In the H2 evolution assay, sodium dithionite is used as the electron source, and with both enzymes dithionite alone supported a significant rate of H2 production without any intermediate electron carrier (Table 2). These rates are at least 50-fold greater than those measured with P. furiosus ferredoxin as the electron donor. The latter mimics a possible physiological reaction, where pyruvate is the electron source and ferredoxin is reduced by P. furiosus POR. Note that H2 production with ferredoxin as the electron donor is not measurable under the usual conditions used to assay H-I (17), but the data shown in Table 2 using ferredoxin were obtained with much higher amounts of the two enzymes (>250 μg per assay) than that typically used (20 μg per assay [17]). The question is, does electron transfer from ferredoxin to either hydrogenase have any physiological relevance? This would seem unlikely for H-II since the rate of H2 production is so low and, as shown in Fig. 3, the presence of ferredoxin only marginally increases the rate compared to that seen by direct POR-hydrogenase interaction. Similar results were obtained with H-I (Fig. 3), although in this case the rates are somewhat higher. With high enzyme concentrations, it is also possible to demonstrate the H2-dependent reduction of ferredoxin by both hydrogenases, as shown in Fig. 4, but again the overall rates are very low (0.0044 to 0.018 U/mg) and are not deemed physiologically significant.
FIG. 3.
H2 production from pyruvate by H-II (A) and H-I (B). H2 production was measured using ferredoxin (Fd) reduced by POR as the electron donor to H-I and H-II as described in Materials and Methods. Abbreviations: + Fd, with ferredoxin; − Fd, without ferredoxin; − H-I, without H-I; − H-II, without H-II.
FIG. 4.
Reduction of P. furiosus ferredoxin by H-I and H-II. The reactions were carried out as described in Materials and Methods.
For H-I, it was proposed that NADP is the physiological electron carrier (17). The enzyme shows high H2 evolution activity with NADPH as the electron donor (10 U/mg), and as shown in Fig. 5, high rates of H2 production are measured in the physiologically relevant, coupled assay system involving POR, ferredoxin, FNOR, NADP, and hydrogenase, where pyruvate is the source of reductant. In contrast, while H-II also used NADPH as an electron donor, the rate (0.2 U/mg) was much lower than that measured with H-I. Similarly, the rate of H2 production in a coupled POR-FNOR system was more than 20-fold less than that seen with H-I (Fig. 5). A full kinetic analysis of the two enzymes in both H2 evolution and H2 oxidation assays with various substrates is summarized in Table 3. Note that H-II also differs from H-I in its ability to use NAD(H) and NADP(H) with comparable efficiencies and has in general a much higher affinity for these nucleotides than does H-I. Interestingly, the affinities of the two enzymes for H2 (with methyl viologen as the electron acceptor) are comparable, but the relatively high values of approximately 0.14 mM (∼17% [vol/vol] H2 in the gas phase) suggest that these enzymes are unlikely to function physiologically to oxidize H2 within the cell.
FIG. 5.
H2 production from pyruvate using a coupled system of P. furiosus enzymes. The reactions were carried out as described in Materials and Methods and the legend to Fig. 3. (A) H-II; (B) H-I. Where indicated, the reaction mixtures also contained P. furiosus FNOR and NADP.
TABLE 3.
Kinetic parameters for H-II and H-I from P. furiosus
| Hydrogenase | Substratea (mM) | Cosubstratea (mM) | Apparent Km (mM) | kcat (min−1) | kcat/Km (min−1 mM−1) | kcat (I)/kcat (II) |
|---|---|---|---|---|---|---|
| H-II | MV+ (0–4.0) | DT (10.0) + H+ (10−5) | 1.25 ± 0.2 | 6,400 | 5,120 | |
| H-II | MV2+ (0–4.0) | H2b | 1.0 ± 0.2 | 1,760 | 1,760 | |
| H-II | H2 (0–0.836) | MV2+ (1.0) | 0.13 ± 0.01 | 960 | 7,385 | |
| H-II | NADPH (0–0.5) | H+ (10−5) | 0.063 ± 0.004 | 24 | 381 | |
| H-II | NADH (0–0.5) | H+ (10−5) | 0.071 ± 0.005 | 22 | 315 | |
| H-II | NADP (0–0.2) | H2b | 0.017 ± 0.003 | 43 | 2,541 | |
| H-II | NAD (0–0.5) | H2b | 0.125 ± 0.015 | 96 | 768 | |
| H-II | S0 (0.25–1.0%)e | H2b | 0.2 ± 0.02c | 41 | NAd | |
| H-II | PSe (0–3.0 mM) | NADPH (0.4 mM) | 0.67 ± 0.10 | 10 | 15 | |
| H-I | MV+ (0–4.0) | DT (10.0) + H+ (10−5) | 0.2 ± 0.02 | 42,900 | 214,500 | 6.7 |
| H-I | MV2+ (0–10) | H2b | 5.0 | 37,500 | 7,500 | 21.3 |
| H-I | H2 (0–0.836) | MV2+ (1.0) | 0.14 | 1,575 | 11,250 | 1.6 |
| H-I | NADPH (0–2.0) | H+ (10−5) | 0.2 | 1,500 | 7,500 | 62.5 |
| H-I | NADH (0–8.0) | H+ (10−5) | 3.0 | 240 | 80 | 11 |
| H-I | NADP (0–0.4) | H2b | 0.04 | 11,250 | 281,000 | 262 |
| H-I | NAD (0–1.0) | H2b | NA | 0 | NA | NA |
| H-I | S0 (1–5%)e | H2b | 1.8 ± 0.2e | 1,665 | NA | 4.5 |
| H-I | PSe (0–3.0 mM) | NADPH (0.4 mM) | 2.0 ± 0.2 | 390 | 195 | 38 |
Abbreviations: MV, methyl viologen; DT, sodium dithionite; Fd, ferredoxin.
Pure H2 gas was used at a pressure of 120 kPa.
Taken from reference 17.
NA, not applicable.
The concentration of S0 is expressed as percent (weight/volume), not a millimolar concentration. PS, polysulfide.
The relatively low activity of H-II in these assays compared to that of H-I is evident from the activity ratios listed in Table 3. This is not a result of enzyme instability, however, as the activity (H2-dependent benzyl viologen reduction) of H-II increased with increasing temperature from 30 to 90°C with an optimum above 90°C, and the half-life of the pure enzyme (1.2 mg/ml in 75 mM EPPS buffer [pH 8.0]) at 95°C was about 6 h. Similar results have been reported for H-I (6).
It has been proposed previously (16) that the physiological role of H-I, in addition to H2 production, is to reduce S0 to H2S, where S0-derived polysulfide is the substrate of the reaction. Sulfide production from H2 and S0 and H2 and polysulfide has been demonstrated previously (16). However, the finding that NADPH is the likely physiological electron donor to H-I raises the issues of whether the reduced nucleotide will also serve as the electron donor for S0 reduction, how electron flow is partitioned between S0 or polysulfide and protons, and how H-II compares with H-I in these reactions. As shown in Fig. 6, when polysulfide (2 mM) is added to a mixture of H-II and NADPH, the rate of NADPH oxidation increases by about 50% and the rate of H2 production decreases by about 60%. The same is true for H-I, where NADPH oxidation increases almost fourfold and H2 production decreases by about 70% (Fig. 6). With both enzymes, the amount of sulfide produced from polysulfide (2 mM) over the assay period was more (over threefold in the case of H-II) than the amount of H2 produced (Table 4).
FIG. 6.
Stimulation of NADPH oxidation and inhibition of H2 production from NADPH by polysulfide. (A) H-II. (B) H-I. The 2-ml reaction mixture contained 100 mM EPPS (pH 8.0), 0.4 mM NADPH, polysulfide as indicated, and 250 μg of H-II (A) or 78 μg of H-I (B). The rate of NADPH oxidation was measured spectrophotometrically over 2 min. The amount of H2 produced (expressed as a percentage) was determined after 10 min.
TABLE 4.
Production of H2 and H2S with NADPH as the electron donor catalyzed by H-I and H-II of P. furiosus
| Hydrogenase | Additiona | H2 (μmol) | H2S (μmol) | NADPH oxidized (μmol) | Ratio of H2S (μmol)/H2 (μmol) |
|---|---|---|---|---|---|
| H-II | None | 0.28 ± 0.03 | 0 | 0.6 ± 0.1 | 0 |
| H-II | + PS | 0.14 ± 0.02 | 0.47 ± 0.15 | 1.2 ± 0.15 | 3.4 |
| H-II | + S0 | 0.27 ± 0.03 | 0.16 ± 0.05 | 0.8 ± 0.1 | 0.6 |
| H-I | None | 1.2 ± 0.1 | 0 | 1.2 ± 0.15 | 0 |
| H-I | + PS | 0.56 ± 0.05 | 0.86 ± 0.25 | 1.9 ± 0.2 | 1.5 |
| H-I | + S0 | 1.28 ± 0.1 | 0.16 ± 0.05 | 1.5 ± 0.1 | 0.13 |
The 2-ml reaction mixture at 80°C contained 100 mM EPPS (pH 8.0), 1 mM NADPH, and 250 μg of H-II or 78 μg of H-I. Polysulfide (PS; 2 mM) or S0 (5% [wt/vol]) was added as indicated. NADPH oxidation and the production of H2 and H2S were measured as described in Materials and Methods.
Thus, both hydrogenases preferentially reduce polysulfide to sulfide rather than protons to H2 using NADPH as the electron donor (Table 4). Much less sulfide and much more H2 were produced when the source of sulfur was S0 (5% [wt/vol]) rather than polysulfide (Table 4). This was expected since there is a pronounced lag phase in the rate of sulfide production from S0 as the latter is first converted to polysulfide (16). Whether S0 or polysulfide is used in these assays, the amount of NADPH oxidized is slightly more than the amount of sulfide and H2 produced (Table 4), presumably due to the abiotic oxidation at the high temperature (15). From kinetic analyses, the approximate apparent Km values for S0 and polysulfide were about 10- and 3-fold lower with H-II than they were for H-I (Table 3).
Sequence analyses.
The N-terminal sequences of four subunits of H-II were determined after they were separated from each other by SDS-polyacrylamide gel electrophoresis. They are as follows (where X is an unknown residue): α, MIXELDEFTRVEGNXKAEIV-; β, MRYVKLHSEYFPEFFNRLKE-; γ, MNPYRSYDARII-; and δ, MKLGVFELTDXGG-. They do not correspond to the N-terminal sequences of the four subunits of H-I, the complete sequences of which are known (18). The sequences of the H-II subunits were used to search the genome sequence of P. furiosus (http://combdna.umbi.umd.edu.genemate.html). Each one corresponded exactly to the translated N-terminal sequences of four distinct open reading frames, and as shown in Fig. 7, these appear to constitute a single operon. The calculated molecular masses of the encoded proteins (β; 39,178 Da; γ, 32,943 Da; δ, 26,282 Da; and α, 46,177 Da) are in agreement with the subunit sizes of H-II determined by SDS-polyacrylamide gel electrophoresis (Fig. 1) and, as shown in Table 4, are comparable to those reported for H-I.
FIG. 7.
DNA and amino acid sequences of the genes proposed to encode H-II from P. furiosus. The amino acid sequence of each open reading frame is given in one-letter code above the DNA sequence. Amino acids given in boldface represent putative motifs that bind the Ni-Fe binding site in the α subunit (400 to 408), the [4Fe-4S] clusters in the β subunit (229 to 240 and 306 to 317) and the δ subunit (11 to 133, 160 to 178, and 188 to 202), and the [2Fe-2S] cluster in the γ subunit (250 to 274). The motifs are extended to include the identity in sequence between H-II and H-I (20). The dashed lines above the amino acid sequence of the γ subunit indicate binding regions for two nucleotide cofactors. Nucleotide sequences in boldface indicate possible RNA polymerase binding sites; underlined sequences indicate putative ribosome binding sites. =>, start of open reading frame; ===, transcription termination signal.
DISCUSSION
The molecular properties of H-II of P. furiosus are remarkably similar to those of the enzyme of this type previously characterized from this organism, H-I (6, 16) (Table 5). Although the size of the holoenzyme of H-II is twice that of H-I, both consist of four subunits and contain Ni, multiple Fe/S centers, and FAD (6, 20). The genes encoding H-I had been previously cloned and sequenced (18). The four subunits of the two enzymes are not only of comparable size but also show significant sequence similarity (55 to 63%). In fact, the gene order (βγδα) for H-II is the same as that for H-I, and in both cases, the four genes are tightly linked. For H-II, those encoding β and γ, and γ and δ, have 4 and 1 overlapping nucleotide, respectively, while δ and α are separated by only 2 nucleotides (Fig. 7). Each translational start codon is preceded by a typical archaeal ribosome binding site (5), while the start codon of the gene encoding the β subunit is TTG, which is rarely used in members of the domain Archaea. In addition, there is a transcription termination signal (T-rich region) beginning 9 nucleotides following the stop codon of the last gene which encodes the α subunit. As shown in Fig. 7, the gene encoding the β subunit is preceded by two possible RNA polymerase binding sites, suggesting that a regulatory mechanism may be involved in H-II expression. What such effectors might be remains unknown.
TABLE 5.
Predicted properties of the subunits of H-I and H-II of P. furiosus
| Subunit | H-II (kDa) | H-I (kDa) | Sequence similarity (%) | Cys residues (II/I) | Proposed cofactors |
|---|---|---|---|---|---|
| α | 46.2 | 48.7 | 55 | 4/4 | Ni-Fe site |
| δ | 26.2 | 29.6 | 63 | 12/13 | 3× [4Fe-4S] |
| β | 39.2 | 41.8 | 58 | 17/18 | 2× [4Fe-4S] |
| γ | 32.9 | 33.2 | 55 | 10/7 | 1× [2Fe-2S] FAD |
| αβγδ | 144.5 | 153.3 | 43/42 |
The EPR properties of reduced H-II indicate that it contains at least two 4Fe and one 2Fe center (per heterotetramer). These would account for only about half of the Fe present in the enzyme, suggesting that additional FeS centers are present but that they are not EPR active under the experimental conditions, presumably due to incomplete reduction. This is substantiated by the presence of numerous cysteines and of several cysteine-containing motifs in the sequences of the subunits. Specifically, the results of our analysis of the genes of H-II are in agreement with those recently proposed by Silva et al. (20) for H-I, and these are summarized in Table 5. Thus, the α and δ subunits of H-II correspond to the so-called large and small subunits of conventional NiFe-hydrogenases, the prototypical example of which is the crystallographically characterized enzyme from Desulfovibrio gigas (21). In H-II, by analogy, the α subunit contains the binuclear NiFe catalytic site while the δ subunit contains three [4Fe-4S] clusters with all cysteinyl ligation. In the D. gigas enzyme, one of the 4Fe clusters is of the [3Fe-4S]-type while another (the distal cluster) has a His ligand. This is not the case in H-II or in H-I. In fact, as indicated in Fig. 7, the cluster-binding motifs show high sequence identity between these two enzymes for all four subunits. These also include those for two [4Fe-4S] clusters in the β subunit and one [2Fe-2S] cluster in the γ subunit.
The β and γ subunits of H-II also contain a significant number (10 and 4, respectively) of cysteinyl residues, most of which are conserved in H-I (7 and 4, respectively). It seems likely that these coordinate additional FeS clusters, since the proposed cluster content (Table 5) accounts for 22 Fe/heterotetramer, which is in good agreement with what is found by chemical analysis of purified H-II. Whether these additional residues are involved in S0 reduction remains to be seen. The γ subunit of H-II also contains two putative nucleotide-binding sites analogous to those previously identified in H-I (18). These are consistent with the presence of FAD in H-II and with its ability to use NAD(P)(H) as electron carriers. The kinetic data presented herein demonstrate that ferredoxin cannot be considered as a physiological electron carrier for either H-II or H-I.
Hence, P. furiosus contains two hydrogenase-type enzymes that are extremely similar in their molecular properties, including their gene organization. Both are capable of reducing S0 and polysulfide, as well as protons, using electrons supplied by NADPH. Indeed, both have a preference for sulfide production (from polysulfide) rather than H2 production. In fact, polysulfide both stimulates NADPH oxidation and leads to a decrease in H2 production. Whether the latter reaction is specifically inhibited by polysulfide, or whether the lower rate results from the diversion of electron flow to polysulfide rather than protons, remains to be elucidated. In any event, it is clear that both hydrogenases have the potential to catalyze sulfide production in vivo.
The two hydrogenases of P. furiosus appear to differ primarily in their relative catalytic activities, with H-II being approximately an order of magnitude less active than H-I in most of the assays investigated (Table 3). The two enzymes are present in the cytoplasm at roughly similar concentrations, and it seems reasonable to conclude that they serve different functions. H-II has a higher affinity for both S0 and polysulfide in the standard assays, and perhaps this enzyme becomes physiologically relevant at low S0 concentrations. In the absence of S0, the role of H-I is proposed to be H2 production using NADPH as the electron donor (16, 17), a reaction in which it is about 60 times more efficient than H-II (Table 3). The only reaction catalyzed by H-II that is very poorly carried out by H-I is the H2-dependent reduction of NAD (Table 3). However, the apparent Km value for NAD (125 μM) seems high for this to be physiologically relevant, and H-II appears to have a much higher affinity for NADP (Km, 17 μM). Hence, in spite of the fact that H-II contains FAD and has a putative nucleotide-binding site, it is hard to imagine that NAD(P) reduction has any physiological significance. Clearly, insight into the function of this enzyme will require additional analyses such as proteomics and mRNA studies, and these are in progress.
ACKNOWLEDGMENTS
This research was supported by grants from the Department of Energy (FG05-95ER20175 and under contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corp.).
We thank Andrea Hutchins for kindly providing P. furiosus POR.
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