Download CO2 reduction to acetate in anaerobic bacteria

FEMS Microbiology Reviews 87 (1990) 391-396
Published by Elsevier
391
FEMSRE 00191
CO 2
:eduction to acetate in anaerobic bacteria
Gabriele Diekert
institut flir Mikrobiologie, Universitiit Stuttgart, Stuttgart, F.R.G.
Key words: Homoacetogenic bacteria; Acetate formation from CO2; Sodium dependent acetate
formation; Methylene-tetraltydrofolate reduction; Energy conservation in homoaeetogens
1. S U M M A R Y
The re,ruction of 2 CO 2 to acetate is catalyzed
in the energy metabolism of homoacetogenic
bacteria, which couple acetate formation to the
synthesis of ATP. The carboxyl group of acetate is
formed from CO 2 via reduction to a bound
carbonyl {[CO]), a reaction that requires the input
of metabolic energy when hydrogen is used as the
electron donor. The methyl group of acetate is
formed via formate and tetrahydrofolate b o u n d
C t intermediates including methyl tetrahydrofolate as the intermediates. The methyl group is
then 'condensed' with the carbonyl and CoA to
acetyl-CoA, which is converted to acetate in the
energy metabolism or to cell carbon in the anabolism of the bacteria. The mechanism of ATP
synthesis coupled to CO x reduction to acetate is
still unclear. The only reaction sufficiently exergonic is the reduction of methylene tetrahydrofolate to methyl tetrahydrofolate. Indirect evidence was presented that this reaction in homoacetogens might be coupled to the electrogenic
transport of sodium across the cytoplasmic membrane. The sodium gradient formed via metbylene-THF reduction could be transformed into a
proton gradient via a s o d i u m / p r o t o n antiporter.
Correspondence to: O. Diekcrt, Institut FOrMikrobiologie,Universifiit Stuttgart, Azenbergstrasse 18, D-7000 Stuttgart 1,
F.R.G.
ATP would then be synthesized by a proton translocating ATP synthase.
2. I N T R O D U C T I O N
Several groups of strictly anaerobic bacteria
catalyze the reduction of 2 CO 2 to an acetyl moiety
in their metabolism. A m o n g them are the homoacetogenic bacteria, which mediate the CO 2 reduction as a multifunctional pathway operative b o t h
in energy metabolism and autotrophic cell carbon
synthesis [1-5]. In the catabolism of homoacetogens this pathway can serve as an electron
sink for reducing equivalents released in the
oxidative degradation of different energy substrates such as sugars (Fig. 1A) or, for instance
upon growth of the bacteria on H 2 plus C O 2 or
carbon monoxide as the energy sources, for energy
conservation coupled to acetate formation from
these substrates (Fig. 1B, C). In anabolism, the
acetyl CoA formation from C O 2 sepses as a carbon
assimilation pathway, which is not only used by
homoa¢etogens, b u t also by o t h e r strictly
anaerobic autotrophic microorganisms such as
sulfidogenic and methanogenic bacteria. For a recent detailed review on this autotrophic CO 2 fixation pathway the reader is referred to [1].
The acetate formation from CO 2 can be reversed in different obligate anaerobes. Some bomoacetogens are able to catalyze acetate oxidation
to COg and use the reduction of protons to hyaro-
0168-6445/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
392
(~
" T "--~ Acetate + CO2
',-----i----, Py. . . . te--~ ---~Acetate
41HI 2 IHI
ICOI?
[
CO2
(~)
co2
Hexoses" - ~
L
formate
formyI-THF
, CH,-X ~ - ~ . ~
methenyI-THF
~21Hi
me th ylene- THF
21HP
3 Hz-~ 61HI
CO2
~
L . CH3-Xx
C02~
CH~-THF
ICOI /
CO.....
CH/-×
H2 ,-~2 IH)
.)...
3 CO2
3CO-~61H)
acetate cell carbon
Fig. 3. Scheme of acetate and cell carbon synthesis in homoacetogcns. The "framed' reaction is catalyzed by melhytcne
tetrahydrofolate reductase.
CO /
Fig. 1. Simplified scheme of acetate formation from (A) hexoses, (B) H 2 plus CO2, and (C) from CO in homoacctogenic
bacteria.
gen as the electron sink [6-8] (Fig. 2). Several
sulfidogens also degrade acetate oxidatively and
use sulfate as the electron acceptor (for a recent
review see [9]). Some of the sulfidogens oxidize
acetate via the citric acid cycle; others involve the
acetyl-CoA pathway (carbon monoxide dehydrogenase pathway) [10,11], which can be considered
Acetate oxidation to CO2
(Sullidogenic and acelogenic bacleria)
61HI
Acetate
/ - . CH3-X --, ._~~
AcetyI-CoA - ~
ICOI
CO2
COz
2 IHI
SO~ + 81HI ~
H2S
to be an inversed acetate formation from CO 2 as
mediated by homoacetogenic bacteria (Fig. 2).
The reduction of CO2 to acetate in homoacetogens involves the formation of a methyl group
(a methyl +) and of a carboxyl group, i.e. a CO2
plus 2 reducing equivalents. The methyl group is
synthesized via tetrabydrofolate (THF)-bound
Cl-intermediates, the carboxyl group via a bound
carbon monoxide ([CO]), which is formed from
CO2 by the key enzyme in this pathway, namely
the carbon monoxide dehydrogenase [12,13]. The
methyl and the carboxyl group are then combined
together with coenzyme A in a reaction, the mechanism Of which is not yet understood. This latter
reaction is probably also catalyzed by the carbon
monoxide dehydrogenase [14]. A scheme of acetate
and cell carbon formation from CO2 in homoacetogens is given in Fig. 3.
(Sulfldogenlc bacter,a)
8 IHI ~
4 H2
(Acetogen,c bacter|a)
Fig. 2. Anaerobic acetate oxidation by homoacetogenic and
some sulfidogenic bacteria.
3. ENEROETICAL ASPECTS OF CO2 REDUCTION TO ACETATE IN ACETOGENS
Most of the homoacetogenic bacteria are able
to grow on H e plus CO2 as the sole energy sources.
This implies that CO2 reduction to acetate must
be coupled to the synthesis of ATP. One ATP is
393
required for the activation of formate to formyl
tetrahydrofolate (formyloTHF). ATP can be
formed by a substrate level phosphorylation
mechanism in the acetate kinase reaction, which is
involved in acetate formation from acetyl-CoA. It
must be considered, however, that part of the
acetyl-CoA is channelled into cell carbon synthesis rather than into acetate formation. Moreover,
the reduction of CO 2 to the bound carbonyl with
H 2 as the electron donor according to
gous reaction in their energy metabolism, namely
the reduction of methylene tetrahydromethanopterin (methylene-THMP) to methyl-THMP:
methylene-THMP + 2[HI --, methyl-THMP
Evidence was presented that this reaction in
methanogens is coupled to the electrogenic transport of sodium across the membrane [19]. Therefore, the role of sodium in the COx reduction to
acetate was tested in acetogenic bacteria.
CO 2 + H 2 ~ [COl + H20
AG o" = + 2 0 kJ/mol
is an endergonic reaction, which is energy driven
in homoacetogens [15] and methanogens [16,17].
For these reasons it is still unclear how homoacetogenic bacteria synthesize ATP. The only reaction in acetyl-CoA formation from CO 2 that is
sufficiently exergonic to be coupled to energy conservation via a chemiosmotic mechanism is
the reduction of methylene tetrahydrofolate to
methyl-THF (see 'framed' reaction in Fig. 3):
methylene-THF + 2[H] --* methyl-THF
With H 2 (or ferredoxin) as the electron donor the
AG O' value of this reaction is - 57.3 kJ/mol, with
N A D H - 3 9 . 4 kJ/tool. The enzyme has been
purified from Clostridium formicoaceticum by Clark
and Ljungdahl [18]. It mediates the reduction of
methylene-THF with reduced ferredoxin as the
electron donor. In the carbon monoxide-utilizing
homoacetogen Peptostreptococcus productus the
reducing equivalents in this reaction are provided
by N A D H (Wohlfarth et al., in press). The enzyme of the latter organism differs considerably
from the clostridial enzyme also in other respects.
Whereas the clostridial enzyme is an octamer
(cf4•4) with a molecular weight of Mr = - 237000
and is oxygen sensitive [18], the P. productus enzyme is not sensitive against oxygen and is an
octamer (as) with an M r of ~ 250000 (Wohlfarth
et al., in press).
From the energetical considerations described
above the methylene reduction to the methyl group
is most likely the reaction, which is coupled to net
ATP formation in homoacetogens growing on H2
plus CO 2. The mechanism of coupling is still
unclear. Methanogenic bacteria catalyze an anal(>-
4. THE ROLE OF SODIUM IN ACETATE
FORMATION FROM CO2
The influence of sodium on acetate formation
from CO 2 was investigated with cell suspensions of
P. productus (strain Marburg). It was found that
the formation of acetate from H 2 plus CO 2, from
formate plus CO2, and frcm CO was significantly
stimulated by the addition of sodium. The apparent K m for sodium in acetate formation from
CO was near 2 mM. In the absence of added
sodium formate excretion was increased [20]. The
findings were interpreted to indicate that one of
the steps involved in the formation of the methyl
group of acetate from formyl-THF was the
sodium-dependent reaction. Since formyl-THF
formation from formate has never been found to
depend on sodium, and in analogy to the
methanogenic bacteria (see above), it was concluded that the reduction of methylene-THF to
methyl-THF was the sodium dependent step [20].
This was further substantiated by recent findings
of G. Gottschalk and collaborators, that in
Acetobacterium woodii CO 2 reduction to acetate is
coupled to sodium extrusion and that acetate formation from formaldehyde, hydrogen, and carbon
monoxide is also dependent on sodium [21].
If the methyl tetrahydrofolate formation from
methylene.THF is coupled to the electrogenie
transport of sodium across the membranes of homoacetogens, the enzyme must be associated with
the membrane. Indirect evidence for a membrane
association of the methylene-THF reductase was
recently presented for Clostridium thermoauto.
trophicum [22]. Evidence for a location of the
enzyme in the particulate fraction is also available
394
Na"
(
c°2~ ' ~ IHCHOI ICH3OHI
H+
H+
Fig. 4. Scheme of energy conservation in homoacetogenic
bacteria upon growth on H 2 plus CO . No stoichiometriesof
cations translocated per reaction are given.
for P. productus (Wohlfarth et al., in press), where
up to 60~ of the enzyme can be demonstrated to
be present in the membrane. The methylene-THF
dehydrogenase served as a cytoplasmic marker
enzyme; about o~ ,. ~ of the latter enzyme was
located in the ~ytovlasmic fraction after ultracentrifugation Of crude extracts. From the data it
was deduced that the methylene-THF reduction is
a m e m b r a n e - a s ~ i a t e d process and is coupled to
the transport of sodium ions across the membrane.
•
5. CONCLUSIONS: H O W IS E N E R G Y CONSERVED IN H O M O A C E T O G E N S ?
The mechanism of energy conservation in homoacetogenic bacteria growing on H 2 plus CO2 a~
the sole energy sources is summarized in Fig. 4. i~
should be mentioned that no stoichiometries of
monovalent cations transported per reaction are
given in the figure. The sodium gradient generated
in the methyle.,.,¢-THF reductase reaction is probably i,ot utrectly used for the synthesis of ATP,
b u t it is converted to a proton gradient by a
s o d i u m / p r o t u n antiporter. The presence of such
a n electrogenic antiporter was recently reported
for Clostridium thermoaceticum [23]. The electrochemical proton potential then is used for the
energy conservation by the action of a proton
translocating A T P synthase, which has also been
demonstrated for C. thermoaceticum [24]. Part of
the proton gradient is consumed for the energy
requiting CO 2 reduction to carbon monoxide [15].
Interestingly, from the findings it can b e deduced that homoacetogenic bacteria are able to
involve three different primary energy-rich 'intermediates' depending on the C t substrates th.tt
they use as the energy source. With H 2 plus CO 2
the energy yielding reaction is the methylene-TEIF
reduction, and thus an electrochemical sodium
gradient is used for the energy conservation. With
carbon monoxide as the substrate a major p~.rt of
the energy is derived from the proton gradient
generated in the CO oxidation to CO2. Finally,
when using methanol as the energy source, homoacetogens synthesize A T P via substrate level phosphorylation in both the acetate kinase reaction
and the formyl-THF synthetase reaction; part of
the ATP must be required for the generation of a
proton gradient for the reduction of CO2 to [CO]
and for the formation of a sodium gradient, which
most probably drives the endergonic oxidation of
methyl tetrahydrofolate to methylene-THF. The
latter conclusions, however, remain speculative a n d
require further investigations.
ACKNOWLEDGEMENTS
This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Oodesberg, and
by the F e n d s der chemischen Industrie.
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