Báo cáo khóa học: Two distinct heterodisulfide reductase-like enzymes in the sulfate-reducing archaeon Archaeoglobus profundus pptx

Eur J Biochem 271, 1106–1116 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04013.x Two distinct heterodisulfide reductase-like enzymes in the sulfate-reducing archaeon Archaeoglobus profundus Gerd J Mander1, Antonio J Pierik2, Harald Huber3 and Reiner Hedderich1 Max-Planck-Institut for Terrestrial Microbiology, Marburg, Germany; 2Laboratory for Microbiology, Department of Biology, Philipps University Marburg, Germany; 3Department of Microbiology and Archaeenzentrum, University of Regensburg, Germany Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea Two types of Hdr have been identified and characterized from distantly related methanogens Here we show that the sulfate-reducing archaeon Archaeoglobus profundus cultivated on H2/sulfate forms enzymes related to both types of Hdr From the membrane fraction of A profundus, a two-subunit enzyme (HmeCD) composed of a b-type cytochrome and a hydrophilic iron–sulfur protein was isolated The amino-terminal sequences of these subunits revealed high sequence identities to subunits HmeC and HmeD of the Hme complex from A fulgidus HmeC and HmeD in turn are closely related to subunits HdrE and HdrD of Hdr from Methanosarcina spp From the soluble fraction of A profundus a six-subunit enzyme complex Heterodisulfide reductase (Hdr) is a unique disulfide reductase, which has a key function in the energy metabolism of methanogenic archaea The enzyme catalyses the reversible reduction of the mixed disulfide (CoM–S–S–CoB) of the two methanogenic thiol-coenzymes, called coenzyme M (CoM-SH) and coenzyme B (CoB-SH) This disulfide is generated in the final step of methanogenesis [1] Two types of Hdr have been identified and characterized from distantly related methanogens [2–6] One type of Hdr, which was purified and characterized from Methanothermobacter marburgensis, is a soluble iron– sulfur flavoprotein composed of the three subunits HdrA, Correspondence to R Hedderich, Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch Str., D-35043 Marburg, Germany Fax: + 49 6421 178299, Tel.: + 49 6421 178230, E-mail: hedderic@staff.uni-marburg.de Abbreviations: HdrABC, soluble flavin–iron–sulfur heterodisulfide reductase from Methanothermobacter spp.; HdrDE, heme-containing membrane-bound heterodisulfide reductase from Methanosarcina spp.; Hdl, HdrABC-like enzyme from Archaeoglobus spp.; Hme, HdrDE-like menaquinol-oxidizing enzyme from Archaeoglobus spp.; Mvh, F420-nonreducing hydrogenase (methylviologen-reducing hydrogenase) from Methanothermobacter spp.; Mvh:Hdl, F420nonreducing hydrogenase:heterodisulfide reductase-like enzyme complex; APS, adenosine 5¢-phosphosulfate; DMN, 2,3-dimethyl-1,4-naphtoquinone Enzyme: heterodisulfide reductase (EC 1.99.4.-) (Received 24 September 2003, revised 10 December 2003, accepted 26 January 2004) (Mvh:Hdl) containing Ni, iron–sulfur clusters and FAD was isolated Via amino-terminal sequencing, the encoding genes were identified in the genome of the closely related species A fulgidus in which these genes are clustered They encode a three-subunit [NiFe] hydrogenase with high sequence identity to the F420-nonreducing hydrogenase from Methanothermobacter spp while the remaining three polypeptides are related to the three-subunit heterodisulfide reductase from Methanothermobacter spp The oxidized enzyme exhibited an unusual EPR spectrum with gxyz ¼ 2.014, 1.939 and 1.895 similar to that observed for oxidized Hme and Hdr Upon reduction with H2 this signal was no longer detectable Keywords: Archaeoglobus; heterodisulfide reductase; Hmc complex; iron-sulfur proteins; sulfate-reducing bacteria HdrB and HdrC [2,3] For clarity this enzyme will be called HdrABC throughout this paper From sequence data it has been deduced that HdrA contains an FAD-binding motif and four binding motifs for [4Fe)4S] clusters HdrC was shown to contain two binding motifs for [4Fe)4S] clusters while in subunit HdrB no characteristic binding motif of any known cofactor could be identified However, this subunit contains 10 highly conserved cysteine residues present in two Cx31)38CCx33)34Cx2C motifs The second type of Hdr, designated as HdrDE, is found in Methanosarcina species [4,6] This enzyme is tightly membrane bound It is composed of two subunits, a membrane anchoring b-type cytochrome (HdrE) and a hydrophilic iron–sulfur protein (HdrD) The amino-terminal part of HdrD contains two characteristic binding motifs for [4Fe)4S] clusters also conserved in subunit HdrC of the Mt marburgensis enzyme The carboxy-terminal part of HdrD harbours the two Cx31)38CCx33)34Cx2C motifs also present in HdrB Subunit HdrD of the Methanosarcina enzyme can be regarded as a hypothetical fusion protein of subunits HdrC and HdrB of Mt marburgensis Hdr [5] The catalytic centre must be located on Ms barkeri HdrD and Mt marburgensis HdrCB, which are conserved in both enzymes [3,5] A detailed spectroscopic characterization showed that the active site harbours a [4Fe)4S] cluster [7,8], which is most probably coordinated by some of the cysteine residues present in the Cx31)38CCx33)34Cx2C motifs With both HdrABC and HdrDE a reaction intermediate is trapped when only coenzyme M is added to the oxidized enzyme (in the absence of coenzyme B) It is characterized by a unique S ¼ 1/2 EPR spectrum with Ó FEBS 2004 Heterodisulfide reductase-like enzymes in A profundus (Eur J Biochem 271) 1107 principal g values ¼ 2.013, 1.991 and 1.938 for HdrDE and gxyz ¼ 2.011, 1.993, 1.944 for HdrABC observable at temperatures below 50 K [7] In this paramagnetic species, which was designated as CoM–Hdr, coenzyme M was shown to be directly bound to the cluster via its thiol group [9] Hence, the active site iron–sulfur cluster is directly involved in the disulfide cleavage reaction The two types of Hdr differ with respect to their physiological electron donor HdrDE receives reducing equivalents from the reduced methanophenazine pool via its b-type cytochrome subunit The enzyme is part of an energy-conserving membrane-bound electron transport chain with H2 or reduced coenzyme F420 as electron donor and the heterodisulfide as terminal electron acceptor [10,11] HdrABC forms a tight complex with the F420-nonreducing hydrogenase (Mvh) This six-subunit complex catalyses the reduction of the heterodisulfide by H2 After cell lysis, this complex is almost completely localized in the soluble fraction It is yet unknown how the exergonic reduction of the heterodisulfide is coupled to energy conservation in Mt marburgensis [12] An interesting result obtained from the analysis of the genome sequence of the sulfate-reducing archaeon Archaeoglobus fulgidus was the presence of several genes encoding enzymes closely related to heterodisulfide reductase from methanogens [13] The isolation of one of these enzymes, called Hme, has recently been reported [14] Hme, when purified, is composed of four subunits HmeACDE The encoding gene cluster predicts the presence of a fifth subunit (HmeB) One of the Hme subunits (HmeC) is a b-type cytochrome, a second subunit (HmeD) is closely related to subunit HdrD of Ms barkeri Hdr HmeD also contains one copy of the Cx31)38CCx33)34Cx2C motif, which was found to be characteristic for Hdr However, in HmeD this cysteine-rich motif is composed of only four cysteine residues, an aspartate residue replaces the last cysteine residue Oxidized Hme exhibited an unusual EPR spectrum with g-values at 2.031, 1.995, and 1.951 The paramagnetic species could be reduced in a one-electron transfer reaction, but could not be oxidized further It thus shows EPR and redox properties similar to a paramagnetic species formed when the active-site iron–sulfur cluster of Hdr from Ms barkeri or Mt marburgensis binds one of its thiol substrates as an extra ligand during the catalytic cycle [9] Based on the spectroscopic properties of Hme and based on the presence of the Cx31)38CCx33)34Cx2C/D motif in A fulgidus HmeD, this subunit was proposed to have a catalytic site similar to that of Hdr [14] Enzymes related to Hme have also been identified in the sulfate-reducing bacterium Desulfovibrio vulgaris, the green sulfur bacterium Chlorobium tepidum and the purple sulfur bacterium Allochromatium vinosum [15–18] One of the major open questions in understanding the energy metabolic pathways of sulfate reducing bacteria and archaea concerns the path of reducing equivalents generated in the oxidative branch of the metabolic pathway to the enzymes of sulfate reduction This electron transfer process is thought to be coupled with energy conservation The A fulgidus Hme protein has been proposed to participate in this electron transfer reaction Evidence has been provided that this enzyme functions as a menaquinol-acceptor oxidoreductase mediating the electron transfer from the quinone pool to a yet unidentified electron carrier in the cytoplasm which in turn could function as an electron donor of the enzymes of sulfate reduction, adenosine 5¢-phosphosulfate (APS) reductase and sulfite reductase [14] In this communication we address the question whether Hme or one of its homologues is also involved in sulfate reduction when H2 is the electron donor Although A fulgidus has been reported to grow with H2 as sole electron donor, growth under these conditions is very poor Lactate-grown A fulgidus cells not exhibit hydrogenase activity [19] Therefore, the hydrogenotrophic Archaeoglobus species, A profundus, was used in this study Materials and methods Materials Unless otherwise stated, chemicals were from Merck (Darmstadt, Germany) and chromatographic materials and columns were from Amersham Biosciences Organism growth A profundus (DSMZ 5631) was grown in a 300-L fermenter at 85 °C as described previously [20] Cells were harvested after shock cooling to °C in a continuous flow centrifuge (Z61, Padberg Lahr, Germany) at 17 000 g; the pellet was frozen in liquid nitrogen and stored at )80 °C prior to use Purification of HmeCD All purification steps were carried out under strictly anaerobic conditions under an atmosphere of N2/H2 (95 : 5; v/v) at 18 °C Cells were lysed by sonication and then centrifuged at 6400 g for h The supernatant was ultracentrifuged at 150 000 g for h The pellet was resuspended in 50 mM Mops/KOH, pH 7.0 (buffer A) using a Teflon homogenizer Protein was solubilized from the membrane with 15 mM dodecyl-b-D-maltoside (2 mg dodecyl-b-D-maltosidmg)1 protein) at °C for 12 h Proteins not solubilized after 12 h were removed by ultracentrifugation as described above Solubilized protein was loaded to a Q-Sepharose column (2.6 · 10 cm) equilibrated with buffer A containing mM dodecyl-b-D-maltoside (buffer A1) Protein was eluted in a stepwise NaCl gradient (80 mL each in buffer A1): mM, 300 mM, 400 mM, 500 mM, 600 mM and M The fractions were checked for their heme-content by UV/visible spectroscopy The majority of the heme-containing proteins eluted at 600 mM NaCl These fractions were applied to a Superdex 200 gel-filtration column (2.6 · 60 cm) equilibrated in buffer A1 with 100 mM NaCl Protein was eluted with the same buffer The only heme-containing fraction eluted after 180 mL (peak maximum) These fractions were loaded on a MonoQ column (1.0 · 10 cm) equilibrated with buffer A1 Protein was eluted using a linear NaCl gradient (0–1 M, 100 mL) Heme-containing protein(s) eluted at 600 mM NaCl These fractions were pooled and concentrated by ultrafiltration (100-kDa cut off, Molecular/Por ultrafiltration membranes, Houston) and stored in buffer A1 at °C Ó FEBS 2004 1108 G J Mander et al (Eur J Biochem 271) under N2 Protein was judged to be > 95% pure by SDS/ PAGE Purification of the Mvh:Hdl enzyme complex from A profundus All purification steps were performed as described above for the purification of HmeCD The 150 000 g supernatant of cell-free extracts was applied to a Q-Sepharose (2.6 · 10 cm) anion exchange column equilibrated with buffer A Protein was eluted in a stepwise NaCl gradient in buffer A (see above) The majority of the hydrogenase activity eluted at 400 mM NaCl (Table 2) These fractions were pooled, the buffer was changed to 10 mM Na-phosphate buffer pH 7.0 by ultrafiltration (50-kDa cut-off) and protein was loaded on a hydroxyapatite column (1.6 · 10 cm) equilibrated with 10 mM Na-phosphate buffer pH 7.0 Protein was eluted in a linear Na-phosphate gradient (10 mM to M, 350 mL) The majority of the hydrogenase activity eluted at 150–180 mM Na-phosphate These fractions were pooled and the buffer was changed to buffer A by ultrafiltration The resulting fraction was loaded on a MonoQ column (1.0 · 10 cm) Protein was eluted in a linear NaCl gradient in buffer A (0–700 mM, 150 mL) The majority of the hydrogenase activity eluted at 400 mM NaCl These fractions were concentrated by ultrafiltration and stored in buffer A at °C under N2 a field modulation frequency of 100 kHz and a modulation amplitude of 0.6 mT The sample was cooled by an Oxford Instrument ESR 900 flow cryostat with an ITC4 temperature controller Spin quantifications were carried out under nonsaturating conditions using copper perchlorate as standard (10 mM CuSO4, M NaClO4, 10 mM HCl) When EPR signals overlapped with other signals, e.g radical signals from flavins, the signals were simulated, and the simulations were double integrated to obtain the spin intensity Temperature dependence studies were carried out under nonsaturating conditions where possible For all signals, the peak amplitude was measured at different temperatures These values were used to obtain Curie plots describing the temperature behaviour of the respective signal EPR signals were simulated using noncommercial programs supplied by S.P.J Albracht based on formulas described previously [21] Determination of amino-acid sequences For determination of amino-terminal amino acid sequences, polypeptides were separated by SDS/PAGE and blotted on to poly(vinylidene difluoride) membranes (Applied Biosystems) as described previously [5] Sequences were determined using an Applied Biosystems 4774 protein/peptide sequencer and the protocol given by the manufacturer Determination of enzyme activities Analytical methods Enzyme assays were routinely carried out under anoxic conditions in 1.5-mL quartz cuvettes at 65 °C One unit of enzyme activity corresponds to lmol H2 consumedỈmin)1 Hydrogen uptake activity with benzylviologen as electron acceptor was determined by following the reduction of benzylviologen at 578 nm (e ¼ 8.6 mM)1Ỉcm)1) The 0.8mL assays contained mM benzylviologen and 0.1 mM sodium dithionite in 50 mM Mops/KOH pH 7.0) One unit of H2-oxidation activity is defined as the reduction of lmol benzylviologenỈmin)1 Iron was quantified colorimetrically with neocuproin (2,9-dimethyl-1,10-phenanthroline) and ferrozine[3-(2-pyridyl)-5,6-bis-(4-phenylsulfonate)-1,2,4-triazine] as described previously [22] Acid labile sulfur was analysed with the methylene blue method [23] Protein concentration was routinely measured by the method of Bradford (Rotinanoquant; Roth, Karlsruhe, Germany) using BSA as standard [24] Nickel was determined by atomic absorption spectroscopy on a 3030 Perkin Elmer atomic absorption spectrometer fitted with a HGA-600 graphite furnace assembly and an AS-60 autosampler For identification of the flavin and determination of the flavin content of the Mvh:Hdl complex, protein (200 lL, 8.9 mgỈmL)1) was denatured by exposure to 10% (m/v) trichloroacetic acid Denatured protein was removed by centrifugation, the resulting supernatant was adjusted to pH with K2HPO4 and analysed by chromatography using a reverse-phase HPLC column (LiChrospher 60 RP 18, lm, 125 · mm, Merck, Germany) equilibrated with 50 mM ammonium formate containing 25% methanol Flavins were eluted isocratically with the equilibration buffer FAD and FMN standards were used to identify and quantify the flavin Hemes were characterized by their pyridine hemochrome spectra [25] Protein (500 lL, mgỈmL)1) was mixed with 500 lL of a stock solution of 200 mM NaOH in 40% (v/v) pyridine/H2O and lL of 0.1 M K3Fe(CN)6 in a 1.5-mL cuvette to record the oxidized spectrum Solid sodium dithionite was then added (2–5 mg) and several successive spectra of the reduced pyridine hemochromes were recorded UV/visible spectroscopy Spectra of samples in 1.5-mL Quartz cuvettes in an anaerobic chamber under N2/H2 (95/5, v/v) were recorded using a Zeiss Specord S10 diode array spectrophotometer connected to a quartz photoconductor (Hellma Muhlheim, ă Germany) Sodium dithionite was added to an enzyme solution (0.7 mg proteinỈmL)1 in buffer A) to obtain the spectrum of the fully reduced enzyme The spectrum of the oxidized enzyme was obtained after oxidation by air The oxidation of the heme groups by DMN (2,3-dimethyl1,4-naphtoquinone) was followed spectrophotometrically DMN was added to the enzyme solution (1 mg proteinỈmL)1 in 50 mM Mops/KOH pH 7.0), mM dodecyl-b-D-maltoside) to a final concentration of 150 lM and spectra were recorded every s EPR spectroscopy measurements EPR spectra at X-band (9.45 GHz) were obtained with a Bruker EMX spectrometer All spectra were recorded with Ó FEBS 2004 Heterodisulfide reductase-like enzymes in A profundus (Eur J Biochem 271) 1109 aggregation of the protein upon boiling, which is frequently observed for integral membrane proteins From g of wet cell mass ( 500 mg protein)  mg of purified enzyme were obtained Determination of the amino-terminal sequences and identification of homologous genes in A fulgidus Fig SDS/PAGE of purified HmeCD Proteins were separated in a 14% slab gel (8 · cm), which was subsequently stained with Coomassie Brilliant Blue R250 The molecular masses of marker proteins are given on the right side, the apparent molecular masses of the polypeptides in lanes and are given on the left side M, Lowmolecular-mass marker (Amersham Biosciences) Lane 1, 10 lg A profundus HmeCD denatured for 30 at room temperature in SDS sample buffer (Laemmli buffer containing mM dithiothreitol and 2% SDS); lane 2, 10 lg Hme complex denatured for at 100 °C in SDS sample buffer The polypeptide with an apparent molecular mass of 32 kDa, identified as a b-type cytochrome by N-terminal sequencing, is nondetectable in the boiled sample; it probably forms aggregates that not run into the gel (lane 2) This behaviour is typical for integral membrane proteins Results Purification of a heme-containing protein from the membrane fraction of A profundus To purify heme-containing enzymes possibly related to the Hme complex from A fulgidus, the characteristic UV/ visible spectrum of heme proteins was followed throughout the purification This resulted in the isolation of the major heme-containing protein from the membrane fraction of A profundus cells cultivated on H2/sulfate For analysis of the protein by SDS/PAGE samples were either boiled for in SDS sample buffer or incubated in SDS sample buffer for 30 at room temperature (Fig 1) The sample incubated at room temperature yielded two major polypeptide bands with apparent molecular masses of 53 kDa and 32 kDa (Fig 1; lane 1) In the boiled sample, the 32-kDa polypeptide was undetectable in Coomassie Brilliant blue-stained gels (Fig 1, lane 2) This could be due to The amino-terminal sequences of the two polypeptides were determined by Edman degradation (Table 1) Using these sequences, the genome of A fulgidus was searched for corresponding genes [13] The amino-terminal sequence of the 53-kDa polypeptide shows 45% sequence identity to the gene product of AF502, the amino-terminal sequence of the 32-kDa polypeptide shows 50% sequence identity to the polypeptide encoded by AF501 (Table 1) In A fulgidus both gene products are part of the Hme complex, which has recently been described [14] AF501 (HmeC) and AF502 (HmeD) were shown to share sequence identity with the two subunits HdrE and HdrD of Hdr from Methanosarcina species Based on its similarity to subunits of the A fulgidus Hme complex the A profundus enzyme was designated as HmeCD Characterization of the heme groups by UV/visible and EPR spectroscopy The enzyme purified under anaerobic conditions generally contained the heme groups in the reduced state Fig shows the dithionite-reduced minus air-oxidized absorbance difference spectrum The absorbance maxima at 426 nm (c band), 530 nm (b band) and a split a band at 557 nm and 562 nm are characteristic for hemes of the b-type [26] A similar splitting of the a band has been observed for other heme proteins, for example the cytochrome bL of the cytochrome bc1 complex form Rhodopseudomonas sphaeroides GA [27] Pyridine hemochrome reduced–oxidized difference spectra show maxima for the a and b band at 553 and 521 nm These values are blue-shifted by nm relative to the published values for protoheme IX [28] The same result was obtained for the heme present in Hme from A fulgidus [14] This suggests that Hme in both organisms contains a modified protoheme IX as prosthetic group Addition of DMN to the reduced enzyme resulted in a rapid oxidation of the heme groups present in the enzyme The rates were too rapid to be resolved In oxidized HmeCD and at temperatures below 10 K a sharp absorption-shaped signal with g-values at 6.06 and 5.83 characteristic for ferric high-spin (S ¼ 5/2, Table Amino-terminal sequences of the membrane-bound heme containing enzyme of A profundus Amino-terminal sequences of the A profundus enzyme were derived by Edman degradation, amino-terminal sequences of A fulgidus were derived from the genome sequence [13] In both sequences, identical amino acids are underlined X, No clear assignment to an amino acid could be made; –, gaps inserted to allow an alignment Amino-terminal sequences Sequence identity (%) Corresponding gene A fulgidus A profundus A fulgidus LEALYIFY-ALPYIXFAIFVI 45 AF501 (HmeC) A profundus A fulgidus EVPEELXIKQKFPNWXYXL 55 AF502 (HmeD) MIGV-IFGVIVPYIAVAIFVI MEEMPERIEIKQKFPSWREML Ó FEBS 2004 1110 G J Mander et al (Eur J Biochem 271) Fig Room temperature reduced/oxidized difference spectrum of the purified HmeCD from A profundus The spectrum of the reduced enzyme was recorded after reduction of Hme (0.7 mg protein per mL in 50 mM Mops/KOH, pH 7.0) with sodium dithionite The oxidized spectrum was obtained after oxidation by air The arrows indicate the maxima of the split a-band at 557 and 562 nm E/D < 0.01) heme was observed as described previously for Hdr from M thermophila [6] The third g-value (g  region) could not observed due to the presence of other signals (see below) The oxidized enzyme at pH showed a additional signal with g-values at 2.87 and 2.28 which is characteristic for low-spin (S ¼ 1/2) ferric heme [29] Oxidized HmeCD also showed an intense signal at a g-value of 4.3 characteristic for adventitiously bound high spin ferric iron Characterization of the iron-sulfur clusters of HmeCD by EPR spectroscopy HmeCD was shown to contain 107 ± nmol nonheme iron and 117 ± nmol acid-labile sulfurỈmg)1 protein From the SDS/PAGE an apparent molecular mass of the enzyme of 85 kDa was calculated, this corresponds to 9.2 ± 0.2 mol nonheme iron per mol enzyme and 10 ± 0.3 mol acid-labile sulfur per mol enzyme indicating the presence of two to three [4Fe)4S] clusters These clusters were further characterized by EPR spectroscopy The sodium dithionite reduced enzyme showed at temperatures below 15 K a broad featured spectrum around g ¼ 1.93 indicative of spin–spin coupling between different [4Fe)4S]1+ clusters In ferricyanide or duroquinone oxidized samples a paramagnetic species was detected with gxyz values at 2.031, 1.995 and 1.948 (Fig 3) The total spin concentration of this species was 2.8 lM, corresponding to 0.15 spinỈmol)1 enzyme This signal was detectable without significant broadening from 15 to 35 K At temperatures below 15 K the signal was readily power saturated and at temperatures higher than 35 K the signal started to broaden and was broadened beyond detection at 60 K The EPR properties of this paramagnetic species are very similar to that of the paramagnetic species recently described for the oxidized A fulgidus Hme complex [14] Fig EPR spectrum of A profundus HmeCD EPR spectrum obtained after oxidation of HmeCD (2 mgỈmL)1 with mM K3Fe(CN)6 (thin black line) EPR conditions: temperature, 20 K; microwave power, mW; microwave frequency, 9.458 GHz; modulation amplitude, 0.6 mT; modulation frequency, 100 kHz The spin concentration was 0.15 spinỈmol)1 enzyme as determined by double integration of the simulated EPR signal (thick grey line) Simulation parameters: g1,2,3 ¼ 1.948, 1.995 and 2.031; W1,2,3 ¼ 1.25, 1.15 and 1.325 mT Purification of a six-subunit [NiFe] hydrogenase from the soluble fraction of A profundus Starting from cell-free extracts of A profundus, hydrogenase was purified by following the hydrogen uptake activity using benzylviologen as artificial electron acceptor The majority (97%) of the hydrogenase activity was found in the soluble fraction (Table 2) Further purification resulted in an enzyme preparation consisting of six major polypeptides with apparent molecular masses of 72, 50, 35, 31, 22 and 15 kDa (Fig 4) It exhibited a specific hydrogen uptake activity of 420 mg)1 protein at 65 °C From g wet cells ( 500 mg protein) 12 mg of the purified enzyme were obtained The amino-terminal sequences of the six polypeptides showed highest sequence identity to proteins encoded by the A fulgidus genome (Table 3) [13] These genes are organized in a putative transcriptional unit (AF1377 to AF1372) (Fig 5) Only the amino-terminal sequence of the AF1376 gene product Table Purification of Mvh:Hdl enzyme complex from A profundus Hydrogenase-uptake activity was measured after each chromatographic step as described in Materials and methods One unit of enzyme activity corresponds to the reduction of two lmol of benzylviologen per minute Purification step Fraction Total activity [U] 150 000 g supernatant Q-Sepharose Hydroxyapatite MonoQ – 400 mM NaCl 150–180 mM PO43– 400 mM NaCl 30 000 21 000 4200 4000 Ó FEBS 2004 Heterodisulfide reductase-like enzymes in A profundus (Eur J Biochem 271) 1111 22-kDa polypeptide did not show any significant sequence similarity to proteins in the databases The AF1374 to AF1372 gene products revealed high sequence identity to the three subunits of F420-nonreducing hydrogenase (Mvh) from Methanothermobacter spp and related methanogens [12,30], the AF1377 to AF1375 proteins showed high sequence identity to subunits HdrA, HdrB and HdrC of Hdr from Methanothermobacter species and related methanogens [3] Due to these sequence identities the proteins of the A profundus enzyme complex were designated as MvhA (AF1372) MvhG (AF1373) and MvhD (AF1374), HdlA (AF1377), HdlC (AF1376) and HdlB (AF1375) Hdl stands for HdrABC-like A detailed sequence analysis revealed the following data (Table 4) HdlA shows sequence similarity to subunit HdrA of heterodisulfide reductase and shares four binding motifs for [4Fe)4S] clusters (Cx2Cx2Cx3C) and one binding motif for FAD [GxGx2Gx16)19(D/E)] with HdrA HdlC corresponds to subunit HdrC of Hdr and shares two binding motifs for [4Fe)4S] clusters with HdrC A multiple sequence alignment of various members of the HdrC protein family showed that the amino terminus of these proteins is poorly conserved This may explain why the determined amino terminus of the 22-kDa polypeptide could not be assigned to the AF1376 gene product HdlB shows sequence similarity to subunit HdrB of Hdr The two CX31)39CCX35)36CX2C sequence motifs present in HdrB are also conserved in HdlB For Hdr it has been proposed that some of these cysteine residues ligate the active-site iron–sulfur cluster [8,9] MvhD from Mt marburgensis binds a [2Fe)2S] cluster [31] It contains five cysteine residues also conserved in the AF1374 gene product MvhG (AF1373) was identified as hydrogenase small subunit with highest sequence identity to MvhG of Mt thermoautotrophicus This protein contains 14 cysteine residues, 12 of these are highly conserved among the hydrogenase small subunits of several [NiFe] hydrogenases and are predicted to ligate three [4Fe)4S] clusters MvhA (AF1372) was identified as Fig SDS/PAGE of the Mvh:Hdl enzyme complex from A profundus Proteins were separated in a 14% slab gel (8 · cm), which was subsequently stained with Coomassie Brilliant Blue R250 Lane 1, 25 lg of purified Mvh:Hdl complex; M, low-molecular-mass marker (Amersham Pharmacia Biotech) The molecular masses of the marker peptides are given on the right side The apparent molecular masses of the polypeptides of lane are given on the left side did not correspond to one of the amino-terminal sequences determined for the subunits of the purified enzyme, however, its molecular mass corresponds to the apparent molecular mass of the 22 kDa subunit of the purified enzyme The amino-terminal sequence of the Table Amino-terminal sequences of the soluble hydrogenase of A profundus Amino-terminal sequences of the A profundus enzyme were derived by Edman-degradation, amino-terminal sequences of A fulgidus were derived from the genome sequence [13] In both sequences, identical amino acids are underlined Annotations made are based on sequence identities of the respective polypeptides (see text) X, No clear assignment to an amino acid could be made; –, gaps inserted to allow an alignment Amino-terminal sequences Sequence identity Gene A fulgidus Annotation A profundus A fulgidus GKYGLFLGCNISFNRPDVEV MFMKYALFPGCKIAFERPDLEL 55% AF1375 HdlB A profundus A fulgidus SEEWEPNII-VAANWXTYQ -MKIIGFACQWCAYQ 50% AF1374 MvhD A profundus A fulgidus MKKIEIEPMTRLEGHXKIAI M-KIEINPVSRIEGHAKVTI 63% AF1372 MvhA A profundus A fulgidus GEEEPKIGVYIXH MKIGVYVCH 67% AF1377 HdlA A profundus A fulgidus LKLA-YLLVXGCGGCDM IDVAFYIA-HGCSGCTM 44% AF1373 MvhG A profundus A fulgidus MEMHEEGVPDVINLSYLAER - – – HdlC Ó FEBS 2004 1112 G J Mander et al (Eur J Biochem 271) Fig Genomic organization of the genes encoding the subunits of the Mvh:Hdl enzyme complex and a putative membrane-bound hydrogenase in A fulgidus The ORFs annotated by TIGR are given above the arrow representing the genes and their direction of transcription The gene names of genes encoding the Mvh:Hdl complex are given below the gene symbols The genes have almost no intergenic regions or they overlap The AF1371 gene product was not found in the enzyme preparation It is predicted to encode a hydrogenase maturation protease The AF1381–AF1379 genes encode a putative membrane-bound hydrogenase closely related to the F420-nonreducing hydrogenases (Vho and Vht) from Mt mazei [37] AF1378 encodes a putative hydrogenase maturation protease Table Features of the subunits of the Mvh:Hdl complex from A profundus TIGR annotation Calculated molecular mass Cofactor binding sites Mt thermoautotrophicus Sequence identity to Annotation AF1377 AF1376 AF1375 AF1374 AF1373 AF1372 72.1 kDa 18 kDa 33.9 kDa 15 kDa 32.1 kDa 50.9 kDa [4Fe)4S], FAD [4Fe)4S] · (Cx31)39CCx35)36Cx2C) [2Fe)2S] 3[4Fe)4S] [Ni–Fe] HdrA (46%) HdrC (32%) HdrB (35%) MvhD (30%) MvhG (31%) MvhA (36%) HdlA HdlC HdlB MvhD MvhG MvhA hydrogenase large subunit carrying the four cysteine ligands of the binuclear [Ni–Fe] centre None of the polypeptides reported above has extended hydrophobic regions, which could form membrane-spanning helices This agrees well with the finding that the enzyme was purified from the soluble fraction Cofactor analysis and characterization of the iron–sulfur clusters by EPR spectroscopy The enzyme preparation contained 3.7 ± 0.5 nmol NiỈ mg)1 protein, 214 ± nmol acid-labile sulfurỈmg)1 protein and 207 ± 11 nmol ironỈmg)1 protein As predicted from the primary structure it contains a flavin identified as FAD ) 3.0 ± 0.2 nmol FADỈmg)1 protein were found A densitometric analysis of Coomassie Brilliant blue-stained SDS gels indicated the presence of all subunits in stoichiometric amounts in the complex From the genome sequence of the close relative A fulgidus the molecular mass of the complex was calculated to be 220 kDa Per mol the enzyme complex thus contains 0.83 ± 0.12 mol Ni, 0.73 ± 0.05 mol FAD, 47 ± mol acid-labile sulfur and 45 ± mol nonheme iron From the primary sequence the enzyme is predicted to harbour one FAD, one [Ni–Fe] centre, one [2Fe)2S] cluster and nine [4Fe)4S] clusters and one active-site [Fe–S] cluster in HdlB A characterization of the iron–sulfur clusters by EPR spectroscopy revealed the following results: the H2 reduced enzyme exhibited a spectrum dominated by a signal with g-values at 2.036, 1.933 and 1.912 (Fig 6A) This signal was detectable without significant broadening at temperatures up to 80 K The g-values, temperature behaviour and redox properties are reminiscent of a signal detected in purified Mvh from Mt marburgensis where this signal was attributed to a [2Fe)2S]1+ cluster [31] In the spectrum of the H2-reduced enzyme a radical signal around g ¼ was also visible The intensity of this signal increased upon further reduction of the enzyme by sodium dithionite (not shown) The line width of the radical signal is 1.5 mT as determined from a spectrum recorded at 120 K (data not shown) In the absorption spectrum there is no maximum around 600 nm, which would be indicative for a neutral (blue) semiquinone This all points to an anionic (red) flavinsemiquinone radical (line width  1.5 mT) [32] At temperatures below 20 K additional signals in the reduced enzyme were detectable as a shoulder of the much more intensive [2Fe)2S]1+ signal at g ¼ 1.890 (Fig 6A) These signals are indicative of spin–spin coupling between the different [4Fe)4S]1+ clusters in the enzyme [33,34] The duroquinone (2,3,5,6-tetramethyl-p-benzoquinone)oxidized enzyme exhibited a rhombic EPR signal with gxyz values at 2.014, 1.939 and 1.895 The line shape of this spectrum was similar to the spectrum observed for oxidized HmeCD (Fig 3), however, the g-values are shifted to lower values (Fig 6B) This paramagnetic species could be measured under nonsaturating conditions at 20 K and was detectable without significant broadening up to 60 K The signal broadened beyond detection at 110 K The total spin concentration was 13 lM corresponding to 0.35 spinỈmol)1 enzyme When the oxidized sample was incubated under 100% H2 the signal was no longer detectable and again the [2Fe)2S]+ signal described above was observed Experiments with heterodisulfide (CoM-S-S-CoB) as electron acceptor and hydrogen as electron donor showed that the complex has no detectable activity with these substrates (data not shown) When the enzyme was oxidized with K3Fe(CN)6 a Ơg ¼ 2.02Õ-EPR signal indicative of a [3Fe)4S]+ cluster was observed This cluster was most probably formed by the oxidative degradation of a [Fe)4S] cluster at high redox potentials Ó FEBS 2004 Heterodisulfide reductase-like enzymes in A profundus (Eur J Biochem 271) 1113 In addition, signals derived from the [NiFe] centre were observed with gxyz ¼ 2.338, 2.174 and 2.007 in the duroquinone oxidized enzyme (data not shown) This signal most probably corresponds to the Ni(III) ready form of the enzyme [35] The identification of this Ni(III) derived signal clearly shows that the enzyme is an oxidized state Discussion Fig EPR spectra of the H2-reduced and duroquinone-oxidized A profundus Mvh:Hdl enzyme complex (A) EPR spectra obtained after reduction of the Mvh:Hdl complex (8.9 mg proteinỈmL)1 at pH 7.0) with hydrogen (1.2 · 105 Pa) at 10 K (power ¼ 0.2 mW) and 25 K (power ¼ mW) The upper spectrum shows a simulation of the [2Fe)2S]1+ signal at 25 K Simulation parameters: g1,2,3 ¼ 1.912, 1.933 and 2.036; W1,2,3 ¼ 2.0, 3.6 and 4.6 mT The flavin radical signal is saturated under these conditions (B) EPR spectrum obtained after oxidation of the Mvh:Hdl enzyme complex (8.9 mg proteinỈmL)1) with mM duroquinone (thin black line) The total spin concentration was 0.35 spinỈmol)1 enzyme as determined by double integration of the simulated EPR signal (thick grey line) Simulation parameters: g1,2,3 ¼ 1.895, 1.939 and 2.014; W1,2,3 ¼ 2.54, 1.62 and 1.00 mT EPR conditions: temperature, 20 K; microwave power, mW; microwave frequency, 9.458 GHz; modulation amplitude, 0.6 mT; modulation frequency, 100 kHz In the present study two Hdr-like enzymes, HmeCD and the Mvh:Hdl-complex, were isolated from H2/sulfate-grown cells of A profundus Each enzyme contains a subunit (HmeD or HdlB) with sequence similarity to the proposed catalytic subunit of Hdr (HdrD from Ms barkeri or HdrB from Mt marburgensis) The EPR signals observed for both, HmeCD and the Mvh:Hdl complex from A profundus are reminiscent to the CoM–Hdr signal However, the two enzymes from A profundus form this paramagnetic species already when oxidized with either ferricyanide or duroquinone in the absence of any added thiol The same result was recently obtained with the A fulgidus HmeACDE complex [14] One possible explanation could be that the enzymes contain substoichiometric amounts of a tightly bound thiol, which becomes ligated to the active-site [Fe–S] cluster upon oxidation This could also explain why the spin concentration obtained is much lower than spin per molecule With the Mt marburgensis enzyme the CoM– Hdr EPR signal could also be obtained when oxidized in the presence of nonsubstrate thiols such as b-mercaptoethanol or cysteine With these thiols the midpoint potential of the signal was, however, shifted to rather nonphysiological, high values [7] Also in the A profundus enzymes a nonsubstrate thiol might induce the paramagnetic species observed by EPR spectroscopy The architecture of A profundus HmeCD resembles that of HdrDE from Ms barkeri (Fig 7) [4,5] It contains a b-type cytochrome as membrane anchor and a hydrophilic iron–sulfur protein, presumably carrying the active-site for the reduction of a yet unidentified substrate In Ms barkeri Hdr together with a membrane-bound [NiFe] hydrogenase and the membrane-bound electron carrier methanophenazine forms an electron transport chain catalysing the reduction of CoM-S-S-CoB by H2 This reaction is coupled to the formation of a proton motive force [10,36] Two isoenzymes of the membrane-bound hydrogenase, called Vho and Vht, are present in Ms barkeri Both enzymes contain a membrane anchoring b-type cytochrome in addition to the hydrogenase large and small subunit The two latter subunits are predicted to extrude into the extracytoplasmic side of the membrane [37] Interestingly, a closely related hydrogenase is encoded by the genome of A fulgidus (AF1381–1379) [13] (Fig 5) The genes are directly upstream of the genes encoding the Mvh:Hdl complex However, only  3% of the hydrogenase activity present in cell extracts of A profundus were localized in the membrane fraction This could be due to the lability of the enzyme Also the Vho/Vht hydrogenases from Ms barkeri rapidly dissociate from their membrane anchor [37,38] It is thus reasonable to assume that the proposed membranebound hydrogenase of A profundus became detached from its membrane anchoring b-type cytochrome subunit upon cell lysis and thus was released into the soluble fraction 1114 G J Mander et al (Eur J Biochem 271) Ó FEBS 2004 Fig Schematic presentation of (A) HmeCD from A profundus in comparison to HdrDE from Ms barkeri and the HmeABCDE complex from A fulgidus and (B) the Mvh:Hdl complex from A profundus in comparison to the Mvh:Hdr complex from Methanothermobacter spp The scheme is based on the sequence analysis of the encoding genes from A fulgidus The physiological substrate of the A profundus enzyme is yet unknown, but based on the similarity to Hdr a disulfide is proposed MP, methanophenazine; MQ, menaquinone During the purification of the Mvh:Hdl complex no other major fraction with hydrogenase activity was detected However, chromatography on hydroxyapatite resulted in a significant loss of hydrogenase activity This could be due to the inactivation or irreversible binding of this second hydrogenase during chromatography on hydroxyapatite During chromatography of solubilized membrane proteins on Q-Sepharose, in addition to the HmeCD-containing fraction further heme-containing fractions were observed which contained  20% of the total heme present in the membrane fraction These fractions, which might contain the b-type cytochrome of the membrane-bound hydrogenase, have not yet been analysed further This proposed membrane-bound hydrogenase and HmeCD could be part of an electron transport chain very similar to that present in Ms barkeri with the exception that methanophenazine is replaced by the modified menaquinone described for A fulgidus [39] Unlike HmeCD from A profundus the A fulgidus HmeACDE complex contains the two additional subunits HmeA and HmeC Both subunits are predicted to extrude into the extracytoplasmic side of the membrane These subunits have recently been proposed to form a distinct module, which may mediate the electron transfer from the menaquinone pool to alternative electron acceptors or oxidoreductases [14] We can currently not exclude that these subunits are also formed in A profundus and in vivo form a complex with HmeCD but are lost during the purification The Mvh:Hdl complex from A profundus is closely related to the Mvh:Hdr complex from Methanothermobacter species (Fig 7) The sequences deduced from the AF1377–1372 (hdlACB and mvhAGD) genes of A fulgidus not only show high sequence identities to the corresponding subunits of the Mvh:Hdr complex from Methanothermobacter spp but also contain all cofactor binding sites present in the Mt marburgensis enzyme complex This was also confirmed by the biochemical characterization of the A profundus enzyme complex A putative transcription unit encoding all six subunits was identified in the genome of A fulgidus In the genome of Mt thermoautotrophicus the Ó FEBS 2004 Heterodisulfide reductase-like enzymes in A profundus (Eur J Biochem 271) 1115 genes encoding the six subunits of the Mvh:Hdr complex are located at three different loci [40] Also the sulfate-reducing bacterium D vulgaris contains a putative six-gene operon (ORF2976–2967) encoding an enzyme complex, designated as H2-heterodisulfide oxidoreductase complex, closely related to the Mvh:Hdl complex described here These genes are expressed in D vulgaris as was shown by macroarray RNA hybridizations [41] Expression of these genes was found to be downregulated in a Fe-only hydrogenase mutant strain (Dhyd) Downregulation was also observed in a strain carrying a deletion of the adh gene, encoding an alcohol dehydrogenase One possible function discussed for the H2-heterodisulfide oxidoreductase complex in D vulgaris is the formation of H2 with reducing equivalents generated by the oxidation of ethanol to acetate, as part of a H2-cycling system [41] Thus far, the Mvh:Hdl genetic organization is unique to sulfate reducers The complete understanding of the process of dissimilatory sulfate reduction is hampered by the high complexity of the systems studied thus far Many of the organisms studied are able to utilize several electron donors for growth and contain many different electron transfer components in parallel In contrast, A profundus is obligatory hydrogenotrophic [20] For this organism H2 is the ultimate electron donor and hence reducing equivalents generated upon H2 oxidation have to be channelled to the enzymes of sulfate reduction Our finding that a major hydrogenase present in cell extracts of A profundus forms a tight complex with an Hdr-like enzyme strongly supports previous assumptions that Hdr-like enzymes play an essential role in the electron transport chain(s) of sulfate reducing archaea and bacteria The Mvh:Hdl enzyme complex accounts for at least 2.5% of the total cell protein of A profundus indicating an important catabolic function In analogy to the Mvh:Hdr complex from Methanothermobacter species, the A profundus enzyme complex is proposed to reduce an electron acceptor which in turn could function as electron donor of the enzymes of sulfate reduction Several questions remain to be answered What is the nature of the physiological electron acceptor of the two Hdrlike enzymes present in A profundis? The similarity to Hdr suggests a disulfide Do both enzymes reduce the same electron acceptor or are different substrates used? If both systems reduce the same substrate, why are two different enzyme systems operating? Is the Mvh:Hdl enzyme complex in vitro bound to the cytoplasmic membrane via additional membrane subunits and is the reaction catalysed by this complex coupled to energy conservation, as has been proposed for the Mvh:Hdr complex from Methanothermobacter marburgensis [12] In future studies the identification of the substrate(s) of both enzymes will be attempted A possible way this could be done is the addition of low molecular mass fractions of cell extracts to purified HmeCD or Mvh:Hdl in the presence of an oxidant This could result in higher spin concentrations of the paramagnetic species, assuming that the substrate binds to an iron–sulfur cluster in the active site Acknowledgements This work was supported by the Max-Planck-Gesellschaft, by the Deutsche Forschungsgemeinschaft, and by the Fonds der Chemischen Industrie We thank D Linder for amino-terminal sequencing References Thauer, R.K (1998) Biochemistry of methanogenesis: a tribute to Marjory Stephenson Microbiology 144, 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enzyme [33,34] The duroquinone... amino-terminal sequence of the Table Amino-terminal sequences of the soluble hydrogenase of A profundus Amino-terminal sequences of the A profundus enzyme were derived by Edman-degradation, amino-terminal