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FEMS Microbiology Letters 121 (1994) 165-170 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 165 FEMSLE 06109 Properties of a thermostable 4Fe-ferredoxin from the hyperthermophilic bacterium Thermotoga maritima Jenny M. Blamey, Swarnalatha M u k u n d and Michael W.W. A d a m s * Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA (Received 5 May 1994; revision received 7 June 1994; accepted 8 June 1994) Abstract: A ferredoxin has been purified from one of the most ancient and most thermophilic bacteria known, Thermotoga maritima, which grows up to 90°C. The reduced protein (M r approx. 6300) contains a single S = 1/2 [4Fe-4S] 1÷ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95°C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria. Key words: Thermotoga maritima; Fermentation; Thermostability; Hyperthermophilic Archaea; Evolution Introduction Thermotoga maritima [1] represents one of the two most thermophilic and most ancient genera within the bacterial domain [2], and is usually referred to as a hyperthermophile. It grows up to 90°C by the fermentation of carbohydrates with lactate, acetate, CO 2 and H 2 as the products. If elemental sulfur (S°) is also added to the growth medium, the organism reduces it to H2S [1]. T. maritima therefore resembles many of the hyperthermophilic archaea such as species of Pyrococcus and Thermococcus [3]. These organisms also * Corresponding author. Tel.: (706) 5422060; Fax: (706) 542 0229. SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 2 5 9 - T grow at 90°C, have S%reducing, saccharolytic metabolisms, and are the most slowly evolving organisms within the their domain [2,3]. T. maritima differs from the archaea, however, in that it couples glucose oxidation to H 2 production via a conventional Embden-Meyerhof-Parnas pathway, pyruvate ferredoxin oxidoreductase [4] and an Fe-hydrogenase [5]. In contrast, hyperthermophilic archaea are proposed to contain an unusual type of ferredoxin-dependent EntnerDoudoroff pathway [6], a novel type of pyruvate ferredoxin oxidoreductase (see [41) and a Ni-hydrogenase [7]. The ferredoxins from the archaea P. furiosus and T. litoralis are extremely thermostable and contain a single 4Fe-cluster [8,9]. In contrast, mesophilic fermentative bacteria typically contain thermally labile 8Fe-type ferredox- 166 ins. Herein we report the purification and characterization of the ferredoxin from the hyperthermophilic bacterium, T. maritima. Materials and Methods Growth of the bacterium and purification of ferredoxin Thermotoga maritima (DSM 3109) was grown as previously described [5]. The ferredoxin was routinely purified from 200 g of cells (wet weight) at 23°C under anaerobic conditions [5]. The procedure was the same as for the purification of T. maritima hydrogenase, up to and including the first Q Sepharose (Pharmacia LKB) column [5]. The ferredoxin started to elute when 0.38 M NaC1 was applied to the column. Fractions (100 ml) with an m390/A280 ratio greater than 0.13 (600 ml) were then applied to a column (5 x 30 cm) of hydroxyapatite (Behring) previously equilibrated with 50 mM Tris. HCI (pH 8.5). A gradient from 0 to 0.3 M potassium phosphate (1.5 I) in the same buffer was applied at 2.5 ml rain -1 and the ferredoxin started to elute as soon as the gradient began. Fractions (20 ml) with a A39o/A28 o ratio above 0.28 were combined (160 ml) and concentrated to approximately 20 ml using a column (HR 10/10) of Q Sepharose equilibrated with 50 mM Tris (pH 8.0). The sample was applied directly to the column and eluted at 0.5 ml min-~ in 3-ml fractions with a gradient (40 ml) from 0 to 1 M NaC1. The concentrated ferredoxin was applied to a column (6.0 × .60 cm) of Superdex 75 (Pharmacia LKB) equilibrated with buffer containing 0.2 M NaC1. Fractions (15 ml) with a A390/Azso value above 0.70 were combined (60 ml, 80 rag) and concentrated using a Q Sepharose column. The ferredoxin was stored as pellets in liquid N 2. used as standards. SDS-electrophoresis, analyses for iron, acid-labile sulfide, tryptophan and cysteine, and the preparation of reduced apoferredoxin were all carried out as previously described [8]. The concentrations of samples of pure ferredoxin were estimated by a heat-modified version of the Lowry method (see [8]). The results agreed (_+ 8%) with the amounts of protein recovered by quantitative recovery of amino acids (see [9]). E P R spectroscopy [4] and the assays of hydrogenase [9] and of pyruvate ferredoxin oxidoreductase [4] of T. maritima were carried out as indicated. Results and Discussion Molecular properties Purified T. maritima ferredoxin gave rise to a single protein band after SDS-electrophoresis (using 20% (w/v) acrylamide) and eluted as a single peak from a gel filtration column. The M r value obtained using both techniques was 7300 + 1500, indicating that it is a monomeric protein. The UV-visible absorption spectra of the airoxidized and dithionite-reduced forms (for exampie, see Fig. 1) were typical of this class of protein (see [10]). The m39o/A280 ratio was 16.3 mM -1 c m - ' and the molar absorbance at 390 nm was 0.74. The protein contained 4.2 +_ 0.6 and 3.9 + 1.2 g atoms (mol i r o n ) - ' and acid-labile sulfide, respectively, suggesting the presence of a single 4Fe-center. The amino acid composition of the ferredoxin was (in residues mol-1): B, 8; T, 2; S, 2; Z, 7; P, 4; G, 6: A, 7; C, 6; V, 5; M,1; I, 2; L, 4; F, 2; K, 4; R, 1 (from four separate determinations), for a total of 61 residues. Tyrosine, histidine and tryptophan were not detected. From these data, the ferredoxin has a M r value of 6331 (including the iron and sulfide), comparable to that of P. furiosus ferredoxin [9]. Analytical methods' The molecular mass was estimated using a column (16/30) of Superdex 75 using 50 mM Tris. HC! (pH 8.0) containing 1.0 M NaC1 as eluent. Bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa) and P. furiosus rubredoxin (5.3 kDa; see [3]) were Thermal stability T. maritima ferredoxin showed no change in its U V / v i s i b l e absorption properties or in its electron acceptor activity for pyruvate ferredoxin oxidoreductase (see below) after heating the protein (1.5 mg m1-1 in 50 mM Tris " HCI, pH 8.0) for 12 167 h at 95°C. After 24 h at this temperature, the decreased by about 20% indicating some destruction of the FeS chromophore. The thermal stability of this protein is therefore similar to that previously reported for P. furiosus ferredoxin [8]. For comparison, the ferredoxin from the moderate thermophile Clostridium thermocellum, has a half life (tl/2) at 80°C of about 60 min [11]. A39o/A280 ratio g = 2.06 i g = 1,93 I- g-189 Electron carrier activity Dithionite-reduced T. maritima ferredoxin did not function as an electron donor for H e evolution by purified T. rnaritima hydrogenase [5], even at concentrations up to 0.5 mM and temperatures up to 90°C, but the oxidized protein did replace methyl viologen as an electron acceptor for pyruvate oxidation in the routine assay of T. maritirna pyruvate ferredoxin oxidoreductase [4]. Using pyruvate (5 raM) and CoA (0.2 mM), the apparent K m and Vmax values were approximately 26 /xM and 280 /~mol of pyruvate oxidized min -~ mg -1, respectively, at 80°C. Similarly, the oxidized ferredoxin was readily reduced at 80°C by 1.5 1.0 ,5 < ",, I 360 P 420 1 I I 325 350 375 Magnetic Field / m T Fig. 2. EPR spectrum of reduced T. maritima ferredoxin. The ferredoxin (6 mg ml - t in 50 mM Tris.HCl, pH 8.0) was reduced with sodium dithionite (2 mM). The conditions were: temperature, 6K; microwave power, 10 mW; modulation amplitude, 0.25 mT; microwave frequency, 9.42 GHz. pyruvate ferredoxin oxidoreductase in the presence of CoA and pyruvate, as its visible absorption spectrum (Fig. 1) was identical to that obtained after reduction of the oxidized protein with sodium dithionite (data not shown). Fermentative, H2-evolving anaerobic bacteria typically utilize ferredoxin as the electron carrier between pyruvate oxidoreductase and an H z-evolving hydrogenase (see [5]). However, T. maritima must have an additional intermediate electron carrier(s). Nature of the iron-sulfur cluster T. maritima ferredoxin reduced by sodium a 0.5 0.0 I 300 [ 480 I 540 600 Wavelength (nm) Fig. 1. Visible absorption spectra of T. maritirna ferredoxin. The ferredoxin (0.29 mg m1-1) was in 50 mM N-(2-hydroxyethyl)piperazine N'-3-morpholinopropane sulfonic acid (EPPS; pH 8.4) containing 0.1 M NaCI. (a) After air oxidation; (b) after incubation with T. maritima pyruvate ferredoxin oxidoreductase (5 U) in the presence of coenzyme A (0.1 mM), MgCI 2 (1 raM), thiamine pyrophosphate (0.05 mM) and pyruvate (5 raM) at 80°C for 2 min under anaerobic conditions (see [4]). Spectra were recorded at 23°C. dithionite exhibited a rhombic E P R spectrum with a gz = 2.06, gy = 1.93 and g~ = 1.89 (Fig. 2). This signal was observed up to 20 K and represented 0.95 _+ 0.14 spins mol-1. No additional absorption was observed at lower magnetic fields. The spectrum is typical of a magnetically isolated S = 1 / 2 [4Fe-4S] ~+ cluster (see [10]). Together with the above results, these data show that the ferredoxin is a monomeric protein containing a single 4Fecluster which in the reduced form exists exclusively with a S = 1 / 2 ground state. Almost all ferredoxins containing 4Fe-clusters exhibit similar E P R properties. A notable exception is the pro- 168 Tm: TI: Pf: Ct: Mt: Dg: Bt: Da: Av: Pa: .MKVR~DADA .MKVSVDKDA AWKVSVDQDT .MKVTVDGDL .MPALVNADE ..PIE VNDD. PKYTIVDKET ARKFYVDQDE ...AFVVTDN ...AYyINDS CIGC..GVCE CIGC..GVCA CIGD..AICA CIAC..QT~I CSGC..GSCV ~MAC..EACV C IAC..~ACG C IAC..ESCV C!K~KYTDCV C__~IAC..~ACK NLCPDVFQLG SICPDVFEMD SLCPDVFEMN DLCPSVEDWD DECPSEAITL EICPDVFEMN AAAPDIYDYD EIAPGAFAMD EV~P.VDCFY PECP.VNIQ. D D G K A K .... _____QPETDLPC D D G K A K .... A L V A E T D L E C DEGKAQPKVE VIEDEELYN~ pE~SLHVIVD EVPEGAEDS~ DEEKGI.AV. yDQDECVE C EE~D..KAV. VINPDSDLD~ EDGIAQGIVE V..PDILIDD ............. PEIEKAY E.HPN...FL VIHPDECID~ Q . H S .... IY A I D A D S C I D ~ AK-(100) AK- (74) AK- (58) AR- (44) GA- (38) VE- (35) MM- (35) VK- (28) AL- (28) GS- (24) Fig. 3. N-terminal amino acid sequences of T. maritima ferredoxin and representative archaeal and bacterial ferredoxins. Gaps (.) have been inserted to maximize homology. The abbreviations used and the number of iron atoms each ferredoxin contains are: Tm, T. maritima (4Fe); Pf, Pyrococcus furiosus (4Fe); TI, Thermococcus litoralis (4Fe); Dg, Desulfovibrio gigas Fd I (3Fe); Bt, Bacillus thermoproteolyticus (4Fe); Da, D. africanus Fd I (7Fe); Ct, Clostridium thermoaceticum (4Fe); Mt, Methanosarcina thermophila (8Fe); Av, Azotobacter vinelandii (7Fe); Pa, Peptococcus aerogenes (8Fe). The asterisks denote the cluster-binding residues for the 4Fe-ferredoxins. Numbers in parentheses indicate identity (%) with Tm ferredoxin. Data taken from [9,13,14]. tein from P. furiosus whose reduced 4Fe-center has a predominantly S = 3 / 2 ground state which gives rise to an approx, g = 5 EPR signal [12]. This unusual property is thought to be related to the partial non-cysteinyl coordination to the cluster, a property not shared by the ferredoxin of the hyperthermophile T. litoralis (see Fig. 3). N-terminal amino acid sequence analysis The sequence of the first 44 amino acids of T. maritima ferredoxin was determined and this is aligned in Fig. 3 with the corresponding sequences of the two 4Fe-ferredoxins from hyperthermophilic archaea ( P. furiosus and Ts. litoralis; [9]), together with representatives of the more than 30 sequences of mesophilic ferredoxins that are known (see [13]). From the consensus sequence of cysteinyl residues found in these proteins, the single [4Fe-4S] cluster in T. maritima ferredoxin appears to have complete cysteinyl coordination, in agreement with the spectroscopic analysis. Remarkably, the amino acid sequence of T. maritima ferredoxin (45/61 residues) shows very high homology with the two ferredoxins (both of the 4Fe-type) from the hyperthermophilic archaea, e.g. 74% identity with the Thermococcus protein, but significantly lower homology with ferredoxins from bacteria and methanogenic archaea (from 19 to 44% comparing all known sequences; [13,14]). Interestingly, the highest identities with the non-hyperthermophilic proteins are with the ferredoxins from the moderately thermophilic bacterium, C. thermoaceticum (44% identity) [11] and from the moderately thermophilic, methanogenic archaeon, Methanosarcina thermophila (38% identity) [14]. However, systematic analyses of the amino acid sequences of analogous mesophilic, moderately thermophilic and hyperthermophilic proteins, even in the case of small redox proteins, have as yet offered no clue as to the mechanisms of protein 'hyperthermostability' (see [3]). The great similarity between the ferredoxins from hyperthermophilic archaea and the hyperthermophilic bacterium, T. maritima, in amino acid sequence, thermal stability and cluster content, obviously suggests a close evolutionary relationship between these proteins. The homology is surprising, however, for two reasons. First, the ferredoxin-dependent metabolic pathways that these two groups of organisms use for sugar oxidation are very different (see [3]). Second, these organisms appear to be the most ancient of all known life forms [2], yet the ancestral ferredoxin is thought to be of the 8Fe-type and to contain two [4Fe-4S] clusters (see [13]). Clearly, the resuits presented here suggest that a revision might be necessary in the evolutionary mechanisms of these simple iron-sulfur proteins. Acknowledgements This research was supported by grants from the Department of Energy (FG09-88ER13901) and by a National Science Foundation Training 169 Group Award to the Center for Metalloenzyme Studies of the University of Georgia (DIR 9014281). 7 8 References 9 1 Huber, R., Langworthy, T.A., K6ning, H., Thomm, M., Woese, C.R., Sleytr, U.B. and Stetter, K.O. (1986) Thermotoga maritirna sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C. Arch. Microbiol. 144, 324-333. 2 Olsen, G.J., Woese, C.R. and Overbeek, R. (1994) The winds of (evolutionary) change: breathing new life into microbiology. J. Bacteriol. 176, 1-6. 3 Adams, M.W.W. (1993) Enzymes and proteins from organisms that grow near and above 100°C. Annu. Rev. Microbiol. 47, 627-658. 4 Blamey, J.M. and Adams, M.W.W. (1994) Characterization of an ancestral-type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga rnaritima. Biochemistry 33, 1000-1007. 5 Juszczak, A., Aono, S. and Adams, M.W.W. (1991) The extremely thermophilic eubacterium, Thermotoga maritima, contains a novel iron-hydrogenase whose cellular activity is dependent upon tungsten. J. Biol. Chem. 266, 13844-13841. 6 Sch~ifer, T. and Sch6nheit, P. (1992) Maltose fermentation to acetate, CO 2 and H 2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the 10 11 12 13 14 operation of a novel sugar fermentation pathway. Arch. Microbiol. 158, 188-202. Bryant, F.O. and Adams, M.W.W. (1989) Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus. J. Biol. Chem. 264, 5070-5079. Aono, S., Bryant, F.O. and Adams, M.W.W. (1989) A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus. J. Bacteriol. 171, 3433-3439. Busse, S.A., La Mar, G.N., Yu, L.P., Howard, J.B., Smith, E.T., Zhou, Z.H. and Adams, M.W.W. (1992) Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis. Biochemistry 31, 11952-11962. Cammack, R. (1992) Iron-sulfur clusters in enzymes: themes and variations. Advs. Inorg. Chem. 38, 281-322. Bruschi, M., Cambillau, C., Bovier-Lapierre, G., Bonicel, J. and Forget, P. (1986) Sequence determination and three-dimensional modeling of Clostridium thermocellum ferredoxin: structural considerations for its high thermal stability. Biochim. Biophys. Acta 873, 31-37. Conover, R.C., Kowal, A.T., Fu, W., Park, J.-B., Aono, S., Adams, M.W.W. and Johnson, M.K. (1990) Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin. J. Biol. Chem. 265, 8533-8541. Matsubara, H. and Saeki, K. (1992) Structural and functional diversity of ferredoxins and related proteins. Adv. Inorg. Chem. 38, 223-280. Clements, A.P. and Ferry, J.G. (1992) Cloning, nucleotide sequence, and transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina thermophila. J. Bacteriol. 174, 5244-5250.