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Arch. Microbiol. 119, 215-218 (1978)

9 by Springer-Verlag 1978

Function of Fumarate Reductase in Methanogenic Bacteria (Methanobacterium) Georg Fuchs, Erhard Stupperich and Rudolf K. Thauer Fachbereich Biologie- Mikrobiologieder Philipps-Universit/it,Lahnberge,D-3550 Marburg, Federal Republicof Germany

Abstract. Methanogenic bacteria contain high activities

of fumarate reductase. An interesting hypothesis has recently been advanced that this enzyme, in cooperation with a succinate dehydrogenase, functions in a fumarate-succinate cycle for ATP synthesis. This hypothesis was tested by determining whether [2,3-3H] succinate loses 3H when taken up by growing cells. Methanobacterium thermoautotrophicum was grown on H 2 plus C O 2 in the presence of [U-14C, 2,3-3H] succinate. The double labelled dicarboxylic acid was found to be incorporated into cell material with the loss of only 30 % of tritium. Neither was 3H released into H20 in significant amounts. This finding excludes a catabolic oxidation of succinate to fumarate in the growing cells and thus the operation of a fumaratesuccinate cycle. It is shown that the function of fumarate reductase in M. thermoautotrophieum is to provide the cells with succinate for the synthesis of 0~ketoglutarate, an intermediate in glutamate, arginine and proline synthesis. Key words: Methanobacterium therrnoautotrophicum ATP synthesis - Fumarate reductase - Menaquinone - Cytochrome b - Succinate dehydrogenase - aKetoglutarate synthesis - Succinate incorporation Fumarate incorporation.

Methanogenic bacteria are strict anaerobic organisms (Wolfe, 1971; Taylor, 1975; Zeikus, 1977; Mah et al., 1977). They can grow on H 2 and CO 2 as sole energy source: 4H2 + CO2 ~ CH4 + 2 H 2 0 AG O, = -131 kJ/mol. CO 2 reduction to methane is coupled with the synthesis of ATP from ADP and inorganic phosphate. The mechanism of ATP synthesis is still unknown. It is generally assumed, however, that in methanogenic bacteria ATP is formed via electron transport phosphorylation since no mechanisms are known for substrate

level phosphorylation coupled to the oxidation of H 2 (Thauer et al., 1977). The only system which has been directly shown to yield ATP by electron transport phosphorylation in chemotrophic anaerobes is the fumarate reductase system (Kr6ger, 1975, 1977b). It is composed of specific dehydrogenases, electron carriers (menaquinone and cytochromes b) and fumarate reductase, and mediates the reduction of fumarate to succinate by various electron donors; the components of the system are all membrane bound. The fumarate reductase system is involved in ATP production in e.g. species of Bacteroides, Clostridium, Desulfovibrio, Desulfuromonas, Escherichia, Haemophilus, Propionibacterium, Proteus, Veillonella, and Vibrio (for literature see Thauer et al., 1977). As a unifying concept, the interesting hypothesis has recently been advanced that ATP synthesis in methanogenic bacteria might also be coupled to the reduction of fumarate (Gottschalk et al., 1977; see also Grossmann and Postgate, 1955). Since these organisms grow o n H 2 plus CO 2 and do not produce succinate it was proposed that methanogenic bacteria operate a fumarate-succinate cycle (Fig. 1). In agreement with the hypothesis, high activities of fumarate reductase have been found in methanogenic bacteria; also succinate dehydrogenase activity could be detected in M. ruminantiurn and Methanobacterium M. O. H. (Gottschalk et al., 1977). In the following investigation experiments with Methanobacterium thermoautotrophicum have been performed to prove or disprove the operation of the proposed fumarate-succinate cycle in methanogenic bacteria.

Materials and Methods

Gases and a gas mixture of H2/CO2 (80 %/20%) were obtained from Messer Griesheim (Diisseldort). [1,4-14C] succinic acid

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Arch. Microbiol., Vol. 119 (1978) c92

25 mg protein was approximately 10 pmol aspartate and 20 pmol glutamate. The isolated amino acids were quantitated by using the ninhydrin method in the modification of Stegemann (1960). The amino acid composition of the protein was determined by amino acid analysis in a Beckman Multichrom C Modell 4255 amino acid analyser.

PartialDegradation of Glutamate. Glutamate was decarboxylated at r

Fig. 1. A tentative scheme of ATP formation in methanogenic bacteria (according to Gottschalk et al., 1977). For thermodynamic reasons the fumarate-succinate cycle cannot be coupled to every CO2 reduction step. Succinate can function as hydrogen donor only in the last step of methane formation. Fumarate/succinate: E ~ = + 33 mV; CHaOH/CH4: E ~ = + 165 mV; CH20/CHaOH: E ~ = -185 mV; HCOOH/CHzO: E ~ --540 mV; CO2/HCOOH: E ~ = - 430 mV

(45 mCi/mmol), [2,3-14C] succinic acid (17.5 mCi/mmol), [2,3-14C1 fumaric acid (38.1 mCi/mmol), and [2, 3-3H] succinic acid (prepared by catalytic reduction of fumaric acid with tritium gas; TR 3/459; 50 mCi/25 ml; > 100 mCi/mrnol) were from Amersham Buchler GmbH (Braunschweig). Tritiated water (2.48 x 106 dpm/g) and [14C]-toluene (1.118 x 106 dpm/g) as standards were from Packard Instruments (Zfirich).

GrowthofM. thermoautotrophieum.The bacterium was isolated from the anaerobic sewage digestor in Marburg (West Germany) using the roll tube technique (Hungate, 1969; Zeikus and Wolfe, 1972). The Marburg strain was enriched, isolated and grown at 65 ~ C on a gas mixture of 80 ~ H2/20 ~ CO2/0.3 ~ H2S in an exclusively mineral salts medium at pH 6.5 (Fuchs and Stupperich, 1978). For the experiments described here the autotrophic organism was cultivated in a small fermentor on 400 ml of the mineral salts medium supplemented with either 1 mM [U-14C] succinate (0.1 mCi/ 0.4retool), or I mM [2,3-~4C] fumarate (0.05 mCi/0.4 retool), or 1 mM [U-14C] succinate (0.04 mCi/0.4 retool) plus [2, 3-aH] succinate (0.067 mCi/0.4 mmol). The pH of the weakly buffered medium was kept constant at pH 6.5 by automatic titration with 1 N NHa. When the uptake of fumarate was studied the culture was allowed to drop to more acid pH values (see Fuchs and Stupperich, 1978) (due to NH a uptake during growth) to facilitate fumaric acid uptake (succinic acid: pK1 = 4.3; pK 2 = 5.6; fumaric acid: pK1 = 3.0; pK2 = 4.4). The gassing rate was 250 ml/min. A 5 ~ inoculum of an exponentially growing culture was used. Bacterial growth was determined by measuring the absorbance difference at 578 rim. Samples of the culture were diluted I :4 with water and measured in a cuvette (d = 1 cm) in a Zeiss PL 4 spectrophotometer against a 1:4 diluted medium blank. The organism grew exponentially with a doubling time of 2.5 h. At the end of exponential growth the cells were harvested by eentrifugation at 25,000 x g for 15 rain, washed twice in 50 ml cold medium, weighed, and stored at -80~ 4 - 6 g cells (wet weight) were obtained per liter culture (4 g of wet cells were equivalent to approximately 1 g dry cells). Isolation of Aspartate and Glutamate. Protein was isolated from 1 g cells as described previously (Fuchs et al., 1978). 25 mg samples of the protein fraction were hydrolysed in 1 ml of 6 N HCI under nitrogen gas phase at 110 ~ C for 24 h. The hydrolysate was flash-evaporated almost to dryness, dissolved in 1 ml distilled water, flash-evaporated again and redissolved in 1 ml distilled water. The pH was adjusted to pH 5.3 with 1 N of LiOH. The precipitated, brown material was removed by centrifugation. Aspartate and glutamate were isolated by ionic exchange chromatography (Fuchs et al., 1978). The yield per

C-I by Chloramin T (Hoare, 1963), and at C-5 by Schmidt degradation (Simon and Floss, 1967). The CO2 formed was deter. mined gaschromatographically, 14CO2 was quantitated by liquid scintillation counting after absorption in 0.2 N KOH (Fuchs et al., 1978).

Separationof3HzO and[14C,3I-1]Succinate.0.5 ml supernatant of the [U-14C; 2,3-3H] succinate grown culture was applied to a small column filled with 1 g (wet weight) Dowex 1 x 8, acetate, 100/200 mesh, and washed with distilled water, aH20 was completely recovered in the pass-through plus 2 ml H20; succinate was not eluted under these conditions.

Determination of Radioactivity, The radioactivity of cells, protein, amino acids, CO 2, and water was determined by liquid scintillation counting in 15 ml Bray solution (Bray, 1960) (Liquid scintillation counter Tri-Carb Model 2425, Packard Instruments). [14C] toluene and [3H] water were used as internal standards. Cells and protein were dissolved in Soluene TM 100 sample solubilizer (Packard Instruments, Zfirich) prior to counting,

Determination of Fumarate )~eduetask, Quinones, and Cytoehrome b. Cell extracts were prepared and fumarate reductase activity was determined as described byZeikus et al. (1977). One unit of fumarate reductase activity corresponds to 2 ~mol of reduced methyl viologen oxidized by fumarate per min. The cells were analysed for quinones and cytochrome b as described by Krrger (in press) and Krrger and Innerhofer (1976). Soluble protein was determined by the method of Bradford (1976).

Results R e c e n t l y it was s h o w n t h a t Methanobacterium thermoautotrophicum t a k e s u p s i g n i f i c a n t a m o u n t s o f succ i n a t e w h e n g r o w n at p H 6.5 o n H E p l u s C O 2 in the presence of 1 mM [14C] s u c c i n a t e ( F u c h s a n d S t u p p e r i c h , 1978). T h e s u c c i n a t e was specifically incorporated via succinyl CoA and ~-ketoglutarate into glutamate, arginine and proline. Based on these finding the following experiments were performed:

[u-Xr Cells

2,3-3H] Succinate Incorporation by Growing

M. thermoautotrophicum was grown at pH 6.5 on H z plus CO2 in the presence of I mM [U-14C, 2,3-3H] succinate. The organism grew exponentially with a doubling time of 2.5 h. The radioactivity in the culture (medium plus cells) remained constant indicating that succinate was not converted to CO 2 or CH 4. Neither did tritium exchange into HEO in significant amounts (Table 1). 14C- and 3H-incorporation into cells paralleled growth. At the end of exponential growth 7 ~tmol of succinate was incorporated into I g cells (dry weight). The cells contained 1,920 dpm 14C and 2,650 dpm 3H p e r m g d r y w e i g h t . T h e 3 H / 1 4 C r a t i o w a s by 12 ~ l o w e r t h a n t h a t o f t h e s u c c i n a t e a d d e d to t h e m e d i u m . M o s t o f t h e label ( > 80 ~ ) was f o u n d in the p r o t e i n f r a c t i o n

217

G. Fuchs et al. : ATP Synthesisin MethanogenicBacteria Table 1. Growth of Methanobacteriumthermoautotrophicumin the presence of [U-14C, 2,3-3H] succinate: Incorporation of label into cells, glutamate and H20. The culture was inoculated at t = 0 and harvested after 14 h (t = 14) at the end of exponential growth Labelled compound Succinate (t = 0) Succinate (t = 14) H20 (t = 0)

Specific radioactivity 1~C 3H dpm/gmol dpm/gmol

Ratio

220,000

370,000

1.6

220,000

370,000

1.6

from cells grown in the presence of [2,3-14C] fumarate or [U-14C] succinate Aspartate dpm/gmol

Glutamate dpm/gmol

180

4,400

100

5,640

3H/14C

160b

Fumarate grown cells Succinate grown cells

Fumarate Reductase, Menaquinone and Cytochrome b Content of Cells Cell extracts of M. thermoautotrophicum contained

H20

(t = 14) Cells (t = 14) Glutamate (t = 14) Aspartate (t = 14)

Table 2. Specificradioactivityof glutamate and of aspartate isolated

180b 1,920

2,650"

1.4

1,270

1,390

1.1

25

28

" dpm/mg b dpm/O.l ml

and, after hydrolysis of the protein, in glutamate, arginine and proline. Glutamate was isolated from the protein and the specific radioactivity of the amino acid was determined (Table 1). Glutamate was partially degraded. C-1 of glutamate was found to be unlabelled and therefore must be derived from CO2. C-5 contained approximately 20 ~ of the 14C.

[2,3-14C] Fumarate Incorporation by Growing Cells M. thermoautotrophicum was grown on H 2 plus CO 2 in the presence of 1 m M [2,3-14C] fumarate. The initial p H of the batch culture was 6.5, the p H of the culture at the end of growth was near 5 as a result of ammonium assimilation. Approximately 30 gmol of fumarate were incorporated into 1 g cells (dry weight). Most of the label was found in the protein fraction ( > 8 0 % ) . Glutamate and aspartate were isolated from the protein and the specific radioactivities of the two amino acids were determined. Only glutamate was found to be labelled (Table 2). The specific radioactivity of aspartate was only 2 ~ of that of glutamate. Glutamate was partially degraded. C-I and C-5 were unlabelled indicating that a-ketoglutarate was synthesized via reductive carboxylation of succinyl CoA.

Glutamate, Arginine and Proline Content of Cells Ceils of M. thermoautotrophicum (protein fraction) were hydrolyzed with 6 N HC1 at 110 ~ C for 24 h. The hydrolysate from 1 g cells (dry weight) was found to contain approximately 0,6 mmol glutamate, 0.2 mmol arginine and 0.2 mmol proline.

360 m-units of fumarate reductase activity per mg protein at 60~ Essentially all the activity was found in the 100,000 g supernatant. The cells were extracted with petroleum ether for the determination of quinones. The difference spectra (reduced versus oxidized) did not indicate the presence of menaquinone, ubiquinone, or other lipid soluble redox components in significant amounts. Cytochrome b was looked for by difference spectroscopy but not detected. Discussion

It was found that low amounts of succinate and fumarate were taken up by Methanobacterium thermoautotrophicum when growing at a pH of 6.5 and lower, 7 ~tmol to 30 gmol per g cells (dry weight). The data obtained, however, are highly significant as shown by the specific incorporation o f the two dicarboxylic acids into the amino acids derived from e-ketoglutarate and into C-2 to C-5 of e-ketoglutarate ( = glutamate). Published growth yields indicate that approximately 2.5 g of cells are synthesized per tool of methane formed (Roberton and Wolfe, 1970). Assuming the proposed fumarate-succinate cycle to be operative in M. thermoautotrophicum, then 0.4 mol of succinate should be oxidized to fumarate during the synthesis of 1 g cells. Since only 1 mmol of succinate is required per g cells for the synthesis of the amino acids glutamate, arginine and proline, the fumarate succinate cycle would turn over 400 times for every succinate incorporated into ~-ketoglutarate. As a consequence, [ 2 , 3 - 3 H ] labelled succinate should be incorporated into e-ketoglutarate ( = glutamate) with the loss of more than 99 % of 3H, even assuming a large isotope effect to occur (R6tey et al., 1970; Bentley, 1970). The finding that the 3H/14C ratio of glutamate isolated from [U-14C, 2, 3-3H] succinate grown cells was only by 30% lower than that of the added succinate thus excludes the operation of the proposed fumaratesuccinate cycle in M. thermoautotrophicum.

218

The 30 ~ loss of tritium during synthesis of glutamate from [2,3-3H] succinate can be explained by a fumarate reductase catalyzed exchange of hydrogen between succinate and H~O (Rbtey et al., 1970; Krezdorn et al., 1977). Also, an exchange of hydrogen between hydrogen of ~-ketoglutarate and water may have occurred (Simon and Floss, 1967). It was confirmed that M. thermoautotrophicum contains high activities of fumarate reductase but no menaquinone and cytochrome b (Zeikus et al., 1977). The reductase appears to be non-membrane bound. These findings indicate that in M. thermoautotrophicum fumarate reductase has an anabolic rather than a catabolic function. In most anaerobic bacteria the anabolic function is to provide the cells with succinate for the synthesis of tetrapyrroles; these organisms contain only low activities of fumarate reductase. In M. thermoautotrophicum succinate is additionally required for the synthesis-of a-ketoglutarate, a precursor of glutamate, arginine and proline. In consequence, the organism must contain higher activities of fumarate reductase. The finding that [2,3-14C] fumarate was specifically incorporated into the amino acids derived from ~-ketoglutarate directly demonstrates the anabolic function of fumarate reductase. [U-14C] fumarate was incorporated into c~ketoglutarate (--glutamate) rather than into oxaloacetate ( = aspartate) by growing cells of M. thermoautotrophicum. Since in the methanogen oxaloacetate is converted to fumarate via malate dehydrogenase and fumarase (Zeikus et al., 1977; Fuchs et al., 1978) at least one of the two reactions between oxaloacetate and fumarate must operate only in the direction of fumarate synthesis. This was surprising as the malate dehydrogenase reaction and the fumarase reaction are generally considered to proceed reversibly. Acknowledgements. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, and by the Fonds der Chemischen Industrie, Frankfurt. We thank Prof. N. P. Wood for reading the manuscript and Dr. L6ffler for performing the amino acid analysis.

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Arch. Microbiol., Vol. 119 (1978) Gottschalk, G., Schoberth, S., Braun, K.: Energy metabolism of anaerobes growing on Cl-compounds. Abstract of the symposium "Microbial growth on Cl-eompounds", pp. 157-158, Scientific Center for Biological Research. Pushchino: USSR Academy of Sciences 1977 Grossmann, J. P., Postgate, J. R.: The metabolism of malate and certain other compounds by Desulphovibrio desulphuricans. J. Gen. Microbiol. 12, 429-445 (1955) Hoare, D. S. : The photo-assimilation of acetate by Rhodospirillum rubrum. Bioehem. J. 87, 284-301 (1963) Hungate, R. W.: A roll tube method for cultivation of strict anaerobes. In: Methods in microbiology, Vol. 3B (J. R. Norris, D. W. Ribbons, eds.), pp. 117-132. New York: Academic Press 1969 Krezdorn, E., Hrcherl, S., Simon, H.: Preparation of (S) 2methylsuccinate and (2S, 3S) [2,3-all] 2-methylsuccinate by biohydrogenation of 2-methylfumarate. Hoppe-Seyler's Z. Physiol. Chem. 358, 945-948 (1977) Krrger, A. : The electron transport-coupled phosphorylation of the anaerobic bacterium Vibrio succinogenes. In; Electron transfer chains and oxidative phosphorylation (E. Quagliariello, S. Papa, F. Palmieri, E. C. Slater, N. Siliprandi, eds.), pp. 265-270. Amsterdam: North-Holland 1975 Kr/~ger, A. : Determination of contents and redox state ofubiquinone and menaquinone. In: Methods in enzymology (S. P. Colowick, N. O. Kaplan, eds.). New York: Academic Press (in press) Krfger, A. : Phosphorylative electron transport with fumarate and nitrate as terminal hydrogen acceptors. In: Microbial energetics (B. A. Haddock, W. A. Hamilton, eds.), pp. 61 -93. Cambridge: Cambridge University Press 1977 Krfger, A., Innerhofer, A. : The function of the b cytochromes in the electron transport from formate to fumarate of Vibrio succinogenes. Eur. J. Biochem. 69, 497-506 (1976) Mah, R. A., Ward, D. M., Baresi, L., Glass, T. L.: Biogenesis of methane. Annu. Rev. Microbiol. 31, 309-341 (1977) Rrtey, J., Seibl, J., Arigoni, D., Cornforth, J. W., Ryback, G., Zeylemaker, W. P., Veeger, C. : Stereochemical studies of the exchange and abstraction of succinate hydrogen on succinate dehydrogenase. Eur. J. Biochem. 14, 232-242 (1970) Roberton, A. M., Wolfe, R. S. : Adenosine triphosphate pools in Methanobacterium. J. Bacteriol. 102, 43-51 (1970) Simon, H., Floss, H. G.: Anwendung von lsotopen in der Organischen Chemie und Biochemie, Vol. 2, pp. 23ff, pp. 50 ff, pp. 8-10. Berlin-Heidelberg-New York: Springer 1967 Stegemann, H. : Bestimmung von Aminos/iuren mit dithionitreduziertem Ninhydrin. Hoppe-Seyler's Z. Physiol. Chem. 319, 102 109 (1960) Taylor, G. T.: The formation of methane by bacteria. Process Biochem. 29-33 (1975) Thauer, R. K., Jungermann, K., Decker, K. : Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, 100-180 (1977) Wolfe, R. S.: Microbial formation of methane. Adv. Microbial Physiol. 6, 107-146 (1971) Zeikus, J. G. : The biology of methanogenic bacteria. Bact. Rev. 41, 514- 541 (1977) Zeikus, J. G., Fuchs, G., Kenealy, W., Thauer, R. K.: Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum. J. Bacteriol. 132, 604-613 (1977) Zeikus, J. G., Wolfe, R. S. : Methanobacterium thermoautotrophicum sp. n., an anaerobic autotrophic extreme thermophile. J. Bacteriol. 109, 707-713 (1972)

Received July 14, 1978