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The methyl-tetrahydromethanopterin: Coenzyme M methyltransferase of Methanosarcina strain Gö1 is a primary sodium pump
FEMS Microbiology Letters 91 (1902) 239-244 © 1992 Federation of European Microbiological Societies 11378-1097/92/$05.00 Published by Elsevier
239
FEMSLE 04808
The methyl-tetrahydromethanopterin • coenzyme M methyltransferase of Methanosarcina strain G61 is a primary sodium pump Burkhard Becher, Volker Miiiler and G e r h a r d Gottschaik hlstitut fiir Mikrobiologie tier Georg-August-Unit ersitiit. (;i~ttingen. F.R.G. Received 27 December It,~91 Accepted 6 JanuaD' I992
Key words: Methanogenic bacteria; Formaldehyde conversion; Sodium pump; Methyl-tetrahydromethanopterin :coenzyme M methyltransferase; Methanosarcina
1. SUMMARY Washed inverted vesicle preparations of Methanosarchla strain G61 catalyzed the formation of methyI-CoM from formaldehyde, H, and CoM in the presence of tetrahydromethanopterin and 2-bromoethanesulfonate. The reaction was associated with the translocadon of sodium ions into the lumen of the vesicles. This translocation was abolished by the Na + ionophore ETH 157 but it was insensitive to the addition of the un-
coupler SF6847 and the Na+/H + ?.ntiport inhibitor amiloride and, therefore, is the result of a primary Na ~ pump. Since the translocation of Na + was also observed when formaldehyde +tetrahydromethanopterin was replaced by methy[-tetrahydromethanopterin, it follows that the methyl transfer from tetrahydromethanopterin to CoM is the sodium-motive reaction. Methyl-tetrahydromethanopterin could be replaced by methyl-tetrahydrofolate.
2. INTRODUCTION Correspondence to: G. Gottschalk. lnstitut fiir Mikrobiologie tier Georg-August-Universitfit, Grisebachstr. 8, W-3400 G6ttingen, F.R.G. Abbreviations: Amiloride. 3,5-diamino-6-chlorooyrazinoylguanidine; F4,,~, the (N-L-lactyl-y-t.-glutamyl)-t.-glutamic acid phosphodiester of 7.8-didemethyl-8-hydroxy-5-deazariboflavin 5' phosphate; CoM, 2-mercaptoethanesulfonate: DTE, dithioerythritol; SF6847, 3,5-Di-tert-butyl-4-hydroxybenzylidenemalonitrile; ETH 157, N,N'-dibenzyI-N,N'-diphenyl-phenylenedioxydiaeetamide: H4MPT, tetrahydromethanopterin: H4F, tetrahydrofolate; heterodisulfide, disulfide of CoM and 7-mercaptoheptanoylthreonine phosphate; methyl-CoM, 2(methyltbio)ethanesulfonate.
In methanogenic bacteria the path of CO, reduction to CH 4 is coupled to a primary proton pump as well as a sodium pump. The proton translocation system has been characterized as a heterodisulfide reductase which is coupled either to H, or to F42oH2 as electron donors [1,2]. Little is known about the reactions leading to the generation of the primary electrochemical sodium gradient [3]. Using whole cells of different methanogenic bacteria it was shown that the con-
240 version of formaldehyde to the formal redox level of methanol is coupled to a primary sodium ion translocation [4,5]. The enzymes involved in this conversion are methylene-H4MPT reductase and methyl-H4MPT: HS-CoM methyltransferase; they have been localized in considerable amounts at the cytoplasmic membrane of strain G61 (Becher, B., Miiller, V. and Gottschalk, G., unpublished). Using an inverted vesicle system of this organism, we demonstrate here for the first time a substrate-dependent 2-'Na+ translocation in vitro and that the generation of the primary sodium gradient is catalyzed by the methylH4MPT : CoM methyltransferase.
3. MATERIALS AND METHODS
.t. 1. Organism and growth conditions Methanosarchla strain G61 (DSM 3647) was grown on methanol (final concentration, 150 mM) in the medium described by Hippe et al. [6] supplemented with 1 g/I of sodium acetate.
.t.9_. Membrane preparation Harvesting of ceils and preparation of membrane vesicles were done as described before [7].
3.3. Enzyme assays Methylene-tetrahydromethanopterin reductase was determined in 40 mM potassium phosphate, pH 6.9, containing 20 mM MgSO4, 0.4 M sucrose and 5 mM DTE by monitoring the H4MPT-dependent oxidation of F4,_,H_, spectrophotometrically at 420 nm (E4,0= 32 mM - l . c m -I at pH 7.0).
3.4. l:~'perimental conditions The experiments were performed at 37°C in 2.3 ml stoppered glass tubes containing threetimes washed vesicles diluted in 40 mM potassium phosphate buffer, pH 6.9, containing 20 mM MgSO4, 0.4 M sucrose, 5 mM DTE, 5 mM 2bromoethanesulfonate and 1 mg resazurine/I under an atmosphere of molecular hydrogen. The substratcs formaldehyde, methyl-H4MPT and methyl-H4F as well as F420 and H4MPT were added as specified in the figure legends.
Formaldehyde [8] and CoM [9] were determined as described.
3.5. Preparation of H4MPT, methyI-H4MPT and F420 H4MPT [10] and F420 [11] were isolated as described. MethyI-H4MPT was prepared by chemical reduction of methylene-H4MPT with sodium borohydride at pH 8.0 under an atmosphere of N 2. KBH 4 was added stepwise until methylene-H4MPT was completely reduced as monitored spectrophotometrically (Am~,x methylH4MPT: 295, 238 and 218 nm). After acidification of the solution, the supernatant was carefully removed and the pH was adjusted to 7.0. The methyl-H4MPT solution was stored at 4°C under N 2.
3.6. Measurement of sodium transport The experiments were done in the buffer system described above containing in addition 2 mM 22NaCI (0.65/xCi/ttmoi) in a final volume of 350 p.I under an atmosphere of molecular hydrogen. After addition of vesicles the suspension was incubated for 20 min to assure equilibration of -'2Na+ before the reaction was started by the addition of the substrates. Samples of 30/.d were withdrawn and passed over a small column (0.5 × 3.2 cm) of Dowex 50 WX8, K ÷ (100-200 mesh). Tile vesicles were collected by washing the column with 1 ml of a 400 mM sucrose solution. After the addition of 10 ml Rialuma (J.T. Baker B.V., Deventer, The Netherlands) the radioactivity in the eluate was determined in a liquid scintillation counter (model PW 4700, Philips, Hamburg, F.R.G.). Free 22Na+ was retained by more than 99.9% by the resin. The intravesicular Na ÷concentration was calculated after correction for Na ÷ not retained using an intracellular volume of 3/.tl/mg protein.
4. RESULTS Washed inverted vesicles of Methanosarcina strain G61 incubated in the presence of H4MPT and CoM converted formaldehyde + H 2 to methyi-CoM when methanogenesis was inhibited
241 by the addition of 2-bromoethanesulfonate (data not shown). Upon the addition of formaldehyde, CoM was consumed with a rate of 86 nmolm i n - i , mg p r o t e i n - t; concomitantly, -'2Na+ was actively transported into the lumen of the vesicles with a rate of 391 n m o l . m i n - I. mg p r o t e i n - I. This resulted in a 2.5-fold accumulation of Na + as compared to !he medium (Fig. I). Sodium transport was completely abolished by the Na + ionophore ETH 157 and the addition of ETH 157 to a pre-existing sodium gradient resulted in its collapse. The extent o f Na + accumulation was increased by the addition of amiloride, an inhibitor o f the N a + / H + antiporter present in G61 (M~ller, V. and Gottschalk, G., unpublished), and by the uncoupler SF6847. These results are
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Fig. 2. CoM-dependent sodium transport as catalyzed by inverted vesicles of Methano.~arcina strain G(il during the conversion of formaldehyde+ H 2 + CoM to methyl-CoM. Washed vesicle suspensions (I.6 mg pmtein/ml)containing 211 ,u.M 1:42o. 40 aM tl4MPT and Ill #M SF6847 were incubated in the absencc( • ) or presence (o) of 411/.tM CoM. Sodium transport was initiated by addition of formaldehyde (final concentration. 40 p.M) at the time indicated by the open arrow. One vesicle suspension ( • ) received CoM at the time indicated by the clnsed arrow.
4
0
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10 20 Time ~,min) Fig. 1. Sodium transport as catalyzed by inverted vesicles of Methanosarc#za strain Gi~i during the conversionof formaldebyde+Hz+CoM to methyI-CoM and effect of inhibitors. Washed vesicle suspensions (I.7 mg protein/ml) containing 20/zM Fa2o, 40 p,M H4MPT and 6 mM CoM were preincubaled for 20 min in the presence of 10 #M SF6847 (o). 50(1 nmol amiloride.mg protein-t (o), 4 nmol ETH 157.mg protein- ~ ( • ) or in the absence of inhibitors ( I , 13). At the time indicated by the open arrow, sodium transport was started by addition of formaldehyde to a final concentration of 6 mM. One vesicle suspension (t3) received ETH 157 at the time indicated by the closed arrow.
in accordance with a primary, electrogenic sodium ion translocation in the course of formaldehyde conversion to methyI-CoM via methylene- and methyI-H4MPT. In order to identify the reaction responsible for Na + translocation, further experiments were performed. In the presence of 40 # M formaldehyde and 4 0 / ~ M H 4 M P T but in the absence of CoM, the methylene-H-4 MPT reductase exhibited an activity of 82 n m o l . m i n - n , mg protein-n; although this reaction proceeded for several minutes, no sodium ion translocation was observed. In the presence of CoM (final concentration, 40 p,M), the reductase activity was not altered but methyI-CoM was produced and, most importantly, -'2Na+ transport was restored (Fig. 2). These experiments indicate that the methyltransferase might be the site of Na + translocation.
242 10
5. D I S C U S S I O N
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Fig. 3. MethyI-H4MPT or methyI-H4F-dependent Na 4 transport as catalyzed by inverted vesicles of Methanosarcinastrain G61. Washed vesicle suspensions (I.6 mg protein/ml) were incubated in the absence ( t2, • ) or presence (a, o) of 6 mM CoM. At the time indicated by the open arrow, sodium transport was initiated by addition of methyI-H4MPT (m, o) or methyI-H4F (o) to final concentrations of 21)0v.M and 1.5 mM. respectively. One vesicle suspension ( • ) received 6 mM CoM at the time indicated by the closed arrow. One vesicle suspension ([]) did not receive any substrate.
This assumption was confirmed by adding the ultimate substrate of the methyltransferase, methyI-H4MPT, to the Na ÷ translocation assay. U p o n addition of m e t h y I - H 4 M P T to washed vesicles incubated in the presence of CoM, a disappearance of CoM was observed at a rate of 98 nmol • m i n - ~ • m g p r o t e i n - ~; concomitantly, 22Na÷ was transported into the lumen generating a t r a n s m e m b r a n e sodium gradient of 1 : 5 (Fig. 3); in the absence of CoM, addition of methylH 4 M P T did not lead to a sodium transport but it was restored by addition of CoM as the methylgroup acceptor. Interestingly, methyi-tetrahydrofolate also functioned as a substrate for Na ÷ transloeation but the rate of CoM disappearance (43 n m o l - min -~ • mg protein -~) as well as the rate of 22Na+ transport and the accumulation factor was decreased (Fig. 3). This reaction was also d e p e n d e n t on the presence of CoM.
It was not before 1981 that the r e q u i r e m e n t of methanogenesis for sodium ions was discovered [12]. A first clue as to the role of Na ÷ was the observation that during methanogenesis from methanol as carried out by Methanosarcina barkeri the oxidation of methanol to the formal redox level of formaldehyde was N a + - d e p e n d e n t , in fact was driven by the electrochemical sodium ion gradient [13]. Later on it was shown that this reaction is reversible and that the reduction of a C r i n t e r m e d i a t e of the redox level of formaldehyde to that of methanol was coupled with Na + extrusion [4]. Thus, evidence was provided that during C O 2 reduction to m e t h a n e two different ion pumps operate in m e t h a n o g e n i c bacteria, a sodium p u m p in addition to the proton p u m p which recently was identified as heterodisulfide reductase [1]. The reduction of the C l-intermediate of the formal redox level of formaldehyde to the one of methanol requires the action of at least two enzymes as can be seen from Fig. 4. MethyleneH 4 M P T is first reduced to methyl-HaMPT. We originally considered this reaction as being responsible for Na + translocation. However, the corresponding reductase was purified to homogeneity from various organisms [14-16]: its localization in the cytoplasm and the low A G o' value of - 5 . 2 k J / m o l with F42oH 2 as reductant argued against a participation of this enzyme in energy transduction. T h e second reaction is a methyl group transfer reaction which actually may be catalyzed by two enzymes: a m e t h y I - H 4 M P T : hydroxybenzimidazolyl cobamide methyltransferase and a methyl-hydroxybenzimidazolyl cobamide: CoM methyltransferase. T h e methyltransferase activities are found in considerable amounts at the cytoplasmic m e m b r a n e of strain G61 (Becher, B., Miiller, V. and Gottschalk, G., unpublished); furthermore, an immunological cross reactivity of a polyclonal antibody against a m e m b r a n e - b o u n d corrinoid protein of unknown function from Mett~anobacterium thermoautotrophicum strain Marburg with a m e t h y I - H 4 M P T : hydroxybenzimidazolyl cobamide transferase isolated from the soluble fraction of Methanobacterium ther-
243 B 6 r n e r a n d R.K. T h a u c r for t h e i r h e l p f u l advice in isolating H 4 M P T . T h i s w o r k w a s s u p p o r t e d by a grant from the Deutsche Forsehungsgemeinschaft.
(.,.- ] 14MP'F Methylene-114Mlrl"
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-~. t7420 MelhyI-II4MPT
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{BI2_IIBI I
REFERENCES
[14Mlrf
•
[I] Dcppenmeier,
IMethyl-B12-1IBII 3~
IIS-CoM IBt2-IIBII
I MethyI-S-CoM I Fig. 4. Reactions involved in the conversion of methylcneH4MPT to methyI-CoM and the possible site of Na * translocation. [Bt2-1tBI], enzyme-bound 5-hydroxybenzimidazolyleobamide; [MethyI-Bi2-HBl]. enzyme-bound methyl-5-hydroxybenzimidazolyleobamide; 1. mcthylene-ll4MPT reductase: 2, methyI-H4MPT:BIz-HBI methyltransferase: 3. BIztlBI : CoM methyltransferase.
moautotrophicum s t r a i n A H i n d i c a t e t h a t t h e corrinoid p r o t e i n m i g h t f u n c t i o n as a m e m b r a n e b o u n d m e t h y l t r a n s f e r a s e [17]. F r o m t h e r e s u l t s p r e s e n t e d in this p u b l i c a t i o n it is n o w clear t h a t this m e t h y l t r a n s f e r a s e r e a c tion is t h e driving force for N a + t r a n s l o c a t i o n . So far p r i m a r y N a + p u m p s a r e t h e result o f d e c a r boxylation o r o f r e d o x r e a c t i o n s [18,19]; t h e Na + p u m p o f m e t h a n o g e n i c b a c t e r i a r e p r e s e n t s a novel s y s t e m b u t it is c o m p l e t e l y u n k n o w n yet h o w a m e t h y l - g r o u p t r a n s f e r c a n be m e c h a n i s t i c a l l y c o u p l e d to s u c h an ion t r a n s l o c a t i o n r e a c t i o n .
ACKNOWLEDGEMENTS T h e e x p e r t t e c h n i c a l a s s i s t a n c e o f S. B o w i e n is g r a t e f u l l y a c k n o w l e d g e d . W e a r e i n d e b t e d to G.
U.. Bhtut,
M., Mahlmann,
A. and
Gottschalk, (i. (1991)) Proc. Natl. Acad. Sci. USA 87. 9449-9453. [2] Deppenmeicr. U.. Blaut, M. and Gottschalk. G. (1991) Arch. Microbiol. 155, 272-277. [3] Miillcr. V.. Blaut. M., lleise, R.. Winner. C. and Gottschalk, G. ( ltlt)(O FEMS Microbiol. Rev. 87, 373-377. [4] Miillcr. V.. Winner, C. and Gonschalk, G. (1989) Eur. J. Biochem. 178. 51t~-525. [51 Kaesler, B. and Sch~inheit. P. (1989) Eur. J. Biochem. 184, 223-232. [61 ilippe, I-I., Caspari. D.. Fiebig. K. and Gouschalk. G. (1979) Proc. Natl. Acad. Sci. USA. 76, 494-498. [7] Deppcnmeicr, U., Blaut, M. and Gonsehalk, G. (1989) Eur. J. Biochcm. 186. 317-323. [8] Nash. T. (1953) Biochem. J. 55. 416-421. [91 EIIman, G.L. (1958) Arch. Biochem. Biophys. 74. 44345(I. [10] Biirner, G. (1988) Diploma thesis. Philipps-Universit:,it Marburg. [11] Deppenmeier. U., Blaut. M.. Mahlmann. A. and Gottschalk, G. (199(I) FEBS Len. 261, 199-203. [12] Pcrski, lI.J.. Moll, J. and Thaucr, R.K. (1981) Arch. Microbiol. 130, 319-321. [13] Miiller, V.. Blaut, M. and Gottschalk, G. (1988) Eur. J. Biochem. 172. 601-606. 1i4] tcBriimmelstroct. B.W.. tlcnsgens. C.M.H., Keltjens. J.T.. van der Drift. C. and Vogels, G.D. (1990) J. Biol. Chem. 265, 185-187. [15] Ma, K. and Thaucr, R.K. (19911) Eur. J. Biochem. 191. 187-193. [16] Ma, K. and Thaucr, R.K. (1990) FEMS Microbiol. Lett. 7(1, 119-124. [17] Stupperich, E.. Juza, A., Eckerskorn. C. and Edelmann, L. (1990) Arch. Microbiol. 155, 28-34. [18] Dimroth, P. (1987) Microbiol. Rev. 5 I. 32(I-34(I. [19] Tokuda, I-I. and Unemoto, T. (1985) Microbiol. Sci. 2, 65-71.
239
FEMSLE 04808
The methyl-tetrahydromethanopterin • coenzyme M methyltransferase of Methanosarcina strain G61 is a primary sodium pump Burkhard Becher, Volker Miiiler and G e r h a r d Gottschaik hlstitut fiir Mikrobiologie tier Georg-August-Unit ersitiit. (;i~ttingen. F.R.G. Received 27 December It,~91 Accepted 6 JanuaD' I992
Key words: Methanogenic bacteria; Formaldehyde conversion; Sodium pump; Methyl-tetrahydromethanopterin :coenzyme M methyltransferase; Methanosarcina
1. SUMMARY Washed inverted vesicle preparations of Methanosarchla strain G61 catalyzed the formation of methyI-CoM from formaldehyde, H, and CoM in the presence of tetrahydromethanopterin and 2-bromoethanesulfonate. The reaction was associated with the translocadon of sodium ions into the lumen of the vesicles. This translocation was abolished by the Na + ionophore ETH 157 but it was insensitive to the addition of the un-
coupler SF6847 and the Na+/H + ?.ntiport inhibitor amiloride and, therefore, is the result of a primary Na ~ pump. Since the translocation of Na + was also observed when formaldehyde +tetrahydromethanopterin was replaced by methy[-tetrahydromethanopterin, it follows that the methyl transfer from tetrahydromethanopterin to CoM is the sodium-motive reaction. Methyl-tetrahydromethanopterin could be replaced by methyl-tetrahydrofolate.
2. INTRODUCTION Correspondence to: G. Gottschalk. lnstitut fiir Mikrobiologie tier Georg-August-Universitfit, Grisebachstr. 8, W-3400 G6ttingen, F.R.G. Abbreviations: Amiloride. 3,5-diamino-6-chlorooyrazinoylguanidine; F4,,~, the (N-L-lactyl-y-t.-glutamyl)-t.-glutamic acid phosphodiester of 7.8-didemethyl-8-hydroxy-5-deazariboflavin 5' phosphate; CoM, 2-mercaptoethanesulfonate: DTE, dithioerythritol; SF6847, 3,5-Di-tert-butyl-4-hydroxybenzylidenemalonitrile; ETH 157, N,N'-dibenzyI-N,N'-diphenyl-phenylenedioxydiaeetamide: H4MPT, tetrahydromethanopterin: H4F, tetrahydrofolate; heterodisulfide, disulfide of CoM and 7-mercaptoheptanoylthreonine phosphate; methyl-CoM, 2(methyltbio)ethanesulfonate.
In methanogenic bacteria the path of CO, reduction to CH 4 is coupled to a primary proton pump as well as a sodium pump. The proton translocation system has been characterized as a heterodisulfide reductase which is coupled either to H, or to F42oH2 as electron donors [1,2]. Little is known about the reactions leading to the generation of the primary electrochemical sodium gradient [3]. Using whole cells of different methanogenic bacteria it was shown that the con-
240 version of formaldehyde to the formal redox level of methanol is coupled to a primary sodium ion translocation [4,5]. The enzymes involved in this conversion are methylene-H4MPT reductase and methyl-H4MPT: HS-CoM methyltransferase; they have been localized in considerable amounts at the cytoplasmic membrane of strain G61 (Becher, B., Miiller, V. and Gottschalk, G., unpublished). Using an inverted vesicle system of this organism, we demonstrate here for the first time a substrate-dependent 2-'Na+ translocation in vitro and that the generation of the primary sodium gradient is catalyzed by the methylH4MPT : CoM methyltransferase.
3. MATERIALS AND METHODS
.t. 1. Organism and growth conditions Methanosarchla strain G61 (DSM 3647) was grown on methanol (final concentration, 150 mM) in the medium described by Hippe et al. [6] supplemented with 1 g/I of sodium acetate.
.t.9_. Membrane preparation Harvesting of ceils and preparation of membrane vesicles were done as described before [7].
3.3. Enzyme assays Methylene-tetrahydromethanopterin reductase was determined in 40 mM potassium phosphate, pH 6.9, containing 20 mM MgSO4, 0.4 M sucrose and 5 mM DTE by monitoring the H4MPT-dependent oxidation of F4,_,H_, spectrophotometrically at 420 nm (E4,0= 32 mM - l . c m -I at pH 7.0).
3.4. l:~'perimental conditions The experiments were performed at 37°C in 2.3 ml stoppered glass tubes containing threetimes washed vesicles diluted in 40 mM potassium phosphate buffer, pH 6.9, containing 20 mM MgSO4, 0.4 M sucrose, 5 mM DTE, 5 mM 2bromoethanesulfonate and 1 mg resazurine/I under an atmosphere of molecular hydrogen. The substratcs formaldehyde, methyl-H4MPT and methyl-H4F as well as F420 and H4MPT were added as specified in the figure legends.
Formaldehyde [8] and CoM [9] were determined as described.
3.5. Preparation of H4MPT, methyI-H4MPT and F420 H4MPT [10] and F420 [11] were isolated as described. MethyI-H4MPT was prepared by chemical reduction of methylene-H4MPT with sodium borohydride at pH 8.0 under an atmosphere of N 2. KBH 4 was added stepwise until methylene-H4MPT was completely reduced as monitored spectrophotometrically (Am~,x methylH4MPT: 295, 238 and 218 nm). After acidification of the solution, the supernatant was carefully removed and the pH was adjusted to 7.0. The methyl-H4MPT solution was stored at 4°C under N 2.
3.6. Measurement of sodium transport The experiments were done in the buffer system described above containing in addition 2 mM 22NaCI (0.65/xCi/ttmoi) in a final volume of 350 p.I under an atmosphere of molecular hydrogen. After addition of vesicles the suspension was incubated for 20 min to assure equilibration of -'2Na+ before the reaction was started by the addition of the substrates. Samples of 30/.d were withdrawn and passed over a small column (0.5 × 3.2 cm) of Dowex 50 WX8, K ÷ (100-200 mesh). Tile vesicles were collected by washing the column with 1 ml of a 400 mM sucrose solution. After the addition of 10 ml Rialuma (J.T. Baker B.V., Deventer, The Netherlands) the radioactivity in the eluate was determined in a liquid scintillation counter (model PW 4700, Philips, Hamburg, F.R.G.). Free 22Na+ was retained by more than 99.9% by the resin. The intravesicular Na ÷concentration was calculated after correction for Na ÷ not retained using an intracellular volume of 3/.tl/mg protein.
4. RESULTS Washed inverted vesicles of Methanosarcina strain G61 incubated in the presence of H4MPT and CoM converted formaldehyde + H 2 to methyi-CoM when methanogenesis was inhibited
241 by the addition of 2-bromoethanesulfonate (data not shown). Upon the addition of formaldehyde, CoM was consumed with a rate of 86 nmolm i n - i , mg p r o t e i n - t; concomitantly, -'2Na+ was actively transported into the lumen of the vesicles with a rate of 391 n m o l . m i n - I. mg p r o t e i n - I. This resulted in a 2.5-fold accumulation of Na + as compared to !he medium (Fig. I). Sodium transport was completely abolished by the Na + ionophore ETH 157 and the addition of ETH 157 to a pre-existing sodium gradient resulted in its collapse. The extent o f Na + accumulation was increased by the addition of amiloride, an inhibitor o f the N a + / H + antiporter present in G61 (M~ller, V. and Gottschalk, G., unpublished), and by the uncoupler SF6847. These results are
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Fig. 2. CoM-dependent sodium transport as catalyzed by inverted vesicles of Methano.~arcina strain G(il during the conversion of formaldehyde+ H 2 + CoM to methyl-CoM. Washed vesicle suspensions (I.6 mg pmtein/ml)containing 211 ,u.M 1:42o. 40 aM tl4MPT and Ill #M SF6847 were incubated in the absencc( • ) or presence (o) of 411/.tM CoM. Sodium transport was initiated by addition of formaldehyde (final concentration. 40 p.M) at the time indicated by the open arrow. One vesicle suspension ( • ) received CoM at the time indicated by the clnsed arrow.
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0
10 20 Time ~,min) Fig. 1. Sodium transport as catalyzed by inverted vesicles of Methanosarc#za strain Gi~i during the conversionof formaldebyde+Hz+CoM to methyI-CoM and effect of inhibitors. Washed vesicle suspensions (I.7 mg protein/ml) containing 20/zM Fa2o, 40 p,M H4MPT and 6 mM CoM were preincubaled for 20 min in the presence of 10 #M SF6847 (o). 50(1 nmol amiloride.mg protein-t (o), 4 nmol ETH 157.mg protein- ~ ( • ) or in the absence of inhibitors ( I , 13). At the time indicated by the open arrow, sodium transport was started by addition of formaldehyde to a final concentration of 6 mM. One vesicle suspension (t3) received ETH 157 at the time indicated by the closed arrow.
in accordance with a primary, electrogenic sodium ion translocation in the course of formaldehyde conversion to methyI-CoM via methylene- and methyI-H4MPT. In order to identify the reaction responsible for Na + translocation, further experiments were performed. In the presence of 40 # M formaldehyde and 4 0 / ~ M H 4 M P T but in the absence of CoM, the methylene-H-4 MPT reductase exhibited an activity of 82 n m o l . m i n - n , mg protein-n; although this reaction proceeded for several minutes, no sodium ion translocation was observed. In the presence of CoM (final concentration, 40 p,M), the reductase activity was not altered but methyI-CoM was produced and, most importantly, -'2Na+ transport was restored (Fig. 2). These experiments indicate that the methyltransferase might be the site of Na + translocation.
242 10
5. D I S C U S S I O N
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20
Fig. 3. MethyI-H4MPT or methyI-H4F-dependent Na 4 transport as catalyzed by inverted vesicles of Methanosarcinastrain G61. Washed vesicle suspensions (I.6 mg protein/ml) were incubated in the absence ( t2, • ) or presence (a, o) of 6 mM CoM. At the time indicated by the open arrow, sodium transport was initiated by addition of methyI-H4MPT (m, o) or methyI-H4F (o) to final concentrations of 21)0v.M and 1.5 mM. respectively. One vesicle suspension ( • ) received 6 mM CoM at the time indicated by the closed arrow. One vesicle suspension ([]) did not receive any substrate.
This assumption was confirmed by adding the ultimate substrate of the methyltransferase, methyI-H4MPT, to the Na ÷ translocation assay. U p o n addition of m e t h y I - H 4 M P T to washed vesicles incubated in the presence of CoM, a disappearance of CoM was observed at a rate of 98 nmol • m i n - ~ • m g p r o t e i n - ~; concomitantly, 22Na÷ was transported into the lumen generating a t r a n s m e m b r a n e sodium gradient of 1 : 5 (Fig. 3); in the absence of CoM, addition of methylH 4 M P T did not lead to a sodium transport but it was restored by addition of CoM as the methylgroup acceptor. Interestingly, methyi-tetrahydrofolate also functioned as a substrate for Na ÷ transloeation but the rate of CoM disappearance (43 n m o l - min -~ • mg protein -~) as well as the rate of 22Na+ transport and the accumulation factor was decreased (Fig. 3). This reaction was also d e p e n d e n t on the presence of CoM.
It was not before 1981 that the r e q u i r e m e n t of methanogenesis for sodium ions was discovered [12]. A first clue as to the role of Na ÷ was the observation that during methanogenesis from methanol as carried out by Methanosarcina barkeri the oxidation of methanol to the formal redox level of formaldehyde was N a + - d e p e n d e n t , in fact was driven by the electrochemical sodium ion gradient [13]. Later on it was shown that this reaction is reversible and that the reduction of a C r i n t e r m e d i a t e of the redox level of formaldehyde to that of methanol was coupled with Na + extrusion [4]. Thus, evidence was provided that during C O 2 reduction to m e t h a n e two different ion pumps operate in m e t h a n o g e n i c bacteria, a sodium p u m p in addition to the proton p u m p which recently was identified as heterodisulfide reductase [1]. The reduction of the C l-intermediate of the formal redox level of formaldehyde to the one of methanol requires the action of at least two enzymes as can be seen from Fig. 4. MethyleneH 4 M P T is first reduced to methyl-HaMPT. We originally considered this reaction as being responsible for Na + translocation. However, the corresponding reductase was purified to homogeneity from various organisms [14-16]: its localization in the cytoplasm and the low A G o' value of - 5 . 2 k J / m o l with F42oH 2 as reductant argued against a participation of this enzyme in energy transduction. T h e second reaction is a methyl group transfer reaction which actually may be catalyzed by two enzymes: a m e t h y I - H 4 M P T : hydroxybenzimidazolyl cobamide methyltransferase and a methyl-hydroxybenzimidazolyl cobamide: CoM methyltransferase. T h e methyltransferase activities are found in considerable amounts at the cytoplasmic m e m b r a n e of strain G61 (Becher, B., Miiller, V. and Gottschalk, G., unpublished); furthermore, an immunological cross reactivity of a polyclonal antibody against a m e m b r a n e - b o u n d corrinoid protein of unknown function from Mett~anobacterium thermoautotrophicum strain Marburg with a m e t h y I - H 4 M P T : hydroxybenzimidazolyl cobamide transferase isolated from the soluble fraction of Methanobacterium ther-
243 B 6 r n e r a n d R.K. T h a u c r for t h e i r h e l p f u l advice in isolating H 4 M P T . T h i s w o r k w a s s u p p o r t e d by a grant from the Deutsche Forsehungsgemeinschaft.
(.,.- ] 14MP'F Methylene-114Mlrl"
1:420
-~. t7420 MelhyI-II4MPT
I?? "~ 2~
AIINa+
{BI2_IIBI I
REFERENCES
[14Mlrf
•
[I] Dcppenmeier,
IMethyl-B12-1IBII 3~
IIS-CoM IBt2-IIBII
I MethyI-S-CoM I Fig. 4. Reactions involved in the conversion of methylcneH4MPT to methyI-CoM and the possible site of Na * translocation. [Bt2-1tBI], enzyme-bound 5-hydroxybenzimidazolyleobamide; [MethyI-Bi2-HBl]. enzyme-bound methyl-5-hydroxybenzimidazolyleobamide; 1. mcthylene-ll4MPT reductase: 2, methyI-H4MPT:BIz-HBI methyltransferase: 3. BIztlBI : CoM methyltransferase.
moautotrophicum s t r a i n A H i n d i c a t e t h a t t h e corrinoid p r o t e i n m i g h t f u n c t i o n as a m e m b r a n e b o u n d m e t h y l t r a n s f e r a s e [17]. F r o m t h e r e s u l t s p r e s e n t e d in this p u b l i c a t i o n it is n o w clear t h a t this m e t h y l t r a n s f e r a s e r e a c tion is t h e driving force for N a + t r a n s l o c a t i o n . So far p r i m a r y N a + p u m p s a r e t h e result o f d e c a r boxylation o r o f r e d o x r e a c t i o n s [18,19]; t h e Na + p u m p o f m e t h a n o g e n i c b a c t e r i a r e p r e s e n t s a novel s y s t e m b u t it is c o m p l e t e l y u n k n o w n yet h o w a m e t h y l - g r o u p t r a n s f e r c a n be m e c h a n i s t i c a l l y c o u p l e d to s u c h an ion t r a n s l o c a t i o n r e a c t i o n .
ACKNOWLEDGEMENTS T h e e x p e r t t e c h n i c a l a s s i s t a n c e o f S. B o w i e n is g r a t e f u l l y a c k n o w l e d g e d . W e a r e i n d e b t e d to G.
U.. Bhtut,
M., Mahlmann,
A. and
Gottschalk, (i. (1991)) Proc. Natl. Acad. Sci. USA 87. 9449-9453. [2] Deppenmeicr. U.. Blaut, M. and Gottschalk. G. (1991) Arch. Microbiol. 155, 272-277. [3] Miillcr. V.. Blaut. M., lleise, R.. Winner. C. and Gottschalk, G. ( ltlt)(O FEMS Microbiol. Rev. 87, 373-377. [4] Miillcr. V.. Winner, C. and Gonschalk, G. (1989) Eur. J. Biochem. 178. 51t~-525. [51 Kaesler, B. and Sch~inheit. P. (1989) Eur. J. Biochem. 184, 223-232. [61 ilippe, I-I., Caspari. D.. Fiebig. K. and Gouschalk. G. (1979) Proc. Natl. Acad. Sci. USA. 76, 494-498. [7] Deppcnmeicr, U., Blaut, M. and Gonsehalk, G. (1989) Eur. J. Biochcm. 186. 317-323. [8] Nash. T. (1953) Biochem. J. 55. 416-421. [91 EIIman, G.L. (1958) Arch. Biochem. Biophys. 74. 44345(I. [10] Biirner, G. (1988) Diploma thesis. Philipps-Universit:,it Marburg. [11] Deppenmeier. U., Blaut. M.. Mahlmann. A. and Gottschalk, G. (199(I) FEBS Len. 261, 199-203. [12] Pcrski, lI.J.. Moll, J. and Thaucr, R.K. (1981) Arch. Microbiol. 130, 319-321. [13] Miiller, V.. Blaut, M. and Gottschalk, G. (1988) Eur. J. Biochem. 172. 601-606. 1i4] tcBriimmelstroct. B.W.. tlcnsgens. C.M.H., Keltjens. J.T.. van der Drift. C. and Vogels, G.D. (1990) J. Biol. Chem. 265, 185-187. [15] Ma, K. and Thaucr, R.K. (19911) Eur. J. Biochem. 191. 187-193. [16] Ma, K. and Thaucr, R.K. (1990) FEMS Microbiol. Lett. 7(1, 119-124. [17] Stupperich, E.. Juza, A., Eckerskorn. C. and Edelmann, L. (1990) Arch. Microbiol. 155, 28-34. [18] Dimroth, P. (1987) Microbiol. Rev. 5 I. 32(I-34(I. [19] Tokuda, I-I. and Unemoto, T. (1985) Microbiol. Sci. 2, 65-71.