FEMS MicrobiologyLetters 24 (1984) 103-107
Published by Elsevier
103
FEM 01848
Protonmotive force-driven synthesis of ATP during methane
formation from molecular hydrogen and formaldehyde or carbon
dioxide in Methanosarcina barkeri
(Protonmotive force; ATP synthesis; uncoupler; reverse electron transport; methanogenesis)
Michael Blaut and Gerhard Gottschalk
lnstitut ff~r Mikrobiologie der Unioersitfit GOttingen, Grisebachstrasse 8, D - 3400 GOttingen, F.R.G.
Received 30 April 1984
Accepted 2 May 1984
1. S U M M A R Y
Methane formation from formaldehyde and H2
or from carbon dioxide and H2, as performed by
cell suspensions of M e t h a n o s a r c i n a b a r k e r i , was
coupled to A T P synthesis. In correspondence with
this, methane formation was inhibited by N, N ' - d i cyclohexylcarbodiimide (DCCD), which at the
same time, caused a decrease of the intracellular
A T P concentration but only a slow decrease of the
membrane potential. Addition of the uncoupler
tetrachlorosalicylanilide(TCS) led to a relief of the
inhibition of methane formation from C H 2 0 + H z,
but not from CO 2 + H 2.
2. I N T R O D U C T I O N
In a study on the energy metabolism of M.
b a r k e r i we have shown recently that the formation
of methane from methanol and H 2 according to
the equation
CH3OH + H 2 "-~ CH 4 + H 2 0
(1)
was coupled to A T P synthesis [1]. The uncoupler
TCS caused a simultaneous decline of the proton-
motive force and of the intracellular A T P content
without affecting methanogenesis. Methane formation from the above substrates was inhibited by
DCCD, an inhibitor of the proton-translocating
A T P synthase [2], but its rate was restored by the
addition of TCS. It also was shown that D C C D
caused a decrease of the intracellular A T P content
without affecting the protonmotive force, A ~ H + ,
which essentially consisted of the electrical transmembrane potential A~b. All these results were in
accordance with a chemiosmotic mechanism of
A T P synthesis. Since methanol has been shown to
be convertible into 2-(methylthio)ethanesulfonic
acid (methyl-coenzyme M) by a transmethylase [3],
the reduction of methyl-coenzyme M must be coupled to a chemiosmotic energy conservation. We
have now extended this work and report here that
an analogous coupling exists between A T P synthesis and methane formation from formaldehyde
and molecular hydrogen according to Eqn. 2:
C H 2 0 + 2 H E --* CH 4 + n 2 0
(2)
CO: + 4 H 2 ~ CH 4 + 2 H 2 0
(3)
Methane formation from carbon dioxide and
molecular hydrogen according to Eqn. 3, however,
differs from the processes in Eqns. 1 and 2 in that
0378-1097/84/$03.00 © 1984 Federation of European MicrobiologicalSocieties
104
it is inhibited by uncouplers [4-6]. A possible
explanation for this finding is presented.
3. M A T E R I A L S A N D M E T H O D S
M. barkeri, strain fusaro (DSM804) was grown
on methanol as described before [7]. Cells were
harvested by centrifugation, washed once with 100
m M P i p e s - N a O H buffer, pH 6.7, containing 1 mg
resazurin and 2 ml titanium (Ti)(III)citrate solution/1 and resuspended in the same buffer. The
Ti(III)citrate solution was prepared as described
[1]. The resulting cell suspension contained 10-20
mg of p r o t e i n / m l and was stored on ice in a
stoppered tube until needed. All manipulations
were done in an anaerobic glove box. Protein was
determined according to [8]. The experiments were
carried out in stoppered 58-ml bottles containing 9
ml of the above Pipes buffer. The gas atmosphere
was oxygen-free hydrogen. An aliquot of the cell
suspension was added to give a final protein concentration of 1 - 2 mg of protein per ml. The
suspension was then preincubated for 10 min at
37 o C on a rotary shaker; additions were made as
indicated for each experiment. D C C D , TCS, and
[14C]tetraphenylphosphonium bromide (TPP +Br ) were added as ethanolic solutions; the controls received only ethanol. The formaldehyde
solution was freshly prepared by heating paraformaldehyde to 180°C. The formaldehyde gas
formed was collected by passage through distilled
water.
Methane was determined by gas chromatography as described [9]. For the determination of the
ATP content, a 0.5-ml sample of the cell suspension was withdrawn anaerobically by syringe and
transferred into a 10-ml centrifuge tube containing
0.2 ml ice-cold 3 M perchloric acid. After 2 h on
ice, the p H was brought to 7.4 by the addition of
0.2 ml 3 M K O H and 0.1 ml 0.4 M T e s - N a O H
buffer, pH 7.4. The KC103 formed was removed
by centrifugation, and ATP was determined with
the luciferin-luciferase assay according to [10]. The
membrane potential Ag, was estimated from the
distribution of [14C]TPP+ according to [11]. 1 /~Ci
(10 /~M) of [14C]TPP+ was added to 10 ml cell
suspension. Separation of the cells from the
medium was done by centrifugation through silicone oil [12]. The density of the silicone oil used
was d = 1.05. The internal and total water spaces
of M. barkeri cells were determined by silicone oil
centrifugation as described [13] using 3 H 2 0 and
[14C]sucrose as markers. The internal water space
was 3.2 _+ 0.1 ~ l / m g protein, the total water space
was 7.8 _+ 0.3 / d / m g protein [1]. Correction for
non-specific binding of [14C]TPP+ was done as
described [14].
4. RESULTS A N D D I S C U S S I O N
Previous experiments had shown that M. barkeri
contained a proton-translocating ATP synthase
and that the activity of this enzyme was inhibited
by D C C D [1,15]. When methanol, formaldehyde
or carbon dioxide were added to a resting cell
suspension of M. barkeri under a H 2 atmosphere,
methane formation started and proceeded with a
linear rate (Fig. 1). Formaldehyde could only be
added in small amounts (3-10 n m o l / m g protein),
otherwise it was inhibitory. The concentration
tolerated by the cells was determined for each cell
suspension. The addition of D C C D resulted in an
inhibition of methane formation from all three
substrates. The subsequent addition of the uncoupler TCS, however, had different effects. The inhibition of methane formation from methanol +
H 2 o r f o r m a l d e h y d e + H 2 was relieved (Fig. 1A
and 1B), whereas the inhibition of methanogenesis
from CO 2 + H 2 was not (Fig. 1C). With all three
substrate combinations, the inhibition of methanogenesis by D C C D was associated with a decline of
the intracellular ATP content of the cells. The
membrane potential Ag,, on the other hand, increased temporarily upon addition of D C C D and
decreased thereafter comparatively slowly. This is
shown for C H 2 0 + H 2 in Fig. 2 and for CO 2 + H 2
in Fig. 3; it had been previously reported for
C H 3 O H + H 2 [1].
The inhibitory effect of D C C D may be explained as follows: methane could only be formed
if ATP was synthesized simultaneously or, in case
that the ATP synthase was blocked by DCCD, if
methane formation was uncoupled from ATP
synthesis by TCS. The role of Zig, in this process is
105
A
30
20
10
0
m
i,°
5
0
C
,o
t/
not clear yet. Blocking of the ATP synthase by
D C C D and continued proton translocation during
electron flow from molecular hydrogen to the respective acceptor resulted in a transient increase of
A+ (Figs. 2B and 3B). This might have been the
signal which switched off methanogenesis. That
methane formation remained inhibited even after
A+ had decreased cannot be explained at the
moment, especially because the addition of the
uncoupler TCS did reverse the inhibition of
methanogenesis. A stringent coupling between
electron flow and ATP synthesis as observed here
is typical for mitochondria [16], but is normally
not seen with bacterial systems.
That the methane formation from CO 2 + H 2,
but not that from formaldehyde+ H z or from
methanol + H 2 was inhibited by uncouplers is in
agreement with the observation that the inhibition
of methanogenesis by D C C D could only be reversed by TCS with the latter substrate combinations but not if CO 2 + H 2 served as substrates.
These differences can be explained on the basis of
the redox potentials of the formal couples which
play a role in the process of CO 2 reduction to CH 4
(Fig. 4). The redox potentials of the formal couples C O 2 / H C O O H and H C O O H / C H z O are
- 4 3 2 mV and - 5 3 5 mV, respectively [17]. It is
obvious from these values that the second step of
CO 2 reduction, the reduction of H C O O H to CH20,
with electrons derived from H 2 ( H + / H 2 ; E~ =
414 mV), is thermodynamically unfavorable, but
may be driven by the terminal energy conserving
reaction (methylcoenzyme-M methylreductase) via
a reverse electron transport mechanism as proposed by Kell et al. [17]. It is in agreement with
this view that methane formation from CO 2 and
H 2 has not been observed in cell extracts of M.
thermoautotrophicurn free of intact vesicles [18] and
that Gunsalus and Wolfe observed a stimulation
of methane formation from these substrates by
methyl-coenzyme M [19]. In accordance with the
redox potentials of the terminal reduction steps
(CH20/CH3OH;
E o = - 1 8 2 mV and CH3OH /
-
0
30
Time
60
(min)
Fig. 1. Effect of DCCD and TCS on methane formation from
H 2 and methanol (A), H 2 and formaldehyde (B), or H 2 and
CO 2 (C) by resting cells of Methanosarcina barker•. 10-ml cell
suspensions (1.85 mg protein/ml) were incubated under H 2. At
the times indicated by open arrows methanol (A), formaldehyde (B), or N a H C O 3 (C) were added to give final concentrations of 30 raM, 15 mM, and 30 mM, respectively. •
•,
methane produced from the substrate and H 2, no further
additions; •
• , addition of DCCD (44 n m o l / m g protein) at the times indicated by closed arrows; •
•,
addition of DCCD (44 n m o l / m g protein) at the times indicated by closed arrows followed by addition of TCS (final
concentration of 10 /~M) at the times indicated by semi-open
arrows.
106
,ol
_1o
,q
Oo ~ ~ "~
io
Time
3b
o
o°
~b
{rain)
2'0
Time
3'0
o
(rain)
Fig. 2. Effect of D C C D on methane formation from formaldehyde and H2: (A) methane formation and intracellular ATP content; (B)
methane formation and A~. Formaldehyde, 4 m M (open arrow) and DCCD, 44 n m o l / m g protein (closed arrow) were added to 10-ml
cell suspensions (1.15 mg protein/ml). (A) methane (v) and ATP (zx) in the presence of DCCD; methane (O) and ATP ( ~ ) in the
absence of DCCD; (B) methane (v) and A~ ('7) in the presence of DCCD; methane (O) and A~ ((3) in the absence of DCCD.
B
A
I
--150
10'7
C
o-3
/
2
CI.
~-100 >E
2
,.._.J
,q
._£
I
o..
0:
0
10
20
Time
30
(min)
0
-50
51
0
0
1'o
3'0
2'0
Time
0
(rain)
Fig. 3. Effect of D C C D on methane formation from CO 2 and H2: (A) methane formation and intracellular ATP content; (B) methane
formation and Aft. NaHCO3, 10 m M (open arrow) and DCCD, 44 n m o l / m g protein (closed arrow) were added to 10-ml cell
suspensions (1.04 mg protein/ml). (A) methane (v) and ATP (A) in the presence of D C C D ; methane (o) and ATP ( ~ ) in the absence
of DCCD; (B) methane (v) and A~ (,7) in the presence of DCCD; methane (O) and A~b (©) in the absence of DCCD.
107
E~
REFERENCES
(mV]
-600
-500
I~HCOOH
~
CH20
endergomc
400
exergomc
300
200
~CH30H
100
0
+100
2H~
"200
.300
Fig. 4. Scheme showing endergonic and exergonic electron
transfers from molecular hydrogen to the respective formal
acceptors involved in the CO z reduction to CH 4. For the sake
of clarity only the reduced forms of the formal redox couples
are given.
C H 3 O H / C H 4 ; E~ = + 169 mV), methane formation from methanol + H 2 or f o r m a l d e h y d e + H z
was not inhibited by the uncoupler TCS.
ACKNOWLEDGEMENTS
This work was supported by a grant from the
Deutsche Forschungsgemeinschaft and by the
Fonds der Chemischen Industrie.
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