Ion Transport and Methane Production in Methanobacterium Thermoautotrophicum FRANK D

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Proc. Natl. Acad. Sci. USA Vol. 91, pp. 4466-4470, May 1994 Biochemistry Ion transport and methane production in Methanobacterium thermoautotrophicum FRANK D. SAUER*t, BARBARA A. BLACKWELLO, AND JOHN K. G. KRAMER* *Centre for Food and Animal Research and *Plant Research Centre, Research Branch, Agriculture Canada, Ottawa, ON, Canada KlA 0C6 Communicated by Ralph T. Holman, January 3, 1994 (receivedfor review April 28, 1993)

ABSTRACT In Methanobacterium thermoautotrophicum, contributed another 50 mV to Adi but without net HI extru- the protonmotive force for the HW-translocating ATPase con- sion. sists mainly of a transmembrane electrical gradient (A*r). These ceils do not establish a s cnt pH gradient (inside alkaline) and, in fact, if the suspending me- MATERIALS AND METHODS dium is of pH > 7.0, the pH gradient may be reversed-i.e., Measurement of Bacterial Cell Parameters. Cells of Mb. inside acid with respect to the extracellular pH. These studies thermoautotrophicum were grown and harvested as de- show by both 23Na NMR and =Na+ distribution that Na+ scribed (1, 5). Cell transfers were carried out in an anaerobic extrusion with the generation of Ad precedes methanogenesis chamber (Coy Laboratory Products, Ann Arbor, MI). Cells in Mb. thermoautotrophicum. It is calculated that the newly (-7.5 mg of protein) were incubated in 50 mM Tris/5 mM established Na+ gradients increase Ad by =50 mV (inside potassium phosphate buffer (pH 7.0) with 50 mM Tris car- negative). There is no detectable H+ extrusion during methane bonate (pH 7.0) under, H2 at 600C in a final volume of 1.5 ml synthesis; instead there is a high rate of H+ consumption for with constant shaking in 5-ml sealed rubber-stopped flasks. methane synthesis and an increase in internal pH. This was The ATP content of cells was assayed by the luciferin/ supported by 31P NMR experiments, which showed an internal luciferase assay (1, 6). pH shift from 6.8 to 7.6. With the cells maintain at an Aqd was determined by measuring the transmembrane dis- external pH of 7.2, the initial transmembrane pH gradient of tribution at equilibrium of either 86Rb+ (with 67 ,uM valino- -0.4 (inside acid) at 60VC is equivalent to Aeof +27 mV (inside positive); after 20 min of incubation, the membrane pH mycin) or tetraphenylphosphonium ion ([14C]Ph4P+), essen- gradient is +0.4 (inside alkali), which at 60°C is equivalent tially as described (7). Na+ transport was measured from the to Ad of -27 mV (inside negative). Actively respiring cells transmembrane distribution of 22Na+ as described (6). generated a protonmotive force of -198 mV. It is proposed that Cells loaded with oxidized benzyl viologen (BV2+) were energy for CO2 reduction to the level offormaldehyde (the first prepared by transferring cells (-50 mg ofprotein) into 50 mM step in methane synthesis) in Mb. thernmautotrophicum is Tris/5 mM potassium phosphate (pH 7.0) with 10 mM NaCl, derived from the Adgenerated by electrogenic Na+ extrusion. 10 mM KC1, and 5 mM BV2+ in a total volume of6.0 ml. The The protonmotive force required for ATP synthesis consists mixture was incubated in a 10-ml sealed vial for 10 mini at 600C primarily of Ad and appears to be the result of both an under H2 and chilled in ice, and the H2 replaced by Ar. These electrogenic Na+ extrusion and a pH gradient (inside alkale) cells had very active hydrogenase activity but were incapable which develops during methanogenesis. of synthesizing methane from CO2 and H2- 23Na and 31p NMR. 23Na and 31P NMR spectra were The mechanism for the generation ofprotonmotive force (Ap) recorded on a Bruker AM500 NMR spectrometer operating in methanobacteria remains controversial. There is no de- at 132.3 MHz and 202.5 MHz, respectively. 23Na NMR tectable proton translocation from inside to outside in Meth- spectra were acquired at "'600C in 10-mm NMR tubes using anobacterium thermoautotrophicum cells synthesizing meth- a 50-kHz sweep width, 900 (26-pusec) pulse, 8000 data points, ane at pH 2 7.0, but these cells are nonetheless competent to and 0.08-sec acquisition time. One thousand scans were generate ATP and establish an energy charge of 0.6 (1). collected, making the total time per spectrum 80 sec. The first Nonetheless, acid-pulse experiments indicate that ATP syn- spectrum represents an average time of 100 sec from the time thesis in Mb. thermoautotrophicum is catalyzed by a H+- the sample was inserted in the magnet. Spectra were acquired translocating ATPase (FoF1 ATPase) (2). While methanobac- in unlocked mode. Free-induction decays (FIDs) were pro- teria have been reported to translocate Na+ during metha- cessed by eliminating the first 18 points from each FID, nogenesis (3), these organisms do not appear to possess a zero-filling, and using a line broadening of4 Hz. NaCl at 100 Na+-translocating ATPase. Na+-pumping ATP synthase, mM was used as a chemical-shift standard (0 ppm). Spectra which responds specifically to Na+ but not to protons, has so were plotted in absolute intensity mode for integration. far been found only in Propionigenium modestum (4). It is The concentration of the paramagnetic-shift reagent dys- likely that methanobacteria must generate Ap required for prosium tripolyphosphate [Dy(P3010)1-] was adjusted to give ATP synthesis primarily from a transmembrane electrical a chemical-shift difference of =10 ppm between the intracel- potential (Aq*). lular and extracellular Na+ pools (8, 9). Na+ concentrations This paper confirms an earlier finding (1) that Mb. ther- were calculated by comparison ofintegrated peak intensity to moautotrophicum cells do not develop a large transmem- a resonance of 1 mM NaCl solution acquired under the same brane pH gradient (ApH) (inside alkaline) and shows that Na+ conditions. Intracellular Na+ concentrations were deter- efflux precedes the onset of methanogenesis and that this mined by correcting for average cell volume and cell con- increases Aqkby -50 mV. During methanogenesis, an internal centrations, assuming no "invisible" Na+ (10). pH shift toward alkaline was detected by 31P NMR, which Abbreviations: BV2+, oxidized benzyl viologen; TCS, 3,5,3',4'- The publication costs of this article were defrayed in part by page charge tetrachlorosalicylanilide; Ap, protonmotive force; ApH, transmem- payment. This article must therefore be hereby marked "advertisement" brane pH gradient; A*, transmembrane electrical gradient. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4466 Downloaded by guest on October 3, 2021 Biochemistry: Sauer et aL Proc. Natl. Acad. Sci. USA 91 (1994) 4467 31P NMR spectra were acquired in 10-mm tubes with 6000 There appears to be no net H+ extrusion in these cells once scans, 16,000 data points, 700 (15-,usec) pulse, 0.4-sec acqui- methane synthesis has been initiated. What happens to the sition time, and 20-kHz sweep width. Spectra were refer- internal pH is less clear. Attempts to monitor internal pH enced to 85% H3PO4 at 0 ppm. The probe was maintained at changes by radiolabeled marker distribution present techni- 40C to reduce the effects of the high concentration of para- cal difficulties, not the least of which is the maintenance of magnetic ions found in these cells. strict anaerobiosis during sampling. Changes in internal pH, however, can be measured noninvasively by 31P NMR. Fig. 2 shows the partial 31P NMR spectra of a cell suspension in RESULTS 5 mM potassium phosphate buffer before and after incubation Mb. thermoautotrophicum incubated under CO2/H2 (1:4) with Tris carbonate. The sample of Fig. 2B showed substan- showed a period of rapid H+ uptake once methane synthesis tial methane production. Before incubation, a resonance was initiated (Fig. 1). This raised the pH of the external bulk assigned to the cytoplasmic inorganic phosphate appeared at phase from 7.5 to >8.3. The pH change was equivalent to 105 2.12 ppm, corresponding to a pH of 6.8. This resonance ,umol of H+ taken up by the cells in 20 min. The addition of shifted downfield upon incubation to disappear under the TCS resulted in only a slight decrease in extracellular pH external inorganic phosphate resonance at 2.90 ppm, corre- (Fig. 1). Methane production after 30 min was 102.5 pmol. sponding to a pH of7.6. This internal pH shift of0.8 unit was These results indicate that during methanogenesis H+ flux is verified by repeating the experiment in the absence of exter- directed inward and, as was shown previously (1), there nal potassium phosphate in the buffer. Within experimental appears to be no net H+ extrusion. error, there was no shift in the external inorganic phosphate To show that the Mb. thermoautotrophicum cytoplasmic resonance. The other notable change upon incubation was membrane is impermeable to H+ and that H+ extrusion, if the appearance of a new sugar phosphate resonance at 4.0 present, would be detectable in the external bulk aqueous ppm. phase, BV2+-loaded cells were prepared. Only BV+ will In what appears to be the absence of an effective ApH cross the cytoplasmic membrane (11) as BV2+ is membrane- (inside alkaline), Mb. thermoautotrophicum depends on the impermeant. When exposed to H2, Mb. thermoautotrophi- generation of A*as the principal component of Ap. With this cum cells with 67 mM BV2+ inside rapidly reduce the in mind, the time course and magnitude of Na+ transmem- oxidized species according to reaction 1. brane gradients were measured either by cell filtration with 22Na+ or by 23Na NMR as described by Castle et al. (8, 10). H2 + 2BV2+ -- 2H+ + 2BV+ [1] 23Na NMR has been used to measure intracellular Na+ (12). This technique was applied to these obligate anaerobes As shown in Fig. 1, protons from this reaction were in the extracellular phase and acidified the suspending medium A Pext from pH 6.8 to pH 6.2. The final pH did not change with time-i.e., there was no evidence of H+ reentering the cells. From BV2+ reduction measured spectrophotometrically (ex- tinction coefficient = 8.19 mM'1cm-1), the stoichiometry for AH+/e- was 1.14. The release of H+ into the exterior Pint aqueous phase with a coincident reduction of internal BV2+ SP suggests that the functional hydrogenase is a membrane NP protein able to oxidize H2 in the external phase and that the membrane is relatively impermeable to HW. 6 4 2 0 B P ext + P int

IQ0L SP l

Time, min FIG. 1. pH changes in suspending medium when cells synthesize 6 4 2 0 methane (A) or reduce BV2+ (o). BV2+-loaded cells (13.5 mg of ppm protein), prepared as described in the text, were suspended in 0.1 M KCl under Ar in a volume of 11.5 ml. H2 replaced Ar in both FIG. 2. Portions of the 31P NMR spectra (202 MHz, 4°() of Mb. experiments when indicated. One micromole of H+ decreased the thermoautotrophicum cell suspensions (78 mg of protein) in 5 mM buffer pH by 0.151 unit. For methane synthesis, cells (71 mg of potassium phosphate/0.1 M KCI/20 mM EDTA/33 mM Tris car- protein) were suspended in 0.1 mM KCI/50 mM NaCl under C02/H2 bonate, pH 7.2. Final volume was 3 ml with 2.2 x 1012 cells per ml. (1:4) in a volume of 11.5 ml. 3,5,3',4'-Tetrachlorosalicylanilide (TCS, 31P NMR spectra were acquired under Ar (A) and after the gas phase 8 ,uM) was added when indicated (arrow). One micromole of H+ was changed to H2 (B). After acquisition of the spectrum in A, the decreased buffer pH by 0.028 unit. Cells were incubated at 60°C with NMR tube was incubated at 60°C for 20 min and then cooled to 40C constant stirring. The methane-producing cells had low activity (103 for acquisition of the spectrum in B. NP, nucleotide phosphate; SP, ,umol/30 min), but the rate of synthesis was linear for 40 min (y = sugar phosphate; P ext, external inorganic phosphate; P int, internal 3.51x - 2.73; r2 = 0.97). inorganic phosphate. Downloaded by guest on October 3, 2021 4468 Biochemistry: Sauer et al. Proc. Natl. Acad. Sci. USA 91 (1994)

where the high interior salt concentration provides the ad- 140 - -28 vantage of greater time resolution in the 23Na NMR spectra, -24 but this advantage is offset by a high interior paramagnetic 120 ion concentration (mostly Ni2+), which broadens the reso- E 100 -20 5 nance lines. The intracellular Na+ concentration also can be + : increased by Na+ loading. Anionic paramagnetic-shift re- z 80 16 a) agents such as Dy(P3O10)17 permit the measurement of co internal and external sodium pools simultaneously. This 60

technique, first used in Escherichia coli (8, 10, 13), has 0 8 2 c -8 E proved useful here, since H2 diffusion in the spinning sample - kept the cells active. Fig. 3 shows the flux ofinternal (+2 ppm) and external (-6 ppm) Na+ pools as a function oftime after the addition ofTris 10 carbonate for cells incubated in an H2 atmosphere. Each Time, min spectrum represented an 80-sec time average about the time after the addition of carbonate. Only three representative FIG. 4. Cells were equilibrated with 10 mM 22NaCl (specific spectra are shown in Fig. 3, which are not plotted relative to activity, 8.9 x 104 dpm/pmol) as described. After equilibration, an each other in order to better show the internal Na+ reso- aliquot of cells (6.0 mg of protein) was incubated under H2 at 60WC nance. The external Na+ resonance at -6 ppm was probably in 50 mM Tris HCl/5 mM potassium phosphate/S0 mM Tris carbon- due to incomplete rinsing of the cells from the Na+-loaded ate/10 mM KCI/10 mM NaCl, pH 7.0, in a final volume of 1.5 ml. Rates of Na+ efflux (o) and methane formation (n) are shown. medium. The internal Na+ resonance was always broader than that ofthe external due to cytoplasmic viscosity and high concentration of Ni2+. of the external Na+ resonance, and thus no evidence was Measurable Na+ equilibration took place before the addi- observed to indicate that bound Na+ was present in substan- tion ofcarbonate. The initial 23Na NMR spectrum of a freshly tial quantities (8, 10). Throughout all experiments, the total prepared cell suspension taken from ice to =60°C in the NMR integrated intensity ofthe two resonances remained constant, probe showed an intensity ratio of approximately 1:1 for within the error of measurement of integrals. The absolute intracellular vs. extracellular Na+. As the cells became values of intracellular Na+ concentration were calculated by activated in the H2 atmosphere, the resonance intensity of comparison with a 1 mM NaCl standard, using cell density intracellular Na+ dropped within 2 min to an equilibrium ratio and average cell volume, and were 3 mM for the external pool of0.5:1 (data not shown). On addition ofcarbonate, there was and =90 mM for the internal pool for initial conditions. These a further decrease of intracellular Na+ to a ratio of 0.34:1 by values closely agreed with the 22Na results. 2 min and to 0.13:1 by 20 min (Fig. 3). This net reduction of In actively metabolizing cells, the rate ofNa+ extrusion was the intracellular Na+ pool could be correlated with measured too rapid to measure by the 23Na NMR technique. The results methane production and generally agreed with results ob- with 22Na+ show that Na+ extrusion precedes methane syn- tained with the cell filtration technique. thesis in highly active cells (Fig. 4). After a 1-min lag, methane When spectra were plotted in absolute intensity mode, the synthesis was linear for 20 min and internal Na+ decreased reduction in peak intensity ofthe cytoplasmic Na+ resonance from 121 mM to 4 mM within 10 min ofincubation against a 10 was compensated by a proportional increase in the intensity mM external Na+ gradient. There was no evidence of Na+ reentry into the cells during the period of methane synthesis. 2 min 10 min 20 min The intracellular Na+ concentration was correlated to the external Na+ concentration of the medium (r2 = 0.93) at the time of equilibration (Table 1). The rate of Na+ extrusion, expressed as a percent of the starting (time zero) concentration, however, was relatively independent of the initial Na+ concentration. The change in A attributable to Na+ transfer out of the cell during incubation, when expressed as -RT/F .lII ln [Na+Iext/[Na+]Ijt, increased by =40 mV with 1 mM ex- Table 1. Intracellular Na+ concentration and changes in A4v attributable to Na+ extrusion in Mb. thermoautotrophicum cells A .i - Na+ * mM ,4-. V, Intracellular Changes in Aq, from Extracellular After 20 Na+ transfer after Na+, mM Time 0 -10 0 -10 0 -10 0 min incubation, mV ppm ppm ppm 1 22.2 ± 2.3 5.3 ± 1.7 -41.2 10 ± ± FIG. 3. 23Na NMR 71.0 6.8 10.7 1.6 -54.0 spectra (132 MHz, 50°C) of Mb. thermoau- 50 137.0 ± 4.4 23.3 ± 3.2 totrophicum showing the response of intracellular Na+ to the addi- -51.0 tion ofTris carbonate as a function oftime. Cells (18.6 mg ofprotein) After equilibration for 30 min on ice under Ar, the gas phase was were equilibrated with 50 mM NaCl by 15 min of preincubation at changed to H2 and cells (7.6 mg ofprotein) were incubated for 20 min 60°C under Ar and then stored on ice for 30 min. The cells were then at 600C in 50 mM Tris-5 mM potassium phosphate, pH 7.0/10 mM suspended in 1.5 ml of 0.1 M Tris (pH 7.0) and 1.5 ml of 24 mM KCl/50 mM Tris carbonate, pH 7.0, final volume of 1.5 ml, with 1, K7Dy(P3010)2 for a final concentration of5.3 x 1011 cells per ml. The 10, or 50 mM NaCl as indicated. sample was gassed with H2. After 10 min of equilibration during *Values are expressed as means ± SEM where n = 10-15 experi- which initial spectra were acquired, Tris carbonate (50 mM) was ments. added. Each spectrum represents an 80-sec time average about the tDerived from the difference in the electrical potential of the Na+ time shown after addition of carbonate. Spectra are plotted with gradient before incubation (time 0) and after incubation (time 20) respect to 100 mM NaCl at 0 ppm. The resonances at 2 ppm and -6 expressed by the relationship -66.2ApNa+, where ApNa+ = pNaAt ppm are assigned to internal and external Na+, respectively. - pN4:t. Downloaded by guest on October 3, 2021 Biochemistry: Sauer et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4469 tracellular Na+ and =50 mV with 10 mM and 50 mM Table 2. Changes in ATP concentration in Mb. extracellular Na+. thermoautotrophicum cells with different Na+ The apparent A4fras measured by [14C]Ph4P+ distribution in levels in the incubation medium cells under Ar was 98 ± 3.51 mV (n = 9), inside negative. This Extracellular ATP, nmol/mg of Methane synthesis, value combines the Aqi of cells at rest and any nonspecific TCS Na+, mM protein umol binding of The subsequent changes with time of [14C]Ph4P+. - 1 6.27 ± 0.62 5.71 ± 0.63 incubation in an H2 atmosphere are recorded in Fig. 5. In 10 10 9.03 ± 0.14 6.24 ± 0.31 mM NaCl, actively a respiring cells rapidly established Aqiof 20 12.77 ± 2.60 6.94 ± 0.59 -198 mV in <10 min. 50 11.31 + 0.54 5.93 ± 0.29 High concentrations of the protonophore TCS did not + 1 2.75 ± 0.18 1.69 ± 0.29 interfere significantly with Na+ transfer (data not shown) or 50 12.48 ± 0.72 6.23 ± 0.08 Adk development (Fig. 5). These observations are consistent with the presence of a primary electrogenic Na+ pump in Cells (7.6 mg of protein) were incubated for 20 min at 600C in H2 methanobacteria (14). in the buffer described in Table 1 with the NaCl concentration as indicated, in the absence or presence of 10 AMM TCS. Values are Although there is compelling evidence against a Na+- expressed as means ± SEM where n = 5 experiments. translocating ATPase in Mb. thermoautotrophicum, Na+ ions nevertheless significantly stimulated ATP synthesis (Ta- small but significant increase in the internal pH of the cells. ble 2). Before incubation, the ATP concentration of cells If the functional hydrogenase is active primarily at the stored on ice was 0.38 + 0.032 nmol/mg of protein (n = 28). external side of the cytoplasmic membrane, methane pro- After 20 min of incubation, the ATP levels were positively duction may be accompanied by an inwardly directed H+ correlated to the NaCl concentration of the suspending flow (together with e- transfer through the Fe4-S4 centers of medium up to a concentration of20 mM NaCl (r2 = 0.99), but the hydrogenase). The shift in internal pH (+0.8 pH unit) with no additional effect for 50 mM NaCl. suggests that H+ consumption outstrips the rate of H+ entry Experiments with TCS, a proton-conducting agent, indi- into the cell. From the present results, one can calculate that cate that ATP was synthesized by a H+-translocating in 20 min of incubation, one cell synthesizes 9 x 106 mole- ATPase. In the presence ofTCS and low Na+ concentration, cules of methane, for which 7.2 x 107 H+ ions are required. ATP synthesis decreased by 78% and methane synthesis by From the buffering capacity of lysed cells (see below), it was 73%. The TCS inhibitory effect was fully reversible by high determined that 2.6 x 107 protons per cell must be removed (50 mM) Na+ levels in the medium (Table 2). from the internal water phase to account for the rise in internal pH of0.8 pH unit. This means that ifthe hydrogenase DISCUSSION functions solely at the external membrane surface, during periods of methane formation approximately two-thirds of In the absence of substrate-level phosphorylation, energy for the protons are derived from the external aqueous phase and ATP synthesis (AG"' 44 kJ/mol) in Mb. thermoautotrophi- one-third from the internal aqueous phase. cum must be derived from the generation of a Ap = Aid - In the present work, the average cell was estimated to be RT/F In ApH. The magnitude of Ap depends on A* (inside 3 j&m in length with a diameter of 0.25 ,um. The surface area, negative) and ApH (inside alkaline). In most organisms, ApH then, is 2.4 x 10-8 cm2 and the internal volume 1.5 x 10-13 (inside alkaline) is generated by means ofH+ pumping, which cm3 per cell. From these data, one can calculate H+ fluxes is driven by the energy derived from specific metabolic across the cell membrane (1 mg of protein = 7.4 x 1010 cells) reactions. In Methanosarcina barked, H+ translocation has (this study). During the short-term incubation (20 min), one been observed when methanol is converted to methane in the cell synthesizes 7.5 x 103 methane molecules per sec, which presence of H2 (15, 16). However, as demonstrated here and utilizes 6 x 104 H+ ions per sec per cell. During the same previously (1), with intact cells ofMb. thermoautotrophicum period, the steady-state level of ATP increases by 80 ATP there is no demonstrable H+ extrusion during methanogen- molecules per sec per cell to a final concentration of 1.1 x esis. Further, with standard chemical techniques (17) it has 10-3 M. If, for the sake of simplicity, we assume that for each not been possible to detect any significant ApH (inside mole of methane 1 mol of ATP is hydrolyzed for diverse alkaline) in this organism (1, 18). However, by 31P NMR, it energy-requiring reactions (including CO2 activation), then is possible to detect after 20 min of methane production a ATP synthesis is at least 7.6 x 103 molecules of ATP per sec per cell. With a stoichiometry of3H+/ATP, 2.3 x 104 H+ ions -80 per sec per cell are utilized for ATP synthesis. Total proton consumption, then, is 8.3 x 104 H+ ions per sec per cell. -100 Titration experiments with lysed and intact cells have shown that between pH 6.0 and 7.0, 3.3 x 106 H+ ions per cell will -120- lower the pH by 0.1 pH unit. The 0.8 pH-unit shift over a 20-min incubation interval, then, is equivalent to a net >E -140- decrease of2.2 x 104 H+ ions per sec per cell (approximately -160- that amount required for ATP synthesis). From the results obtained with BV2+-loaded cells, it appears that the hydro- -180- genases span the cytoplasmic membrane and release H+ derived from H2 in the extracellular space. From this it -200- follows that during a 20-min incubation interval, the H+ ions that are not derived intracellularly must move across the 0 2 4 6 8 10 12 cytoplasmic membrane at the rate of 6.1 x 104 per sec per Time, min cell. This rate would require an apparent permeability coef- FIG. 5. Changes in Adk with incubation. To the buffer (10 mM ficient for the cytoplasmic membrane of =4 x 10-12 cm/sec. NaCl/10 mM KCl/5 mM potassium phosphate/50 mM Tris4HCl/50 Vesicles made of phospholipid bilayers have given apparent mM Tris carbonate, pH 7.0) were added cells (6.3 mg of protein), 0.2 permeability coefficients of 5 x 10-12 to 10-9 cm/sec (19) or uCi tetraphenylphosphonium bromide. TCS additions: *, none; o, 10-4 cm/sec in the absence of diffusion potentials (20). 0.53 mM. Final volume was 1.5 ml. Although membranes composed of biphytanyl ether lipid Downloaded by guest on October 3, 2021 4470 Biochemistry: Sauer et aL Proc. Nat!. Acad. Sci. USA 91 (1994) chains, as in Mb. thermoautotrophicum, are generally con- sidered to be less permeable to H+ than phospholipid membranes, the rate of H+ translocation required to maintain methane synthesis could still be met easily. During methanogenesis, Mb. thermoautotrophicum cells shift their internal pH from 6.8 to 7.6. Whether or not this shift in internal pH results in ApH of significant magnitude depends on the pH of the external bulk phase. Localized transmembrane pH gradients not in equilibrium with the bulk phase may also factor into establishing ApH. At 60TC, a ApH = 0.8 (i.e., if made equal to the shift in internal pH) contributes -53 mV to Ap. The development of a charge separation ....-.aNo--_... | I . * need not be accompanied by a large shift in internal pH. For ADP.~~~~~~~~~~~~I example, the electric capacitance of a single cell, calculated as a parallel plate capacitor, C = K"0A/d, where K is the .x, P 6 ., dielectric constant (taken as 3; ref. 21), eo is the permittivity A-Nc~T constant, A = 2.4 x 10-12 m2, and d = 7 x 10-9 m, is =1 x 10-14 F. From the relation V = qIC (charge q in coulombs), it can be calculated that at this low capacitance, the transfer Cytopiasm CM exterior of570 charges will change the membrane potential by 10 mV. Because of the internal buffering capacity of the cell at pH FIG. 6. Ion flow in Mb. thermoautotrophicum at the onset of 6-7, the net number of charges required to change Aqiby 100 methanogenesis. CM, cell membrane. mV would be <0.01 pH unit. In Mb. thermoautotrophicum, Aiz shifts from -100 mV here, a A* of approximately -200 mV is generated with an (resting state) to -170 mV within 2 min-i.e., before the inwardly directed H+ flow (Fig. 6). These results suggest that onset of methanogenesis-and stabilizes at -195 mV within A*/is the principal component ofAp in Mb. thermoautotrophi- 4 min. The kinetics of Na+ extrusion, as presented here, cum. correspond closely to the development of A+. Na+ extrusion The contribution number for the Centre for Food and Animal contributes approximately -50 mV to the shift in A+, irre- Research is 2023; the contribution number for the Plant Research spective of the starting internal and external Na+ concentra- Centre is 1478. tions. A primary electron transport-driven Na+ pump is present 1. Sauer, F. D., Erfle, J. D. & Mahadevan, S. (1981) J. Biol. in Vibrio which Chem. 256, 9843-9848. alginolyticus (22, 23), generates Ad (at 2. Mountfort, D. 0. (1978) Biochem. Biophys. Res. Commun. 85, alkaline pH) by electron transfer-driven Na+ extrusion but 1346-1351. which does not require transmembrane H+ transfer. A pri- 3. Becher, B., Mfiller, V. & Gottschalk, G. (1992) J. Bacteriol. mary Na+ pump has recently also been reported for Meth- 174, 7656-7660. anosarcina strain Go 1 (3). As shown here in Mb. thermoau- 4. Laubinger, W. & Dimroth, P. (1988) Biochemistry 27, 7531- totrophicum, Na+ translocation and Adk induction can occur 7537. even when methane formation is blocked by protonophores. 5. Sauer, F. D., Blackwell, B. A., Kramer, J. K. G. & Marsden, This strongly suggests that Na+ pumping may also be linked B. J. (1990) Biochemistry 29, 7593-7600. to H2 oxidation and electron transport via the membrane- 6. Kaesler, B. & Sch6nheit, P. (1989) Eur. J. Biochem. 184, bound hydrogenase. 223-232. 7. Kaesler, B. & Sch6nheit, P. (1989) Eur. J. Biochem. 186, The first step in methane synthesis is the reduction of CO2 309-316. to the level of formaldehyde according to reaction 2, with a 8. Castle, A. M., Macnab, R. M. & Shulman, R. G. (1986) J. Biol. AG0' = + 16 kJ. Chem. 261, 3288-3294. 9. Gupta, R. K. & Gupta, P. (1982) J. Magn. Res. 47, 344-350. H2 + CO2 + methanofuran 10. Castle, A. M., Macnab, R. M. & Shulman, R. G. (1986)J. Biol. Chem. 261, 7797-7806. -- formylmethanofuran + H20 [2] 11. Jones, R. W. (1980) Biochem. J. 188, 345-350. 12. Hutchinson, R. B. & Shapiro, J. I. (1991) Concepts Magn. With a two-electron transfer (n = 2) and from the relationship Reson. 3, 215-236. AG' = -nFAE6, a potential difference of =85 mV can be 13. Pan, J. W. & Macnab, R. M. (1990) J. Biol. Chem. 265, calculated for reaction 2. This series of events may follow. 9247-9250. Na+ extrusion Adk increases to approximately -150 mV, 14. Muller, V., Winner, C. & Gottschalk, G. (1988) Eur. J. Bio- which initiates the formation of chem. 178, 519-525. formylmethanofuran (reac- 15. Blaut, M., Mfiller, V. & Gottschalk, G. (1987) FEBS Lett. 215, tion 2) from CO2 and H2. The reductive reactions from 53-57. formylmethanofurane to methane are exergonic (AG"' = 16. Deppenmeier, U., Blaut, M., Mahlmann, A. & Gottschalk, G. -146 kJ/mol of methane) and require no additional energy (1990) Proc. Nat!. Acad. Sci. USA 87, 9449-9453. input. 17. Rottenberg, H. (1979) Methods Enzymol. 55, 547-569. Results presented here emphasize the importance of Na+ 18. Kaesler, B. & SchOnheit, P. (1988) Eur. J. Biochem. 174, ion in the bioenergetics of Mb. thermoautotrophicum. As 189-197. summarized in the model (Fig. 6), Na+ extrusion is the first 19. Nozaki, Y. & Tanford, C. (1981) Proc. Nat!. Acad. Sci. USA detectable event when the cell temperature is increased to 78, 4324-4328. 60"C as shown by both 22Na filtration and NMR. In 20. Deamer, D. W. & Nichols, J. W. (1983) Proc. Nat!. Acad. Sci. 23Na these USA 80, 165-168. cells, therefore, Ali appears to be generated initially by a 21. Michel, H. & Oesterhelt, D. (1980) Biochemistry 19,4607-4614. primary Na+ pump which drives Na+ extrusion as the initial 22. Tokuda, H. & Unemoto, T. (1984) J. Biol. Chem. 259, 7785- response to H2 oxidation. With methane synthesis, additional 7790. Na+ is extruded via the methyltetrahydromethanopterin: 23. Tokuda, H. & Unemoto, T. (1982) J. Biol. Chem. 257, 10007- coenzyme M methyltransferase reaction (3) and, as reported 10014. Downloaded by guest on October 3, 2021