INTRODUCTION

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The methanoarchaea are strictly anaerobic microbes that evolve methane as a product of their energy-yielding metabolism and are classified within the Archaea domain (1). These microbes utilize two distinct pathways for most of the methane produced in the biosphere. In the acetate fermentation pathway (Reaction 1), the methyl group is reduced to methane with electrons derived from oxidation of the carbonyl group to CO₂. In the CO₂ reduction pathway, CO₂ is reduced to methane at the expense of electrons provided by either hydrogen (Reaction 2) or formate (Reaction 3). Energy is conserved in both pathways by an electron transport-coupled phosphorylation of ADP. Although the understanding of electron transport in both of these pathways is incomplete, flavoproteins are of major importance. A flavoprotein has been described that appears to function in electron transport pathways of CO₂-reducing methanoarchaea (2, 3). The oxidations of H₂ and formate are catalyzed by the Fe-S flavoproteins, hydrogenase, and formate dehydrogenase (4-7). The final electron transfer step for both pathways (Reaction 4) is catalyzed by heterodisulfide reductase (HDR),¹ which is a FAD/Fe-S protein in the CO₂ reducer, Methanobacterium thermoautotrophicum (8), and a heme/Fe-S in the acetate fermenters, Methanosarcina thermophila (9) and Methanosarcina barkeri (10). This reaction regenerates the active sulfhydryl forms of the coenzymes, CoB and CoM (8). The substrate for HDR, the heterodisulfide CoB-S-S-CoM, is the product of the methane-forming reaction (Reaction 5), which is also common to both pathways and catalyzed by methyl-CoM reductase. Recently, a homodimeric Fe-S flavoprotein Isf (iron-sulfur flavoprotein) was identified from the acetate-fermenting methanoarchaeaon M. thermophila (11). The isf gene was cloned and sequenced, and Isf was produced in Escherichia coli and partially characterized. Comparisons of the deduced Isf sequence with sequences in the available protein data bases suggest that Isf is a novel Fe-S flavoprotein. Reconstitution experiments suggest Isf is a required component of the electron transport chain in the pathway for the fermentation of acetate (11). The UV-visible absorption spectrum and cofactor and elemental analyses indicate that Isf contains one FMN and either one [4Fe-4S] or one [3Fe-4S] center per monomer. The sequence of Isf contains several cysteine residues; however, none of the typical cysteine motifs known to accommodate [4Fe-4S] or [3Fe-4S] centers are recognizable. Thus, the identity of the Fe-S center is uncertain. Here we report on EPR and Mössbauer studies of Isf from M. thermophila, which demonstrate that the Fe-S center is of the [4Fe-4S] type. Potentiometric titrations of the [4Fe-4S] and FMN centers predict a role for Isf in the electron transport pathway leading to the formation of CH₄ in acetate-fermenting methanoarchaea. Additional results are presented that indicate Isf homologs also have a role in electron transport pathways of CO₂-reducing methanoarchaea.

	EXPERIMENTAL PROCEDURES

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Materials-- M. thermophila Isf was produced in E. coli strain BL21(DE3) and purified as described previously (11). Isf was stored at 80 °C in 50 mM potassium phosphate buffer at pH 7.0. For production of ⁵⁷Fe-labeled Isf, E. coli cells were in medium containing 20 µM ⁵⁷Fe (Penwood Chemicals, Inc.), which was prepared by dissolving the iron metal in 2 N H₂SO₄ at 60 °C for 1 week. M. thermophila Fd (12) was purified as described. M. thermoautotrophicum strain Marburg was grown as described (13). The following redox dyes were used: phenosafarin (Sigma), benzyl viologen (Sigma), methyl viologen (Sigma), 1,1'-trimethylene-2,2'-bipyridyl, and 4,4'-dimethyl-2,2'-dipyridyl (Aldrich). All experiments utilized NANOpure deionized water. N₂ (99.98%) and helium (99.998%) were obtained from Linweld (Lincoln, NE). Helium was deoxygenated by passing through an Oxisorb column and a heated column containing BASF catalyst.

Methods-- Protein manipulations were performed under strictly anaerobic conditions at 20 °C in a Vacuum Atmospheres chamber maintained between 1 and 5 ppm oxygen. The concentrations of the oxidized Isf were determined spectrophotometrically with the extinction coefficients previously determined (11) and by the Rose Bengal assay (14).

UV-visible Spectroscopy and Potentiometric Titrations-- Potentiometric measurements were performed as described previously (15, 16). All electrochemical potentials are reported relative to the standard hydrogen electrode. Isf (3-4 µM) was titrated at 20 °C in 50 mM potassium phosphate buffer (pH 7.0-7.05) in a solution containing methyl viologen (0.1 mM) as the mediator dye with phenosafarin (E_m = 0.252 V, pH 7.0) (5 µM) and benzyl viologen (E_m = 0.362 V, pH 7.0) (5 µM) as the indicator dyes. The pH measured after the experiment was recorded as the pH for the titration. The visible spectra in each experiment were obtained and stored on an Olis-14 interfaced Cary spectrophotometer. The absorbance at 480 nm was used to monitor the amount of oxidized and reduced FMN after correcting the spectra for turbidity. The reduction potentials reported were determined by potentiometric measurements in the reductive direction. After each potentiometric titration of the FMN chromophore, the iron-sulfur flavoprotein was reoxidized completely using ferrocyanide (0.1 mM) as the mediator dye. Equilibrium of the system in the UV-visible potentiometric measurements was considered to be obtained when the measured potential drift was less than 1 mV in 5 min; this was typically around 1-2 h. The midpoint potentials (E_m) and n values were calculated using the Nernst equation (Equation 1),

(Eq. 1)

where E is the measured equilibrium potential at each point in the titration, and n is the number of electrons. The typical error in the reported reduction potential values was ±2-3 mV. All midpoint potential value determinations exhibited Nernstian behavior as indicated by their n values.

EPR Spectroscopy and Potentiometric Titrations-- The EPR spectra in each experiment were recorded on a Bruker ESP 300E spectrometer equipped with an Oxford ITC4 temperature controller, a Hewlett Packard model 5340 automatic frequency counter, and Bruker gaussmeter. The spectroscopic parameters are given in the figure legends. Double integration of the EPR signals was performed with copper perchlorate (1 mM) as the standard. Isf was frozen in liquid nitrogen prior to EPR analyses.

(Eq. 2)

(Eq. 3)

Mössbauer Spectroscopy-- Mössbauer spectra were recorded on a constant acceleration spectrometer, model MS-1200D from Ranger Scientific, using a Janis SuperVaritemp cryostat (model 8DT), a Lakeshore temperature controller (model 340), and a ⁵⁷Co source in a rhodium foil purchased from Isotope Products Laboratory. All isomer shifts are quoted relative to iron metal at room temperature.

	RESULTS

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Sequence Analysis-- Resequencing of M. thermophila genomic DNA revealed an error in the previously reported isf sequence (11). The corrected isf and deduced Isf sequences are shown in Fig. 1. The corrected Isf contains 191 residues, 81 fewer than previously reported. Additionally, the C-terminal residues ¹⁷⁷KLCDVLELIQKNRDK¹⁹¹ in the corrected Isf sequence replace ¹⁷⁷NSVMSWNLFRKIEIN¹⁹¹ in the previously reported Isf. Resequencing revealed the correct isf sequence in pML701 used for the heterologous production of Isf reported here and previously (11).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Nucleic acid sequence and predicted amino acid sequence of isf from M. thermophila. The DNA is presented in the 5' to 3' direction. The predicted amino acid sequence of Isf is shown in single-letter code directly below the first base of each codon. *, initial base of translation stop codon.

Evidence for Isf Homologs in Phylogenetically and Physiologically Diverse Methanoarchaea-- M. thermophila is physiologically distinct from Methanococcus jannaschii and M. thermoautotrophicum, which are unable to ferment acetate to CH₄ and instead oxidize H₂ and reduce CO₂ to CH₄. The genome of M. jannaschii (19, 20) contains two ORFs (MJ1083 and MJ0731) encoding predicted proteins with 194 and 192 residues that have high identity to the corrected M. thermophila Isf sequence (Fig. 2). Inspection of the genomic sequence of M. thermoautotrophicum (21) identified three ORFs that also share significant identity to Isf (Fig. 2). An N-terminal cysteine motif in Isf from M. thermophila is strictly conserved in the M. jannaschii and M. thermoautotrophicum sequences. These results suggest a previously unrecognized electron transfer role for Isf homologs in electron transport pathways of CO₂-reducing methanoarchaea. M. jannaschii, M. thermoautotrophicum, and M. thermophila represent all three currently described families of methanoarchaea, suggesting that Isf homologs are present in phylogenetically diverse methanoarchaea, which further supports a general function for this electron carrier.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   Multiple amino acid sequence alignment of Isf from M. thermophila with sequences deduced from open reading frames identified in the genomic sequences of M. jannaschii and M. thermoautotrophicum. Accession numbers for the M. jannaschii (2) and M. thermoautotrophicum (21) sequences and percentage identity (in parentheses) with M. thermophila Isf are as follows: MTH1350 (41%), MTH1473 (34%), MTH1595 (30%), MJ1083 (49%), and MJ0731 (40%). Asterisks indicate conserved cysteine residues.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Time course for reduction of Isf with extract from M. thermoautotrophicum. The assay mixture (700 µl) contained cell extract (180 µg protein), M. thermophila ferredoxin (13.5 µg), 50 mM Tris (pH 7.6), and 2 mM dithiothreitol. Ferredoxin was omitted in two of the assays (, ). The assay mixtures were anaerobically equilibrated with 1.0 atmosphere of CO ( ), H2 (,), or N2 () in a stoppered 1.0-ml cuvette maintained at 35 °C. After a 10-min incubation, the reaction was initiated by the addition of 180 µg of Isf.

EPR Spectroscopy of the Fe-S Center-- The EPR spectra of the reduced Fe-S center (Fig. 4) exhibited g values of 2.06 and 1.93 typical of [4Fe-4S]¹⁺ clusters; however, the signal in the region of g_min shows two, instead of one, negative absorption features with g values at 1.86 and 1.82. The relaxation properties also are indicative of a [4Fe-4S] cluster, since the g = 1.93 signal was not observable above 25 K (22). An average of 1.3 spins/mol of dimeric Isf was determined from different samples by double integration of the EPR signal at 10 K referenced to a copper perchlorate standard. The existence of two negative features in the spectrum of the cluster is unexpected, since rhombic spectra should exhibit only three g values. The extra features did not derive from the viologen dyes, since reduction of Isf with sodium dithionite alone yielded an EPR spectrum identical to that shown in Fig. 4. In some Fe-S proteins, complex spectral features can be simplified by incubation with low concentrations of urea; however, dithionite reduction of Isf in the presence of 2 M urea had no effect on the spectrum. The possibility that the two negative absorption features derived from strong hyperfine interaction between the unpaired electron on the cluster and a strongly coupled proton (would produce a two-line splitting because it has a nuclear spin of 1/2) also was ruled out. When Isf was extensively exchanged with D₂O and reduced with dithionite, the spectrum was identical to that shown in Fig. 4. Often, complex EPR spectra are observed when two paramagnetic centers are close enough to undergo dipolar interactions, including wings in the g_max and g_min regions. In addition, there apparently is not another paramagnet, since the flavin is diamagnetic in the oxidized and reduced states. Therefore, the peculiar morphology of the EPR spectrum derives from a unique characteristic of the reduced [4Fe-4S] cluster tentatively attributed to microheterogeneity within the population of Isf molecules. This hypothesis is supported by the Mössbauer results described below.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   EPR spectroscopy of the [4Fe-4S] cluster in Isf poised at various redox potentials in 50 mM potassium phosphate buffer (pH 7.0). Experimental conditions were as follows: temperature, 10 K; microwave power, 1.26 milliwatts; microwave frequency, 9.43 GHz; receiver gain, 2 × 104; modulation amplitude, 10 G; modulation frequency, 100 kHz. The derivative feature at g = 2.0 results from the mediator dyes.

1

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Semilogarithmic plot of P1/2 versus 1/T, which shows a linear relationship according to Equation 3. The slope (/k = 1.5 ± 0.73) yields an estimate of 11.5 cm1 for the the zero field splitting value (). Inset, a nonlinear plot of P1/2 versus 1/T, which includes the data at 5 K. The slope (/k = 14.1 ± 1.8) yields an estimate for  of 9.8 cm1.

Mössbauer Spectroscopy-- Mössbauer spectra of the oxidized iron-sulfur center (Fig. 6A) exhibited a single broad quadrupole doublet with an average isomer shift () of 0.45 mm/s and an average quadrupole splitting (E_Q) of 1.22 mm/s at 4.2 K, with the quadrupole splitting decreasing slightly (E_Q = 1.12 mm/s) at 100 K. These parameters are typical for [4Fe-4S] clusters in the 2+ oxidation state. The line shape of the quadrupole doublet indicates either that the irons within the cluster are not identical (one possible deconvolution: E_Q(1) = 1.6 mm/s, E_Q(2) = 1.3 mm/s, E_Q(3) = 1.16 mm/s, E_Q(4) = 0.85 mm/s) or that there is a microheterogeneous population of iron-sulfur clusters in the Isf molecules, each having slightly different quadrupole splittings.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Mössbauer spectra recorded at 100 K. A, oxidized Isf protein. The solid line is a least-squares fit, assuming a single quadrupole doublet and a Voigt line shape (EQ = 1.12 mm/s,  = 0.43 mm/s). B, reduced Isf protein. The solid line is a least-squares fit, assuming four quadrupole doublets and a Voigt line shape (EQ(1) = 0.74 mm/s, (1) = 0.52 mm/s; EQ(2) = 1.07 mm/s, (2) = 0.54 mm/s; EQ(3) = 1.47 mm/s, (3) = 0.55 mm/s; and EQ(4) = 1.89 mm/s, (4) = 0.59 mm/s).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Mössbauer spectra of reduced Isf protein recorded at 4.2 K and 450 G applied parallel (A) or 450 G applied perpendicular (B) to the  beam. The solid line is a theoretical fit of an S = 1/2 Hamiltonian using the parameters cited in Fig. 6 and A1 = 27.4, 41.2, 32.9 MHz; A2 = 27.4, 27.4, 35.0 MHz; A3 = +2.7, +6.2, or +17.8 MHz; A4 = +11.0, +27.4, +2.7 MHz; 1 = 0, 2 = 0.9, 3 = 0.7, 4 = 0.9.

Potentiometric Titrations of FMN and the [4Fe-4S] Cluster-- The visible spectrum of Isf was monitored between 300 and 700 nm during potentiometric titration (Fig. 8). The FMN absorption dominates the spectrum of the oxidized protein. At potentials of 0.305 and 0.342 V, the flavin is almost completely reduced and the spectrum of the oxidized [4Fe-4S] cluster is dominant. The ratio of the oxidized:reduced flavin was determined by monitoring the FMN absorbance peak at 480 nm, instead of 452 nm, to minimize possible spectral interference from the [4Fe-4S] cluster. Since [4Fe-4S] clusters have their maximum absorbance between 390 and 420 nm, the cluster exhibits a negligible contribution to the 480 absorbance value. The difference extinction coefficient is calculated to be 48 mM¹ cm¹ for the dimeric protein. An E_m value of 0.277 ± 0.003 V was determined for the FMN/FMNH₂ couple of Isf (Fig. 8, inset). A Nernst plot gave a 34-mV slope for the potentiometric measurement, which is near the theoretical value of 29 mV for a two-electron transfer. Neither the red nor blue forms of the FMN semiquinone, which have characteristic spectra, were observed during the titration. The FMN semiquinone also was not observed in titrations of Isf with sodium dithionite (data not shown) (11). Thus, the individual formal potential value of E₁^o' (FMN/FMNH·) must be below 0.380 V, and the formal potential value of E₂^o' ⁽FMNH·/FMNH₂) must be above 0.175 V. 

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Potentiometric titration of the FMN in Isf (3.2 µM) in 50 mM potassium phosphate buffer (pH 7.0) at 20 °C (curves 1-7, fully oxidized, 0.262, 0.272, 0.281, 0.290, 0.305, and 0.342 V, respectively). Inset, Nernst plot of the potentiometric data.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   EPR spectrum of Isf (170 µM dimer) was recorded at 10 K following incubation for 17 min with CO and CODH (45 µg) at 25 °C. The amount of FMN hydroquinone and reduced FeS cluster at this time point were 46 and 3% (0.03 spin/mol), respectively. After the sample was frozen in liquid nitrogen, the spectrum was recorded using the conditions described in Fig. 4.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   A fit of the Isf midpoint potential data to a theoretical curve generated from the Nernst equation for two redox centers with reduction potentials of 0.277 V (n = 2) and 0.394 V (n = 1).

	DISCUSSION

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The methanogenic fermentation of acetate by the methanosarcina involves cleavage of acetyl-CoA into carbonyl and methyl groups, the latter of which is reduced to CH₄ with an electron pair derived from oxidation of the former to CO₂ (1). Cleavage of acetyl-CoA and oxidation of the carbonyl group is catalyzed by a five-subunit CODH/ACS complex. The methyl group is transferred to tetrahydrosarcinapterin and then to coenzyme M. Reductive demethylation of the methyl group of CH₃-S-CoM to CH₄ requires the electron donor coenzyme B (HS-CoB), which produces the heterodisulfide CoB-S-S-CoM as a second reaction product. The heterodisulfide is reduced to the corresponding active sulfhydryl forms of the cofactors by HDR. The electron pair for this reduction originates from oxidation of the carbonyl group of acetyl-CoA by the CODH/ACS complex. Oxidation of either exogenous CO or the carbonyl group of acetyl-CoA is proposed to take place at the nickel/Fe-S center (center C) in the CdhA subunit of the CODH/ACS complex (28, 29), which also contains a low potential [4Fe-4S] center (28) that is proposed to shuttle electrons from center C to a low potential 8Fe ferredoxin (12). Electron transfer from ferredoxin to HDR apparently involves membrane-bound electron carriers consistent with the formation of a transmembrane proton gradient (9, 30). Possible membrane-bound electron carriers include methanophenazine (31) and/or b-type cytochromes. Cytochromes of the b-type are reduced by CO and oxidized by CoB-S-S-CoM, which suggests that they could play a role in electron transport during the fermentation of acetate (30). Acetate-grown methanosarcina are reported to contain three b-type cytochromes with midpoint potentials of 330, 250, and 182 mV (32). In addition, the purified HDRs from M. thermophila (9) and M. barkeri (10, 33) contain two b-type cytochromes.

Isf is reducible by ferredoxin, and Isf stimulates electron flow from CO to CoB-S-S-CoM in a reconstituted system containing CODH/ACS, ferredoxin, membranes, and HDR, indicating that Isf is a component of the electron transport pathway of M. thermophila (11); however, it was not possible to propose with confidence a specific role for Isf in electron transport without further characterization of the redox centers. The sequence of Isf reveals a four-cysteine spacing that is perfectly conserved with Isf-like sequences from phylogenetically distant methanoarchaea (Fig. 2); however, the cysteine spacing is atypical of motifs coordinating [4Fe-4S] or [3Fe-4S] centers. The EPR and Mössbauer spectroscopic results presented here unequivocally demonstrate the presence of a [4Fe-4S] center in Isf. Additional experiments are required to conclusively identify the coordinating ligands to the [4Fe-4S] center. The presence of two negative absorption features in the EPR spectra of the reduced cluster is unusual. The results presented here indicate that microheterogeneity within the population of Isf molecules accounts for this atypical feature. This situation is similar to that of a class of corrinoid/Fe-S proteins from methanoarchaea and homoacetogenic anaerobes from the bacteria domain in which the reduced [4Fe-4S] cluster exhibits a broad absorption feature in the same g value region (34-36). The electrochemical properties of the [4Fe-4S] cluster in Isf are highly similar to those of other low potential [4Fe-4S] clusters like the clostridial ferredoxins (37). Since the E_m of M. thermophila ferredoxin is 0.407 V (12), reduced ferredoxin would be expected to efficiently donate electrons to the Isf [4Fe-4S] cluster (Fig. 11). It is likely that both redox centers of Isf are involved in electron transfer reactions coupled to CO metabolism, since both the [4Fe-4S] cluster and the flavin of Isf are reduced by CO in the presence of ferredoxin and the CODH/ACS complex (11). The E_m values presented here suggest that intramolecular electron transfer is from the [4Fe-4S] center to FMN. The E_m values presented here for Isf, combined with published E_m values for other electron transfer components, suggest that the [4Fe-4S] and FMN redox centers in Isf mediate electron transfer between ferredoxin and membrane-bound b-type cytochromes.

View larger version (85K): [in this window] [in a new window]   Fig. 11.   Proposed electron transport pathway for oxidation of CO or the carbonyl group of acetyl-CoA and reduction of CoB-S-S-CoM. Cyt b, cytochrome b; CdhA, subunit of the CO dehydrogenase/acetyl-CoA synthase; FdxA, ferredoxin; CoB, coenzyme B; CoM, coenzyme M; ?, postulated unknown electron carrier. Midpoint potentials are shown in mV.

Most flavin/Fe-S proteins stabilize four redox states: flavin_ox:FeS_ox, flavin semiquinone:FeS_ox, flavin semiquinone:FeS_red, and flavin_red:FeS_red (38, 39). Kinetic or thermodynamic stabilization of the semiquinone allows the versatility of mediating both one-electron and two-electron transfer reactions. In contrast, the flavin semiquinone of bound FMN in Isf is thermodynamically unstable. Therefore, the physiological electron acceptor for Isf (which is unknown) could be a two-electron carrier; Isf would then function as a one-electron/two-electron switch. Involvement of the obligate two-electron carrier coenzyme F₄₂₀ in the electron transport chain has been excluded (30); however, other two-electron carriers, like methanophenazine, cannot be ruled out. Another possibility is that the semiquinone radical, which is indeed transiently formed, does transfer electrons to one-electron accepting b-type cytochromes. It is plausible that the semiquinone state in Isf may become even more stabilized when Isf is complexed with other physiological electron donors/acceptors. For example, in nitrate reductase, the semiquinone is highly stabilized upon reduction by its physiological electron donor NADH although the amount of semiquinone species formed during potentiometric titrations is just 1% of the FAD present (40). This allows one electron transfer from the flavin to the b-type cytochrome in nitrate reductase. Thus, despite the instability of the flavin semiquinone in Isf during the potentiometric titrations, it is still feasible that the flavin mediates one-electron transfers from the Fe-S cluster in vivo.

The H₂- or CO-dependent reduction of Isf by extracts of M. thermoautotrophicum and the occurrence of Isf homologs in the genomes of M. thermoautotrophicum and M. jannaschii suggest that Isf functions in CO₂-reducing methanoarchaea. Cells of M. thermoautotrophicum grow and produce CH₄ with CO as the sole energy source (41), indicating a physiological role for CODH in the energy metabolism. Furthermore, this methanoarchaeon involves a CODH in the synthesis of CO for incorporation into acetyl-CoA for cell carbon (42). The CODHs from either M. thermoautotrophicum or M. jannaschii have not been purified, and therefore, the electron acceptor is unknown. The results presented here are consistent with ferredoxin as the electron acceptor; however, purification of the CODH is necessary to prove this hypothesis. The gene organization in M. jannaschii (20) provides additional support for diverse functions of Isf homologs in the methanoarchaea. In this organism, ORF MJ731 has a deduced sequence 40% identical to Isf from M. thermophila and is located in a gene cluster containing an ORF (MJ728) identified as CODH (20). The sequence of this putative enzyme has greatest identity to the CODH from Rhodospirillum rubrum, which catalyzes the oxidation of CO but not acetyl-CoA synthesis (43). MJ731 and MJ728 are also located near an ORF (MJ722) identified as an 8Fe ferredoxin (20), consistent with a role for this electron carrier as an electron donor to Isf. However, none of the deduced sequences of ORFs with identity to Isf in the genome of M. thermoautotrophicum (MTH1350, MTH1474, and MTH1595) are organized near CODH or any other redox proteins (21).