INTRODUCTION

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Bacteria which utilize CO as a growth substrate include the aerobic carboxidotrophs and the anaerobic acetogens, sulfate-reducers, methanogens, and phototrophs (16, 34, 42, 44, 47). Carboxydothermus hydrogenoformans is a strictly anaerobic, thermophilic, gram-positive eubacterium which was isolated from a volcanic hot spring (55). Phylogenetically, C. hydrogenoformans falls into the group of the low-G+C subphylum of the gram-positive bacteria and shows highest 16S rRNA gene sequence homology to Thermoterrabacterium (52). C. hydrogenoformans utilizes CO under chemolithoautotrophic conditions (55). The bacterium couples the oxidation of CO to CO₂ (E₀' = 0.52 V) to the reduction of protons to H₂ (E₀' = 0.41 V) in the energy-conserving reaction CO + H₂O  CO₂ + H₂, G⁰' = 20 kJ mol¹ (54, 55). The metabolism of C. hydrogenoformans is strictly fermentative because the bacterium is obligately anaerobic and utilizes protons as the characteristic intracellular electron acceptor. On the basis of the formation of H₂ as the ultimate fermentation product by C. hydrogenoformans, we propose the terms "hydrogenogenic," "hydrogenogens," and "hydrogenogenesis" to refer to the type of metabolism, the physiological group, and the process of H₂ formation, respectively. Although C. hydrogenoformans is not a phototroph, it is in many ways similar to the phototrophic bacteria Rhodospirillum rubrum and Rhodocyclus gelatinosus, which utilize CO anaerobically in the dark (34, 58, 59). C. hydrogenoformans is also able to ferment pyruvate to acetate and H₂ (56).

Although carbon monoxide dehydrogenases (CODHs) formally catalyze the same reaction (CO + H₂O  CO₂ + 2 e + 2 H⁺), different types of enzymes serving different metabolic functions operate in the various groups of CO-oxidizing bacteria (15, 16, 42, 43, 44, 47). Aerobic CODHs are MoFeS flavoproteins containing [2Fe-2S] clusters, while anaerobic CODHs are NiFeS proteins containing [4Fe-4S] clusters. The high-resolution crystal structures of the MoFeS CODHs from Oligotropha carboxidovorans (12, 26, 43) or Hydrogenophaga pseudoflava (27, 43) show a dimer of two heterotrimers in an (LMS)₂ subunit structure. Each heterotrimer is composed of a molybdoprotein (L subunit), a flavoprotein (M subunit), and an iron-sulfur protein (S subunit). The molybdoprotein carries the active site, which contains a 1:1 molar complex of molybdopterin cytosine dinucleotide and a molybdenum atom. The iron-sulfur protein contains the type I and type II [2Fe-2S] centers. The flavoprotein contains the flavin adenine dinucleotide (FAD) cofactor and shows a new flavin-binding type (25, 26). The NiFeS CODHs are either monofunctional or bifunctional (15, 16, 47). The latter are associated and operate in a complex with acetyl coenzyme A (acetyl-CoA) synthase (ACS). The monofunctional CODH from the phototrophic bacterium R. rubrum (6, 7) is inducible in the dark under anaerobic conditions in the presence of CO, shows a micromolar K_m for CO (6, 7, 34), and contains a proposed nickel-iron-sulfur cluster (cluster C) (30, 32, 53) and a conventional [4Fe-4S] cluster (cluster B) (32, 47). Radiolabeling studies suggested a catalytically essential nonsubstrate CO ligand (CO_L) to the Fe atom in the putative [Fe-Ni] center of cluster C (31). Acetogens and methanogens employ the bifunctional CODH-ACS (15, 16, 47). The enzymes are tetramers of two different subunits or pentamers of five different subunits. The subunits harboring the CODH activity contain cluster C and cluster B (16, 47), which is similar to the situation in R. rubrum CODH.

Significant CO:oxidized benzyl viologen (BV) oxidoreductase activity was identified in cytoplasmic fractions of strains of C. hydrogenoformans (46). The CODH structural genes cooF and cooS from C. hydrogenoformans have been sequenced (23). cooS was identified directly downstream of cooF. The genes showed the highest similarity to the cooF genes from the archeon Archaeglobus fulgidus and the cooS gene from the bacterium R. rubrum, respectively.

In this investigation we have identified, purified, and characterized two distinct homodimeric NiFeS CODHs from C. hydrogenoformans. Immunoelectron microscopy showed both enzymes attached to the inner aspect of the cytoplasmic membrane. The N-terminal sequences of CODH II and a 49-kDa chymotryptic peptide match the sequence of CooS. This is the first study describing Ni CODHs from an anaerobic CO-oxidizing hydrogenogenic bacterium.

	MATERIALS AND METHODS

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Organism and cultivation. C. hydrogenoformans Z-2901 (DSM 6008) (55) was grown under strictly anaerobic conditions in 50-liter fermentors (Biostat U; Braun Biotech, Melsungen, Germany) at 60°C and pH 6.8 in the following medium (units are milligrams per liter): 1,500 NH₄Cl; 200 MgSO₄ · 7H₂O; 20 CaCl₂ · 2H₂O; 300 KH₂PO₄; 300 K₂HPO₄; 500 NaHCO₃; 500 Na-thioglycolate; 500 yeast extract; 1 resazurin; 2 NiCl₂ · 6H₂O; 10 ammonium ferric(III) citrate; 1 FeSO₄ · 7H₂O; 1 ZnSO₄ · 7H₂O; 5 MnSO₄ · H₂O; 0.1 H₃BO₃; 1 CoCl₂ · 6H₂O; 0.1 CuSo₄ · 5H₂O; 0.1 Na₂MoO₄ · 2H₂O; 0.2 Na₂SeO₃ · 5H₂O; 0.1 KAl(SO₄)₂ · 12H₂O; 1 ml of vitamin solution (61). The fermentors were continuously supplied with 0.5 liters of CO min¹ and stirred at 500 rpm. Bacteria were harvested by centrifugation under N₂ and kept frozen at 20°C under N₂ until use.

Anaerobic procedures. All procedures for the preparation of cell extracts, cell fractions, and enzyme purifications were carried out anaerobically under a flow of N₂ or in an anoxic glove box chamber (model 1024 anaerobic system; Forma Scientific, Marrietta, Ohio) under an atmosphere of pure N₂. All buffers were repeatedly degassed by evacuation, flushed with N₂, supplied with 2 mM Na-dithionite, and maintained under a slight overpressure of N₂.

Preparation and purification of CODH. For enzyme purifications, C. hydrogenoformans was harvested at an approximate optical density at 436 nm of 2. About 100 g of bacterial cell mass was suspended in 200 ml of 50 mM Tris-HCl (pH 8.0) containing 2 mM Na-dithionite (buffer A), 0.1 mg of lysozyme per ml, 0.05 mg of DNase I per ml, and 0.2 mM phenylmethylsulfonyl fluoride and incubated for 30 min at 37°C with gentle stirring. The protoplasts thus obtained were subjected to osmotic lysis followed by low-spin centrifugation. The resulting cell-free extracts were subjected to ultracentrifugation for 2 h at 120,000 × g, yielding cytoplasmic and membrane fractions. Intact protoplasts were prepared from bacteria suspended in the above buffer and supplied with 0.6 M sucrose.

Enzyme assays. CODH activity was assayed at 70°C, which is the optimal growth temperature for C. hydrogenoformans (55), by following the CO-dependent reduction of oxidized methyl viologen (MV) in a spectrophotometer employing an ₅₇₈ of 9.7 mM¹ cm¹ (6). For the assays, 1-ml volumes containing 20 mM MV and 2 mM dithioerythritol (DTE) in buffer B (50 mM HEPES-NaOH [pH 8.0]) were flushed with CO in screw-cap cuvettes sealed with a rubber septum. Reactions were initiated by injecting enzyme with a syringe. H₂ oxidation activity was assayed under the same conditions except that CO was replaced by H₂. One unit of CO or H₂ oxidation activity is defined as the reduction of 2 µmol of MV min¹, which is equivalent to 1 µmol of CO or H₂ oxidized min¹.

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Electron acceptor specificity. Electron acceptor specificity of CO oxidation catalyzed by CODH was tested spectrophotometrically under the conditions of the CODH activity assay with the exception that DTE was omitted from the reaction mixture. The following compounds (100 µM concentrations of each) were examined (extinction coefficient units are per millimolar per centimeter) (11): BV, ₅₆₀ = 8.7; NAD⁺ or NADP⁺, ₃₄₀ = 6.2; FAD, ₄₅₀ = 11.3; flavin mononucleotide (FMN), ₄₅₀ = 12.2; 1-phenyl-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride (INT)-20 µM 1-methoxy-phenazine methosulfate (MPMS), ₄₉₆ = 18.0 (35); ubiquinone Q₁₀, ₂₇₆ = 14.7; methylene blue, ₆₁₅ = 37.1; phenazine methosulfate (PMS), ₃₈₇ = 25.0; 2,6-dichlorophenol-indophenol (DCPIP), ₆₀₀ = 16.1; horse heart cytochrome c, ₅₅₀ = 29.5.

Kinetic measurements. The K_m for CO was determined by varying the CO concentration in the reaction mixture under MV saturation (20 mM). The different CO concentrations were established by adding appropriate amounts of CO-saturated reaction mixture composed of buffer B, 20 mM MV, and 2 mM DTE to assays containing the same reaction mixture saturated with N₂. At 70°C and 1 atm pressure, the CO concentration in CO-saturated reaction mixtures was taken to be 645 µM (38). The actual starting CO concentration was calculated from the final concentration of reduced MV, considering that 1 µmol of CO reduces 2 µmol of MV. The K_m for MV was determined in a CO-saturated reaction mixture composed of 50 mM buffer B, 2 mM DTE, and different concentrations of MV.

Peptide mapping by limited proteolysis. Purified CODH in 50 mM Tris-HCl (pH 8.0) containing 0.5% (wt/vol) sodium dodecyl sulfate (SDS) was denatured by boiling for 2 min. Assay mixtures were diluted to 0.1% SDS by employing 100 mM acetate buffer (pH 4.0) (pepsin digestion) or 100 mM Tris-HCl (pH 8.0) (-chymotrypsin digestion). Limited proteolysis was carried out at 23°C (pepsin) or 37°C (-chymotrypsin) at protein-to-protease ratios (by mass) of 4:1 for pepsin and 80:1 for -chymotrypsin. Proteolytic fragments were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 12 or 15% (wt/vol) running gels.

Immunological methods. Polyclonal immunoglobulin G (IgG) antibodies directed against CODH I (Sequence Laboratories GmbH, Göttingen, Germany) or CODH II (Eurogentec Bel S.A., Herstal, Belgium) were obtained from immunized rabbits. The antibodies were purified from polyclonal rabbit antisera by affinity chromatography on protein A-Sepharose CL-4B (Pharmacia) according to the instructions of the manufacturer. For Western blotting, alkaline phosphatase-labeled goat anti-rabbit IgGs (Sigma, Deisenhofen, Germany) were employed.

Miscellaneous methods. Protein estimation employed conventional methods (5, 10). Analytical PAGE was carried out according to Laemmli (37). For SDS-PAGE, 7.5% (wt/vol) stacking gels and 12 or 15% (wt/vol) running gels were used. Native PAGE was performed under anaerobic conditions employing 5% (wt/vol) stacking gels and 7.5% (wt/vol) running gels. Proteins were stained with Coomassie brilliant blue G-250. Protein transfer from acrylamide gels to polyvinylidene difluoride membranes and N-terminal amino acid sequencing were carried out as described previously (24). Isoelectric focusing was carried out on IsoGel agarose plates (FMC Bioproducts, Rockland, Maine) according to the manufacturer's instructions.

Chemicals. All chemicals employed were obtained from the usual commercial sources. Gases were purchased from Riessner-Gase (Lichtenfels, Germany).

	RESULTS

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CO oxidation in subcellular fractions. Under the conditions detailed in Materials and Methods, C. hydrogenoformans grew with a generation time of 4.9 h and a yield of 1.2 mg of protein per ml. During growth, the specific CO oxidation activity in cell-free extracts increased continuously from 20 to 1,800 U mg of protein¹. About 71% of the total CO oxidation activity in extracts appeared to be soluble in the cytoplasmic fraction (Table 1). Less than 1% of the total CO oxidation activity showed up outside protoplasts. CODH is apparently only loosely associated with the cytoplasmic membrane, since washing of the membranes with buffer mobilized about 50% of the membrane-associated CODH fraction. The H₂-evolving hydrogenase (74.7 U mg of membrane protein¹) of C. hydrogenoformans is entirely membrane bound and showed only a little H₂ oxidation activity (0.9 U mg of membrane protein¹).

Identification of two distinct CODHs. We managed to establish a protocol for the purification of two CODHs from C. hydrogenoformans (Table 1). Upon anion exchange chromatography (step 3) CODH activity eluted as a single peak at 0.25 M NaCl. Upon hydrophobic interaction chromatography (steps 4 and 8) two distinct peaks of CODH activity appeared, one at 0.6 M ammonium sulfate (designated CODH I) and a second one at 0.35 M ammonium sulfate (designated CODH II). CODH I and CODH II were purified separately (Table 1). Upon hydrophobic interaction chromatography (step 5) CODH I eluted at 0.05 M ammonium sulfate. Upon anion exchange chromatography (steps 6 and 9) CODH I and CODH II eluted at 0.17 and 0.14 M NaCl, respectively. CODH I was purified 14-fold with a specific activity of 15,756 U mg of protein¹ and a yield of 29%. CODH II was purified 12-fold with a specific activity of 13,828 U mg of protein¹ and a yield of 13%. Both enzymes had the same isoelectric point (pI) of 5.5.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Analysis of CODH I and CODH II by PAGE. CODH I and CODH II were subjected to native PAGE (A), SDS-PAGE (B), peptide mapping (C), and Western blot analysis (D). (A) Native PAGE. Lane 1, molecular mass markers; lane 2, 10 µg of CODH I; lane 3, 10 µg of CODH II. (B) SDS-PAGE. Lane 4, 5 µg of CODH I; lane 5, 5 µg of CODH II; lane 6, molecular mass markers. (C) Peptide mapping of 10 µg of CODH I or CODH II by limited proteolysis with pepsin or -chymotrypsin for 30 min. Lane 7, pepsin digest of CODH I; lane 8, pepsin digest of CODH II; lane 9, -chymotrypsin digest of CODH I; lane 10, -chymotrypsin digest of CODH II (the number 49 refers to the 49-kDa peptide which has been sequenced). (D) Western blot analysis with purified polyclonal IgG antibodies directed against CODH I or CODH II (10 µg of each). Lane 11, CODH I and antibodies directed against CODH I; lane 12, CODH II and antibodies directed against CODH I; lane 13, CODH I and antibodies directed against CODH II; lane 14, CODH II and antibodies directed against CODH II.

Catalytic properties. The two CODHs catalyze the CO-dependent reduction of various oxidized electron acceptors and show similar electron acceptor specificities (CO oxidation activities at 70°C and pH 8.0, in units per milligram) for CODH I (BV, 5,200 [set at 100%]; MV, 1,400 [27%]; methylene blue, 1,248 [24%]; PMS, 572 [11%]; FAD, 364 [7%]; FMN, 104 [2%]; DCPIP, 52 [1%]) and for CODH II (BV, 3,100 [set at 100%]; MV, 1,000 [32%]; PMS, 341 [11%]; methylene blue, 155 [5%]; FAD, 31 [1%]; FMN, 31 [1%]; DCPIP, 31 [1%]). NADP⁺, NAD⁺, INT-MPMS, ubiquinone Q₁₀, and horse heart cytochrome c were not reduced by the two enzymes. At saturating electron acceptor concentrations (20 mM), the following specific CO oxidation activities (in units per milligram) were obtained for CODH I: 29,400 (BV) and 15,800 (MV). For CODH II the activities were 27,000 (BV) and 13,800 (MV).

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Spectral characterization. The UV-visible absorption spectra of purified CODH I (Fig. 2A) and CODH II (Fig. 2B) were similar under the various conditions examined. Treatment of either enzyme with CO or dithionite resulted in bleaching of the FeS-like shoulder extending from 350 to 550 nm and centered around 419 nm, referring to a reduced type of spectrum (Fig. 2A and B, traces c and d). The shoulder reappeared when the enzymes treated with CO (Fig. 2A and B, trace c) were exposed to air (Fig. 2A and B, trace b). When dithionite was removed from the as-isolated enzymes kept under N₂, the spectra shown in Fig. 2A and B (trace a) were obtained. The spectra are similar to those of the air-oxidized enzymes (Fig. 2A and B, trace b) and therefore are believed to represent an oxidized state. Corresponding oxidized-minus-reduced-difference spectra are shown in the insets. The following extinction coefficients (, per millimolar per centimeter) were calculated for the two CODHs in the CO-reduced (Fig. 2A and B, trace c) or oxidized (Fig. 2A and B, trace a) state: CODH I, ₂₈₀ = 191.9 (oxidized) or 172.8 (reduced), ₄₁₉ = 74.0 (oxidized) or 35.5 (reduced); CODH II, ₂₈₀ = 200.4 (oxidized) or 203.7 (reduced), ₄₁₉ = 83.8 (oxidized) or 42.8 (reduced). Assuming an extinction coefficient of about 4 per mM per Fe in a 4Fe cluster, the calculated approximate number of 4Fe clusters is 4.6 in CODH I and 5.2 in CODH II.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   UV-visible absorption spectra of CODH I (A) and CODH II (B). The enzymes (0.2 mg ml1) were in 50 mM Tris-HCl (pH 8.0). Conditions for each curve: a, under N2 as isolated, dithionite was removed by gel filtration; b, oxidized with air; c, reduced with pure CO; d, reduced with 2 mM dithionite under N2. Insets: difference spectra of condition a minus condition c.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   EPR spectra of CODH I (A) and CODH II (B). CODH I (3.8 mg ml1) or CODH II (3.1 mg ml1) were in 50 mM Tris-HCl (pH 8.0). Traces: (a and e) as-isolated freshly prepared enzyme was frozen under N2; (b and f) air-oxidized as-isolated enzyme was kept under air for 12 h at 4°C; (c and g) CO-reduced as-isolated enzyme was kept under pure CO for 60 min at 50°C; (d and h) dithionite-reduced as-isolated enzyme was treated with 4 mM dithionite under pure N2. The relevant g values are indicated in the spectra. General conditions: 10 mW microwave power; 10 G modulation amplitude; 9.47 GHz microwave frequency; temperatures (in degrees K) as indicated.

Intracellular location of the two CODHs. Osmotic lysis, which is a gentle method of cell breakage, revealed 71% of the total CODH activity was in the cytoplasmic fraction and 29% was in the membrane fraction of C. hydrogenoformans (Table 1). Western blotting indicated that all fractions contained CODH I as well as CODH II. It was, therefore, of interest to examine the intracellular location of the two CODHs in intact cells of C. hydrogenoformans. For this purpose, immunogold labeling of ultrathin sections of CO-grown bacteria was employed (Fig. 4). Low-temperature embedding in Lowicryl K4M resin resulted in good preservation of the bacterial ultrastructure (Fig. 4 A). The distribution of the gold label was the same for the specific IgG antibodies directed against CODH I or CODH II and was independent of the growth phase (Table 2; Fig. 4C to H). Longitudinal and cross-sections showed the gold label nearly exclusively close to the cytoplasmic membrane (Fig. 4C, D, F, and G; Table 2). Tangential sections, which leave a major part of the inner aspect of the cytoplasmic membrane exposed, revealed significant labeling (Fig. 4E and H), which is also indicative of a location of the two enzymes at the membrane. The preference of the gold label for the cytoplasmic side of the membrane (Fig. 4C, D, F, and G) suggests a position of both CODHs at the inner side of the membrane. The specificity of labeling is apparent from control sections treated with preimmune sera showing virtually no gold particles (Fig. 4B).

View larger version (150K): [in this window] [in a new window]   FIG. 4.   Localization of CODH I and CODH II on immunogold-labeled ultrathin sections of CO-grown C. hydrogenoformans. (A) Ultrastructure of the cell envelope after low-temperature embedding. CM, cytoplasmic membrane; S, surface layer; WL1, first wall layer; WL2, second wall layer; WL3, third wall layer. (B) Control labeling was with preimmune serum and gold-labeled secondary IgG antibodies. IgG antibodies directed against CODH I or CODH II were absent. (C and F) Longitudinal and cross-sections after labeling with IgG antibodies directed against CODH I and the gold-labeled secondary IgG antibodies. (D and G) Treated as for panels C and F, except that IgG antibodies directed against CODH II were used. (E) Tangential section parallel to the long cell axis; all other conditions were as for panels C and F. (H) Tangential section through a cell pole; all other conditions were as for panels C and F. Bar, 0.1 µm.

Reconstitution of the CO-dependent production of H₂ by cytoplasmic membranes. As already mentioned, cytoplasmic membrane fractions contained less than 30% of the total CO-oxidizing activity present in cell-free extracts (Table 1) along with the entire H₂-evolving hydrogenase activity. The experiments reported in Table 3 examined the conditions of the CO-driven formation of H₂ by cytoplasmic membranes of C. hydrogenoformans. H₂ evolution increased considerably when both CODH I and protein B were present. Under the same conditions, CODH II or protein B alone had no effect. Apparently, protein B functions as a specific electron acceptor of CODH I, transferring the electrons to the hydrogenase. Although oxidized flavins are able to accept electrons from both CODHs, they could not functionally replace protein B. Protein B refers to a greenish-brown-colored protein fraction which eluted at 0.67 M NaCl from the Macro-Prep High Q anion exchanger in step 3 of the CODH purification scheme (Table 1). The fraction presumably contains one or several ferredoxins, according to the negative charge, and a broad shoulder centered around 390 nm and extending from 360 to 500 nm in the oxidized UV-visible absorption spectrum. We have not been able to identify a fraction from the Macro-Prep High Q anion exchanger that would couple the electron transfer from CODH II to the hydrogenase.

CO-dependent production of NADPH. Other than the purified CODHs, cytoplasmic fractions of C. hydrogenoformans catalyzed the CO-dependent reduction of NADP⁺ (2 mM) with a rate of 0.31 µmol of NADP⁺ reduced min¹ mg¹. NADPH formation increased 1.5-fold upon addition of CODH II (0.5 mg of CODH II per mg of cytoplasmic protein). CODH I or NAD⁺ had no effect.

	DISCUSSION

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C. hydrogenoformans contains two distinct CODHs. CODH I and CODH II of C. hydrogenoformans are distinct proteins according to the results which we have obtained. CODH I (subunit mass, 62.5 kDa; holoenzyme mass, 125 kDa) and CODH II (subunit mass, 64.5 kDa; holoenzyme mass, 129 kDa) differ slightly in their subunit and holoenzyme masses (Fig. 1A and B) and are immunologically unrelated to each other (Fig. 1D). Their N-terminal sequences and proteolytic peptide patterns (Fig. 1C) indicate different primary structures. The enzymes show different temperature optima and catalytic properties. CODH I and CODH II from C. hydrogenoformans, as well as the CODH from R. rubrum, are very similar in sharing high CO-oxidizing activity, high affinity for CO, and comparable holoenzyme and subunit masses (6, 7).

Both CODHs are membrane-associated homodimeric NiFeS proteins. Intact CO-grown cells of C. hydrogenoformans contain more than 92% of the total CO dehydrogenase population in association with the inner aspect of the cytoplasmic membrane (Fig. 4 and Table 2). The intracellular location of both CODHs was independent of the growth phase. Upon cell disintegration, a high proportion (~ 70%) of both CODHs became solubilized (Table 1), reflecting rather weak noncovalent interactions of the enzymes with the membrane.

Presumed functions of the two CODHs in the metabolism of C. hydrogenoformans. According to the scheme presented in Fig. 5, CODH I is involved in the generation of energy, and CODH II is involved in the assimilation of carbon. CODH I generates the electrons from CO which are subsequently channelled via the ferredoxin-like protein B to a hydrogenase, which is the site where intracellular protons are reduced to H₂. Ferredoxins couple the electron transport from CODH to hydrogenase in phototrophs (14), acetogens (48), and methanogens (57). The CO-driven proton respiration in C. hydrogenoformans is coupled to the translocation of H⁺ across the cytoplasmic membrane. This is indicated by experiments where the application of CO pulses to resting cells led to a transient acidification of the bacterial environment from which H⁺/CO ratios of 0.5 could be extrapolated under conditions where the membrane potential was dissipated by thiocyanate (KSCN). In R. rubrum (4, 20) or methanogenic Archaea (4, 29, 36, 41), the complex I-related H₂-evolving hydrogenases were proposed to be the site of proton translocation. We are, therefore, assuming the same function for the membrane-bound hydrogenase of C. hydrogenoformans, which shows sequence similarities of 80, 95, 72, and 67% (TIGR database) to the large (CooH), small (CooL), proposed H⁺-translocating (CooK), and TYKY (CooX) subunits of the corresponding [Ni-Fe] hydrogenase of R. rubrum (20).

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Hypothetical scheme showing the function of the two CODHs in C. hydrogenoformans. CODH I is involved in energy generation and CODH II serves anabolic functions. For details refer to the text. The scheme is not intended to give correct stoichiometries. Abbreviations: B, ferredoxin-like protein B; H2ase, membrane-bound [NiFe] hydrogenase.

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