Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage.

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.

The CO/acetyl-CoA exchange reaction is a valuable analytical tool since it requires the disassembly of acetyl-CoA into its three components and its re-synthesis from bound methyl, carbonyl, and CoA moieties. It does not require a corrinoid protein or a methyltransferase, like Reaction 2. Furthermore, this exchange reaction does not involve net redox chemistry, since CO is at the same redox state as the carbonyl group of acetyl-CoA. Reactions 2 and 3 include an intermediate metalcarbonyl species that is paramagnetic and has been called the NiFeC species (4). Studies of this EPR signal led to the first discovery of a metal cluster in biology containing nickel and iron (4). The acetyltransferase reaction between acetyl-CoA and free CoASH (Reaction 4) measures the ability of the enzyme to cleave and re-synthesize only the C-S bond. These reactions have been studied extensively with CODH/ACS from the acetogenic bacterium, Clostridium thermoaceticum (see Refs. 5 and 6 for review). The acetyl-CoA/CoA exchange reaction of the Methanosarcina barkeri (1,7) and Methanosarcina thermophila (8) CODH/ACS complex also has been studied in great detail. The CO/acetyl-CoA exchange activity of the CODH/ACS complex also has been previously studied (8).
The CODH/ACS complexes from M. thermophila (9,10), M. barkeri (11), and Archaeoglobus fulgidus (12) (a sulfate reducing archaeon) contain five subunits (␣, ␤, ␥, ␦, and ⑀) with molecular masses of 89, 71, 60, 58, and 19 kDa. The ␣⑀ dimer contains one [Fe 4 S 4 ] cluster (Cluster B) and a center called Cluster C, which appears to be a Ni-X-[Fe 4 S 4 ] cluster (where X is an unknown bridging ligand) and serves as the site of CO oxidation (13). These clusters have very similar properties to those of Clusters B and C in the clostridial CODH subunit (14 -17). The ␥␦ dimer with one corrinoid and one [Fe 4 S 4 ] * This work was supported by Department of Energy Grant ER20053 and National Institutes of Health Grant GM39451 (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.
Evidence suggests that the ␤ subunit contains the molecular machinery to catalyze the first steps in acetyl-CoA utilization, the disassembly of acetyl-CoA to bound methyl, carbonyl, and CoA groups. The M. thermophila ␤ subunit shares 42% sequence identity with the large ␣ subunit (acsB) of the C. thermoaceticum enzyme (18), which harbors Cluster A, the site of acetyl-CoA synthesis (19,20). However, there is only 16% identity between the ␤ subunit and the small subunit (acsA) of the acetogenic enzyme (18). Furthermore, when the CODH/ACS complex is subjected to limited proteolysis, acetyl-CoA synthesis activity declines in parallel with loss of the intact ␤ subunit; yet the truncated ␤ subunit retains the ability to catalyze the CoA/acetyl-CoA exchange reaction (7). On the other hand, spectroscopic studies of an apparently purified ␣⑀ dimer indicated that Cluster A is a component of this complex (13).
As reported here, the location of the acetyl-CoA cleavage site was investigated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electron paramagnetic resonance (EPR) spectroscopy, and kinetic methods. Our results indicate that acetyl-CoA cleavage/synthesis site is located in the ␤ subunit and that interaction between the ␣⑀ dimer and the ␤ subunit is necessary for breaking and forming the C-C bond of acetyl-CoA.
Cell Growth and Enzyme Purification-M. thermophila TM1 cells were grown, harvested, and lysed as described (21). The CODH/ACS complex was purified essentially as outlined by following the oxidation of CO to CO 2 (21). M. thermophila cells were suspended into Buffer A (50 mM Tris/HCl, pH 7.6, 10% glycerol, 0.6% Triton X-100, and 2 mM DTT) and were lysed by sonication. Triton X-100 did not affect the CODH/ACS activities and was needed for purification of heterodisulfide reductase from the same batch of cells. After ultracentrifuging at 32,000 rpm for 2 h, the cell-free extract was loaded on a Q-Sepharose high performance column (2.5 ϫ 40 cm) and washed with Buffer A containing 0.1 M KCl. The CODH/ACS complex was eluted with a 0.1-0.8 M KCl linear gradient. The CODH/ACS complex fractions were pooled, diluted 3-fold with Buffer B (50 mM Tris/Cl, pH 6.8, 10 mM MgCl 2 , 10% glycerol, and 2 mM DTT), and applied to a second Q-Sepharose HP column (2.5 ϫ 15 cm). After washing the column with Buffer B containing 0.2 M KCl, the CODH/ACS complex was eluted with the same buffer containing 0.4 M KCl. Fractions containing CODH activity were pooled, concentrated on a YM-30 membrane, and loaded on a Superose 6 column (2.5 ϫ 60 cm). The CODH/ACS complex appeared to be homogeneous based on analysis by SDS-PAGE (11.25% polyacrylamide). The purified enzyme exhibited specific activities of CO oxidation and CO/acetyl-CoA exchange of 286 and 0.206 mol min Ϫ1 mg Ϫ1 , respectively. The CODH/ ACS complex was labeled with radioactive nickel by adding 50 Ci of 63 Ni (NiCl 2 ) to 5 liters of media containing a total nickel concentration of 20 M.
Enzyme Assays-Protein concentration was determined colorimetrically using a Rose Bengal dye-binding method (22) with bovine serum albumin as the standard. CO oxidation activity was measured using methyl viologen as the electron acceptor (23). The CO/acetyl-CoA exchange activity was assayed using [1-14 C]acetyl-CoA (24).
Separation of the Components-To separate the components of the CODH/ACS complex, a cationic detergent, dodecyltrimethylammonium bromide (DTAB), and Triton X-100 were added to the purified CODH/ ACS complex to give final concentrations of 1.0 and 0.3%, respectively. After incubating at room temperature for 10 min and at 4°C, the solution was applied to a Q-Sepharose HP column (2.5 ϫ 15 cm). After washing with Buffer B containing 0.1% DTAB and 0.2 M KCl, the components were eluted with a linear gradient from 0.2 to 0.8 M KCl in the same buffer.
Bromelain Treatment-Limited proteolysis was performed by mixing purified CODH/ACS complex (final concentration of 25 mg/ml) with bromelain from pineapple stem (90% purity, Sigma catalog number B-5144, 8.8 units/mg protein, final concentration of 0.1 mg/ml) in a solution containing 50 mM Tris/HCl, pH 7.2, 0.2 M KCl, 10 mM MgCl 2 , 2 mM DTT, 1 mM L-cysteine, and 10% glycerol. The ratio of the CODH/ ACS complex to bromelain was 250:1 (w/w). To examine the effect of bromelain on the NiFeC EPR signal, both the CODH/ACS complex and bromelain solutions were preincubated with 1 atm CO.
Chymotrypsin Treatment-CODH/ACS complex (800 l of 20 mg/ml) in Buffer B was mixed with ␣-chymotrypsin (Sigma catalog number C-3142) from bovine pancreas (37.5 l of 10 mg/ml at 60 units/mg protein) at a ratio of the complex to chymotrypsin of 43:1 (w/w). The proteolysis reaction was quenched by adding 42.5 l of 2 mg/ml soybean chymotrypsin inhibitor (Sigma catalog number T-9777) into 150 l of the mixture at different times. The chymotrypsin inhibitor did not affect the NiFeC EPR signal, CO oxidation, or CO/acetyl-CoA exchange activity.
Other Methods-EPR spectra were recorded on a Bruker ESP300E spectrometer equipped with a temperature controller (Oxford ITC4) and automatic frequency counter (Hewlett-Packard Co., model 5340A). Homology searches were performed using BLAST and PSI-BLAST (25).

Nickel Content of the ␣ and ␤ Subunits of the CODH/ACS
Complex-CODH/ACS complex was isolated from M. thermophila cells grown with 63 Ni. The purified complex contains 2.1 g atm of nickel per mol, assuming an ␣␤␥␦⑀ stoichiometry, in agreement with earlier measurements (2). To determine which subunit(s) contains nickel, the complex was treated with 1% DTAB and 0.3% Triton X-100 (conditions that dissociate the multiprotein complex (2)) and chromatographed on a Q-Sepharose HP column. Two radioactive peaks were observed (Fig. 1). The first peak was highly enriched in the ␣ and ⑀ subunits, whereas the second peak contained a mixture of the ␤, ␥, and ␦ subunits with traces of the ␣ and ⑀ subunits (Fig. 1, inset). The first peak contained 1.2 g atm of nickel per mol of the ␣⑀ dimeric unit. Since the ⑀ subunit does not contain metals, the nickel must be associated with the ␣ subunit, as concluded earlier (2). The ␤ subunit was further purified by pooling the fractions in the second peak and treating again with 1% DTAB and 0.3% Triton X-100. After diluting 5-fold, the enzyme solution was loaded on the Q-Sepharose HP column and eluted with a linear gradient of 0.2-0.8 M KCl. The protein and 63 Ni radioactivity profiles obtained from SDS-PAGE analysis are shown in Fig. 2. Nickel was clearly associated with the pure ␤ subunit; for example, fraction 16 contains highly purified ␤ subunit with FIG. 1. 63 Ni radioactivity profile after dissociation of the complex by DTAB. 100-l aliquots from each fraction from the Q-Sepharose HP column were taken for SDS-PAGE analysis and for measuring radioactivity by liquid scintillation counting. The SDS gels from the two peaks and the complex before the detergent treatment are shown. no apparent traces of the ␣ subunit and 0.4 g atm of nickel per mol of ␤ subunit. Apparently, some nickel was lost during the purification. Both Clusters A (4, 26, 27) and C (15, 28) contain a NiFeS cluster. Since Cluster C is unarguably associated with the ␣ subunit (1, 13), these results suggest that the nickel in the ␤ subunit is a component of Cluster A, which is responsible for acetyl-CoA cleavage and synthesis.
Demonstration That the ␤ Subunit Elicits the NiFeC EPR Signal-When the methanogenic (13,29) or acetogenic (4, 30) CODH/ACS is treated with CO, a characteristic EPR signal is observed. This spectrum has been assigned to a metal-carbonyl species at Cluster A, the active site of acetyl-CoA synthesis (19). Therefore, our objective was to identify the subunit that elicits this EPR signal. This would provide strong evidence that Cluster A and the acetyl-CoA cleavage activity are present on that subunit.
When the salt concentration of the DTAB-treated CODH/ ACS complex is decreased below 0.1 M KCl, the ␤, ␥, and ␦ subunits precipitate (Fig. 3). Densitometric analysis of the intensities of the stained protein bands on the SDS-PAGE gel show that 95% of the ␤ subunit and 60% of the ␥ and ␦ subunits had precipitated. EPR spectra were obtained at 80 K of the DTAB-treated samples that were incubated with CO. The NiFeC signal was observed for the DTAB-treated sample; however, the spin quantitation only yielded 0.01 spins/mol. In comparison, the CO-incubated complex that had not been treated with DTAB elicited 0.3 spins/mol. The congruence between the proportion of ␤ subunit and NiFeC EPR signal indicates that Cluster A is located on this subunit. For the detergent-treated as-isolated sample in the absence of CO, a characteristic low spin Co 2ϩ EPR signal of the CFeSP (31) was observed with a signal intensity amounting to 40% of that from the intact enzyme complex. These results agree with earlier studies demonstrating that the corrinoid is present in the ␦ subunit (31,32).
Alteration of the NiFeC EPR Signal by Chymotrypsin Treatment-Limited protease digestion dissociates the components of the M. barkeri CODH/ACS complex and cleaves the ␤ subunit into two fragments (1). The isolated truncated ␤ subunit retains the ability to catalyze an exchange reaction between CoA and acetyl-CoA but is unable to synthesize acetyl-CoA (1). CODH/ACS complex was treated with chymotrypsin at a ratio of 43:1 (CODH/chymotrypsin, w/w) to dissociate the complex and truncate the ␤ subunit. The protease reaction was quenched at different times by adding chymotrypsin inhibitor, which we found to quench rapidly the protease reaction without adversely affecting the NiFeC EPR signal, CO oxidation, or the CO/acetyl-CoA exchange activity.
The NiFeC EPR signal (Fig. 4), SDS-PAGE-stained protein patterns (Fig. 5), and CO/acetyl-CoA exchange activity of samples quenched at different times after chymotrypsin treatment were measured (Fig. 6). The CO oxidation activity was not affected by the chymotrypsin treatment over the time course followed. The shape of the NiFeC signal underwent a marked change in morphology. The peak at g ϭ 2.08 decreased, and the peak at g ϭ 2.05 increased at the same rate constant of 0.16 min Ϫ1 (Figs. 4 and 6). This modified signal was first observed in N-bromosuccinimide-inhibited ACS (33) and has been called the "pseudo NiFeC signal" (34). Essentially all of the NiFeC signal was converted to the "pseudo" form within 1 h of reaction with chymotrypsin, but the total spin concentration did not change significantly, even after 24 h. At approximately the same rate that the EPR signal changed, the CO/acetyl-CoA exchange activity decayed (0.11 min Ϫ1 ) and the ␤ subunit underwent degradation (0.08 min Ϫ1 ) (Fig. 6B).
Western hybridization analysis was performed using an antibody specific to the ␤ subunit. The decrease in band intensity at the position of the ␤ subunit matches the SDS-PAGE results described above. This band disappeared at the same rate as the NiFeC EPR signal intensity and the CO/acetyl-CoA exchange activity (data not shown). Western analysis allowed us to follow degradation of the ␤ subunit. These results are fully consistent with those reported earlier (1). One of the peptides produced overlaps with the ␦ subunit (58 kDa), which explains why the intensity at the position of the ␦ subunit increases with time of chymotrypsin treatment (see Fig. 5). A smaller fragment (26 kDa) also was observed. The first 10 amino acids of the 26-kDa cleavage product were sequenced and perfectly matched the N-terminal sequence of the ␤ subunit. These results strongly support the concept that the acetyl-CoA cleavage/synthesis site is associated with the ␤ subunit.
Disruption of the NiFeC EPR Signal by Bromelain-We examined the effect of bromelain, another protease, on the integrity of Cluster A. The CODH/ACS enzyme complex was digested with bromelain in the presence of CO, and aliquots of the reaction were freeze-quenched in liquid nitrogen at different times. Unfortunately, we were unable to use bromelain inhibitor to quench the reaction because it alone degraded the ␤ subunit (data not shown). For each time point, the NiFeC EPR signal was measured; SDS-PAGE was performed, and the EPR and SDS-PAGE band patterns were compared (Fig. 7). Whereas the chymotrypsin treatment converted the EPR signal from the NiFeC to the pseudo form, bromelain treatment led to the disappearance of the EPR signal. The degradation of the ␤ subunit and the loss of NiFeC signal intensity followed the same time course, indicating that the EPR signal arose from the ␤ subunit. Since it is known that the NiFeC signal derives from a CO adduct of Cluster A (34 -36), these results bolster the hypothesis that the acetyl-CoA cleavage/synthesis site is associated with the ␤ subunit. In C. thermoaceticum, Clusters A and C and their related acetyl-CoA synthase and CO oxidation activities are also located on separate subunits (19,20,37).
Requirement of CODH Component for Acetyl-CoA Cleavage/ Synthesis-As described above, limited proteolysis of the CODH/ACS complex leads to alteration or loss of the EPR signal derived from Cluster A and to loss of acetyl-CoA synthesis activity. To determine if the activity loss results from damage to the ␤ subunit or to separation of the components, the enzyme complex was treated with DTAB in the presence of 0.2 M KCl. At higher salt concentrations, the components of the complex do not precipitate. DTAB treatment has no effect on the CO oxidation activity, demonstrating that Cluster C is unaffected by the DTAB treatment. On the other hand, DTAB treatment results in loss of the CO/acetyl-CoA exchange activity and conversion of the NiFeC EPR signal to the pseudo form. Surprisingly, more than 40% of the CO/acetyl-CoA exchange activity recovers, and the standard NiFeC signal returns after removing the detergent from the solution by extensive ultrafiltration (Fig. 8). Thus, the DTAB treatment is reversible. These results suggest that an association between the ␤ subunit and another component of the complex is required for CO/acetyl-CoA exchange and, therefore, for acetyl-CoA cleavage or synthesis.

DISCUSSION
It has been clear since 1984, when the first methanogenic CODH was isolated from M. barkeri (38), that CO oxidation activity resides in a complex of the ␣ and ⑀ subunits. In the acetogenic system, this activity is located in the ␤ subunit (acsA) (19,39), which has high sequence homology to the methanogenic ␣ subunit (cdhA) (18) and Rhodospirillum rubrum CooS (40). The results described here agree with these conclusions. The isolated ␣⑀ dimer generated after detergent or proteolytic treatment retains high levels of CO oxidation activity.
Which component(s) of the methanogenic CODH/ACS complex is required for formation or cleavage of the C-C and C-S bonds of acetyl-CoA? In the acetogenic enzyme, these activities are located in the ␣ subunit (acsB) (19,20). Work by Grahame and DeMoll (1) suggests that the ␤ subunit of the methanogenic

FIG. 3. Correlation between the EPR signals and the SDS-PAGE band intensities after DTAB treatment.
EPR spectra A and C are from intact CODH/ACS complex (top, SDS-PAGE), and spectra B and D are from the sample that lacks 95% of the ␤ subunit and 60% of the ␥␦ dimer (bottom, SDS-PAGE). Spectra A and B show the NiFeC signal obtained after the samples were treated with CO. Spectrum B was magnified 10fold. The spin concentrations for A and B are 0.3 and 0.01 spins/mol, respectively. Spectra C and D show the Co(II) cobamide signal from the CFeSP. EPR conditions; 80 K, 40 milliwatts, and 20,000 gain. CODH/ACS complex binds CoA, cleaves (or forms) the C-S bond, and harbors the acetyl-enzyme intermediate. They showed that limited proteolysis of the CODH/ACS complex truncates the ␤ subunit leading to dissociation of the complex and loss of acetyl-CoA synthesis activity (from CO 2 , methyl-H 4 MPT, and CoA). They also showed that the isolated, truncated ␤ subunit retains the ability to catalyze an exchange reaction between CoA and acetyl-CoA (Reaction 4). Furthermore, the M. thermophila ␤ subunit is homologous to the large ACS subunit of the C. thermoaceticum enzyme, not to the small CODH subunit (18). These results strongly indicate that the ␤ subunit plays an important role in acetyl-CoA synthesis. How-ever, they are in apparent contradiction with two observations. A low level of the NiFeC EPR signal (0.1 spin per mol of homodimer) was observed when the apparently homogeneous ␣⑀ component from M. thermophila was incubated with CO (13). Furthermore, the two-subunit (␣⑀) Methanothrix soehngenii CODH exhibits a low level of CO/acetyl-CoA exchange activity (35 nmol min Ϫ1 mg Ϫ1 ). 3 These results suggest that Clus- The spin concentrations of the EPR spectra and relative SDS band intensities of the ␤ subunit to the ␣ subunit (␤/␣) were plotted against the time after addition of bromelain to CODH/ACS complex. Inset, EPR spectra after limited proteolysis. The final CODH/ACS complex concentration was 25 mg/ml. Spectra were collected at the following time points after mixing with bromelain: 0.6, 1.2, 2.0, 5.0, 10, 20, 30, and 60 min. The spectra at 0.6, 10, and 60 min are shown. EPR conditions were the same as shown in Fig. 3. ter A, hence acetyl-CoA synthesis, is associated with the ␣ subunit. How can one explain these two discordant findings? One suggestion is that both ␣ and ␤ subunits contain an ACSactive site (41). One goal of the studies described in this paper was to resolve this conundrum.
We decided to study the simplest acetyl-CoA synthesis reaction that would presumably only involve the ACS-active site. It is difficult to unambiguously define the component(s) required for cleavage or synthesis of the C-C and C-S bonds of acetyl-CoA by studying the total synthesis of acetyl-CoA from CO 2 , methyl-H 4 MPT, and CoA. This reaction involves CODH (the ␣⑀ component) to reduce CO 2 to CO, the corrinoid iron-sulfur protein (the ␥␦ component) for two transmethylation reactions, and the ACS component(s) to assemble CO, the methyl group, and CoA to form acetyl-CoA. The exchange reaction between CO 2 and acetyl-CoA also requires CODH as well as the ACS component(s). By studying reactions that, in theory, would only require the component of ACS responsible for C-C and C-S bond cleavage, we reasoned that we might be able to define the ACS component(s) and identify the ambiguous role of the ␤ subunit. We focused on two reactions. The first one is formation of the paramagnetic NiFeC species, a metal-carbonyl species that is a catalytically competent intermediate in acetyl-CoA synthesis (see Ref. 42 and references therein). The second is the CO/acetyl-CoA exchange reaction (Reaction 3, above). This is a very convenient reaction for defining ACS activity since it involves only the disassembly of acetyl-CoA, the isotope exchange between CO and the NiFeC intermediate, and reassembly of acetyl-CoA. This was the key analytical tool used to discover that the two-subunit acetogenic CODH is the acetyl-CoA synthase of the Wood-Ljungdahl pathway (24).
Limited proteolysis of the five-subunit complex truncates the ␤ subunit (Fig. 5), disrupts associations among the subunits of the complex (1), and causes loss (with bromelain, Fig. 7) or alteration (with chymotrypsin, Fig. 6) of the NiFeC EPR signal. The NiFeC EPR signal intensity strictly correlates with the amount of intact ␤ subunit. These results strongly indicate that the ␤ subunit catalyzes one of the key steps in acetyl-CoA synthesis, formation of the Cluster A-CO adduct that is the precursor of the carbonyl group of acetyl-CoA.
In the bromelain experiments, the NiFeC EPR signal disappears, whereas in the chymotrypsin experiments, the NiFeC signal is converted into the pseudo NiFeC signal. The pseudo NiFeC signal also is formed when N-bromosuccinimide-inactivated CODH/ACS from C. thermoaceticum is treated with CO (33) or when the C. thermoaceticum ␣ subunit, isolated after SDS treatment, is reacted with CO (34). The pseudo NiFeC species exhibits 61 Ni, 57 Fe, and 13 CO hyperfine interactions (4) and Mössbauer and UV-visible spectroscopic parameters that are very similar to those of the standard NiFeC species (27,43). Thus, the truncated ␤ subunit contains an altered form of Cluster A that is still able to bind CO. Apparently, the pseudo NiFeC EPR signal reflects the CO adduct of a slightly altered and inactive NiFeS cluster, and the standard NiFeC signal characterizes the active cluster. It is interesting that pseudo NiFeC species is formed by such diverse treatments, including modification of proximal tryptophan residues, SDS, or DTAB treatment, or proteolytic truncation of the ␤ subunit.
The results described above provide significant evidence that the ␤ subunit is an essential component of the CO/acetyl-CoA exchange reaction. Why doesn't the truncated ␤ subunit alone catalyze the CO/acetyl-CoA exchange reaction since it can still catalyze the CoA/acetyl-CoA exchange activity (1), contains Cluster A, and can bind CO? Our results indicate that interactions between the subunits harboring the ACS and CODH components markedly affect the structure of the ACS-active site and are important for cleavage/synthesis of the C-C bond of acetyl-CoA. Since the N terminus of the ␤ subunit remains intact, these intersubunit interactions must involve amino acids near the C terminus. Macromolecular interactions during acetyl-CoA synthesis were further probed by treatment of the CODH/ACS complex with the detergent DTAB, which separates the complex into three components. Dissociation of the complex by DTAB treatment causes a reversible loss of CO/ acetyl-CoA exchange activity and alteration of the NiFeC EPR signal. However, DTAB removal causes recovery of the stand- ard NiFeC EPR signal and of the CO/acetyl-CoA exchange activity. This result indicates that DTAB inhibits by separating the components of the CODH/ACS complex, possibly by disrupting essential interactions between the CODH and ACS subunits.
Why would macromolecular interactions be important for formation of the NiFeC signal or for catalysis of the CO/acetyl-CoA exchange? It is obvious why separation of the CODH and ACS subunits would inhibit acetyl-CoA synthesis from methyl-H 4 MPT, CO 2 , and CoA. This reaction involves the CFeSP and the CO 2 reduction (CODH) components. Furthermore, both the CODH and ACS subunits would also be required for the CO 2 / acetyl-CoA exchange reaction. The requirement for the ␣⑀ component in forming the NiFeC EPR signal also can be rationalized. Cluster A has a low midpoint potential of approximately Ϫ530 mV (44), and reduction of the catalytically active form of Cluster A appears to require CO (45). Therefore, as shown in Fig. 9, generation of the active state of Cluster A could require the C red2 form of Cluster C (with a similar midpoint potential (36)). However, the CO/acetyl-CoA exchange reaction (see Reaction 3), which is not a net redox reaction, also requires a component(s) of the complex. As shown in Fig. 9, we propose that a covert intramolecular electron transfer reaction occurs between the CO oxidation catalyst (the ␣⑀ component) and Cluster A (the ACS catalyst) on the ␤ subunit at each catalytic cycle of acetyl-CoA synthesis. This proposal extends the hypothesis offered in 1985 (24), based on the finding that ferredoxin or redox mediators strongly stimulates the CO/acetyl-CoA exchange reaction, that an internal electron transfer reaction occurs during the exchange reaction.
In Fig. 9, the first step of acetyl-CoA synthesis involves CO binding to the reduced state of Cluster A. This step does not require close association between CODH and ACS since the pseudo NiFeC species forms upon incubating the DTAB-or protease-treated complex with CO. This form of Cluster A has been described as the carbonyl adduct of a Ni(I)-X-[4Fe-4S] 2ϩ complex (43). Extensive evidence supports the catalytic compe-tence of the paramagnetic NiFeC species; this intermediate forms and decays at catalytically competent rates and undergoes isotopic exchange with acetyl-CoA (see Refs. 42, 46, and 47 for detailed discussion). In addition, CO binds most tightly to low valent states of transition metals, indicating that CO is bound to the nickel site, as has been proposed earlier (43).
As shown in Fig. 9, the next step is attack by Ni(I) on the methyl group of methylcob(III)amide (bound to the corrinoid iron-sulfur component of the complex) to form methyl-Ni(III) (Reaction 5). Recent results strongly indicate that this methyl transfer is a nucleophilic displacement, not a radical, reaction (46,48). We propose that it is the next step at which the CODH subunit is required. Methyl-Ni(III) is an extremely strong oxidant. For example, the midpoint for the methyl-Ni(III)/(II) couple of F 430 is much more positive than 0 V (49, 50) (Reaction 6). Therefore, capture of one electron from a site on CODH to generate a diamagnetic methyl-Ni(II) species would be extremely favorable. This proposal can explain why the NiFeC EPR signal disappears when the paramagnetic CO adduct of CODH/ACS is reacted with CH 3 -H 4 folate (51) (the acetogenic enzyme) or CH 3 -H 4 MPT (52) (the methanogenic complex) in the presence of the methyltransferase and CFeSP. Fig. 9 assumes that the electron originates and is returned from Cluster C of the CODH subunit; however, Cluster B or the "X" or "S" redox centers (14,53) could also fulfill this role. Another explanation has been offered for the loss of the NiFeC EPR signal upon methylation of the clostridial CODH/ACS (51). It is argued that reaction of a radical with the methyl group donor should generate another FIG. 9. Proposed mechanism for acetyl-CoA synthesis or cleavage. Cluster C is shown as the electron donor that catalyzes the reductive activation of Cluster A and reduction of the methyl-Ni(III) intermediate during catalysis. CH 3 -Co(III) represents the methylated corrinoid iron sulfur protein.
radical. Therefore, it is argued, the "true" carbonylated intermediate on ACS should be diamagnetic, e.g. a Ni(II)-CO species. However, Ni(II) is less nucleophilic than Ni(I), and removal of the methyl group from methyl-Co(III) requires a nucleophile that rivals Co(I).
The next proposed step is formation of the acetyl-enzyme intermediate (see Refs. 6,7,and 47). Since net redox chemistry is not required during the CO/acetyl-CoA exchange reaction, the electron removed from CODH to stabilize the methyl-ACS intermediate must be returned. We speculate that return of the electron to CODH is associated with CoA binding or cleavage of the acetyl-ACS intermediate. Transient kinetic experiments using single turnover conditions are in progress to test this mechanism and, specifically, to identify the one-electron donor. Given the similarity in structure and function of the methanogenic and acetogenic CODH/ACS, the mechanism described in Fig. 9 is expected to apply to both classes of organisms.
The results described here complement those of Grahame and DeMoll (1); however, they are in apparent contradiction with the occurrence of CO/acetyl-CoA exchange activity and the NiFeC signal, albeit at low levels (described above), in apparently purified samples of ␣⑀ dimeric CODH. One possibility is that both ␣ and ␤ subunits contain an ACS-active site (41). This would explain the occurrence of the NiFeC EPR signal and exchange activity in these two samples. However, it is inconsistent with the findings that the amount of intact ␤ subunit consistently correlates with the NiFeC signal intensity and the CO/acetyl-CoA exchange activity. Furthermore, the sequence homology between the ␣ subunit of the methanogenic enzyme and the ACS subunit of the acetogenic enzyme is insignificant. On the other hand, a cysteine-rich region in the C. thermoaceticum ACS subunit has high sequence homology with the 71-kDa subunits of M. thermophila and other methanogens (Fig. 10). We propose that this sequence includes the Cluster A ligands. A Psi-Blast analysis of this sequence against the ␣ subunit of M. thermophila CODH/ACS complex or against the R. rubrum CooS yields no significant homology. Therefore, one possible explanation for the discrepancy noted above is that the M. thermophila and M. soehngenii samples contained small amounts of intact five-subunit complex. The NiFeC EPR signal intensity for the "dimeric enzyme" from M. thermophila was 6-fold lower than that observed for the five-subunit complex (13). CONCLUSIONS Our results indicate that the ␤ subunit (cdhC) of the methanogenic CODH/ACS complex contains the NiFeS cluster known as Cluster A, which is responsible for cleavage of the C-C and C-S bonds of acetyl-CoA. Evidence is described that indicates a requirement for intermolecular interactions between the CODH and the ACS subunits. These interactions are considered to include, but are not limited to, electron transfer reactions associated with methylation or demethylation of Cluster A during acetyl-CoA synthesis. Perhaps this cryptic electron transfer reaction could help explain why CODH subunits are often found in isolation but active ACS subunits have so far only been found tightly bound to CODH.