THE JOURNAL OP BIOI.OGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 4, Issue of February 5, pp. X09-1615.1986 Printed in II. S. A.

The Autotrophic Pathway of Acetogenic Bacteria ROLE OF CO DEHYDROGENASE DISULFIDE REDUCTASE*

(Received for publication, July 15, 1985)

Ewa Pezacka and Harland G. Wood From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

An enzyme from Clostridium thermoaceticum has been isolated which reduces disulfides of carbon mon- oxide dehydrogenase and it has been named CO dehy- drogenase disulfide reductase. The enzyme is a tetra- mer of molecular weight 225,000 made up of four apparently identical monomers. It does not contain methionine or tryptophan and contains 2 calcium and 1 zinc/monomer. NADP or ferredoxin serves as an electron carrier. This enzyme is part of the system that permits certain bacteria to grow with CO or CO2 and Hs as the source of carbon and energy. The portion of the pathway which is being investigated is the conver- sion of methyltetrahydrofolate, CO, and CoASH to ace- tyl-CoA. All the enzymes required for this synthesis have now been purified. In combination with CO de- hydrogenase, CO dehydrogenase disulfide reductase with NADP or ferredoxin catalyzes a reversible ex- change of [3H]CoASH with acetyl-CoA. The disulfide reductase apparently is involved in the portion of the pathway in which CoASH is introduced into the acetyl- CoA. In addition, the reductase activates CO dehydro- genase in the overall synthesis of acetyl-CoA from methyltetrahydrofolate, CO, and CoASH by reducing about one disulfide group/monomer of the a& CO de- hydrogenase. The above exchange reaction in combi- nation with the observation that [14C]acetate is formed from CO and the “CH3-[Co]corrinoid enzyme in the absence of CoASH have permitted ordering of the se- quence of reactions by which CO dehydrogenase plays a central role in the autotrophic synthesis of acetyl- CoA.

A pathway of autotrophic growth has recently been eluci- dated which differs from all others previously described. It involves the reduction of one COZ to a methyl group which is then condensed with CoASH and a carbonyl group formed from a second CO,; the resulting acetyl-CoA serves as the source of carbon for the anabolic processes. This pathway has been elucidated through investigations of the bacterium Clos- tridium thermoaceticum, a thermophilic anaerobe that fer- ments glucose with the formation of 3 mol of acetate/m01 of glucose. Barker and Kamen (1) showed that l*C from l*CO, is fixed in both positions of acetate, and Wood (2) showed that acetate was formed from 13C02 with an atomic mass two greater than normal acetate. Thus it became clear in this

*This research was supported by Grant GM 24913 from the National Institutes of Health. 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.

We wish to dedicate this paper to Professor Louis F. Leloir on his 80th birthday, a creative scientist and a true friend.

fermentation that COZ serves as the electron acceptor and is reduced to the third mole of acetate. The mechanism of this synthesis has been a subject of investigation since that time but progress has been relatively slow until quite recently (for background, see Refs. 3,4, 5).

The pathway involves the reduction of CO, via formate and THF’ to CHsTHF and the conversion of CH,THF plus CoASH and a C1 species to acetyl-CoA. The enzymes catalyz- ing the synthesis of CH,THF from CO, have been isolated and characterized by Ljungdahl and collaborators (3, 6). En- zyme preparations catalyzing the conversion of pyruvate, CoASH, and CH,THF to acetyl-CoA were isolated by Drake et al. (7). Then it was found that CO could replace the requirement for pyruvate as the carboxyl donor and that acetyl-CoA was synthesized by the following overall reaction.

CH,THF + CO + CoASH + CH&OS-CoA + THF (1)

It thus was evident that this was truly an autotrophic type synthesis.

The central enzyme of this synthesis is CO dehydrogenase which catalyzes the conversion of CO to CO,.

CO + H20 + CO, + 2H+ + 26 (2)

It was first described by Diekert and Thauer (8) and has been purified by Ragsdale et al. (9, 10) and shown to be an a& enzyme of molecular weight 440,000 containing 6 ions of nickel, 3 of zinc, 32 of iron, and 42 of acid-labile sulfide. This enzyme was considered to catalyze formation of the C1 inter- mediate which gives rise to a carbonyl group of acetyl-CoA from CO, COZ, or carboxyl group of pyruvate (11). Recently (12) homogeneous CO dehydrogenase was observed to catalyze the exchange of 12C0 with [l-‘4C]acetyl-CoA without a de- crease in the amount of acetyl-CoA. This exchange had pre- viously been observed by Hu et al. (13) with a mixture of enzymes. The exchange is illustrated in Reaction 3 where X, Y, and 2 are acceptor sites of the CO dehydrogenase.

X-CH, X-CH3 CH314COSCoA + XYZ + Y - “CO 2% Y

Z - SCoA Z - SCoA (3)

+ 14C02 + wo

Clearly, as illustrated, the bond between the methyl and carbonyl and between the carbonyl and SCoA must be broken, and then the 14C0 arising from the [l-‘4C]acetyl-CoA must mix with the “CO of the gas phase so that resynthesis gives acetyl-CoA with decreased radioactivity. In addition, the CO dehydrogenase must have binding sites X, Y, and Z since no other acceptor was added in the experiments. Based on these findings, a new scheme for the biosynthesis of acetyl-CoA has

’ The abbreviations used are: THF, tetrahydrofolate; CH,THF, methyltetrahydrofolate.

1609

1610 CO Dehydrogenase Disulfide Reductase and Acetogenesis

TABLE I Purification of CO dehydrogenase disulfide reductase from C. thermoaceticum

See “Experimental Procedures” for details. In the bottom line, the standard deviations for 10 preparations are given in parentheses.

Protein Activity Specific activity Recovery

w units“ unitslmg 76 1. Cell extract 3 100 262 0.084 100 2. DEAE-Sephacel 240 135 0.50 52

4. Phenyl Sepharose 30 85 2.82 32 5. Bio-Gel HTP hydroxylapatite 12.3 47.5 3.89 18 6. Bio-Gel A-0.5mm gel filtration 2.1 20.7 9.94 8

3. (NH&SOl, 38-6076 180 131 0.725 50

(1.8 f 0.5) (19.7 f 2.5) (11.9 f 1.3) (7 f 1.5) The units of disulfide reductase activity are expressed as micromoles of ”SH produced from cystine/min.

been proposed (12) which assigns CO dehydrogenase the central role in the pathway. This role had previously been assigned to the corrinoid enzyme (3,4, 13).

Ragsdale et al. (14) found that when CO dehydrogenase reacts with CO, an electron spin resonance (ESR) signal is observed that originates from a nickel-carbon species, Recent studies (15) using isotope substitution have shown that this ESR signal results from a spin-coupled metal-carbon center containing iron, nickel, and carbon derived from CO. This center probably is the y site referred to in Reaction 3. In further studies (12), an additional ESR signal was observed and when the enzyme was treated with both CO and CoASH or acetyl-coA, a marked change in the spectra was observed demonstrating that CoASH and acetyl-coA bind to CO de- hydrogenase at or near the metal-carbon center.

An enzyme was isolated with an unknown function in the synthesis of acetyl-coA and we called it protein X (11). It has now been shown that this enzyme is a disulfide reductase with high activity on S-S bonds of CO dehydrogenase and is, therefore, called CO dehydrogenase disulfide reductase. All the enzymes required for the synthesis of acetyl-coA from CH3THF, CoASH, CO, or CO, and HP have now been isolated from C. thermoaceticum. They include, in addition to the disulfide reductase, methyltransferase (7), the corrinoid en- zyme (16), CO dehydrogenase (9, lo), ferredoxin (7), and hydrogenase (17, 18). We describe here the isolation and properties of CO dehydrogenase disulfide reductase and its proposed role in the synthesis of acetyl-coA.

EXPERIMENTAL PROCEDURES’

RESULTS

Purification of Disulfide Reductase-The procedure de- scribed under “Experimental Procedures” involves six steps and yields 2 mg of homogeneous enzyme from 50 g of bacteria. The yields of enzyme from one typical preparation are shown in Table I as well as the standard deviation observed with 10 preparations. Occasionally, Step 2 was exchanged with Step 3 without any effect on the purity of the enzyme. Two or more disulfide reductases which reduce low molecular weight disulfide (cystine) are present in C. thermoaceticum but only one enzyme isolated as described in Table I has the capacity to reduce disulfides of the CO dehydrogenase. This enzyme

The “Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-2315, cite the authors, and include a check or money order for $1.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

was separated from the others by step 4 of the purification. With some preparations, a specific activity was obtained greater than 9.94 units/mg. These preparations did not con- tain dithionite during the first two steps of the purification which may irreversibly inhibit enzyme activity. It is known that dithionite forms S-sulfonates with disulfides of en- zymnes, or irreversibly modifies serine or threonine and thus causes physical or chemical changes of the protein (19).

Assuming the best specific activity of 12.6 pmol min” mg” and 8% recovery, approximately 0.7% of the soluble cell protein exits as CO dehydrogenase disulfide reductase.

Stability of the Disulfide Reductase-The purified enzyme is extremely sensitive to oxygen. Additions of air to a final oxygen concentration of 10 pM resulted in immediate loss of 100% of the activity. Under anaerobic conditions, the enzyme loses 95% of its activity in 4 h when diluted with buffer I11 to a concentration of -100 pg/ml; it should be stored in a protein concentration as high as possible. The enzyme is active for 3 days when stored at room temperature in 10% or more glyc- erol. It is inactivated by storage at 4 “C. When stored with approximately equal molar amounts of CO dehydrogenase, the enzyme was stable for about 2 weeks. This stabilizing effect by CO dehydrogenase occurred even when the CO dehydrogenase itself had become inactive. The stabilization is effective even at low temperatures and in the absence of dithioerythritol and glycerol. Other proteins such as methyl- transferase and ferredoxin of C. thermoaceticum or albumin from chicken egg did not stabilize the reductase.

Physical Properties of the Pure Enzyme-Isoelectric focus- ing of the purified enzyme in the presence of 8 M urea gave a single band of protein with an isoelectric point of 6.3 -1- 0.2 when visualized with the highly sensitive silver nitrate stain (20). By polyacrylamide gel electrophoresis in sodium dodecyl sulfate, one protein band was observed, which corresponded to a molecular weight of 50,500 f 2,500 both in the presence and in the absence of &mercaptoethanol, indicating the ab- sence of interchain disulfide bridges (Fig. 1).

The molecular weight of the native disulfide reductase was determined by molecular filtration on Bio-Gel A-0.5 m and by glycerol gradient centrifugation and was found to be 230,000 and 239,000, respectively. These results, in conjunc- tion with those obtained by gel electrophoresis and from the amino acid analysis (below), indicate that the enzyme is an

homotetramer. Chemical Composition of the Pure Enzyme-The amino acid

composition of the purified enzyme is given in Table 11. The results obtained by two methods of amino acid analysis (see “Experimental Procedures”) are similar and reveal a high proportion of Glx and Asx. The disulfide reductase contains a number of cysteines but no methionine. Tryptophan anal- ysis was performed but it was found in an amount lower than

CO Dehydrogenase Disulfide Reductase and Acetogenesis 1611

4-

30K* e

1 4 K c ~

- 1. 2.

FIG. 1. Sodium dodecyl sulfate-gel electrophoresis of CO dehydrogenase disulfide reductase. Lane I contained the molec- ular weight standards and lane 2 0.015 mg of the disulfide reductase after gel filtration (Step 6). The proteins were prepared and run in the sodium dodecyl sulfate-Tris-HC1 system of Weber and Osborn (43) and stained with Coomasssie Brilliant Blue R-250.

TABLE I1 Amino acid analysis of CO dehydrogenase disulfide reductase by ion exchange chromatography and by reverse-phase high pressure liquid chromatography (HPLC) of phenylthiocarbamyl (PTC) derivatives

Amino acids HPLC of PTC Ion exchange derivatives chromatomaphv nmol amino acidlnmol of monomer”

Glx 32.5 36 Pro 17.1 17.3

Ala 20.8 20.8 Cm-Cys’ 11.0 8.0 Val 28.0 30.8 Met‘ Ile 17.8 18.3 Leu 16.6 17.0 TY r 7.0 7.8 Phe 8.3 7.0 His 6.7 7.5 LYS 15.3 15.3 Arg 16.7 16.8 Thr 21.6 20.9 Asx 25.3 28.3 Ser 5.1 5.0 Trp

G ~ Y 38.2 40.3

Based on a molecular weight of 53,500. ‘Cysteine was determined as carboxymethyl derivatives or as

cysteic acid and methionine as methioninesulfone. See “Experimental Procedures” for more details.

0.1 nmol/nmol of the reductase. The calculated molecular weight for the monomer was 56,200. Plasma emission spec- troscopy analysis of the homogeneous enzyme gave values of 1.8 k 0.1 calcium and 0.8 f 0.3 zinc/mol of monomer. The enzyme does not contain iron or acid-labile sulfur or colored coenzymes such as flavin.

Catalytic Properties of the Disulfide Reductase-The CO dehydrogenase disulfide reductase reduces the oxidized forms

cystine but not oxidized glutathione (GS-SG), oxidized lipoic acid, lipoamide, or cystamine. With proteins, activity was found only with CO dehydrogenase, not with the corrinoid enzyme, methyltransferase or pyruvate ferredoxin oxidore- dutase from C. thermoaceticum (7), or with oxidized ribonu- clease A. The enzyme was active with either NADPH or reduced ferredoxin as electron donor but not with NADH or reduced FMN. A comparison of the rates of generation of SH groups from disulfides of CO dehydrogenase with those from CoAS-SCoA and cystine with NADPH as the reductant is shown in Fig. 2. The initial rate was about 5 times faster with CO dehydrogenase than with cystine or CoAS-SCoA, giving a specific activity with CO dehydrogenase of about 50 pmol of sulfhydryl produced min” mg”. The optimum pH is 5.5- 6.2, which is near the optimum pH for the overall synthesis of acetyl-coA from CH3THF, CoASH, and CO.

Requirement of CO Dehydrogenase Disulfide Reductase for Synthesis of Acetyl-coA from CH3THF, CO, and CoASH- The synthesis of acetyl-coA from CH3THF, CO, and CoASH is catalyzed by five enzymes: CO dehydrogenase, methyltrans- ferase, corrinoid enzyme, CO dehydrogenase disulfide reduc- tase, and ferredoxin (Table 111). Both the reductase and ferredoxin are required. NADPH does not replace the require- ment for ferredoxin in the overall synthesis even though it is effective for the reduction of disulfides. Dithioerythritol partly replaced the requirement for the disulfide reductase.

Function of CO Dehydrogenase Disulfide Reductase in the Synthesis of Acetyl-CoA-The effect of the reduction of disul- fides of CO dehydrogenase and the effectiveness of the result- ing CO dehydrogenase in the synthesis of acetyl-coA is illus- trated in Fig. 3. In this experiment, the CO dehydrogenase was incubated with NADPH and various concentrations of the reductase for 15 min. Then an aliquot was taken for determination of the SH groups of the CO dehydrogenase and a second aliquot was tested in the complete system for the synthesis of acetyl-coA. The SH content of the CO dehydro- genase is plotted on the right ordinate. There were 22 nmol of SH group/nmol in the CO dehydrogenase prior to the

50 t 40 4

I “ 2 0 + /

ylo?

U CODH COAS-SCOb Cystine

10 2 0 30 Tline (rnin)

FIG. 2. The reduction of low molecular weight disulfides and Co dehydrogenase disulfide with disulfide reductase. The incubation mixture (0.5 ml) contained potassium phosphate buffer, 50 mM, pH 6.0; NADPH, 1 pmol; dithioerythritol-free disulfide re- ductase, 15 pg; cystine, 250 nmol; or oxidized CoASH (CoAS-SCoA), 250 nmol; or CO dehydrogenase, 1.02 nmol. The gas phase was argon and temperature 55 “C. At indicated times, 2,2’-dithiobis-(5-nitro- pyridine), 1 mM, and EDTA, 1 mM in total volume of 0.5 ml of potassium phosphate buffer, 50 mM, pH 6.3, was added. Because of the presence of air in the added solution, the enzymatic reaction stops at this point. After 15 min at room temperature, 1 ml of 0.4% sodium dodecvl sulfate in Tris-HC1. 0.1 M. DH 9.0. was added and the

of COASH (COAS-SCOA), CoA-glutathione (CoAS-SG) and absorbance was measured at 386 nm ( e = 10,600) (37).

1612 CO Dehydrogenase Disulfide Reductase and Acetogenesis

TABLE I11 Synthesis of acetyl-coA from CO, CoASH, and CH3THF with

purified enzymes: Requirement for CO dehydrogenase disulfide reductase and ferredoxin

The synthesis was measured by determination of the conversion of “CHaTHF, CoASH, and CO to acetyl-coA as described elsewhere (7, 18). The complete reaction mixture in 0.5 ml contained: CO dehydrogenase, 20 units; methyltransferase, 3.8 units; corrinoid en- zyme, 15 pg; disulfide reductase, 7.5 units; potassium phosphate buffer, 50 pmol, pH 6.0; 14CH3THF, 1 pmol (115,000 cpm); CoASH, 1 pmol; and ferredoxin, 60 pg or NADPH, 1 pmol. The gas phase was 100% CO, temperature 55 ‘C, and time of reaction 15 min. All enzymes were dithioerythritol-free.

Reaction mixture Acetyl-coA nmol

1. Complete 118 2. Minus disulfide reductase 3.4 3. Minus ferredoxin 8.3 4. Minus ferredoxin plus NADPH 9.0 5. Minus disulfide reductase and 39.0

ferredoxin plus dithioerythritol (10 mM)

16 2 0 Units of disulfide reductase

FIG. 3. The reduction by disulfide reductase of disulfides of CO dehydrogenase and the effect on the synthesis of acetyl- CoA. The CO dehydrogenase (1.5 nmol) was reduced with different concentrations of disulfide reductase and NADPH, 1 pmol in 500 pl of potassium phosphate buffer, 0.1 M, pH 5.9. The temperature was 55 “C and gas phase, argon. SH groups of the CO dehydrogenase with 2.2’-dithiobis-(5-nitropyridine) as described in the legend to Fig. 2. A separate aliquot of the reduced CO dehydrogenase was added to the system for synthesis of acetyl-coA. The synthesis was measured by determination of the conversion of l4CH3[Co]E, CoASH, and CO to acetyl-coA. The complete incubation mixture contained: reduced CO dehydrogenase, 0.1 nmol; ferredoxin, 25 p; “CH3[Co]E (110,000 cpm), 0.5 pmol; potassium phosphate buffer, 50 mM, pH 6.0. The volume was 300 pl, gas phase 100% CO, and temperatures 55 ‘C. The reaction was stopped after 15 min by addition of 50 pl of 1 M HClO4.

treatment with the reductase. With increasing amounts of reductase (shown on the abscissa), there was an increase in SH groups to 50 nmol/nmol of CO dehydrogenase. With the untreated CO dehydrogenase, 75 nmol of acetyl-coA were synthesized (shown on the left ordinute). The acetyl-coA yield increased to 350 nmol when the SH groups had increased to 33 in the CO dehydrogenase. Thus, about 10 additional SH groups were required for maximum synthesis of acetyl-coA. Since this is an a3P3 enzyme, perhaps the reduction of one disulfide/monomer (or two/monomer if the disulfides are exclusively in either the a or P monomer) is sufficient to give maximum activity. There was little change when the SH

groups increased to 37 but when the number increased to 50, there was a marked decrease in the yield of acetyl-coA.

The reductase in combination with CO dehydrogenase cat- alyzes the exchange of [3H]CoASH with acetyl-coA (Table IV). The experiment consisted of incubating acetyl-coA with [3H]CoASH in a mixture containing CO dehydrogenase, di- sulfide reductase, NADPH, and the corrinoid enzyme and then separating the [3H]CoASH from the acetyl-coA and determining the radioactivity of the acetyl-coA. The nano- moles of CoASH incorporated into the acetyl-coA were cal- culated from its radioactivity and the specific radioactivity of the [3H]CoASH. As a control, the overall synthesis was de- termined using [3H]CoASH, CH3THF and CO, and the re- quired components as in Table 111 except the [3H]CoASH was 40 nmol instead of the 1000 nmol of CoASH. With this low concentration of [3H]CoASH, the yield of acetyl-coA was much lower (11.2 nrnol) than in the experiment of Table 111. Thus the 3.1 nmol observed by exchange of Table IV was 28% of the overall reaction. Both CO dehydrogenase and the disulfide reductase are required for the exchange, omission of the corrinoid enzyme had little effect, and ferredoxin could substitute for NADP as the electron carrier. Dithioerythritol was very effective as a replacement for the reductase and NADP, which is in contrast to its low activity in the overall synthesis of acetyl-coA (Table 111).

Effect of Modifying Agents on the Reductase Activity and the CoASH Exchange Reaction as Catalyzed by the Disulfide Reductase-In the experiments of Table V , the disulfide re- ductase activity was measured using cystine and NADPH and the exchange reaction using [3H]CoASH and acetyl-coA. The results show that both methylglyoxal and CO inhibited the exchange reaction but had no effect on the reductase activity. Perhaps the CO is competing with the acetyl group for the Y site. Dithiobis-(2-nitrobenzoate) inhibited both reactions. EDTA inhibited both reactions 50% at 1 mM concentration but had no effect when the concentration was 0.1 mM. 0- Phenanthroline at 1 mM inhibited the reductase activity 25%. Sodium dithionite (5.0 mM) inhibited the exchange reaction 70%.

DISCUSSION

The first portion of the pathway of autotrophic growth by acetogenic bactria involves the synthesis of CH3THF from COz which can be accomplished by numerous organisms. It is

TABLE IV Requirement of disulfide reductase for the exchange between PHI

CoASH and acetyl-CoA as catalyzed by CO dehydrogenase The exchange reaction was measured by determining the radioac-

tivity of acetyl-coA. The complete reaction mixture contained CO dehydrogenase, 20 units; corrinoid enzyme, 9.5 nmol; disulfide reduc- tase, 10 units; [3H]CoASH, 40 nmol (440,000 cpm); acetyl-coA, 80 nmol; NADPH, 1 pmol. The concentration of dithioerythritol (DTE) was 5 mM and reduced ferredoxin (181, 50 pg. The volume was 300 p1, time of the reaction 30 min, temperature 55 “C, and gas phase argon. The acetyl-coA was separated from unreacted [3H]CoASH on a DEAE-cellulose column (0.5 X 7.5 cm) as described by Gregolin et al. (35).

[‘HICoASH incorporated in acetyl-coA

nmol

Reaction mixture

1. Complete 3.1 2. Minus CO dehydrogenase 0.2 3. Minus disulfide reductase 0.5 4. Minus corrinoid enzyme 2.6 5. Minus NADPH plus reduced ferredoxin 3.1 6. Minus disulfide reductase and NADPH 2.6

plus DTE

CO Dehydrogenase Disulfide Reductase and Acetogemsis 1613

TABLE V Inhibition by different reagents of the disulfide reductase OS measured

with cystine and NADPH and the exchange reaction as measured with PHJCoASH and acetyl-coA

The activity of disulfide reductase was measured by determination of the cysteine formed from cystine as in Fig. 2. The exchange reaction was assayed by incorporation of [3H]CoASH into acetyl-coA as in Table IV.

Reaction mixture

Inhibition Inhibition of

of disulfide reductase PHlCoASH

% . .

%

1. No addition 0 2. Plus 1 mM methylglyoxal 0 3. Plus CO gas phase 0 4. Plus 40 nmol DTNB” 100 5. Plus 1.0 mM EDTA 50 6. Plus 0.1 mM EDTA 0 7. Plus 1.0 mM 0-phenananthroline 25 8. Plus 5.0 mM sodium dithionite ND (I DTNB, 5,5’-dithiobis-(2-nitrobenzoic acid). * ND, not determined.

0 85 80

100 50

0 NDb

70

the second portion of the pathway, the conversion of CH3THF, CoASH, and CO or CO, and HZ to acetyl-coA which is unique. It is this portion which enables c. thermo- aceticum (21) and almost certainly other acetogenic bacteria to grow with CO or COz and Hz as the source of carbon and energy. With the isolation of CO dehydrogenase disulfide reductase, all the enzymes required for this synthesis have been purified.

CO Dehydrogenase Disulfide Reductase-Of the enzymes involved in the synthesis of acetyl-coA from CH3THF, CO, and CoASH, only CO dehydrogenase served as a substrate for the reductase and the reaction was much faster with CO dehydrogenase than with the low molecular weight com- pounds (Fig. 2 ) . We, therefore, have named the enzyme CO dehydrogenase disulfide reductase. It has been purified about 120-fold and is a homotetramer of molecular weight 225,000 which contains 8 mol of calcium and 4 mol of zinc/mol. The only optical absorption peak is at 280 nm. The amino acid composition is somewhat unique; it contains no methionine or tryptophan.

CO dehydrogenase disulfide reductase differs from the mammalian protein disulfide isomerase which catalyzes rear- rangement of non-native disulfide bonds to the native form (22 , 23). In this process, no free additional SH groups can be detected, which is in contrast to our findings (Fig. 3). Ferre- doxin-thioredoxin reductase purified from Clostridiumpasteu- rianum, in contrast to CO dehydrogenase disulfide reductase, contains a flavin tightly bound to the protein (24). This enzyme serves in the regulation of enzyme activities by “thiol- redox” control (25, 26).

CO dehydrogenase disulfide reductase is extremely labile to oxygen; exposure to very low concentrations of air causes almost instant inactivation. Thus far, the only method which we have found for its stabilization over a period of a week has been to mix the reductase in stoichiometric amounts with CO dehydrogenase. This stability may be related to the fact that the reductase forms a complex with CO dehydrogenase. When a mixture of CO dehydrogenase and the reductase without dithioerythritol is passed through a Bio-Gel A-0.5m column, the enzymes do not separate. If the mixture is preincubated with 0.01 M dithioerythritol at room temperature for 0.5 h, then the enzymes separate on the column. Addition of dithio- threitol likewise is required for separation of the two enzymes during purification of the reductase. It appears that the 2

enzymes may combine by a disulfide linkage. In the cell, these two enzymes probably occur as a complex.

Reduction of CO dehydrogenase by disulfide reductase has been found greatly to stimulate its activity in the synthesis of acetyl-coA when combined with the other enzymes of the overall system (Fig. 3). This stimulation occurred when about 1 mol of disulfide/mol of monomer of the a3P3 enzyme was reduced. On further incubation, 50 sulfhydryls/mol of enzyme and a decrease in activity was observed. Possibly, some sulfhy- dryl groups are exposed by unfolding of the enzyme when disulfides are reduced and not all the titrated sulfhydryl groups are formed by reduction. It also has been observed that disulfide reductase reactivated CO dehydrogenase, which had lost activity for the conversion of CO to CO,. For example, the activity of CO dehydrogenase increased from a specific activity of 11.5 units/mg to 45.0 units/mg when incubated with the reductase and NADPH. It is possible that alteration of the activity of CO dehydrogenase by disulfide reductase may provide for metabolic control.

The reductase in combination with CO dehydrogenase cat- alyzes an exchange of CoASH with acetyl-coA (Table IV). This observation together with the observation that acetate is synthesized from CO and the methyl of methylated corri- noid enzyme when CoASH is omitted has aided greatly in determining the sequence of reactions of CO dehydrogenase in the synthesis of acetyl-coA. These observations will be considered and related to the overall scheme of synthesis of acetyl-coA as proposed in Fig. 4.

Pathway of Acetyl-coA Synthesis-A source of low potential electrons is required in any autotrophic pathway for the reduction processes. With CO, and Hz, these electrons are derived by the hydrogenase reaction (Hz + 2H’ + 28) and with CO, they are produced by the conversion of CO to COz by CO dehydrogenase (CO + H20 4 COZ + 2Hf + 2e). The required ATP is probably generated by electron or proton transfer phosphorylation.

Reduction of CO, to CH3THF-The pathway (lower left of Fig. 4) involves reduction of COz to formate by formate dehydrogenase (FDH) conversion to formyltetrahydrofolate by formyltetrahydrofolate synthetase (F-THFS) and the re- duction of the formyl group to the methyl of CHsTHF by a series of reactions. The enzymes of this portion of the pathway have all been purified (3, 6). Hz is shown as the source of electrons for these reductions, but CO is the source when it is the substrate. Hz, likewise, is in the source of electrons for the reduction of CO, to CO by the CO dehydrogenase (see Reaction 1 of Fig. 4).

The methyl of CH3THF is transferred to the corrinoid enzyme ([Co] E ) yielding the methylated corrinoid enzyme. Thereafter, CO dehydrogenase and CO dehydrogenase disul- fide reductase play a central role in the synthesis of acetyl- CoA. The CO dehydrogenase is represented in Fig. 4 by the oval shaped form in which X, Y and Z represent acceptor sites for the CHB, CO, and CoASH groups, respectively. That there are three sites is evident from the C0:acetyl-CoA exchange which was observed by Ragsdale and Wood (12) (see Reaction 3).

Formation of Acetate by CO Dehydrogenase from CO and the Methyl Corrinoid Enzyme-Hu et al. (16) using a fraction containing a mixture of enzymes, showed that [14C]acetate was formed from CO and 14CH3 corrinoid enzyme in the absence of CoASH. S. W. Ragsdale3 has confirmed this syn- thesis using homogeneous CO dehydrogenase, methylated cor- rinoid enzyme, and CO. The acetate was identified by Dowex-

S. W. Ragsdale, unpublished results.

1614 CO Dehydrogenase Disulfide Reductuse and ace to genes^

FIG. 4. Proposed pathway of ace- tyl-coA synthesis and au~otrophic growth with CO or COa and Ha as the source of carbon and energy. The CO dehydrogenase is represented by the oval shaped form in which X, Y, and 2 represent acceptor sites for the CHB, CO, and CoASH groups, respectively, F- THFS, formyl-THF synthetase; FDH, formate dehydrogenase; Hzase, hydro- genase; Fd, ferredoxin; CHSTr, methyl- transferase; [ Co]E, corrinoid enzyme; S- SRd, CO dehydrogenase disulfide reduc- tase.

I chromatography (27). The importance of this observation is that it demonstrates that the acetyl group can be formed by the CO dehydrogenase from CO and the methyl of the corri- noid enzyme prior to the involvement of CoASH. The pro- posed sequence of the reaction for acetate synthesis is illus- trated in Reac t io~ 1 3 a of Fig. 4. In the absence of CoASH and the disulfide reductase, the acetyl group is hydrolyzed to acetate. The dashed arrow to acetate is to indicate that this is a “dead end” conversion. The Exchange of CoASH with Acetyl-CoA-This exchange

helped in determining the role of the CO dehydrogenase disulfide reductase in the overall synthesis. CO dehydrogenase alone catalyzed the exchange of CO with [l-14C]acetyl-l-CoA (12) but for CoASH exchange with acetyl-coA, the CO de- hydrogenase reductase, NADP, or ferredoxin (or dithio- threitol) are also required (Table IV). Since the CoASH remains bound to the CO dehydrogenase during the CO ex- change, clearly the reductase is required for its removal (and addition) from acetyl-coA. We propose that the CoASH is linked to the CO dehydrogenase via an S-SCoA linkage and is released by the reduction of the disulfide bond by CO dehydrogenase disulfide reductase as shown in Reaction 4 of Fig. 4. The CO exchange and CoA exchange both involve Reactions 6 and 7. The dashed lines 6a and 6b represent the additional sequence involved in the CO exchange and are dead end reactions. In writing the dead end reactions, we assume CO associates and dissociates from CO dehydrogenase inde- pendent of the presence of bound CoA and methyl groups. Since arginine modifiers inhibit both the exchange of CO (12) and of CoASH with acetyl-CoA (Table V), we speculate that arginine may be involved in binding acetyl-coA and CoA. Lange et al. (28) have proposed that the guanidino group of arginine binds to the pyrophosphate bridge of NADH. In the present case, the binding would be to the pyrophosphate bridge of the acetyl-coA. To complete the cycle, the reduced ferredoxin produced by the disulfide reductase when CoASH is linked to the Co dehydrogenase is used to regenerate the disulfide form of Co dehydrogenase as shown in Reaction 8 of Fig. 4.

Although the scheme of Fig, 4 is based on substantial

experimental evidence, many gaps remain. We have not es- tablished that the CoASH is bound to the CO dehydrogenase by a disulfide linkage, although this seems likely in view of the results with disulfide reductase. Nor have we established that CoASH and acetyl-coA bind to arginine. Nothing is known about the binding of the methyl group to CO dehydro- genase. The actual role of the Ni-Fe center which binds the CU remains to be estabiished. Nevertheless, with purified enzymes and the schem as a model from which to work, we expect to conduct experiments which will provide further details concerning this intriguing series of chemical reactions.

The Rok of the Autotrophic Pathway in Other Organisms- There are a number of bacteria that grow with CO or COz and H2 as the source of energy including some of the meth- anogens and sulfate reducers and there is considerable evi- dence that a pathway (similar to that of C. thermoaceticum) is used by these microorganisms. These investigations have recently been reviewed by Zeikus et al. (29) (see also Refs. 30- 34). However, these pathways have not been reconstituted using purified enzymes from the bacteria; most of the evidence is based on tracer studies. A critical question is whether or not CO dehydrogenase from these organisms has a role in their ~ a b o ~ s m similar to that of CO dehydrogenase from C. thermoaceticum.

Acknowledgments-We thank Dr. S. W. Ragsdale for providing some of the purified CO dehydrogenase and disulfide reductase and for his helpful suggestions in preparations of this manuscript. We are most grateful to Dr. N. F. B. Phillips for his assistance during the high pressure liquid chromatography studies and to Maurine Nicora (University of Georgia, Athens, Georgia) for determining the concen- trations of metals by plasma emission spectroscopy.

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