Electrochemical and Spectroscopic Properties of the Iron-Sulfur Flavoprotein from Methanosarcina thermophila *

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mössbauer spectroscopy. The midpoint potential (E m ) of the FMN/FMNH2 couple was −0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The E m value for the 2+/1+ redox couple of the cluster was −0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.

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 CO 2 . In the CO 2 reduction pathway, CO 2 is reduced to methane at the expense of electrons provided by either hydrogen (Reaction 2) or formate (Reaction 3). CH  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 CO 2 -reducing methanoarchaea (2,3). The oxidations of H 2 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 CO 2 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.
CoB-S-S-CoM ϩ 2e Ϫ ϩ 2H ϩ 3 HS-CoM ϩ HS-CoB 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 * This work was supported in part by National Institutes of Health Grant 1-R15-GM52666-01 (to K. K. S.) and by Department of Energy Basic Energy Sciences Grants DE-FG02-ER20053 (to S. W. R.) and DE-FG02-95ER20198 (to J. G. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM
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 M) 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 ϭ Ϫ0.252 V, pH 7.0) (5 M) and benzyl viologen (E m ϭ Ϫ0.362 V, pH 7.0) (5 M) 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), 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 Ϯ2-3 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.
For the power saturation studies, Isf (74 M, pH 7.0) was reduced in the presence of 50 mM methyl viologen with a 40-fold excess of sodium dithionite prepared freshly at pH 9.0. The solution was immediately frozen in an EPR tube and stored in liquid nitrogen. Spectra of the reduced [4Fe-4S] cluster were recorded at powers varying from 0.1 to 200 milliwatts at five different temperatures between 5 and 25 K. The power for half saturation (P1 ⁄2 ) at each temperature was determined by a fit to a plot of log (S/P⅐e 0.5 ) versus log P using Equation 2, where S is the signal amplitude, P is the microwave power incident on the cavity, and b is the inhomogeneity parameter (17). Best fits to the data were obtained by using a b value of 1.2.
The zero field splitting constant (⌬) was determined by a linear fit to a plot of ln P1 ⁄2 versus 1/T according to Equation 3, where T is the temperature, P1 ⁄2 is the power for half saturation, k is the Boltzmann constant, and A is a coefficient representative of the phonon spincoupling properties of the [4Fe-4S] cluster (17).
Potentiometric measurements of the [4Fe-4S] cluster were performed in an EPR spectroelectrochemical cell (18). Isf samples (80 -160 M) in 50 mM potassium phosphate buffer (pH 7.0) were titrated at 20°C in the presence of the mediator dyes, 150 . The intensity of the EPR signal with a g value of 1.93 was monitored to determine the redox state of the [4Fe-4S] cluster during the titration. Potentiometric measurements were performed in the reductive and oxidative directions. The system was considered to have reached equilibrium when the measured potential drift was less than 1 mV in 2 min.
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 57 Co source in a rhodium foil purchased from Isotope Products Laboratory. All isomer shifts are quoted relative to iron metal at room temperature.

RESULTS
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 177 KLCDVLELIQKNRDK 191 in the corrected Isf sequence replace 177 NSVMSWNLFRKIEIN 191 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).
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 CH 4 and instead oxidize H 2 and reduce CO 2 to CH 4 . 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 CO 2 -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.
Extracts of H 2 -CO 2 -grown M. thermoautotrophicum cells catalyzed the reduction of M. thermophila Isf with either H 2 or CO as the electron donor (Fig. 3). These results suggest that Isf homologs are components of electron transport chains in CO 2reducing methanoarchaea initiating with either hydrogenase or CO dehydrogenase (CODH). The rate of Isf reduction with CO as the electron donor was greater than with H 2 , indicating that CO-dependent reduction of Isf does not require prior con-version to H 2 and CO 2. The addition of ferredoxin stimulated the rate of Isf reduction 10-fold by CO and 1-fold by H 2 . These results suggest that 8Fe ferredoxins are able to couple the oxidation of either H 2 or CO to the reduction of Isf; indeed, the sequence of the M. thermoautotrophicum genome reveals several ORFs encoding putative 8Fe ferredoxins (21). The ability of H 2 or CO to serve as electron donors for the reduction of M. thermophila Isf in a CO 2 -reducing species is further indicative of a function for Isf homologs in electron transport pathways of phylogenetically and physiologically diverse methanoarchaea. Additional research with purified proteins is necessary to determine the precise role of Isf homologs in electron transport pathways of CO 2 -reducing methanoarchaea.
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] 1ϩ 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 ϭ 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 D 2 O 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.
Power saturation studies of the cluster were performed at five different temperatures. The power for half-saturation (P1 ⁄2 ) ranged from 79 milliwatts (25 K) to 14.4 milliwatts (5 K). A linear dependence of the ln P1 ⁄2 on 1/T was observed at temperatures above 5 K (Fig. 5). The zero field splitting parameter (⌬) was between 10 and 11.5 cm Ϫ1 (Fig. 5, inset).
Mössbauer Spectroscopy-Mössbauer spectra of the oxidized iron-sulfur center (Fig. 6A)  In the Mössbauer spectrum of the reduced Isf protein recorded at 100 K (Fig. 6B), a single, very broad, nonsymmetrical quadrupole doublet is observed with an average isomer shift, ␦ ϭ 0.55 mm/s, and an average quadrupole splitting, ⌬E Q ϭ 1. The spectrum was fit using four distinct ⌬E Q values (see Fig.  6B), which gives a reasonable but not unique deconvolution, since the quadrupole doublets of the four iron sites are not resolved. The line shape of the doublet is Voigt (gaussian distribution of a Lorentzian line shape) rather than Lorentzian, indicative of a microheterogenous environment for the iron sites.
Above 40 K, the electronic spin of the iron-sulfur cluster relaxes fast and only quadrupole interactions are observed in Mössbauer spectra; however, below 40 K paramagnetic hyperfine interactions also are observed. Mössbauer spectra of the reduced Isf protein recorded at 4.2 K (Fig. 7) (24). A detailed data analysis of the paramagnetic hyperfine interactions will require a high field

Mössbauer study.
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 Ϫ0.305 and Ϫ0.342 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 mM Ϫ1 cm Ϫ1 for the dimeric protein. An E m value of Ϫ0.277 Ϯ 0.003 V was determined for the FMN/FMNH 2 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 E 1 o ' (FMN/FMNH⅐) must be below Ϫ0.380 V, and the formal potential value of E 2 o ' ( FMNH⅐/ FMNH 2 ) must be above Ϫ0.175 V.
Although the flavin semiquinone is thermodynamically unstable during the potentiometric titrations, the FMN . radical was formed when Isf was reacted with CODH/ACS from M. thermophila and CO. Samples were frozen at different time points during the reaction and monitored by EPR spectroscopy. Over the course of 50 min, the cluster underwent reduction as the FMN was fully reduced to the hydroquinone state. A radical signal at g ϭ 2.00 ( Fig. 9) that can be attributed to the flavin semiquinone formed to a maximum of 2.5% of the total FMN present at 28 min and then decayed. The EPR signal yielded a line width of 16 gauss, indicative of an anionic or red semiquinone species (1). Thus, although formation of semiquinone is not thermodynamically favorable, these results suggest that a transiently stable semiquinone can exist under physiological conditions during the electron transfer from CODH/ACS to Isf.
The midpoint potential determined for the 2ϩ/1ϩ couple of the [4Fe-4S] cluster was Ϫ0.394 V at pH 7.0 (Figs. 4 and 10). The slope of the log-linear plot was 53 mV, which is close to the theoretical value of 58 mV for a one-electron transfer. The redox reaction was fully reversible, since the reduction was titrated in both the oxidative and reductive directions. Thus, the midpoint potential of the [4Fe-4S] cluster is more than 100 mV lower than that of the FMN/FMNH 2 couple (Figs. 8 and 10) and is similar to the value reported for other low potential [4Fe-4S] clusters (26,27). DISCUSSION 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 CH 4 with an electron pair derived from oxidation of the former to CO 2 (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 CH 3 -S-CoM to CH 4 requires the electron donor coenzyme B (HS-CoB), which pro- duces 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 membranebound 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 Ϫ330, Ϫ250, and Ϫ182 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 Ϫ0.407 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 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 F 420 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 H 2 -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 CO 2 -reducing methanoarchaea. Cells of M. thermoautotrophicum grow and produce CH 4 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).