INTRODUCTION

Biologically produced CH₄ derives from either the reduction of CO₂ or the methyl group of acetate by two separate pathways present in the methanoarchaea. The latter accounts for most of the CH₄ produced in nature. Elucidation of the pathway for CO₂ reduction to CH₄, the first to be investigated, has yielded several novel enzymes and cofactors (1). Methanosarcina thermophila is a moderately thermophilic methanoarchaeon capable of growth with acetate, methanol, and methylated amines (2). The pathway for the fermentation of acetate to CH₄ and CO₂ in M. thermophila (3, 4) is now understood on a biochemical and genetic level comparable with understanding of the CO₂-reducing pathway. Enzymes involved in the fermentation of acetate by M. thermophila have been purified and characterized (3, 4). The genes encoding these enzymes have been cloned, sequenced, transcriptionally mapped, and their regulation defined on a molecular level (5, 6, 7, 8, 9, 10, 11, 12). In the pathway, acetate is first activated to acetyl-CoA by acetate kinase and phosphotransacetylase and then cleaved by the CO dehydrogenase·acetyl-CoA synthase (CODH·ACS)¹ complex yielding enzyme-bound methyl and carbonyl groups. The methyl group is ultimately transferred to HS-CoM, and the CH₃-S-CoM is reduced with electrons from HS-CoB to release CH₄ and the heterodisulfide CoM-S-S-CoB. The carbonyl group is oxidized to CO₂ by the CODH·ACS complex that reduces a ferredoxin. The ferredoxin participates in the transfer of electrons to a membrane-bound electron transport chain (13); however, additional electron carriers may be required. The terminal electron acceptor is heterodisulfide reductase which reduces CoM-S-S-CoB to the active sulfhydryl forms of the cofactors. A carbonic anhydrase is proposed to convert CO₂ to HCO₃ outside the cell membrane, facilitating removal of CO₂ from the cytoplasm (5).

Although the enzymology and molecular biology of carbon flow is well resolved, less is known concerning electron transport in the acetate-fermenting and CO₂-reducing methanoarchaea. Recently, a novel flavoprotein that contains FMN as the prosthetic group was purified from the methanoarchaeon Methanobacterium thermoautotrophicum where it is proposed to function as an electron carrier in the CO₂-reducing pathway (14, 15). Here we report on a novel FMN-binding iron-sulfur protein (Isf) in acetate-grown M. thermophila. The results suggest Isf functions as an electron carrier in the pathway for the fermentation of acetate.

The terms methanoarchaea and methanoarchaeon, first suggested by Ralph S. Wolfe, are used here to indicate the phylogenetic placement of methane-producing microbes in the Archaea domain.

EXPERIMENTAL PROCEDURES

DNA analyses were performed using the Pustell/IBI sequence manager (IBI-A Kodak Co., Rochester, NY) and GenePro 4.2 analysis software (Riverside Scientific, Seattle, WA). Amino acid alignments were performed using the default parameters of the Version 8, BestFit program (Genetics Software Group, Madison, WI). The DNA and deduced amino acid sequences of isf were compared with the non-redundant updated nucleotide and protein data bases at the National Center for Biotechnology Information using the BLAST program (16). The isf sequence (Fig. 2) was submitted to GenBank and assigned the accession number U50189[GenBank].

The coding region of isf was amplified using polymerase chain reaction. The sequence of the upstream primer was 5-GAGCGATCCATATGAAAATAACAGGA-3 that corresponds, in part, to nucleotides 1-15 in Fig. 2 and contains the recognition sequence for NdeI. The sequence of the downstream primer was 5-GCTGTATTGGATCCTGCGATCATAAAC- that corresponds, in part, to nucleotides 890-902 in Fig. 2 and contains the recognition sequence for BamHI. Blunt ends were created on the polymerase chain reaction product with T4 DNA polymerase, and the product was restriction endonuclease digested with NdeI and BamHI. The resulting DNA was cloned into the BamHI and NdeI sites of the pT7-7 overexpression vector of Tabor and Richardson (17). The pT7-7 derivative carrying the isf gene (designated pML701) was transformed into Escherichia coli strain BL21(DE3) (18) that was grown at 37 °C in Luria-Bertani broth (containing 100 µg ml¹ ampicillin) to an A_600 nm of 1.6, at which point the culture was induced to produce Isf by the addition of 1% (final concentration, w/v) Bacto-lactose. After 2 h growth at 30 °C, the cells were harvested by centrifugation at 11,800 × g for 10 min at 4 °C.

All steps in the purification of Isf were performed anaerobically, and all buffers contained 2 mM dithiothreitol. Where applicable, a Coy anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI) was employed. All steps were performed at 21 °C except where indicated. Fast protein liquid chromatography (FPLC) columns and Q-Sepharose Fast Flow anionic exchange resin were obtained from Pharmacia Biotech Inc. Oxygen-impermeable Saran (Pyramid Plastics, Hope, AK) tubing was used with the Pharmacia FPLC system. Protein concentrations in column elution samples were routinely determined using the Bio-Rad microassay (19) with bovine serum albumin as the standard. Spectra were obtained with a model 8452A diode-array spectrophotometer (Hewlett-Packard Corp., Palo Alto, CA).

Cells (approximately 45 g, wet weight) were suspended in a total volume of 130 ml of 25 mM Tris (pH 7.4) and lysed by French pressure cell disruption at 20,000 p.s.i. (138 megapascals). The lysate was centrifuged at 78,400 × g for 20 min at 4 °C. Streptomycin sulfate (final concentration, 1% w/v) was added to the supernatant solution and centrifuged as above. The soluble fraction was applied to a Q-Sepharose Fast Flow column (5 × 10 cm) previously equilibrated with 2 column volumes of 25 mM Tris (pH 7.4). A 500-ml 0-1.0 M NaCl linear gradient was applied at 6 ml min¹. Fractions containing Isf were determined by SDS-PAGE (see below). An equal volume of 1.2 M (NH₄)₂SO₄ in 25 mM Tris (pH 7.4) was added to the pooled fractions containing Isf. The solution was passed over a phenyl-Sepharose Hi-Load 26/10 FPLC column preequilibrated with 600 mM (NH₄)₂SO₄ in 25 mM Tris (pH 7.4). The eluate containing Isf was diluted 1:10 with 50 mM Tris (pH 7.6) and divided into four equal aliquots of approximately 25 mg of protein each. Each of the four aliquots was separately applied to a Mono-Q 10/10 FPLC column preequilibrated with 50 mM Tris (pH 7.6). The column was developed with 50 ml of a 0-1.0 M NaCl linear gradient applied at 2 ml min¹. Pure Isf was recovered in a single symmetrical peak eluting between 220 and 240 mM NaCl.

M. thermophila was grown on acetate to midlog phase and harvested as described (20). Cell extract was prepared from 20 g of cells (wet weight) as described previously (20). The extract (100 mg of protein) was applied to a Mono-Q 10/10 FPLC column equilibrated with 50 mM Tris buffer (pH 7.2). The column was developed with 50 ml of a 0-1.0 M KCl gradient in the same buffer.

The subunit molecular mass of Isf was estimated by 12% SDS-PAGE (21) using low molecular weight protein standards from Bio-Rad. Native molecular mass estimates were based on elution from a Superose-12 gel filtration FPLC column calibrated with the following proteins of known molecular masses: bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), chymotrypsinogen (25 kDa), and RNase A (13.7 kDa). The buffer used was 50 mM Tris (pH 7.6) containing 150 mM NaCl. A flow rate of 0.5 ml min¹ was used.

The flavin cofactor was extracted from purified Isf by acidification of 531 µg of protein with 0.5% (final concentration, v/v) trifluoroacetic acid at 21 °C. A Hewlett-Packard model 1050 HPLC system in conjunction with a Hewlett-Packard LiChrosorb C-18 reversed phase column was used to identify the flavin extracted from Isf. Neutralized samples were injected into a mobile phase consisting of 80% water (containing 5 mM ammonium acetate (pH 6.0)) and 20% methanol running at 2 ml min¹ (22). Elution of flavins was monitored at 450 nm. The amount of FMN released from the protein and purified by HPLC was determined spectrophotometrically using an extinction coefficient for FMN of 12.2 mM¹ cm¹ at 450 nm (23). The concentration of Isf in the original sample (before flavin extraction) was determined using the biuret assay (24) with bovine serum albumin as the standard.

Iron (25) and acid-labile sulfur (26) were determined as described. Protein concentration was determined using the biuret reagent method with bovine serum albumin as the standard. N-terminal sequences were determined on a model 470 gas-phase peptide sequencer (Applied Biosystems, Inc., Foster City, CA) by the Virginia Polytechnic Institute and State University protein sequencing facility. The phenylthiohydantoin derivatives were identified with an on-line Applied Biosystems liquid chromatograph.

Hydrogenase activities were determined as described (27). The reconstitution and activity assay of the CO·CoM-S-S-CoB oxidoreductase system was performed as described (13).

Western blot analysis was performed using polyclonal antibodies raised against heterologously produced Isf in rabbits by Cocalico Biologicals, Inc., Reamstown, PA. SDS-PAGE (12%) was performed as described (21) except some samples (where indicated) were not boiled prior to loading on the gel. Proteins were electroblotted onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 23 V for 14-16 h at 4 °C. Antiserum was diluted 1:20,000. All other procedures were performed as described previously (28).

Restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 DNA polymerase, exonuclease III, exonuclease VII, isopropyl--D-thiogalactopyranoside, and Blue-Gal were purchased from Life Technologies, Inc. -EMBL3 phage arms were from Packagene  packaging system extracts, and all E. coli strains used in cloning were obtained from Promega, Madison, WI. Sequenase 2.0, all sequencing primers, and ultrapure acrylamide were supplied by U. S. Biochemicals Corp. [-³⁵S]dATP (1,417 Ci/mmol) was supplied by DuPont-NEN. Polymerase chain reaction reagents and Taq polymerase were purchased from Perkin-Elmer. Goat anti-rabbit alkaline phosphatase conjugate was purchased from Bio-Rad. Ampicillin, streptomycin sulfate, FAD, and FMN were purchased from Sigma. Purified CODH·ACS complex was a gift from Dr. Madeline Rasche. Purified ferredoxin was a gift from Dr. Andrew Clements. The overexpression plasmid pT7-7 was a gift from Dr. Stanley Tabor. E. coli strain BL21(DE3) was a gift from Dr. William Studier.

RESULTS AND DISCUSSION

Sequence analysis of the previously reported 3.9-kilobase pair EcoRI fragment of M. thermophila genomic DNA containing pta and ack (7) identified a third open reading frame (Figs. 1 and 2) designated isf (ron-ulfur lavoprotein) for reasons described below. The isf gene was located 265 base pairs upstream of pta and oriented in the opposite direction. The AT-rich DNA between pta and isf contained several sequences with identity to the consensus box A archaeal promoter sequence (5-TTTA(T/A)ATA-3) (29). The transcription of archaeal genes typically initiates 22-27 base pairs downstream of box A at a purine-pyrimidine dinucleotide located in a box B sequence (consensus 5-ATGC-3) (29). Three potential box A sequences were located 22-27 base pairs upstream of sequences with identity to the consensus box B (Fig. 2). One of the three potential box A sequences coincided with the putative box A sequence previously determined for the pta-ack operon by primer extension analysis (11). A potential ribosome binding site was identified 10 bases upstream of the putative N-terminal methionine codon of isf. A stretch of thymine bases (Fig. 2) was identified 98 bases downstream. Thymine-rich regions have been shown to function as transcriptional terminators in the Archaea (29). Additional experimentation is needed to determine if isf is monocistronic or part of a larger operon.

The putative polypeptide deduced from isf (Isf) was 273 amino acids in length and had a calculated anhydrous molecular mass of 30,451 Da (Fig. 2). Analysis of the deduced protein sequence revealed a cluster of cysteine residues (Fig. 2) with a spacing (CXXCXXC) reminiscent of proteins that accommodate either a [4Fe-4S] or [3Fe-4S] center (30). Iron-sulfur proteins with [4Fe-4S] centers utilize, in addition to the CXXCXXC motif, a Cys-Pro pair to provide the fourth acid-stable cysteine sulfur ligand to the iron atoms (30). Two Cys-Pro pairs are located in the C-terminal portion of Isf (Fig. 2).

The heterologously produced Isf was purified to apparent homogeneity as determined by SDS-PAGE. N-terminal sequencing of the first five residues confirmed that the protein purified from E. coli was encoded by isf. SDS-PAGE indicated a subunit molecular mass of approximately 29 kDa that is in close agreement with the molecular mass predicted from the amino acid sequence deduced from isf (30.4 kDa). Native gel filtration chromatography using a calibrated Superose-12 column yielded a single peak corresponding to a molecular mass of 65 ± 7 kDa, indicating that Isf was purified as an ₂ homodimer.

The UV-visible spectrum of purified Isf showed absorbance maxima at 484, 452, 430, 378, and 280 nm, typical of iron-sulfur flavo proteins (Fig. 3). No absorbance was observed at wavelengths greater than 550 nm (data not shown). Addition of dithionite decreased absorbance in the 350-550 nm range (Fig. 3). Exposure to air reoxidized the dithionite-reduced Isf yielding the same spectrum (data not shown) obtained for the purified protein (Fig. 3). These results indicate that Isf is a redox-active protein. Although purified anaerobically, exposure to air for 30 min at 21 °C did not change the spectrum (data not shown), suggesting that purified Isf was in the oxidized form. The molar absorption coefficients calculated for the ₂ homodimer at 272, 378, and 452 nm were 140,350, 35,087, and 57,894 M¹ cm¹. Mildly acidified Isf had a visible spectrum typical of flavins (Fig. 3). The flavin released from Isf by acidification was identified as FMN by HPLC. A total of 15 nmol of HPLC-purified FMN was obtained from 8 nmol of native Isf, suggesting 2 FMN per ₂ homodimer. Analyses of heterologously produced Isf indicated 7.4 (± 0.5, n = 3) mol of iron and 6.9 (± 0.6, n = 3) mol of acid-labile sulfur/mol of ₂ homodimer. These results indicated either one [4Fe-4S] or one [3Fe-4S] center was present per subunit, which is consistent with a single CXXCXXC motif in the deduced amino acid sequence.

A search of the available data bases failed to identify any sequence with significant overall identity to Isf including the recently described FMN-binding protein from M. thermoautotrophicum; however, when aligned using the BestFit program, the Isf sequence showed significant identity and similarity to the flavodoxin of Clostridium acetobutylicum (Fig. 4). The results suggest that Isf is a novel iron-sulfur flavoprotein possibly related to flavodoxins from the Bacteria domain. It is conceivable that the genes encoding the two proteins evolved from a common ancestral gene; however, compared to Isf, the C. acetobutylicum flavodoxin is missing an amino acid stretch that contains the CXXCXXC motif as well as the C-terminal sequence of Isf containing two Cys-Pro pairs.

Extract from acetate-grown M. thermophila was fractionated on a Mono-Q column. The whole extract, Mono-Q fractions, and purified heterologously produced Isf were analyzed by Western blotting using polyclonal anti-Isf antisera (Fig. 5). Purified Isf was detected whether boiled or not boiled prior to SDS-PAGE (Fig. 5, lanes 7 and 8). The blots showed a dense band of cross-reacting protein corresponding to the 29-kDa monomeric form of purified Isf and a faint band of 50-kDa cross-reacting protein. Boiling the sample decreased the intensity of the 50-kDa band and increased the intensity of the 29-kDa band suggesting that the 50-kDa protein is a higher molecular mass form of the Isf monomer, possibly the ₂ homodimer. A 45-kDa cross-reacting protein was detected in whole extract and a Mono-Q fraction that eluted between 350 and 375 mM KCl (Fig. 5, lanes 3-6). The band was detected only when the samples were not boiled; apparently when boiled, a factor in extracts and the Mono-Q fraction interacted with the protein that interfered with detection. The 45-kDa cross-reacting protein detected in M. thermophila had a molecular mass that was different from the bands detected in blots of purified Isf. A 45-kDa band was also detected in blots of extract amended with purified heterologously produced Isf; however, the 29-kDa monomeric Isf and faint cross-reacting protein bands corresponding to higher molecular mass forms were also detected (Fig. 5, lane 2). Similar results were obtained when Isf-amended extract was boiled prior to SDS-PAGE except that the 45-kDa cross-reacting band was fainter, the 29-kDa band was more intense, and the higher molecular mass forms of Isf were not detectable (Fig. 5, lane 1). The results indicated that acetate-grown M. thermophila contained Isf. The reason for the anomalous migration of Isf in non-boiled samples of extract and the Mono-Q fraction is unknown. It is conceivable that the Isf monomer associates with another protein yielding a heterodimer resistant to dissociation during SDS-PAGE of non-boiled samples.

An electron transport chain operates in acetate-grown M. thermophila, which is minimally composed of the CODH·ACS complex, ferredoxin, membranes, and a soluble CoM-S-S-CoB reductase (13). Cell extract from acetate-grown cells catalyzed the CO-dependent reduction of Isf (Fig. 6), suggesting it is linked to an electron transport chain initiating with the CODH·ACS complex. No change in absorbance was detected between 500 and 700 nm during the experiment (data not shown), which argues against a flavin semiquinone intermediate. H₂ was unable to replace CO for reduction of Isf by cell extract (Fig. 7) and Isf had no detectable H₂ uptake hydrogenase activity when assayed with methyl viologen (data not shown). These results indicate that Isf is not involved in H₂ uptake activity of acetate-grown cells. Purified CODH·ACS complex slowly reduced Isf with CO (Fig. 7); however, the rate was stimulated severalfold by the addition of ferredoxin, the electron acceptor of the CODH·ACS complex (32). The results suggest that ferredoxin is a physiological electron donor to Isf.

Recently, a CO·CoM-S-S-CoB oxidoreductase system was reconstituted in vitro with purified and partially purified components from acetate-grown M. thermophila (13). The results show that electron flow in vitro is: CO  CODH·ACS complex  ferredoxin  membranes  CoM-S-S-CoB reductase; however, the participation of other electron carriers was not excluded. Addition of Isf resulted in stimulation of CO·CoM-S-S-CoB oxidoreductase activity of the reconstituted system (Fig. 7), which is consistent with Isf functioning as an electron transfer component of the system. Since Isf was reduced by ferredoxin and contains FMN and Fe-S centers, it is possible that Isf functions as a 1-electron/2-electron switch reducing obligate 2-electron carriers. Further research is required to identify electron carriers other than ferredoxin that couple with Isf and to identify whether Isf is a component of the in vivo electron transport chain linking the CODH·ACS complex with the CoM-S-S-CoB reductase in acetate-grown M. thermophila.