- Email: [email protected]
[39] Formate dehydrogenase, a selenium-tungsten enzyme from Clostridium thermoaceticum
360
NONHEMEMETALLOPROTEINS
[39]
Prosthetic Group. The w-hydroxylase contains one atom of iron per polypeptide chain, but heine is not present. 7 The iron is largely removed by treatment with EDTA or other metal chelators provided that a reducing agent, such as dithionite, is also added. Hydroxylation activity is restored by incubating the apohydroxylase preparation with ferrous ions prior to the spectrophotometric assay. The to-hydroxylase contains only one molecule of cysteine per polypeptide chain, as shown by amino acid analysis and sulfhydryl titrations. No labile sulfide is present. Other Properties. The purified ~hydroxylase preparation contains phospholipid, primarily phosphatidylethanolamine, as well as a few carbohydrate residues. Removal of the lipid results in a loss of nearly all hydroxylation activity, which can be restored by the addition of various phospholipids, r The enzyme is reversibly inhibited by cyanide, but is not inhibited by carbon monoxide. Specificity. The ~hydroxylase catalzyes the hydroxylation of a variety of aliphatic hydrocarbons, including n-alkanes having 6-14 carbon atoms, as well as branched-chain structures, such as 2,5-dimethylhexane, and alicyclic compounds, such as cyclohexane and methylcyclohexane. n-Fatty acids with 6-14 carbon atoms also serve as substrates. The enzyme also catalyzes the epoxidation of alkenes, such as octadiene. TM More recent studies in our laboratory show that iron and phospholipid are required for epoxidation as well as for hydroxylation. ,a S. W. May and B. J. Abbott, J. Biol. Chem. 248, 1725 (1973).
[39] F o r m a t e Enzyme
Dehydrogenase, a Selenium-Tungsten from Clostridium thermoaceticum
By LARS G. LJUNGDAHL and JAN R. ANDREESEN Formate dehydrogenase of Clostridium thermoaceticum catalyzes the following reversible reaction a'Z: HCOO- + NADP+ ~,~--CO2 + NADPH
(1)
The only known naturally occurring electron acceptor is NADP, and the enzyme should perhaps be named formate:NADP oxidoreductase. The ' L.-F. Li, L. Ljungdahl, and H. G. Wood, J. Bacteriol. 92, 405 (1966). 2 R. K. Thauer, FEBS Lett. 27, 111 (1972).
[39]
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361
physiological role of the e n z y m e is to catalyze the reduction of COz to formate. Clostridium t h e r m o a c e t i c u m is a thermophilic anaerobic bacterium that ferments sugars to acetate as the only product.3 About one-third of the acetate is formed by reduction of CO2, which serves as electron acceptor of electrons generated during the fermentation. This is shown in the following reactions.
C~H,~O6 + H~O --~ CH:~COCOOH + CH:~COOH + 6H' + CO2
(2)
CH3COCOOH + CO2 + 6H ~ 2 CH3COOH + H20
(3)
Sum: C~H,..,O6~ 3 CH:~COOH
(4)
In reaction (2) the hexose is fermented to 2 tool of pyruvate, of which one is further metabolized to acetate and CO2. Six equivalents of electrons are generated; these are used to reduce CO2 to a methyl group, which is converted to acetate in a transcarboxylation reaction involving the carboxyl group of pyruvate [reaction (3)]. 4 The reduction of CO2 to acetate proceeds with formate, 10-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5-methyltetrahydrofolate, and a Co-methylcobamide as intermediates. ~'6 The first e n z y m e in the pathway is the N A D P - d e p e n d e n t formate dehydrogenase. This e n z y m e is present in a relatively high level in cells of C. t h e r m o a c e t i c u m grown in media containing selenite plus either tungstate or molybdate. Cells grown in media lacking these chemicals have much lower formate dehydrogenase activity. ¢ Culturing M e t h o d s for C l o s t r i d i u m t h e r m o a c e t i c u r n Clostridium t h e r m o a c e t i c u m (DSM 521) is maintained in agar-stab cultures or in liquid broth cultures. It is a strictly anaerobic bacterium, and it fails to grow in the presence of small amounts of oxygen. A g a r - S t a b Cultures. The agar medium has the following composition
3 F. E. Fontaine, W. H. Peterson, E. McCoy, M. J. Johnson, and G. J. Ritter, J. Bacteriol. 43, 701 (1942). 4 M. Schulman, R. K. Ghambeer, L. G. Ljungdahl, and H. G. Wood, J. Biol. Chem. 248, 6255 (1973). 5 L. G. Ljungdahl and It. G. Wood, Annu. Rev. Microbiol. 23, 515 (1969). 6 L. G. Ljungdahl and J. R. Andreesen, in "Symposium on Microbial Production and Utilization of Gases (Hz, CH4, CO)." (H. G. Schlegel, G. Gottschalk, and N. Pfenning, eds.), p. 163. E. Goltze Verlag, G6ttingen, German Federal Republic, 1976. 7j. R. Andreesen and L. G. Ljungdahl, J. Bacteriol. 116, 867 (1973).
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NONHEMEMETALLOPROTEINS
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in grams per liter: agar, 20; glucose, 5; yeast extract (Difco), 5; tryptone (Difco), 5; (NH4)2SO4, 0.5; sodium thioglycolate, 0.5; and MgSO4"7 H20, 0.1. Culture tubes equipped with tight-sealing plastic screw caps are three-quarters filled with the medium. After autoclaving, the tubes are transferred into a wide-mouth bottle filled with COs gas and left until the agar is solidified. After inoculation using a needle, the tubes are sealed and incubated for 2-5 days at 58 °. Bacterial growth should be clearly visible. New stab cultures are prepared at intervals of 6 months, but cultures stored for several years at 4 ° are viable. Old stab cultures assume growth faster if they are activated by heating in a boiling water bath for 10 min.
Liquid Culture. Clostridium thermoaceticum is best grown in an atmosphere of COs at 58 ° in the medium listed in Table I. The medium is prepared in three solutions: A, containing glucose dissolved in 150 ml of distilled water; B, containing yeast extract, tryptone, and metals in 550 ml of water; C, containing sodium bicarbonate and potassium phosphate in 300 ml of water. It is practical to make solution C in the flask that will TABLE I MEDIUM FOR Clostridium thermoaceticum
Solution
A B
C
Ingredient
Glucose Water Yeast extract (Difco) Tryptone (Difco) (NH4)2SO4 MgSO4'7 H20 Fe(NH4)dSO4h '6 H20 Co(NOa)z'6 H20 NazWO4"2 H20 Na~MoO4-2 H20 Na~SeO3 Sodium thioglycolate (Difco) Water NaHCOa K2HPO4 KH2PO4 Water
Amount (g)
Volume (ml)
Final Concentration a (raM)
100
18 150 5 5 1 0.25 0.039 0.030 0.0033 0.0024 0.0002 0.5
7.6 1 0.1 0.1 0.01 0.01 0.001 4.4 550
200 40 40
16.8 7 5.5
a Concentration after mixing of solutions A, B, and C.
300
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FORMATEDEHYDROGENASE
363
be used for the culture. This flask should be fitted with a bubbler consisting of a rubber stopper with two glass tubes, of which one (the inlet) reaches to the bottom of the flask. Cotton plugs are inserted into the tubes of the bubbler, and it and the three solutions are sterilized separately. To prepare the medium for inoculation, the bubbler is inserted into the flask containing solution C. The solution is gassed with CO2 until the pH is below 8.5. Solutions A and B are then combined with solution C, and the medium is placed in an incubator maintained at about 58 °. Gassing with CO2 is continued. After the solution has reached 55 °, it is inoculated with about 50 ml of a broth culture less than a week old. A slow constant stream of CO2 is maintained through the medium during the growth period. The cells are normally harvested within 48 hr by centrifugation at room temperature without exclusion of air. The cells may be stored frozen at - 8 0 ° for several months with only a small loss of formate dehydrogenase activity. The yield is between 10 and 17 g of wet cells per liter of medium. Normally C. thermoaceticum is maintained in l-liter liquid cultures with transfers twice weekly. Large amounts of cells of the bacterium have been produced by cultivation in 20-liter carboys or in 400-liter fermentors. The medium given in Table I can be changed. Thus, either tungstate or molybdate may be excluded without effect on the cell yield. However, the formate dehydrogenase activity is less in cells grown in a medium containing only molybdate. 7 Clostridium thermoaceticum can also be grown without yeast extract and tryptone in a completely synthetic medium. 8 Assay of Formate Dehydrogenase from C. t h e r m o a c e t i c u m In addition to the natural electron acceptor, NADP, C. thermoaceticure formate dehydrogenase reacts with methyl- or benzylviologen.7 The most convenient assay is to follow spectrophotometrically the reduction of NADP at 340 nm (E = 6.3 × 103 M -1 cm-') 9 or of methylviologen at 600 nm (e = 1.3 × 10 4 M -1 c m - 1 ) . TM The assay must be performed using strictly anaerobic conditions and is done under N2. The enzyme has low activity below 40 ° and an assay temperature of 45 ° is recommended. One unit of enzyme is defined as the amount that reduces 1 txmol of NADP (two electrons acceptor) or 2/~mol of methylviologen (one electron ac8 j. R. A n d r e e s e n , u n p u b l i s h e d results. 9 H. V. Bergmeyer, Z. Klin. Chem. Klin. Biochem. 13, 507 (1975). ,o R. N. F. Thorneley~ Biochim. Biophys. Acta 333, 487 (1974).
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NONHEME METALLOPROTEINS
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ceptor) per minute at 45 ° under conditions described below and activity is expressed as units per milligram of protein.
Reagents. All reagents are made up in O2-free water and stored under N2, and all containers are stoppered with serum stoppers. Transfers are made with syringes gassed with N2. (A) Triethanolamine (TEA) maleate buffer, pH 7.5, 1 M. Triethanolamine, 1 mol, is diluted to about 750 ml with water. Solid maleic acid or a concentrated solution is added to obtain a pH of 7.5. The volume is then adjusted to 1 liter. (B) Sodium formate, 0.2 M (C) Sodium ascorbate, 0.5 M, prepared daily (D) Dithiothreitol or dithioerythrol, 30 mM, prepared daily (E) NADP, 20 mM (F) Methylviologen, 0.2 M Water, oxygen-free: The water is boiled for 10 rain and then allowed to cool under a stream of N2. Stored under N2. N2, the gas, best quality available, is freed from 02 by passage through a heated copper column (Sargent-Welch, Skokie, Illinois) and is then saturated with water vapor by passage through a wash bottle. Mix: 1 ml each of solutions A, B, C, D, and E (with NADP as electron acceptor) or F (with methylviologen as electron acceptor) are mixed in a tube previously filled with N2. This mixture is enough for 10 assays. Procedure. The assay is conducted in a 1-ml glass cuvette with 1-cm light path (No. 114, Hellma Cells, Jamaica, New York, or similar). This cuvette has a round opening and can be sealed with a serum stopper (7 × I 1 mm, No. 8826-D32, A. H. Thomas Co., Philadelphia, Pennsylvania). The sealed cuvette is gassed with N2 through hypodermic needles, of which the inlet reaches to the bottom of the cuvette. While still gently gassing, 0.5 ml of the mix is transferred into the cuvette. Water is then added to make a final volume of I ml including the enzyme solution, which is added after the cuvette has been equilibrated to the assay temperature (45°). During the equilibration, the cuvette is continuously gassed with N2. The cuvette is then placed in the spectrophotometer which is equipped with a thermostat and a recorder. The reaction is started by the addition of the enzyme. The reaction should start immediately without a lag period and be linear until an absorbancy of I is reached. If a lag period or a nonlinear reaction is observed, the assay cuvette is not completely anaerobic and then the enzyme is not fully active. The rate of increase in absorbancy is proportional to the amount
[39]
FORMATE DEHYDROGENASE
365
of enzyme in the assay. Calculation of units is conventional. The activity of the enzyme with methylviologen as electron acceptor is about 3 times that obtained with NADP as acceptor.7 Since it is often necessary to determine several samples from chromatographic columns, etc., it is advisable to construct a manifold with outlets to which several cuvettes can be connected and gassed simultaneously. This manifold should be placed over a water bath so that the cuvettes can be placed in the bath and equilibrated to the assay temperature while being gassed with N2. Protein Assay The buffer used in the purification of the formate dehydrogenase from C. therrnoaceticurn contains thioglycolate and iron. These substrates
interfere with the assay of protein if the biuret method or the method by Lowry et al. 11 is used.Therefore, protein is assayed by precipitating with trichloroacetic acid and by determining the turbidity after 10 min at 400 rim.
R e a g e n t s and Procedure. The only reagent is a solution of 26% (w/v) trichloroacetic acid in water. This solution, stored in a refrigerator, is stable for at least a month. Determinations are performed in 1-ml glass cuvettes with 1-cm light path. To 0.2 ml of the protein solution (containing between 0.001 and 0.05 mg of protein) is added 0.8 ml of trichloroacetic acid reagent. The solutions are thoroughly mixed, and the turbidity is determined in a spectrophotometer at 400 nm after 10 min. The protein concentration is calculated from a standard curve obtained using bovine serum albumin as the standard.
Purification of F o r m a t e D e h y d r o g e n a s e from Clostridium thermoaceticum
Formate dehydrogenase from C. thermoaceticum is rapidly inactivated by air. Thus, l ml of French-press extract, prepared as described below, when exposed to air loses 93% of its activity within 2 min, and no activity can be detected after 5 min. Consequently, all work with the enzyme must be performed anaerobically. The partial purification of formate dehydrogenase from 40 g of wet cells of C. thermoaceticum is outlined in Table II. HO. H. Lowry, N. J. Rosebrough, A. L. Fan-, and R. J. Randall,J, Biol. Chem. 193, 265 (1951).
366
NONHEME METALLOPROTEINS
[39]
T A B L E 1I PURIFICATION OF FORMATE DEHYDROGENASE FROM 40 g OF WET CELLS OF Clostridium therrnoaceticum
Step 1. 2. 3. 4. 5.
French-press extract Heat treatment (NH~)2SO4-precipitation Ultrogel AcA 34 DEAE-cellulose
Vol (ml)
Protein (mg)
Formate (total units)
Dehydrogenase (units/mg)
120 120 16 160 34
1800 1440 624 48.8 2.0
564 900 708 206 73,2
0.31 0.63 1.13 4.22 36.6
Anaerobic TEA-Maleate Buffer. The buffer used for preparing cellfree extract and for chromatography using gel filtration (step 4) contains TEA-maleate, pH 7.5, 100 raM; sodium formate, 10 mM; sodium thioglycolate, 25 mM; ferrous ammonium sulfate, 2 mM; and EDTA, 0.1 mM. It is prepared by mixing 850 ml of water, 100 ml of TEA-maleate, 1 M (solution A, see assay), 50 mi of sodium formate, 0.2 M (solution B, see assay), and ! ml 0.1 M EDTA. The mixed solution is freed of oxygen by boiling for 10 min. It is then stored under N2, which is slowly bubbled through the solution. During the cooling and continuous flow of N2, sodium thioglycolate (Difco), 2850 mg, and Fe(NH4)2(SO4)2, 784 mg, are added. The buffer is stable, stored under Nz, for at least a week. A red color due to oxidation or a black precipitate of iron sulfide indicates an unsatisfactory buffer. Buffers of different concentrations of TEA-maleate for gradient chromatography on DEAE-cellulose are prepared with concentrations of the chemicals described above, but by varying the amounts of TEA-maleate. Step 1. French-Press Extract. Frozen cells (40 g) thawed under a stream of Nz are suspended in 120 ml of anaerobic TEA-maleate buffer. The suspension is passed through a French pressure cell at a pressure of 1000 kg/cm 2. The liquid is collected in centrifuge tubes (No. 335348, Beckman Instrument Co., Palo Alto, California) previously filled with N~ and centrifuged for 1 hr at 100,000 g (30,000 rpm in a Beckman L2-65B ultracentrifuge with type 30 fixed-angle aluminum rotor). The supernatant solution containing the enzyme is transferred anaerobically into clean centrifuge tubes. The pellet is discarded. Step 2. Heat Treatment. The tubes with the enzyme from step i are immersed in a water bath at 68 ° for 30 min. After chilling to room
[39]
FORMATEDEHYDROGENASE
367
temperature, the tubes are centrifuged for 15 min at 4000 g to remove denatured protein. During the heat treatment, formate dehydrogenase activity increases substantially. This apparent activation is especially evident when the heat treatment is performed in the presence of formate.
Step 3. (NH4)2SO4 Precipitation. The enzyme solution from step 2 is transferred with a syringe into a 250-ml centrifuge tube (for Sorvall RC2B centrifuge with GSA rotor) stoppered with a rubber stopper and previously gassed with N2. To each 100 ml of enzyme solution, 82 ml of a saturated O2-free (NH4)2SO4 solution (at 25 °) are added to obtain 45% saturation of (NH4)2SO4. After a few minutes of equilibration, the tube is centrifuged at 6000 g for 20 min. The supernatant solution is withdrawn while flushing with N2 gas and discarded. The pellet containing the enzyme is dissolved in about 10 ml of anaerobic TEA-maleate buffer and is stored under N2. Step 4. Ultrogel AcA 34 Gel Filtration. The following types of gels have been and may be used in this step: Bio-Gel A-0.Sm (Bio-Rad Laboratories, Richmond, California), Sepharose 6-B (Pharmacia, Uppsala, Sweden), and Ultrogel AcA 34 (LKB, Bromma, Sweden). All gels give satisfactory results; however, the Ultrogel AcA 34 can be run with a faster flow rate without loss of resolution and is now preferred. The chromatography has to be performed under anaerobic conditions in a system similar to that shown in Fig. I. It may seem elaborate but is actually very easy to operate. Its main parts are a column (1) for ascending flow (K-50/100 with plungers, Pharmacia, Uppsala, Sweden); a cylindrical glove box (7) (Mercaplex GB 1100, Frenchen, Switzerland) con-
0
~
Flo. 1. Anaerobic system. 1, Column plus loading device; 2, elution buffer; 3, IsoVersioric tubing; 4, N2 atmosphere; 5, replacement system; 6, fraction collector; 7, glove box; 8, vacuum pump; 9, heated copper coils; 10, gas train; 11 gas tank.
368
NONHEME METALLOPROTEINS
[39]
taining a fraction collector (6) (ISCO Pup 1200, Lincoln, Nebraska); and a system for keeping the elution buffer anaerobic (2, 4, and 5). The cylindrical glove box has the advantage over squared boxes in that it can be evacuated using a vacuum pump (8). Thus, the atmosphere in the glove box can be quickly replaced with Nz, which is purified by passage through a heated copper column (9) and then saturated with water (10) (see Assay of Formate Dehydrogenase, Reagents, N2). The elution buffer, 0.1 M anaerobic TEA-maleate, is placed in a 2-liter aspirator bottle (2) previously gassed with Nz. To avoid any contact with air, a buffer replacement system (5) is constructed using two 2-liter aspirator bottles. They contain oxygen-free water under a layer of paraffin oil. To further exclude air, a slow stream of CO2 is passed over the liquid in the open aspirator bottle, which also is used to regulate the flow through the column (1) by being lowered or raised using a lab-jack. All connections are of Iso-Versinic tubing (Verneref, France) or stainless steel tubes. Regular rubber or plastic tubings must be avoided since air penetrates them. The loading device is a 25-ml syringe. The glove box is placed on a cart. Before chromatography, the fraction collector in the glove box is filled with test tubes. Extra tubes or collection vessels, as well as serum stoppers for the test tubes, are placed in the box, which is evacuated and filled with N2 repeatedly, at least 3 times. The glove box is then disconnected from the N2 inlet and rolled into a cold room, where it is connected to the column and the rest of the anaerobic system. The loading device is filled with elution buffer. The enzyme solution from step 3 is placed, with the help of a syringe, under the buffer and is allowed to enter the column. Fractions of 8 ml are collected. The enzyme is obtained in fractions 110-135. To take out fractions for analysis from the glove box, selected fractions are stoppered with serum stoppers and passed through the gas lock of the glove box. It is advisable to perform this operation in the laboratory, since working through the gloves in the cold is quite chilly. Typical elution profiles for Sephadex 6-B and Bio-Gel A-0.5m columns have been published.7,12 Very similar results are obtained using Ultrogel AcA 34.
Step 5. DEAE-Cellulose Chromatography. DEAE-cellulose (Schleicher and Schuell Co., Keene, New Hampshire) is first washed with 0.5 N NaOH, distilled water, 0.5 N HC1, distilled water, and then suspended in I M TEA-maleate, pH 7.5. The pH is adjusted, if necessary, to 7.5 by addition of TEA. The purified DEAE-cellulose is stored in the 1 M buffer until used. The anaerobic system described for step 4, appropriately modified, is also used for the DEAE-cellulose chromatography. 12 L. G. Ljungdahl and J. R. A n d r e e s e n , F E B S Lett. 54, 279 (1975).
[39]
FORMATEDEHYDROGENASE
369
A column of DEAE-cellulose (1.5 × 15 cm) is equilibrated with 50 mM anaerobic TEA-maleate buffer by passing about I liter of the buffer through the column. Fractions from the gel filtration (step 4) having a specific activity above three are combined inside the glove box in a small aspirator bottle. It is stoppered with a serum stopper, removed from the glove box, and then connected with the help of a syringe needle to the DEAE-cellulose column. N2 gas is passed over the enzyme solution to keep it anaerobic. As soon as the solution has passed through the column on which the enzyme is held, the column is washed with about 50 ml of 50 mM anaerobic TEA-maleate buffer. The enzyme is then eluted using a gradient consisting of 125 ml of 50 mM and 125 ml of 0.25 M anaerobic TEA-maleate buffer. Fractions of 8 mi are collected in the glove box. The enzyme is found in fractions 7-15. The highest specific activity obtained in this step is 50. Further purification can be obtained by rechromatographing the enzyme on Ultrogel AcA 34 columns. However, the enzymatic activity is rapidly lost at this stage and attempts to prevent this loss have failed up to now. When C. thermoaceticum is grown in media containing 75Se-selenite or '85W-tungstate, the formate dehydrogenase as purified through step 5 contains radioactive metal bound to the protein. 7,12 During gel filtration of radioactive formate dehydrogenase from step 5 on Bio-Gel A-0.5 M, Sephadex G-200, or Ultrogel AcA 34 columns, a constant ratio is observed between radioactivity and enzyme activity. These findings, together with other evidence, suggest that selenium and tungsten are constituents of the formate dehydrogenase. From the '85W-tungstate experiments, assuming 1 g-atom of W per mole of enzyme with a molecular weight of 300,000, it was possible to calculate a turnover number of 48,400 mol of substrate per mole of enzyme per minute. 6 The specific activity of the pure enzyme would then be 160. The best preparations obtained so far have specific activities of 40-50 units/mg.
Properties of Formate Dehydrogenase from Clostridium thermoaceticum Substrate Specificity and Reactions Catalyzed. The naturally occurring electron acceptor for the oxidation of formate catalyzed by C. thermoaceticum formate dehydrogenase is NADP.I~ The following acceptors are inactive: NAD, ferredoxin from several Clostridium species, riboflavin 5'-phosphate, FAD, cytochrome c3 from Desulfovibrio gigas, and cytochrome c from Torula yeast, Of artificial electron acceptors corn'z J. R. Andreesenand L. G. Ljungdahl,J. Bacteriol. 120, 6 (1974).
370
NONHEME METALLOPROTEINS
[39]
monly used, only methyl- and benzylviologen are active. No activity is observed with methylene blue, ferricyanide, and phenazine methosulfate. An isotope exchange between 14CO2 and formate is catalyzed by the enzyme purified through step 3.1 This exchange is dependent on a catalytic amount of NADP. The reduction of CO2 to formate with NADPH as electron donor using a crude enzyme preparation has been investigated by Thauer.2 The reduction requires an appropriate NADPH-regenerating system. Reduction of CO2 with reduced methylviologen has also been demonstrated. In addition to the formate dehydrogenase activity, the enzyme preparation from step 5 catalyzes a reversible electron transfer between methylviologen and NADPH, a reduction of FMN or FAD with NADPH, and oxidation of NADPH with 02.13 Since the enzyme preparation is not pure, the possibility exists that some of these reactions are catalyzed by contaminating enzymes; but, if so, they must be specific for NADPH.
K m Values. Apparent Km values, measured at 55 ° but otherwise at the conditions described for the assay of the enzyme, are as follows: NADP, 1.1 × 10-4 M; methylviologen, 2.35 × 10-3 M; and for formate, 2.3 × 10-4M, using NADP and 0.83 × 10-4M using methylviologen as electron acceptor.13 Apparent K m values for CO2 and NADPH are 1.1 × 10-2 M and 2 x 10 -5, respectively, at 55 ° and pH 7.0. 2 pH Optimum. In the direction of formate oxidation, the enzyme has a broad pH optimum between 7.0 and 9.5, when NADP is the acceptor.'3 With methylviologen, the reaction rate increases from pH 7.0 to 9, at which pH a formate-independent reduction of methylviologen occurs and becomes prominent. The pH optimum in the direction of CO2 reduction is also broad, with a maximum at pH 7.0. 2
Effect of Temperature. Clostridium thermoaceticum formate dehydrogenase has high heat stability and can be incubated for 3 hr at 70 ° with only a slight loss of activity. 14 During heating of crude extracts an apparent activation occurs. This is especially noticeable in the presence of the substrate formate or the inhibitor sodium azide. The optimum temperature using NADP as electron acceptor is approximately 70 °, and with methylviologen, 85 °. The increase of activity with temperature follows a smooth curve to about 55 °, at which temperature a definite break occurs. Below 55 °, straight lines are obtained with both NADP and methylviologen as acceptors in Arrhenius plots. The slopes of the lines ,4 L. G. Ljungdahl, D. W. Sherod, M. R. Moore, and J. R. Andreesen, in "Enzymes and Proteins from Thermophilic Microorganisms" (Proc. Int. Syrup. Zurich, 1975) (H. Zuber, ed.), Experientia Suppl. 26, 237 (1976).
[39]
FORMATEDEHYDROGENASE
371
are the same, indicating the same activation energy of about 10,500 cal/ mol with both electron acceptors.
Molecular Weight. Gel filtration studies and ultracentrifugation in sucrose gradient indicate a molecular weight of between 270,000 and 300,000.'2"a Activators and Inhibitors. Formate dehydrogenase from C. thermoaceticum requires a sulfhydryl compound for activity.a"a Dithiothreitol, cysteine glutathione, or thioglycolate at concentrations of 1 /xM satisfy this requirement. Mercaptoethanol acts inhibitorily, especially with NADP as electron acceptor. Hypophosphite and azide, both structural analogs to formate, act as competitive inhibitors.'3 However, the azide inhibition is only partially reversible by formate. This is especially noticeable if the enzyme has been in contact with azide for a longer period. The enzyme is 50% inhibited by 10 /xM cyanide and 100% by 10 raM. EDTA inhibits the enzyme 50% at 25 mM and 100% at 0.1 M. a3 Effect of Selenium, Tungsten, and Molybdenum. Clostridium thermoaceticum grows without including selenite, tungstate, and molybdate in the medium. However, cells grown in the absence of these chemicals or in the presence of only one of them have very low formate dehydrogenase activity.7 High enzyme activity is obtained only when selenite, in combination with tungstate or molybdate, is present in the medium. It is apparent that there is a dual requirement for either selenite-tungstate or selenite-molybdate in the formation of active formate dehydrogenase in C. thermoaceticum. The demonstration, using 7~Se-selenite and lssWtungstate, that both selenium 7and tungsten '2 are incorporated into protein fractions containing formate dehydrogenase suggests that C. thermoaceticum formate dehydrogenase is a selenium-tungsten or a selenium-molybdenum enzyme. That cyanide and EDTA are inhibitors also indicates a role for metals in the activity of the enzyme. Several molybdenum enzymes are known. Tungsten, which is chemically very similar to molybdenum, has been found to act antagonistically against molybdenum and to prevent the formation of molybdenum enzymes. 15--17Therefore, it is surprising that tungsten and molybdenum are both active and apparently are replacing each other. The question then arises whether tungsten or molybdenum is preferentially incorporated 'aJ. R. Benemann, G. M. Smith, P. J. Kostel, and C. E. McKenna, FEBS Lett. 29, 219 (1973). '~; K.-Y. Lee, R. Erickson, S.-S. Pan, G. Jones, F. May, and A. Nason, J. Biol. Chem. 249, 3953 (1974). 17j. L. Johnson, H. J. Cohen, and K. V. Rajagopalan, J. Biol. Chem. 249, 5046 (1974).
372
NONHEME METALLOPROTEJ.NS
[39]
o v e r the other metal into the formate dehydrogenase. This question was answered by growing C. thermoaceticum in a m e d i u m containing selenite and 10 /zM '85W-tungstate in the p r e s e n c e of varying concentrations (none, 0.1 /zM, 10 taM, and 1 mM) of m o l y b d a t e . 6 The incorporation of tungsten into the f o r m a t e d e h y d r o g e n a s e fraction was not d e p r e s s e d by the m o l y b d a t e , although its concentration was 100 times that of tungstate. Thus, tungsten a p p e a r s to be the preferred metal and f o r m a t e dehydrogenase in C. thermoaceticum seems to represent the first e x a m p l e of an e n z y m e having tungsten as an active metal. H o w e v e r , tungsten in combination with selenium also seems to p r o m o t e formation of formate deh y d r o g e n a s e in Clostridium f o r m i c o a c e t i c u m 18 and Clostridium acidiurici. 19 Clostridium f o r m i c o a c e t i c u m ferments sugars to acetate via a p a t h w a y similar to that used by C. thermoaceticum, whereas C. acidiurici ferments purines to a m m o n i a , CO2, and acetate. M o l y b d e n u m m a y replace tungsten in C. formicoaceticum, but it has little effect on the level of the formate d e h y d r o g e n a s e in C. acidiurici. Finally, tungsten has been implicated in the m e t a b o l i s m of M e t h a n o c o c c u s vannielii. 2o,2~ E x a m p l e s of selenium e n z y m e s other than the formate dehydrogenases from C. thermoaceticum, C. f o r m i c o a c e t i c u m , and C. acidiurici are formate d e h y d r o g e n a s e from Escherichia coli, 22 a protein c o m p o n e n t of glycine reductase from Clostridium sticklandii, 2~ and glutathione peroxidase from bovine blood, 24 and e r y t h r o c y t e s from rats. 25 The formate d e h y d r o g e n a s e from E. coli, judged to be 93-99% pure, has a molecular weight of a b o u t 590,000. The e n z y m e contains 4 hemes (b-type cytochrome), and a tetrameric structure has been suggested. It contains, per mole of heme, 0.95 m o l y b d e n u m , 0.96 selenium, 14 n o n h e m e iron, and 13 acid-labile sulfide. Tungsten, when included in the growth medium, acts antagonistically toward m o l y b d e n u m in the formation of formate d e h y d r o g e n a s e in E. coli. Radioactive 75Se-selenite is incorporated into glutathione p e r o x i d a s e from rat erythrocytes. The e n z y m e has been purified from bovine blood and crystallized. N e u t r o n activation analysis showed that it contains 4 Se per mole of e n z y m e , which is a t e t r a m e r with a molecular weight of 84,000. 18j. R. Andreesen, E. El Ghazzawi, and G. Gottschalk, Arch. Microbiol. 96, 103 (1974). 1~R. Wagner and J. R. Andreesen, Arch. Microbiol. 114, 219 (1977). 20j. B. Jones and T. C. Stadtman, in -Symposium on Microbial Production and Utilization of Gases (H2, CH4, CO)" (H. G. Schlegel, G. Gottschalk, and N. Pfenning, eds.), p. 199. E. Goltze Verlag, Gottingen, German Federal Republic, 1976. 21j. B. Jones and T. C. Stadtman, J. Bacteriol. 130, 1404 (1977). 22H. G. Enoch and R. L. Lester. J. Biol. Chem. 250, 6693 (1975). 23D. C. Turner and T. C. Stadtman, Arch. Biochem. Biophys. 154, 366 (1973). 24L. Flohe, W. A. Giinzler, and H. H. Schock, FEBS Lett. 32, 132 (1973). 25j. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Science 179, 588 (1973).
NONHEMEMETALLOPROTEINS
[39]
Prosthetic Group. The w-hydroxylase contains one atom of iron per polypeptide chain, but heine is not present. 7 The iron is largely removed by treatment with EDTA or other metal chelators provided that a reducing agent, such as dithionite, is also added. Hydroxylation activity is restored by incubating the apohydroxylase preparation with ferrous ions prior to the spectrophotometric assay. The to-hydroxylase contains only one molecule of cysteine per polypeptide chain, as shown by amino acid analysis and sulfhydryl titrations. No labile sulfide is present. Other Properties. The purified ~hydroxylase preparation contains phospholipid, primarily phosphatidylethanolamine, as well as a few carbohydrate residues. Removal of the lipid results in a loss of nearly all hydroxylation activity, which can be restored by the addition of various phospholipids, r The enzyme is reversibly inhibited by cyanide, but is not inhibited by carbon monoxide. Specificity. The ~hydroxylase catalzyes the hydroxylation of a variety of aliphatic hydrocarbons, including n-alkanes having 6-14 carbon atoms, as well as branched-chain structures, such as 2,5-dimethylhexane, and alicyclic compounds, such as cyclohexane and methylcyclohexane. n-Fatty acids with 6-14 carbon atoms also serve as substrates. The enzyme also catalyzes the epoxidation of alkenes, such as octadiene. TM More recent studies in our laboratory show that iron and phospholipid are required for epoxidation as well as for hydroxylation. ,a S. W. May and B. J. Abbott, J. Biol. Chem. 248, 1725 (1973).
[39] F o r m a t e Enzyme
Dehydrogenase, a Selenium-Tungsten from Clostridium thermoaceticum
By LARS G. LJUNGDAHL and JAN R. ANDREESEN Formate dehydrogenase of Clostridium thermoaceticum catalyzes the following reversible reaction a'Z: HCOO- + NADP+ ~,~--CO2 + NADPH
(1)
The only known naturally occurring electron acceptor is NADP, and the enzyme should perhaps be named formate:NADP oxidoreductase. The ' L.-F. Li, L. Ljungdahl, and H. G. Wood, J. Bacteriol. 92, 405 (1966). 2 R. K. Thauer, FEBS Lett. 27, 111 (1972).
[39]
FORMATE DEHYDROGENASE
361
physiological role of the e n z y m e is to catalyze the reduction of COz to formate. Clostridium t h e r m o a c e t i c u m is a thermophilic anaerobic bacterium that ferments sugars to acetate as the only product.3 About one-third of the acetate is formed by reduction of CO2, which serves as electron acceptor of electrons generated during the fermentation. This is shown in the following reactions.
C~H,~O6 + H~O --~ CH:~COCOOH + CH:~COOH + 6H' + CO2
(2)
CH3COCOOH + CO2 + 6H ~ 2 CH3COOH + H20
(3)
Sum: C~H,..,O6~ 3 CH:~COOH
(4)
In reaction (2) the hexose is fermented to 2 tool of pyruvate, of which one is further metabolized to acetate and CO2. Six equivalents of electrons are generated; these are used to reduce CO2 to a methyl group, which is converted to acetate in a transcarboxylation reaction involving the carboxyl group of pyruvate [reaction (3)]. 4 The reduction of CO2 to acetate proceeds with formate, 10-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5-methyltetrahydrofolate, and a Co-methylcobamide as intermediates. ~'6 The first e n z y m e in the pathway is the N A D P - d e p e n d e n t formate dehydrogenase. This e n z y m e is present in a relatively high level in cells of C. t h e r m o a c e t i c u m grown in media containing selenite plus either tungstate or molybdate. Cells grown in media lacking these chemicals have much lower formate dehydrogenase activity. ¢ Culturing M e t h o d s for C l o s t r i d i u m t h e r m o a c e t i c u r n Clostridium t h e r m o a c e t i c u m (DSM 521) is maintained in agar-stab cultures or in liquid broth cultures. It is a strictly anaerobic bacterium, and it fails to grow in the presence of small amounts of oxygen. A g a r - S t a b Cultures. The agar medium has the following composition
3 F. E. Fontaine, W. H. Peterson, E. McCoy, M. J. Johnson, and G. J. Ritter, J. Bacteriol. 43, 701 (1942). 4 M. Schulman, R. K. Ghambeer, L. G. Ljungdahl, and H. G. Wood, J. Biol. Chem. 248, 6255 (1973). 5 L. G. Ljungdahl and It. G. Wood, Annu. Rev. Microbiol. 23, 515 (1969). 6 L. G. Ljungdahl and J. R. Andreesen, in "Symposium on Microbial Production and Utilization of Gases (Hz, CH4, CO)." (H. G. Schlegel, G. Gottschalk, and N. Pfenning, eds.), p. 163. E. Goltze Verlag, G6ttingen, German Federal Republic, 1976. 7j. R. Andreesen and L. G. Ljungdahl, J. Bacteriol. 116, 867 (1973).
362
NONHEMEMETALLOPROTEINS
[39]
in grams per liter: agar, 20; glucose, 5; yeast extract (Difco), 5; tryptone (Difco), 5; (NH4)2SO4, 0.5; sodium thioglycolate, 0.5; and MgSO4"7 H20, 0.1. Culture tubes equipped with tight-sealing plastic screw caps are three-quarters filled with the medium. After autoclaving, the tubes are transferred into a wide-mouth bottle filled with COs gas and left until the agar is solidified. After inoculation using a needle, the tubes are sealed and incubated for 2-5 days at 58 °. Bacterial growth should be clearly visible. New stab cultures are prepared at intervals of 6 months, but cultures stored for several years at 4 ° are viable. Old stab cultures assume growth faster if they are activated by heating in a boiling water bath for 10 min.
Liquid Culture. Clostridium thermoaceticum is best grown in an atmosphere of COs at 58 ° in the medium listed in Table I. The medium is prepared in three solutions: A, containing glucose dissolved in 150 ml of distilled water; B, containing yeast extract, tryptone, and metals in 550 ml of water; C, containing sodium bicarbonate and potassium phosphate in 300 ml of water. It is practical to make solution C in the flask that will TABLE I MEDIUM FOR Clostridium thermoaceticum
Solution
A B
C
Ingredient
Glucose Water Yeast extract (Difco) Tryptone (Difco) (NH4)2SO4 MgSO4'7 H20 Fe(NH4)dSO4h '6 H20 Co(NOa)z'6 H20 NazWO4"2 H20 Na~MoO4-2 H20 Na~SeO3 Sodium thioglycolate (Difco) Water NaHCOa K2HPO4 KH2PO4 Water
Amount (g)
Volume (ml)
Final Concentration a (raM)
100
18 150 5 5 1 0.25 0.039 0.030 0.0033 0.0024 0.0002 0.5
7.6 1 0.1 0.1 0.01 0.01 0.001 4.4 550
200 40 40
16.8 7 5.5
a Concentration after mixing of solutions A, B, and C.
300
[39]
FORMATEDEHYDROGENASE
363
be used for the culture. This flask should be fitted with a bubbler consisting of a rubber stopper with two glass tubes, of which one (the inlet) reaches to the bottom of the flask. Cotton plugs are inserted into the tubes of the bubbler, and it and the three solutions are sterilized separately. To prepare the medium for inoculation, the bubbler is inserted into the flask containing solution C. The solution is gassed with CO2 until the pH is below 8.5. Solutions A and B are then combined with solution C, and the medium is placed in an incubator maintained at about 58 °. Gassing with CO2 is continued. After the solution has reached 55 °, it is inoculated with about 50 ml of a broth culture less than a week old. A slow constant stream of CO2 is maintained through the medium during the growth period. The cells are normally harvested within 48 hr by centrifugation at room temperature without exclusion of air. The cells may be stored frozen at - 8 0 ° for several months with only a small loss of formate dehydrogenase activity. The yield is between 10 and 17 g of wet cells per liter of medium. Normally C. thermoaceticum is maintained in l-liter liquid cultures with transfers twice weekly. Large amounts of cells of the bacterium have been produced by cultivation in 20-liter carboys or in 400-liter fermentors. The medium given in Table I can be changed. Thus, either tungstate or molybdate may be excluded without effect on the cell yield. However, the formate dehydrogenase activity is less in cells grown in a medium containing only molybdate. 7 Clostridium thermoaceticum can also be grown without yeast extract and tryptone in a completely synthetic medium. 8 Assay of Formate Dehydrogenase from C. t h e r m o a c e t i c u m In addition to the natural electron acceptor, NADP, C. thermoaceticure formate dehydrogenase reacts with methyl- or benzylviologen.7 The most convenient assay is to follow spectrophotometrically the reduction of NADP at 340 nm (E = 6.3 × 103 M -1 cm-') 9 or of methylviologen at 600 nm (e = 1.3 × 10 4 M -1 c m - 1 ) . TM The assay must be performed using strictly anaerobic conditions and is done under N2. The enzyme has low activity below 40 ° and an assay temperature of 45 ° is recommended. One unit of enzyme is defined as the amount that reduces 1 txmol of NADP (two electrons acceptor) or 2/~mol of methylviologen (one electron ac8 j. R. A n d r e e s e n , u n p u b l i s h e d results. 9 H. V. Bergmeyer, Z. Klin. Chem. Klin. Biochem. 13, 507 (1975). ,o R. N. F. Thorneley~ Biochim. Biophys. Acta 333, 487 (1974).
364
NONHEME METALLOPROTEINS
[39]
ceptor) per minute at 45 ° under conditions described below and activity is expressed as units per milligram of protein.
Reagents. All reagents are made up in O2-free water and stored under N2, and all containers are stoppered with serum stoppers. Transfers are made with syringes gassed with N2. (A) Triethanolamine (TEA) maleate buffer, pH 7.5, 1 M. Triethanolamine, 1 mol, is diluted to about 750 ml with water. Solid maleic acid or a concentrated solution is added to obtain a pH of 7.5. The volume is then adjusted to 1 liter. (B) Sodium formate, 0.2 M (C) Sodium ascorbate, 0.5 M, prepared daily (D) Dithiothreitol or dithioerythrol, 30 mM, prepared daily (E) NADP, 20 mM (F) Methylviologen, 0.2 M Water, oxygen-free: The water is boiled for 10 rain and then allowed to cool under a stream of N2. Stored under N2. N2, the gas, best quality available, is freed from 02 by passage through a heated copper column (Sargent-Welch, Skokie, Illinois) and is then saturated with water vapor by passage through a wash bottle. Mix: 1 ml each of solutions A, B, C, D, and E (with NADP as electron acceptor) or F (with methylviologen as electron acceptor) are mixed in a tube previously filled with N2. This mixture is enough for 10 assays. Procedure. The assay is conducted in a 1-ml glass cuvette with 1-cm light path (No. 114, Hellma Cells, Jamaica, New York, or similar). This cuvette has a round opening and can be sealed with a serum stopper (7 × I 1 mm, No. 8826-D32, A. H. Thomas Co., Philadelphia, Pennsylvania). The sealed cuvette is gassed with N2 through hypodermic needles, of which the inlet reaches to the bottom of the cuvette. While still gently gassing, 0.5 ml of the mix is transferred into the cuvette. Water is then added to make a final volume of I ml including the enzyme solution, which is added after the cuvette has been equilibrated to the assay temperature (45°). During the equilibration, the cuvette is continuously gassed with N2. The cuvette is then placed in the spectrophotometer which is equipped with a thermostat and a recorder. The reaction is started by the addition of the enzyme. The reaction should start immediately without a lag period and be linear until an absorbancy of I is reached. If a lag period or a nonlinear reaction is observed, the assay cuvette is not completely anaerobic and then the enzyme is not fully active. The rate of increase in absorbancy is proportional to the amount
[39]
FORMATE DEHYDROGENASE
365
of enzyme in the assay. Calculation of units is conventional. The activity of the enzyme with methylviologen as electron acceptor is about 3 times that obtained with NADP as acceptor.7 Since it is often necessary to determine several samples from chromatographic columns, etc., it is advisable to construct a manifold with outlets to which several cuvettes can be connected and gassed simultaneously. This manifold should be placed over a water bath so that the cuvettes can be placed in the bath and equilibrated to the assay temperature while being gassed with N2. Protein Assay The buffer used in the purification of the formate dehydrogenase from C. therrnoaceticurn contains thioglycolate and iron. These substrates
interfere with the assay of protein if the biuret method or the method by Lowry et al. 11 is used.Therefore, protein is assayed by precipitating with trichloroacetic acid and by determining the turbidity after 10 min at 400 rim.
R e a g e n t s and Procedure. The only reagent is a solution of 26% (w/v) trichloroacetic acid in water. This solution, stored in a refrigerator, is stable for at least a month. Determinations are performed in 1-ml glass cuvettes with 1-cm light path. To 0.2 ml of the protein solution (containing between 0.001 and 0.05 mg of protein) is added 0.8 ml of trichloroacetic acid reagent. The solutions are thoroughly mixed, and the turbidity is determined in a spectrophotometer at 400 nm after 10 min. The protein concentration is calculated from a standard curve obtained using bovine serum albumin as the standard.
Purification of F o r m a t e D e h y d r o g e n a s e from Clostridium thermoaceticum
Formate dehydrogenase from C. thermoaceticum is rapidly inactivated by air. Thus, l ml of French-press extract, prepared as described below, when exposed to air loses 93% of its activity within 2 min, and no activity can be detected after 5 min. Consequently, all work with the enzyme must be performed anaerobically. The partial purification of formate dehydrogenase from 40 g of wet cells of C. thermoaceticum is outlined in Table II. HO. H. Lowry, N. J. Rosebrough, A. L. Fan-, and R. J. Randall,J, Biol. Chem. 193, 265 (1951).
366
NONHEME METALLOPROTEINS
[39]
T A B L E 1I PURIFICATION OF FORMATE DEHYDROGENASE FROM 40 g OF WET CELLS OF Clostridium therrnoaceticum
Step 1. 2. 3. 4. 5.
French-press extract Heat treatment (NH~)2SO4-precipitation Ultrogel AcA 34 DEAE-cellulose
Vol (ml)
Protein (mg)
Formate (total units)
Dehydrogenase (units/mg)
120 120 16 160 34
1800 1440 624 48.8 2.0
564 900 708 206 73,2
0.31 0.63 1.13 4.22 36.6
Anaerobic TEA-Maleate Buffer. The buffer used for preparing cellfree extract and for chromatography using gel filtration (step 4) contains TEA-maleate, pH 7.5, 100 raM; sodium formate, 10 mM; sodium thioglycolate, 25 mM; ferrous ammonium sulfate, 2 mM; and EDTA, 0.1 mM. It is prepared by mixing 850 ml of water, 100 ml of TEA-maleate, 1 M (solution A, see assay), 50 mi of sodium formate, 0.2 M (solution B, see assay), and ! ml 0.1 M EDTA. The mixed solution is freed of oxygen by boiling for 10 min. It is then stored under N2, which is slowly bubbled through the solution. During the cooling and continuous flow of N2, sodium thioglycolate (Difco), 2850 mg, and Fe(NH4)2(SO4)2, 784 mg, are added. The buffer is stable, stored under Nz, for at least a week. A red color due to oxidation or a black precipitate of iron sulfide indicates an unsatisfactory buffer. Buffers of different concentrations of TEA-maleate for gradient chromatography on DEAE-cellulose are prepared with concentrations of the chemicals described above, but by varying the amounts of TEA-maleate. Step 1. French-Press Extract. Frozen cells (40 g) thawed under a stream of Nz are suspended in 120 ml of anaerobic TEA-maleate buffer. The suspension is passed through a French pressure cell at a pressure of 1000 kg/cm 2. The liquid is collected in centrifuge tubes (No. 335348, Beckman Instrument Co., Palo Alto, California) previously filled with N~ and centrifuged for 1 hr at 100,000 g (30,000 rpm in a Beckman L2-65B ultracentrifuge with type 30 fixed-angle aluminum rotor). The supernatant solution containing the enzyme is transferred anaerobically into clean centrifuge tubes. The pellet is discarded. Step 2. Heat Treatment. The tubes with the enzyme from step i are immersed in a water bath at 68 ° for 30 min. After chilling to room
[39]
FORMATEDEHYDROGENASE
367
temperature, the tubes are centrifuged for 15 min at 4000 g to remove denatured protein. During the heat treatment, formate dehydrogenase activity increases substantially. This apparent activation is especially evident when the heat treatment is performed in the presence of formate.
Step 3. (NH4)2SO4 Precipitation. The enzyme solution from step 2 is transferred with a syringe into a 250-ml centrifuge tube (for Sorvall RC2B centrifuge with GSA rotor) stoppered with a rubber stopper and previously gassed with N2. To each 100 ml of enzyme solution, 82 ml of a saturated O2-free (NH4)2SO4 solution (at 25 °) are added to obtain 45% saturation of (NH4)2SO4. After a few minutes of equilibration, the tube is centrifuged at 6000 g for 20 min. The supernatant solution is withdrawn while flushing with N2 gas and discarded. The pellet containing the enzyme is dissolved in about 10 ml of anaerobic TEA-maleate buffer and is stored under N2. Step 4. Ultrogel AcA 34 Gel Filtration. The following types of gels have been and may be used in this step: Bio-Gel A-0.Sm (Bio-Rad Laboratories, Richmond, California), Sepharose 6-B (Pharmacia, Uppsala, Sweden), and Ultrogel AcA 34 (LKB, Bromma, Sweden). All gels give satisfactory results; however, the Ultrogel AcA 34 can be run with a faster flow rate without loss of resolution and is now preferred. The chromatography has to be performed under anaerobic conditions in a system similar to that shown in Fig. I. It may seem elaborate but is actually very easy to operate. Its main parts are a column (1) for ascending flow (K-50/100 with plungers, Pharmacia, Uppsala, Sweden); a cylindrical glove box (7) (Mercaplex GB 1100, Frenchen, Switzerland) con-
0
~
Flo. 1. Anaerobic system. 1, Column plus loading device; 2, elution buffer; 3, IsoVersioric tubing; 4, N2 atmosphere; 5, replacement system; 6, fraction collector; 7, glove box; 8, vacuum pump; 9, heated copper coils; 10, gas train; 11 gas tank.
368
NONHEME METALLOPROTEINS
[39]
taining a fraction collector (6) (ISCO Pup 1200, Lincoln, Nebraska); and a system for keeping the elution buffer anaerobic (2, 4, and 5). The cylindrical glove box has the advantage over squared boxes in that it can be evacuated using a vacuum pump (8). Thus, the atmosphere in the glove box can be quickly replaced with Nz, which is purified by passage through a heated copper column (9) and then saturated with water (10) (see Assay of Formate Dehydrogenase, Reagents, N2). The elution buffer, 0.1 M anaerobic TEA-maleate, is placed in a 2-liter aspirator bottle (2) previously gassed with Nz. To avoid any contact with air, a buffer replacement system (5) is constructed using two 2-liter aspirator bottles. They contain oxygen-free water under a layer of paraffin oil. To further exclude air, a slow stream of CO2 is passed over the liquid in the open aspirator bottle, which also is used to regulate the flow through the column (1) by being lowered or raised using a lab-jack. All connections are of Iso-Versinic tubing (Verneref, France) or stainless steel tubes. Regular rubber or plastic tubings must be avoided since air penetrates them. The loading device is a 25-ml syringe. The glove box is placed on a cart. Before chromatography, the fraction collector in the glove box is filled with test tubes. Extra tubes or collection vessels, as well as serum stoppers for the test tubes, are placed in the box, which is evacuated and filled with N2 repeatedly, at least 3 times. The glove box is then disconnected from the N2 inlet and rolled into a cold room, where it is connected to the column and the rest of the anaerobic system. The loading device is filled with elution buffer. The enzyme solution from step 3 is placed, with the help of a syringe, under the buffer and is allowed to enter the column. Fractions of 8 ml are collected. The enzyme is obtained in fractions 110-135. To take out fractions for analysis from the glove box, selected fractions are stoppered with serum stoppers and passed through the gas lock of the glove box. It is advisable to perform this operation in the laboratory, since working through the gloves in the cold is quite chilly. Typical elution profiles for Sephadex 6-B and Bio-Gel A-0.5m columns have been published.7,12 Very similar results are obtained using Ultrogel AcA 34.
Step 5. DEAE-Cellulose Chromatography. DEAE-cellulose (Schleicher and Schuell Co., Keene, New Hampshire) is first washed with 0.5 N NaOH, distilled water, 0.5 N HC1, distilled water, and then suspended in I M TEA-maleate, pH 7.5. The pH is adjusted, if necessary, to 7.5 by addition of TEA. The purified DEAE-cellulose is stored in the 1 M buffer until used. The anaerobic system described for step 4, appropriately modified, is also used for the DEAE-cellulose chromatography. 12 L. G. Ljungdahl and J. R. A n d r e e s e n , F E B S Lett. 54, 279 (1975).
[39]
FORMATEDEHYDROGENASE
369
A column of DEAE-cellulose (1.5 × 15 cm) is equilibrated with 50 mM anaerobic TEA-maleate buffer by passing about I liter of the buffer through the column. Fractions from the gel filtration (step 4) having a specific activity above three are combined inside the glove box in a small aspirator bottle. It is stoppered with a serum stopper, removed from the glove box, and then connected with the help of a syringe needle to the DEAE-cellulose column. N2 gas is passed over the enzyme solution to keep it anaerobic. As soon as the solution has passed through the column on which the enzyme is held, the column is washed with about 50 ml of 50 mM anaerobic TEA-maleate buffer. The enzyme is then eluted using a gradient consisting of 125 ml of 50 mM and 125 ml of 0.25 M anaerobic TEA-maleate buffer. Fractions of 8 mi are collected in the glove box. The enzyme is found in fractions 7-15. The highest specific activity obtained in this step is 50. Further purification can be obtained by rechromatographing the enzyme on Ultrogel AcA 34 columns. However, the enzymatic activity is rapidly lost at this stage and attempts to prevent this loss have failed up to now. When C. thermoaceticum is grown in media containing 75Se-selenite or '85W-tungstate, the formate dehydrogenase as purified through step 5 contains radioactive metal bound to the protein. 7,12 During gel filtration of radioactive formate dehydrogenase from step 5 on Bio-Gel A-0.5 M, Sephadex G-200, or Ultrogel AcA 34 columns, a constant ratio is observed between radioactivity and enzyme activity. These findings, together with other evidence, suggest that selenium and tungsten are constituents of the formate dehydrogenase. From the '85W-tungstate experiments, assuming 1 g-atom of W per mole of enzyme with a molecular weight of 300,000, it was possible to calculate a turnover number of 48,400 mol of substrate per mole of enzyme per minute. 6 The specific activity of the pure enzyme would then be 160. The best preparations obtained so far have specific activities of 40-50 units/mg.
Properties of Formate Dehydrogenase from Clostridium thermoaceticum Substrate Specificity and Reactions Catalyzed. The naturally occurring electron acceptor for the oxidation of formate catalyzed by C. thermoaceticum formate dehydrogenase is NADP.I~ The following acceptors are inactive: NAD, ferredoxin from several Clostridium species, riboflavin 5'-phosphate, FAD, cytochrome c3 from Desulfovibrio gigas, and cytochrome c from Torula yeast, Of artificial electron acceptors corn'z J. R. Andreesenand L. G. Ljungdahl,J. Bacteriol. 120, 6 (1974).
370
NONHEME METALLOPROTEINS
[39]
monly used, only methyl- and benzylviologen are active. No activity is observed with methylene blue, ferricyanide, and phenazine methosulfate. An isotope exchange between 14CO2 and formate is catalyzed by the enzyme purified through step 3.1 This exchange is dependent on a catalytic amount of NADP. The reduction of CO2 to formate with NADPH as electron donor using a crude enzyme preparation has been investigated by Thauer.2 The reduction requires an appropriate NADPH-regenerating system. Reduction of CO2 with reduced methylviologen has also been demonstrated. In addition to the formate dehydrogenase activity, the enzyme preparation from step 5 catalyzes a reversible electron transfer between methylviologen and NADPH, a reduction of FMN or FAD with NADPH, and oxidation of NADPH with 02.13 Since the enzyme preparation is not pure, the possibility exists that some of these reactions are catalyzed by contaminating enzymes; but, if so, they must be specific for NADPH.
K m Values. Apparent Km values, measured at 55 ° but otherwise at the conditions described for the assay of the enzyme, are as follows: NADP, 1.1 × 10-4 M; methylviologen, 2.35 × 10-3 M; and for formate, 2.3 × 10-4M, using NADP and 0.83 × 10-4M using methylviologen as electron acceptor.13 Apparent K m values for CO2 and NADPH are 1.1 × 10-2 M and 2 x 10 -5, respectively, at 55 ° and pH 7.0. 2 pH Optimum. In the direction of formate oxidation, the enzyme has a broad pH optimum between 7.0 and 9.5, when NADP is the acceptor.'3 With methylviologen, the reaction rate increases from pH 7.0 to 9, at which pH a formate-independent reduction of methylviologen occurs and becomes prominent. The pH optimum in the direction of CO2 reduction is also broad, with a maximum at pH 7.0. 2
Effect of Temperature. Clostridium thermoaceticum formate dehydrogenase has high heat stability and can be incubated for 3 hr at 70 ° with only a slight loss of activity. 14 During heating of crude extracts an apparent activation occurs. This is especially noticeable in the presence of the substrate formate or the inhibitor sodium azide. The optimum temperature using NADP as electron acceptor is approximately 70 °, and with methylviologen, 85 °. The increase of activity with temperature follows a smooth curve to about 55 °, at which temperature a definite break occurs. Below 55 °, straight lines are obtained with both NADP and methylviologen as acceptors in Arrhenius plots. The slopes of the lines ,4 L. G. Ljungdahl, D. W. Sherod, M. R. Moore, and J. R. Andreesen, in "Enzymes and Proteins from Thermophilic Microorganisms" (Proc. Int. Syrup. Zurich, 1975) (H. Zuber, ed.), Experientia Suppl. 26, 237 (1976).
[39]
FORMATEDEHYDROGENASE
371
are the same, indicating the same activation energy of about 10,500 cal/ mol with both electron acceptors.
Molecular Weight. Gel filtration studies and ultracentrifugation in sucrose gradient indicate a molecular weight of between 270,000 and 300,000.'2"a Activators and Inhibitors. Formate dehydrogenase from C. thermoaceticum requires a sulfhydryl compound for activity.a"a Dithiothreitol, cysteine glutathione, or thioglycolate at concentrations of 1 /xM satisfy this requirement. Mercaptoethanol acts inhibitorily, especially with NADP as electron acceptor. Hypophosphite and azide, both structural analogs to formate, act as competitive inhibitors.'3 However, the azide inhibition is only partially reversible by formate. This is especially noticeable if the enzyme has been in contact with azide for a longer period. The enzyme is 50% inhibited by 10 /xM cyanide and 100% by 10 raM. EDTA inhibits the enzyme 50% at 25 mM and 100% at 0.1 M. a3 Effect of Selenium, Tungsten, and Molybdenum. Clostridium thermoaceticum grows without including selenite, tungstate, and molybdate in the medium. However, cells grown in the absence of these chemicals or in the presence of only one of them have very low formate dehydrogenase activity.7 High enzyme activity is obtained only when selenite, in combination with tungstate or molybdate, is present in the medium. It is apparent that there is a dual requirement for either selenite-tungstate or selenite-molybdate in the formation of active formate dehydrogenase in C. thermoaceticum. The demonstration, using 7~Se-selenite and lssWtungstate, that both selenium 7and tungsten '2 are incorporated into protein fractions containing formate dehydrogenase suggests that C. thermoaceticum formate dehydrogenase is a selenium-tungsten or a selenium-molybdenum enzyme. That cyanide and EDTA are inhibitors also indicates a role for metals in the activity of the enzyme. Several molybdenum enzymes are known. Tungsten, which is chemically very similar to molybdenum, has been found to act antagonistically against molybdenum and to prevent the formation of molybdenum enzymes. 15--17Therefore, it is surprising that tungsten and molybdenum are both active and apparently are replacing each other. The question then arises whether tungsten or molybdenum is preferentially incorporated 'aJ. R. Benemann, G. M. Smith, P. J. Kostel, and C. E. McKenna, FEBS Lett. 29, 219 (1973). '~; K.-Y. Lee, R. Erickson, S.-S. Pan, G. Jones, F. May, and A. Nason, J. Biol. Chem. 249, 3953 (1974). 17j. L. Johnson, H. J. Cohen, and K. V. Rajagopalan, J. Biol. Chem. 249, 5046 (1974).
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NONHEME METALLOPROTEJ.NS
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o v e r the other metal into the formate dehydrogenase. This question was answered by growing C. thermoaceticum in a m e d i u m containing selenite and 10 /zM '85W-tungstate in the p r e s e n c e of varying concentrations (none, 0.1 /zM, 10 taM, and 1 mM) of m o l y b d a t e . 6 The incorporation of tungsten into the f o r m a t e d e h y d r o g e n a s e fraction was not d e p r e s s e d by the m o l y b d a t e , although its concentration was 100 times that of tungstate. Thus, tungsten a p p e a r s to be the preferred metal and f o r m a t e dehydrogenase in C. thermoaceticum seems to represent the first e x a m p l e of an e n z y m e having tungsten as an active metal. H o w e v e r , tungsten in combination with selenium also seems to p r o m o t e formation of formate deh y d r o g e n a s e in Clostridium f o r m i c o a c e t i c u m 18 and Clostridium acidiurici. 19 Clostridium f o r m i c o a c e t i c u m ferments sugars to acetate via a p a t h w a y similar to that used by C. thermoaceticum, whereas C. acidiurici ferments purines to a m m o n i a , CO2, and acetate. M o l y b d e n u m m a y replace tungsten in C. formicoaceticum, but it has little effect on the level of the formate d e h y d r o g e n a s e in C. acidiurici. Finally, tungsten has been implicated in the m e t a b o l i s m of M e t h a n o c o c c u s vannielii. 2o,2~ E x a m p l e s of selenium e n z y m e s other than the formate dehydrogenases from C. thermoaceticum, C. f o r m i c o a c e t i c u m , and C. acidiurici are formate d e h y d r o g e n a s e from Escherichia coli, 22 a protein c o m p o n e n t of glycine reductase from Clostridium sticklandii, 2~ and glutathione peroxidase from bovine blood, 24 and e r y t h r o c y t e s from rats. 25 The formate d e h y d r o g e n a s e from E. coli, judged to be 93-99% pure, has a molecular weight of a b o u t 590,000. The e n z y m e contains 4 hemes (b-type cytochrome), and a tetrameric structure has been suggested. It contains, per mole of heme, 0.95 m o l y b d e n u m , 0.96 selenium, 14 n o n h e m e iron, and 13 acid-labile sulfide. Tungsten, when included in the growth medium, acts antagonistically toward m o l y b d e n u m in the formation of formate d e h y d r o g e n a s e in E. coli. Radioactive 75Se-selenite is incorporated into glutathione p e r o x i d a s e from rat erythrocytes. The e n z y m e has been purified from bovine blood and crystallized. N e u t r o n activation analysis showed that it contains 4 Se per mole of e n z y m e , which is a t e t r a m e r with a molecular weight of 84,000. 18j. R. Andreesen, E. El Ghazzawi, and G. Gottschalk, Arch. Microbiol. 96, 103 (1974). 1~R. Wagner and J. R. Andreesen, Arch. Microbiol. 114, 219 (1977). 20j. B. Jones and T. C. Stadtman, in -Symposium on Microbial Production and Utilization of Gases (H2, CH4, CO)" (H. G. Schlegel, G. Gottschalk, and N. Pfenning, eds.), p. 199. E. Goltze Verlag, Gottingen, German Federal Republic, 1976. 21j. B. Jones and T. C. Stadtman, J. Bacteriol. 130, 1404 (1977). 22H. G. Enoch and R. L. Lester. J. Biol. Chem. 250, 6693 (1975). 23D. C. Turner and T. C. Stadtman, Arch. Biochem. Biophys. 154, 366 (1973). 24L. Flohe, W. A. Giinzler, and H. H. Schock, FEBS Lett. 32, 132 (1973). 25j. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Science 179, 588 (1973).