Characterization of the Intramolecular Electron Transfer Pathway from 2-Hydroxyphenazine to the Heterodisulfide Reductase from Methanosarcina thermophila

Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in meth-anogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe 4 S 4 ] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a mem-brane-bound cofactor, which can be replaced by a wa-ter-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH 2 ) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH 2 with the following values: k cat 5 74 s 2 1 at 25 °C, K m (HPhenH 2 ) 5 92 m M , K m (CoB-S-S-CoM) 5 144 m M . Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using

The ultimate electron donor to HDR can be the CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex when bacteria grow on acetate. In this case, the electron transfer pathway involves ferredoxin and possibly an iron-sulfur flavoprotein (5,6). When bacteria grow on methanol, the ultimate donor is reduced coenzyme F 420 H 2 , generated by F 420 dehydrogenase. The F 420 dehydrogenases from Methanosarcina mazei (7), Methanolobus tindarius (8), and Archaeoglobus fulgidus (9) have been isolated and shown to contain Fe-S clusters and FAD. The direct electron donor for HDR appears to be a membrane-bound cofactor, methanophenazine (MPhen) (10). This cofactor has been isolated from membranes of M. mazei strain Gö1 (11) and Methanosarcina thermophila. 2 It has a 25-carbon isoprenoid chain attached to position 2 of phenazine via an ether bond, which makes it insoluble in aqueous solution (11). The 2-hydroxyphenazine (HPhen) derivative is a suitable water-soluble substitute for MPhen that can accept electrons from F 420 H 2 and can donate electrons to the purified HDR from M. thermophila (10). Electron transfer from F 420 H 2 to HPhen results in the translocation of two protons per two electrons transferred (12). Another two protons (per two electrons) are translocated during reduction of CoB-S-S-CoM by HPhen (13).
HDR from M. thermophila consists of two subunits. A 53-kDa subunit contains two distinct [Fe 4 S 4 ] clusters with midpoint potentials of Ϫ100 and Ϫ400 mV (6). A 27-kDa membrane-associated subunit contains two b-type hemes, one that is low spin and is hexacoordinate and another that is high spin and is five-coordinate. The midpoint potentials of the low and high spin hemes are Ϫ180 and Ϫ23 mV, respectively (6).
We have used steady-state and pre-steady-state kinetics to answer some key questions about the HDR mechanism. Which of the metal centers in HDR is the initial electron acceptor from reduced HPhen (HPhenH 2 )? The midpoint potentials of some of the metal centers are outside the range of the HPhen/HPhenH 2 and CoB-S-S-CoM/CoB-SH, CoM-SH couples; therefore, are all of the metal clusters involved in the electron transfer reaction? What is the intramolecular electron transfer pathway? Based on our results, we propose that the physiological electron transfer pathway from methanophenazine to the heterodisulfide is: MPhenH 2 3 [Fe 4 S 4 ] high 3 heme low 3 CoB-S-S-CoM. * This work was supported by Department of Energy Grant ER20053 (to S. W. R.). 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.

EXPERIMENTAL PROCEDURES
Materials-HPhen was synthesized as described (11). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo).
Cell Growth and Enzyme Preparation-M. thermophila TM1 was cultured on acetate at 50°C and pH 6.8 in a 5-liter New Brunswick fermentor equipped with a pH auxostat (14) as described (15). HDR was purified as described previously (6), except for enzyme concentration steps. As mentioned by Thauer et al. (16), higher activity was recovered when membranes with molecular/pore molecular mass cut-off of 50 kDa (Spectrum) were used.
Enzyme Assays-HDR activity was measured by monitoring the oxidation of reduced methyl viologen at 604 nm (⑀ ϭ 13.9 mM Ϫ1 cm Ϫ1 ) and 55°C (6,17,18). One unit of HDR activity corresponds to 1 mol of CoB-S-S-CoM reduced per minute. HDR activity was also assayed using 2-hydroxyphenazine as the electron donor. A solution containing HPhen (final concentration of 200 M) in Buffer A (50 mM Tris, pH 7.6, and 10% glycerol) was reduced by bubbling with 100% hydrogen gas for 20 min, adding partially purified hydrogenase from M. thermophila, and incubating at 55°C for 2 h. For steady-state kinetic experiments, varying amounts of the HPhenH 2 stock solution were added to Buffer A. Then, HDR was added and the reaction was started by adding CoB-S-S-CoM. The oxidation of HPhenH 2 was monitored either at 25°C or at 55°C by following an increase in absorbance at 365 nm. The difference (oxidized minus reduced, ⌬⑀) extinction coefficient at 365 nm was measured to be 3.58 mM Ϫ1 cm Ϫ1 . One unit is defined as 1 mol of CoB-S-S-CoM reduced min Ϫ1 mg Ϫ1 . The data were fit to eq. 3 for a ping-pong reaction, where v 0 is initial velocity and A and B are HPhenH 2 and CoB-S-S-CoM, respectively, as follows.
Protein concentrations were determined by the Bio-Rad protein assay (19) using bovine serum albumin as a standard. UV-visible spectra were collected on a Cary-14 spectrophotometer modified by On-Line Instrument Systems, Inc. (Bogart, GA). EPR spectra were recorded on a Bruker ESP300E spectrometer equipped with a temperature controller (Oxford ITC4) and automatic frequency counter (Model 5340A, Hewlett-Packard Co.).
Stopped-flow Experiments-HDR was oxidized by adding thionin (EЈ 0 ϭ ϩ60 mV) until the blue color disappeared. Excess thionin was removed by passing the solution through a Sephadex G-25 column (Amersham Pharmacia Biotech). Oxidized HDR (6 M before mixing)  Reduction of the hemes was followed at 423 nm. Two phases with equal amplitude were observed, a fast phase with k obs ϭ 87 s Ϫ1 (q) and a slow phase with a rate constant of 9.4 s Ϫ1 (E).

FIG. 3. Stopped flow kinetics of the HDR reaction at low concentrations of HPhenH 2 .
A, oxidized HDR (3 M) was mixed with HPhenH 2 (5 M), and the reaction was followed at 420 nm. B, spectra were collected at 0, 70, and 300 ms after mixing by rapid-scanning stopped-flow, and the difference spectrum was generated by subtracting the 300-ms spectrum from the 70-ms spectrum. and varied concentrations of HPhenH 2 (10, 20, 50, 100, 150, and 200 M before mixing) were rapidly mixed at 25°C in a 1:1 ratio. Heme reduction was followed at 423 nm in a rapid-scanning stopped flow instrument (On-Line Instrument Systems, Inc.).
Freeze-quench EPR-Thionin-oxidized HDR (60 M) was rapidly mixed with 200 M HPhenH 2 at room temperature using a chemical/ freeze-quench apparatus (Update Instrument, Inc., Madison, WI). The HDR-containing and HPhenH 2 -containing syringes (2 ml) were connected by a mixer, and the reaction time was controlled using aging hoses of different lengths. The solutions were mixed using four sequential ram displacements of 1.3 mm (83 l per shot) and ram velocity of 8 cm/s. The reaction was quenched by rapidly freezing the mixture in a funnel attached to an EPR tube that was filled with low temperature (Ϫ120°C) isopentane. The frozen snow containing the quenched reaction mixture was then packed tightly in EPR tubes. The 0-ms time point was obtained by mixing the oxidized HDR with buffer.
Mersalyl Acid Treatment-Oxidized HDR (above) was treated with 1 mM (final concentration) mersalyl acid for 30 min. Then, excess mersalyl acid was removed by centrifuging the solution through a Sephadex G-25 column (Amersham Pharmacia Biotech).

RESULTS
Steady State-Steady-state kinetic experiments were performed at 55°C at varying concentrations of HPhenH 2 and CoB-S-S-CoM to determine the overall mechanism of the HDR reaction. The data were fitted to the Michaelis-Menten equation ( Fig. 1), which yielded the K m values for HPhenH 2 and CoB-S-S-CoM of 92 Ϯ 22 and 144 Ϯ 33 M, respectively. The specific activity was 52 mol min Ϫ1 mg Ϫ1 (k cat of 70 s Ϫ1 , assuming a dimeric unit of 80 kDa). In a ternary-complex mechanism, the V/K value for one substrate increases with the concentration of the other substrate. The V max /K m values for CoB-S-S-CoM do not increase with HPhenH 2 concentration ( Fig. 1, inset), indicating that the reaction follows a ping-pong mechanism. At higher concentrations of HPhenH 2 , the V/K value decreases, indicating some degree of substrate inhibition.
Pre-steady-state Kinetics-The electron transfer pathway from HPhenH 2 to HDR was studied by rapid-scanning stoppedflow kinetics at 25°C. The enzyme used in the stopped-flow experiments was highly active, with a turnover number at 25°C of 72 s Ϫ1 . HPhenH 2 was rapidly mixed with oxidized HDR, and heme reduction was monitored at 423 nm. Two phases of equal amplitude were evident, and the data fit well to a biexponential equation, corresponding to reduction of the two b-type hemes of HDR (Fig. 2). Only the first phase of this reaction appears to be kinetically relevant (87 s Ϫ1 ), because the rate constant for the second phase (9.4 s Ϫ1 ) is significantly slower than the turnover number (72 s Ϫ1 ) for the enzyme. Results described below indicate that it is the low potential heme that is reduced at catalytically competent rates. Furthermore, only the first rate constant is dependent on HPhenH 2 concentration; the heme reduced in the second phase was HPhen-independent. These results suggest that one heme (the high potential heme, see below) is not involved in CoB-S-S-CoM reduction by HPhenH 2 .
We had hoped to independently monitor reduction of the heme and the Fe-S cluster; however, the heme absorbance dominated the spectra of HDR. When the HPhenH 2 concentration is similar to that of HDR and is well below its K m value, there are two clearly distinguishable phases: a rapid increase in absorbance (k obs1 ϭ 34 s Ϫ1 ) followed by a slower decay (k obs2 ϭ 2.3 s Ϫ1 ) (Fig. 3). The difference spectrum generated by subtracting the spectrum collected at 300 ms from that at 70 ms (Fig. 3B) matches that of the difference spectrum of the reduced minus oxidized HDR. The amplitudes of the two phases are . The enzyme and substrate were kept in separate syringes at 20°C before mixing. A, reduction of the high potential FeS cluster. The spectra shown from the top to the bottom were collected 0, 7.8, 35, 115, and 215 ms after rapidly mixing HDR with HPhen at 20°C. B, signal intensity (g ϭ 1.95-1.90) was plotted and fitted in a single-exponential curve. C, reduction of the high potential heme. The spectra shown from the top to the bottom were collected 0, 7.8, 215, and 415 ms after mixing. D, signal intensity at g ϭ 6.2 was plotted and fitted in a single-exponential curve. The k cat for the HDR used in this experiment was 48 s Ϫ1 (at 25°C). EPR conditions were: temperature, 10 K; power, 40 milliwatts; gain, 20,000; modulation frequency, 100 kHz; modulation amplitude, 10 G; microwave frequency, 9.4767 GHz. equal and correspond to 0.25 heme. The electron acceptor is likely to be either an FeS cluster or some other redox site on the protein, possibly a redox-active disulfide. If the acceptor is an FeS cluster, the results would be most consistent with reoxidation of the heme group by the high-potential Fe-S cluster. Given the slow rate of heme reoxidation, however, this event is unlikely to be involved in catalysis.
Freeze-quench EPR-Because we are unable to follow the reduction of FeS clusters by stopped-flow due to dominating absorbance of hemes at 400 -450 nm, rapid freeze-quench EPR studies were performed. The FeS cluster is fully reduced within 35 ms (Fig. 4A). The rate constant for Fe-S reduction by HPhen is 73 Ϯ 31 s Ϫ1 at 20°C (Fig. 4B). These results clearly show that the FeS cluster is reduced at catalytically competent rates.
Given the standard error in the freeze-quench measurement, we cannot conclude whether the cluster is reduced before, after, or simultaneously with the low potential heme. It is the high potential cluster that undergoes reduction, because HPhenH 2 reduces only the high-potential FeS cluster; the low potential cluster remains oxidized (see below).
The EPR signal of the low potential heme is observed at very low intensity (6) and could not be detected in the freeze-quench experiments; however, we were able to follow the rate at which HPhenH 2 reduces the high potential high spin heme (Fig. 4C). The high spin heme signal (g ϭ 6.2) remained more than 60% oxidized 415 ms after reaction with HPhenH 2 . The rate constant for reduction of the high spin heme is 1.3 Ϯ 0.1 s Ϫ1 (Fig.  4D). Thus, the freeze-quench EPR and stopped-flow results show that reduction of the high potential heme by HPhenH 2 occurs about 10-fold slower than k cat , indicating that this heme is not involved in electron transfer from HPhenH 2 to CoB-S-S-CoM. This is consistent with stopped-flow and inhibition studies described below.
Effects of CO on the HDR Reaction-We showed earlier that CO binds tightly to one of the hemes (K d ϭ 0.8 M) (6). However, even at 1 mM concentration, CO does not affect the rate of reduction of CoB-S-S-CoM when HPhenH 2 or methyl viologen is the electron donor (Fig. 5). Because only the high spin high potential heme binds CO (6), these results indicate that only the low potential heme is required for CoB-S-S-CoM reduction.
When dithionite is added to HDR, both hemes are reduced [ Fig. 6A]. When excess CoB-S-S-CoM is then added, the UVvisible spectrum shows a broad Soret peak around 420 nm (Fig.  6B, solid line). This spectrum can be fit to the sum of two hemes with 66% in the oxidized and 34% in the reduced state (dashed line). Therefore, the remaining reduced heme is not involved in substrate reduction. To determine which of the two hemes is involved in catalysis, we added CO to the CoB-S-S-CoM treated enzyme. The Soret peak for the reduced heme shifts to 420 nm (dotted line), which corresponds to the CO-bound form of HDR (6) and the spectrum of the oxidized heme was unchanged. This result clearly shows that, when CoB-S-S-CoM is added, the high potential heme remains reduced and the low potential hexacoordinate heme undergoes oxidation.
These combined results strongly indicate that the high potential heme is not involved in CoB-S-S-CoM reduction and that electrons from the reduced low potential heme can reduce CoB-S-S-CoM to the dithiol products at kinetically relevant rates.
Effects of Mersalyl Acid-Mersalyl acid is known to disrupt FeS clusters (20). Addition of mersalyl acid to HDR only slightly affects the heme spectra. This indicates that mercury treatment does not alter the heme environment. This is expected, because mercury only affects hemes with sulfur ligands, not histidine-ligated hemes like those in HDR (6). The difference spectrum between the native and the mersalyl acidtreated HDR showed a broad band around 400 -500 nm that is characteristic of FeS clusters (Fig. 7). There is a small peak at 420 nm above the broad absorption band, which is likely to be from the heme. However, this would constitute less than 10% alteration of the heme. Using a typical extinction coefficient for ferredoxin, which is 16 mM Ϫ1 cm Ϫ1 per cluster (21), these results indicate that the mersalyl acid disrupted 1.8 clusters per dimeric unit. HDR contains two [4Fe-4S] clusters (6).
HPhenH 2 was unable to reduce the hemes of the mersalyl acid-treated oxidized enzyme, whereas dithionite reduced both hemes. The mersalyl acid-treated enzyme was also unable to catalyze the reduction of CoB-S-S-CoM, when either HPhenH 2 or methyl viologen was used as the electron donor. Assuming that mersalyl acid only affects the FeS cluster, these results indicate that an iron-sulfur cluster is the initial acceptor of electrons from HPhenH 2 . Another possibility is that disruption of the FeS cluster damages the HPhen binding site, which would prevent heme reduction. The redox potentials of the two clusters are Ϫ100 and Ϫ400 mV (6). Because the midpoint redox potential for HPhen/HPhenH 2 is ϳϪ250 mV, it seems likely that the high potential cluster is the electron acceptor from HPhenH 2 . Because the results described above indicated that the high potential heme is not involved in CoB-S-S-CoM reduction, we hypothesize that the electron pathway from HPhenH 2 to the heme is: where the H and L subscripts designate the high and low potential centers. However, this is not the most thermodynamic electron transfer pathway, because the midpoint potential of the low potential heme is 80 mV more negative than that of the high potential cluster.
Effects of Diphenylene Iodonium-Diphenylene iodonium (DPI) is a lipophilic reagent that inhibits a variety of flavoproteins such as NAD(P)H-dependent dehydrogenases and oxidases (22)(23)(24). The inhibitor is thought to interact with flavins and low potential b-type cytochromes in these enzymes. DPI also inhibits the reduction of CoB-S-S-CoM by factor F 420 H 2 dehydrogenase in the membrane-bound electron transport chain of M. mazei Gö1 (25). To elucidate the intramolecular electron transfer pathway among the centers of HDR, we studied inhibition of the purified enzyme by DPI.
Inhibition of the HDR Reaction by DPI-When HPhenH 2 is the electron donor, DPI is a strong inhibitor of CoB-S-S-CoM reduction (Fig. 8A). DPI inhibits heterodisulfide reduction in a competitive manner with respect to HPhenH 2 with a K i value below 1 M (Fig. 9). Surprisingly, it increases the rate of methyl viologen oxidation by CoB-S-S-CoM (Fig. 8B). When CoB-S-S-CoM is absent, methyl viologen oxidation is not observed. These results suggest that the mechanism of CoB-S-S-CoM reduction is different with the two electron donors. One possibility is that DPI, in competing with the HPhen binding site,  blocks electron transfer to the high potential Fe-S cluster. However, methyl viologen, which can reduce CoB-S-S-CoM in the presence of DPI, has a different binding site than HPhen; it may transfer electrons directly to the heme.
UV-visible Spectra of DPI-treated HDR-The effects of DPI on the oxidation states of the metal centers of HDR are summarized in Table I. When HPhenH 2 is used as the electron donor, both of the hemes are reduced (Fig. 10). Adding DPI to the HPhenH 2 -reduced enzyme causes the Soret band at 425 nm to shift to 410 nm, corresponding to the oxidized form of HDR. This indicates that both hemes are oxidized by DPI. Dithionite reduces both hemes in the absence of DPI. However, when dithionite is then added to HPhenH 2 -reduced and DPI-oxidized HDR, a composite spectrum is obtained with peaks at 425 and 410 nm. The spectrum fits a mixture of 50% oxidized and 50% reduced heme. These results indicate that, although dithionite has a low enough potential to reduce both hemes, reduction of one of the two hemes is inhibited by prior treatment with DPI. CO shifts the spectrum of the reduced heme in the DPI-treated enzyme indicating that, after treatment of HDR with DPI, only the high potential 5-coordinate heme can be reduced. Reduction of the low potential hexacoordinate heme is inhibited. This constitutes further evidence that the low potential, but not the high potential, heme is involved in electron transfer from HPhenH 2 to the heterodisulfide.
EPR Spectra of DPI-treated Enzyme-EPR spectroscopy was used to evaluate the effects of DPI on the metal centers in HDR (Fig. 11). Oxidized HDR displays an EPR spectrum with g values at 6.2, 5.8, and 2.0 that derives from the high spin (spin ϭ 5/2) heme (6) (Fig. 11A). The low spin heme displays a "large g max " EPR spectrum with very low intensity and values of g max that are Ͼ3. When the oxidized enzyme is reduced with HPhenH 2 (Fig. 11B), the high spin heme spectrum disappears and a new EPR signal with g values at 2.06, 1.95, and 1.90 appears, which is from a singly reduced [4Fe-4S] cluster (6). Unlike dithionite (Fig. 11C), HPhenH 2 is not a strong enough electron donor to reduce the low potential cluster. When DPI is added to HPhenH 2 -reduced HDR (Fig. 11D), the EPR spectrum of the cluster disappears as the g ϭ 6 signal from the high spin heme reappears. These results combined with the UV-visible spectroscopic results described above indicate that the high potential FeS cluster and the two hemes undergo oxidation by DPI. When the thionin-oxidized enzyme is treated with dithionite (Fig. 11C), all the metal centers of HDR are reduced; the characteristic complex spectrum of the doubly reduced protein is observed with g values at 2.03, 1.97, 1.92, and 1.88 (6). The complicated signals result from dipolar coupling between the two clusters. Addition of DPI to the dithionite-reduced enzyme led to the oxidation of one cluster (Fig. 11E). Presumably, DPI does not oxidize the high potential cluster. DISCUSSION Electron transfer from HPhenH 2 through the redox centers of HDR to CoB-S-S-CoM drives the translocation of two protons across the cytoplasmic membrane per two electrons transferred (13). Reduction of HDR by HPhenH 2 has been studied here by kinetic and spectroscopic methods. Stopped-flow and freezequench EPR experiments indicate that only one of the two hemes and one of the two clusters undergoes reduction by HPhenH 2 with rate constants exceeding k cat . Our CO-binding and DPI inhibition experiments indicate that the low potential heme is involved in the electron transfer pathway and that the high potential heme is not involved in catalysis. This is consistent with the redox demands of the reaction; i.e. the midpoint potential for the CoB-S-S-CoM/(RSH) 2 couple is approximately Ϫ200 mV, whereas that of the high spin high potential heme is Ϫ23 mV (6).
Why would HDR retain an unnecessary high potential heme throughout evolution? One possibility is that this heme is involved in stabilizing the protein or in generating the proton gradient associated with the HDR reaction. There is a high potential c-type cytochrome in the membranes of M. mazei with unknown function. It is attractive to consider an electron transfer chain leading from the oxidized heme to cytochrome c that would be coupled to proton translocation. Such a pathway would make the HDR reaction analogous to cytochrome bc 1 .
Which redox center is the direct electron acceptor from HPhenH 2 ? HPhenH 2 reduces the FeS cluster and the low potential heme at similar rates. Thus, the rapid kinetics experiments cannot distinguish between the following electron transfer pathways: HPhenH 2 3 FeS 3 heme L , HPhenH 2 3 heme L 3 FeS, or a simultaneous reduction of heme L and FeS by HPhenH 2 . Inhibition experiments provide further information about the possible electron transfer pathways. HPhenH 2 does not reduce either heme group of mersalyl-treated HDR. Because the heme spectra are not appreciably altered by the treatment and mercury is known to disrupt FeS clusters, we assume that inhibition of heme reduction is due to a direct effect of Hg 2ϩ on the cluster. Therefore, it appears that electrons are transferred directly from HPhenH 2 to an iron-sulfur cluster, indicating that the electron transfer pathway is HPhenH 2 3 FeS 3 heme L .
There are two 4Fe-4S clusters in HDR (6). Which of the two clusters is the initial electron acceptor? DPI oxidizes only the low potential Fe-S cluster, leaving the high potential cluster reduced; however, this form of HDR can still reduce CoB-S-S-CoM. Thus, apparently, the low potential Fe-S cluster does not undergo redox cycling during the catalytic mechanism. Therefore, we propose that the electron transport pathway is HPhenH 2 3 high potential [Fe 4 S 4 ] 3 low potential [heme b] L . If the low potential cluster is not involved in the relay of electrons from HPhenH 2 to CoB-S-S-CoM, why would it be present in HDR? One possibility, described below, is that the oxidized low potential cluster is involved in a catalytic step and not in redox chemistry. Another is that HDR can accept electrons from several sources and that, under some conditions, a redox partner with a more negative midpoint potential than MPhen could donate electrons to the low potential cluster, which would in turn donate electrons to the high potential cluster. The electron transport pathway from CODH and CO, with a midpoint potential below Ϫ500 mV, has not been elucidated.
The experiments described in this report were performed in aqueous solution with the water-soluble HPhen analog of MPhen. It is important to determine whether the membranebound enzyme uses the same electron transfer pathway. If the physiologically relevant order of electron flow is indeed HPhenH 2 3 [Fe 4 S 4 ] H 3 [heme b] L , the high potential cluster is expected to be located near the subunit interface. This is because the large subunit containing the clusters is cytoplasmic and the heme is in the membrane-associated subunit (16). This would be similar to several quinone-coupled enzymes whose FeS clusters are in close contact with a quinone-binding site in membrane, such as quinol:fumarate reductase (26), Me 2 SO reductase (27), and NADH:ubiquinone oxidoreductase (28).
How do electrons passing through one-electron redox centers accomplish the two-electron reduction of CoB-S-S-CoM? Because the two classes of HDRs are heme iron-sulfur or flavin iron-sulfur proteins, Thauer et al. (16) proposed that the ironsulfur subunits harbor the active site of heterodisulfide reduction and that the mechanism of disulfide reduction could resemble that of the ferredoxin:thioredoxin reductases from chloroplasts and cyanobacteria. In the plant thioredoxin reductase mechanism, a sulfur radical, which is formed as an intermediate, appears to be stabilized by binding to a [Fe 4 S 4 ] cluster (29,30). Evidence presented here indicates that the low potential cluster of HDR is not involved in electron transfer reactions. Because the midpoint potential of this cluster (Ϫ400 mV) is much lower than that of HPhen (ϳϪ250 mV) or CoB-S-S-CoM (ϳϪ220 mV), this cluster may play a catalytic role, analogous to that of the cluster in thioredoxin reductase. This putative role is to stabilize a radical anion formed by one electron reduction of the disulfide substrate. Further studies are required to test this hypothesis.