Download Adaptations of anaerobic archaea to life under extreme energy

REVIEW ARTICLE
Adaptations of anaerobic archaea to life under extreme energy
limitation
Florian Mayer & Volker Müller
Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt/Main,
Frankfurt, Germany
€ller,
Correspondence: Volker Mu
Department of Molecular Microbiology &
Bioenergetics, Institute of Molecular
Biosciences, Johann Wolfgang Goethe
University Frankfurt/Main, Max-von-Laue-Str.
9, 60438 Frankfurt, Germany. Tel.:
+49 69 79829507; fax: +49 69 79829306;
e-mail: [email protected]
Received 26 March 2013; revised 30 August
2013; accepted 3 September 2013. Final
version published online 5 November 2013.
DOI: 10.1111/1574-6976.12043
MICROBIOLOGY REVIEWS
Editor: Michael Bott
Keywords
hydrogenase; Rnf; Ech; phosphorylation
potential; electrochemical ion potential;
sodium bioenergetics.
Abstract
Some anaerobic archaea live on substrates that do not allow the synthesis of
1 mol of ATP per mol of substrate. Energy conservation in these cases is only
possible by a chemiosmotic mechanism that involves the generation of an electrochemical ion gradient across the cytoplasmatic membrane that then drives
ATP synthesis via an A1AO ATP synthase. The minimal amount of energy
required is thus depending on the magnitude of the electrochemical ion gradient,
the phosphorylation potential, and the ion/ATP ratio of the ATP synthase. Methanogens, Thermococcus, Pyrococcus, and Ignicoccus have evolved different ways to
energize their membranes, such as methyltransferases, H+, or NAD+ reducing
electron transport systems fueled by reduced ferredoxin or H2-dependent sulfur
reduction that all operate at the thermodynamic limit of life. The structure and
function of the enzymes involved are discussed. Despite the differences in membrane energization, they have in common an A1AO ATP synthase that shows an
extraordinary divergence in rotor composition and structural adaptations to life
under these conditions. In sum, adaptation of anaerobic archaea to energylimited substrates involves chemiosmotic energy coupling, often with Na+ as
coupling ion and a structurally and functionally highly adapted ATP synthase.
Introduction
Ever since archaea have been discovered, their unusual
lifestyle, their metabolic and structural adaptations to
extreme environments, and their use as biocatalyst in
industrial processes have attracted much attention
(Schiraldi et al., 2002; Stetter, 2006; Sharma et al., 2012).
Most of the archaea first discovered grew under extreme
conditions such as temperatures from 80 to 121 °C, high
salt concentrations, acidic or alkaline pHs, and combinations thereof (Stetter et al., 1983; Fiala & Stetter, 1986;
Zillig et al., 1990; Kurr et al., 1991; Bl€
ochl et al., 1997;
Andrei et al., 2012; Mesbah & Wiegel, 2012; Sharma
et al., 2012). Therefore, the name ‘archaea’ is often synonymous with ‘extremophile’, although this is not correct
because a number of archaea are not extremophilic at all.
For example, members of the methanogenic archaea inhabit the gastrointestinal tract of humans or the rumen.
They also inhabit anoxic zones of freshwater lakes or live
in other, nonextreme environments (Miller & Wolin,
1982; Belay et al., 1990; Kulik et al., 2001; Moissl et al.,
FEMS Microbiol Rev 38 (2014) 449–472
2003; Lepp et al., 2004), not to mention the recently discovered phylum of ammonia-oxidizing Thaumarchaeota
(Brochier-Armanet et al., 2008; Schleper & Nicol, 2010).
Methanogens can grow on a limited number of substances that are converted to methane (Zinder, 1993).
The substrate used by the most is H2 + CO2, and the
substrate used by a few species, but with the most impact
on production of this greenhouse gas in nature, is acetate. The pathway of methanogenesis has been worked
out in the last decades (Thauer, 1998; Deppenmeier &
M€
uller, 2008). The question, how this pathway is used to
synthesize ATP, has also been answered to a great extent
(M€
uller et al., 1999; Sch€afer et al., 1999; Deppenmeier &
M€
uller, 2008; Thauer et al., 2008; Schlegel & M€
uller,
2013). The free energy change in methanogenesis from
various substrates is low and often not enough for the
synthesis of 1 mol of ATP. Methanogens can only use
this type of metabolism using sophisticated and unusual
biochemical reactions and a chemiosmotic mechanism of
energy conservation. The reactions that drive the generation of a transmembrane ion gradient are often close to
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450
the minimal amount of energy required to translocate an
ion against an electrochemical ion gradient across the
membrane. Methanogens are the ‘pioneers’ in our studies
to understand the principal mechanisms how cells
synthesize ATP under extreme energy limitation. They
turned out to involve membrane-bound electron transport systems, to use Na+ instead or along with H+, and
to use an unusual ATP synthase (M€
uller et al., 1999;
Deppenmeier & M€
uller, 2008; Thauer et al., 2008;
Schlegel & M€
uller, 2013). Later, it turned out that other
anaerobic archaea use similar, principal strategies to energize their membrane for ATP synthesis or substrate transport. It is the purpose of this review to briefly introduce
the thermodynamic constraints of (archaeal) life under
extreme energy limitation. Next, we will describe the
chemiosmotic mechanisms of energy conservation used in
selected members of the anaerobic archaea to finally conclude
with the structure and function of the most important
enzyme in cellular bioenergetics, the ATP synthase.
F. Mayer & V. Müller
synthase is, thus, the most important and universal
enzyme in cellular bioenergetics. As we shall see later, the
ATP synthases known to date arose from a common
ancestor, have the same principal mode of operation, but
evolved differently in terms of structure and function in
the three domains of life (M€
uller & Gr€
uber, 2003). The
ATP synthase catalyzes the following reaction (Eqn. 1):
ATP þ H2 OADP þ Pi
0
DG0 ¼ 31:8 kJ mol1 (1)
The free energy change in the reaction under standard
conditions is 31.8 kJ mol1 (Thauer et al., 1977). That
means that any reaction that is more exergonic than
31.8 kJ mol1 is sufficient to drive ATP synthesis by a
direct coupling mechanism. Evolution evolved only a few
reactions (Eqns 2–7) in living cells that are more exergonic and used to drive the phosphorylation of ADP
(Thauer et al., 1977; Teague & Dobson, 1999):
Acetate kinase:
Principles of energy conservation or
how much energy is required to make
an ATP
Highly ordered structures such as living cells require
energy to maintain an ordered ‘status’. Energy is supplied
by different ways, either by light or by chemical reactions
(Harold, 1986). Light-driven ATP synthesis is observed
not only in plants under aerobic conditions, but also in
microorganisms under anaerobic conditions. For example,
Rhodospirillaceae, Chromatiaceae, and Chlorobiaceae are
well known for their anaerobic photosynthesis. Interestingly, one prominent family of the Archaea, the Halobacteriaceae, is known for their light-driven proton pump,
bacteriorhodopsin, which translocates protons from the
inside to the outside using a light-driven retinal switch
(Oesterhelt & Tittor, 1989; Krebs & Khorana, 1993;
Mukohata et al., 1999). In contrast, the archaea discussed
here are strictly chemotrophic. However, they differ with
respect to their metabolism, and organoheterotrophic as
well as lithoautotrophic species are known. Anyway, the
primary energy source, be it light or a chemical reaction,
is converted to an energy currency that can be used by the
living cell. Evolution has brought about two different
energy currencies in living cells: ATP and the transmembrane electrochemical ion (H+, Na+) gradient across a
membrane. It is essential that both currencies are usually
freely convertible. This is done by an enzyme that was in
the beginning of bioenergetics called the ‘coupling factor’,
nowadays known as ATP synthase (Mitchell, 1966; Senior,
1988; Cross & M€
uller, 2004). This enzyme is present in
every living cell, despite the different ways in which ATP
is synthesized or the membrane is energized. The ATP
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Acetyl phosphate þ H2 O ! Acetate þ Pi
0
DG0 ¼ 44:8 kJ mol1
(2)
Phosphoglycerate kinase:
1; 3-Bisphosphoglycerate þ H2 O ! Phosphoglycerate þ Pi
0
DG0 ¼ 51:9 kJ mol1
(3)
Pyruvate kinase:
Phosphoenolpyruvate þ H2 O ! Pyruvate þ Pi
0
DG0 ¼ 51:6 kJ mol1
(4)
Creatine kinase:
Creatine phosphate þ H2 O ! Creatine þ Pi
0
DG0 ¼ 43:3 kJ mol1
(5)
Arginine kinase:
Arginine phosphate þ H2 O ! Arginine þ Pi
0
DG0 ¼ 45 kJ mol1
(6)
Carbamate kinase:
Carbamyl phosphate þ H2 O ! Carbamate þ Pi
0
DG0 ¼ 39:3 kJ mol1
(7)
Sugar-fermenting organisms mostly use reactions 2, 3,
and 4 for ATP synthesis. Thus, anaerobic chemoorganotrophs that ferment sugars to acetate gain 4 mol of ATP
if the sugar is converted to:
FEMS Microbiol Rev 38 (2014) 449–472
451
Archaeal life under extreme energy limitation
Hexose þ 4ADP þ 4Pi ! 2 acetate þ 4ATP þ 4H2 þ 2CO2
(8)
Compared with aerobic bacteria, this is little ATP per
mol of sugar, but for an anaerobic lifestyle, this is the
most one gets out of a hexose molecule by substrate-level
phosphorylation. However, the fermentation according to
Eqn. (8) is only possible if the reducing equivalents are
‘blown away’ as hydrogen.
Hydrogen production (from ferredoxin) leads us to the
second energy currency, the transmembrane electrochemilNaþ ). As we shall see later,
cal ion potential (D~
lHþ , D~
proton translocation by proton reduction to hydrogen gas
is one way to energize the cytoplasmic membrane. Aerobic and anaerobic archaea have a beautiful diversity of
membrane integral enzymes that couple exergonic
reactions to the translocation of ions out of the cell.
Translocation of the ion, protons in most cases, leads to
translocation of a positive charge and the translocation of
a substance. Thus, the exergonic reaction drives the establishment of an electrochemical proton gradient (Eqn. 9):
D~
lHþ =F ðmVÞ ¼ Dpmf ¼ DW z DpH
(9)
where pmf = proton-motive force; DW = electrical membrane potential; z = 2.3 9 R 9 T/F; DpH = pHinside
pHoutside; R = gas constant; T = temperature; F = Faraday constant.
As we shall see later, organisms that live under energy
limitation often use Na+ instead of H+. Then, the
Eqn. (10) is:
D~
lNaþ =F ðmVÞ ¼ Dsmf ¼ DW z DpNa
(10)
where smf = sodium-motive force.
If we reconcile that an exergonic reaction drives the
export of an ion against its electrochemical potential, we
can now ask how much energy is required to translocate
an ion. This is given by the following Eqn. (11):
DG ¼ n F D~
lion
(11)
where DG = free energy change in the reaction; n = number of translocated ions; F = Faraday constant;
D~
lion = electrochemical potential of the coupling ion
(Na+ or H+).
lHþ or D~
lNaþ ) has been measured only in
The D~
lion (D~
some bacteria and archaea and seems to be rather constant at around 180 to 220 mV (for simplicity D~
lion
rather than D~
lion =F is used from here onwards). With
Eqn. (11), this calculates to a free energy change of 17
to 21 kJ mol1 for the exergonic reaction to translocate
one proton or sodium ion across the membrane (Schink,
1997; Hoehler et al., 2001). Obviously, the minimal
FEMS Microbiol Rev 38 (2014) 449–472
amount of energy required to translocate an ion depends
on the magnitude of D~
lion . If DG gets smaller than
17 kJ mol1, the membrane may still be energized if
D~
lion is also decreased.
Indirect coupling mechanisms for ATP synthesis
involve an ATP synthase that is driven by D~
lion according
to the following equation:
lion
DGP ¼ n F D~
(12)
where DGP = intracellular phosphorylation potential.
The intracellular phosphorylation potential describes
the ratio of ATP over ADP and Pi as described in the following equation:
0
DGP ¼ DG0 þ R T lnð½ATP=½ADP ½Pi Þ
(13)
Synthesis of ATP is a highly endergonic reaction, and
the phosphorylation potential is a function of the cellular
concentrations of ATP, ADP, and Pi that, again, has been
determined only for a few model organisms (Slater et al.,
1973; Kashket, 1982). These data revealed a value of
around +60 kJ mol1. This value is used throughout in
textbooks and is seen nearly as a ‘standard value’ for the
phosphorylation potential. But there have been only a few
organisms analyzed and only one archaeon. In the latter,
the estimates are lower, for example +45 kJ mol1 (Jetten
et al., 1991). The second, very important variable is n, the
number of ions translocated by the ATP synthase. Let us
consider for the moment a constant DGP value. If the
D~
lion decreases, ATP synthesis requires a larger number
of n, the number of protons translocated. Thus, even at
low D~
lion , ATP synthesis is still possible if n increases.
How much ‘n’ varies between species or within one
species is discussed in detail later. Let us, again for the
moment, turn the argument around. If a chemical reaction linked to metabolism translocates two protons, then
the reaction has to proceed twice to export the four
protons required for ATP synthesis under standard conditions. If only one ion can be pumped by the chemical
reaction, only 0.25 mol of ATP can be formed. How
could this number be increased? Only if DGP is lower as
under standard conditions! For example, a chemical reaction leading to the export of two ions may yield one
ATP, if DGP is not +60, but +30 kJ mol1. Thus, the
magnitude of the phosphorylation potential is another
important variable that determines how much energy is
required to sustain life.
In sum, direct and indirect coupling mechanisms differ
with respect to the amount of energy required to phosphorylate ADP. In the former, the complete amount is
required by one chemical reaction; for the latter,
the energy can be sequestered to the number of ions
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452
F. Mayer & V. Müller
transported across the membrane. There, the minimum
amount is that required to translocate one ion across the
membrane.
After having described the thermodynamic principles,
we will now move on to discuss the different ways that
energy-limited archaea use to drive ATP synthesis.
Because energy limitation always includes a chemiosmotic
way of ATP synthesis, we will describe the beauty of
diversity of membrane energization in selected anaerobic
archaea.
Energy conservation in methanogenic
archaea
Methanogens are a nutritionally unique, but phylogenetically diverse group of organisms that all belong to the
phylum Euryarchaeota (Wolfe, 1992). Methanogens have
attracted much attention because of their unique metabolism. Methanogenic substrates are H2 + CO2, methyl
groups as in methanol or trimethylamine, or acetate. The
pathway of methanogenesis, the biochemistry of
the enzymes involved with their unique coenzymes, and
(a)
the way ATP is synthesized have been the subject of
recent, excellent reviews (Blaut et al., 1992; Gottschalk &
Thauer, 2001; Deppenmeier, 2002a, b; Deppenmeier &
M€
uller, 2008; Thauer et al., 2008). Therefore, we will keep
this paragraph short and focus on the coupling principles.
The conversion of H2 + CO2 to CH4 requires unique
coenzymes with unprecedented reaction mechanisms.
This linear pathway is functional even at high temperatures up to 113 °C as shown for Methanopyrus kandleri
(Kurr et al., 1991). Methanogenesis from H2 + CO2 could
allow for the synthesis of about 2 mol ATP under standard conditions (Deppenmeier & M€
uller, 2008; Thauer
et al., 2008) according to Eqn. (14):
4H2 þ CO2 ! CH4 þ H2 O
0
DG0 ¼ 131 kJ mol1
(14)
Considering the low hydrogen partial pressure in natural environments, the energetics allows for the synthesis
of only about one-third of an ATP (Deppenmeier &
M€
uller, 2008; Thauer et al., 2008). Methanogens use a
chemiosmotic way of energy conservation and couple
(b)
Fig. 1. Energy conservation in methanogens without (a) and with cytochromes (b). Methanogens couple their metabolism to the generation of a
primary Na+ gradient that is established by the methyltetrahydromethanopterin–coenzyme M methyltransferase (Mtr), which transfers the methyl
group from methyltetrahydromethanopterin to coenzyme M. In cytochrome-free methanogens, this Na+ gradient is used by the A1AO ATP
synthase to synthesize ATP. The reduction of the electron acceptor, a heterodisulfide of coenzyme M and coenzyme B, is catalyzed by a soluble
complex of heterodisulfide reductase and the methyl viologen-reducing hydrogenase (Mvh–Hdr complex). Exergonic electron transfer from
hydrogen to the heterodisulfide drives endergonic electron transfer from hydrogen to ferredoxin by electron bifurcation (Kaster et al., 2011). The
reduction of the heterodisulfide is in cytochrome-containing methanogens achieved by a proton-motive electron transport chain (Vho–Hdr
complex), which contains cytochromes and methanophenazine. These H+ and Na+ gradients are then also used by a promiscuous A1AO ATP
synthase that concurrently translocates Na+ and H+. In cytochrome-containing methanogens, the endergonic electron transfer from hydrogen to
ferredoxin is driven by the D~
lHþ catalyzed by the Ech hydrogenase. The ion stoichiometries depicted are mostly based on thermodynamic
calculations and have some uncertainties.
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Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 38 (2014) 449–472
453
Archaeal life under extreme energy limitation
methanogenesis to the generation of two primary ion gradients (Deppenmeier, 2002a). One of these ion gradients
is a primary, electrochemical sodium ion gradient established by the methyltetrahydromethanopterin–coenzyme
M methyltransferase (Mtr; M€
uller et al., 1988; Gottschalk
& Thauer, 2001), a unique enzyme in the bioenergetics
of methanogens. This membrane-bound, multisubunit
enzyme complex transfers the methyl group from methyltetrahydromethanopterin to coenzyme M; this exergonic
reaction is used to drive sodium ion translocation across
the cytoplasmatic membrane (Gottschalk & Thauer, 2001;
Fig. 1a). The DG0′ value of the reaction is 30 kJ mol1
that is sufficient to translocate 2 Na+ (Sch€
onheit &
Beimborn, 1985; Thauer et al., 2008). Methyl-CoM
is then reduced with coenzyme B (CoB, 7-mercaptoheptanoylthreoninephosphate) to CH4, and a heterodisulfide of CoM and CoB, CoM-S-S-CoB, is formed. The
reduction of this heterodisulfide is the ultimate step in
the pathway. The process, by which the heterodisulfide is
reduced, is different in the two groups of methanogens
(Deppenmeier, 2002b; Thauer et al., 2008). The cytochrome-free methanogens such as Methanothermobacter
thermautotrophicus and Methanocaldococcus jannaschii
have a soluble complex of heterodisulfide reductase and
the methyl viologen-reducing hydrogenase (Mvh–Hdr
complex) that use the recently established mechanism of
electron bifurcation (Buckel & Thauer, 2013) to use the
energy liberated during exergonic heterodisulfide reduction to drive endergonic electron transfer from hydrogen
(E0′ = 420 mV) to ferredoxin (E0′ = 500 mV) [the
redox potential of ferredoxin is set to 500 mV, which is
the redox potential of the CO2/formylmethanofuran couple (Thauer et al., 2008)] (Fig. 1a). The reduced ferredoxin is then used as a ‘high-potential’ electron donor to
drive the first reaction in methanogenesis, the endergonic
formylmethanofuran dehydrogenase reaction (Thauer
et al., 2008). These organisms have only one coupling site
that is Na+ motive, and thus, the ATP synthase should be
Na+ selective. This is confirmed by sequence analysis
(M€
uller & Gr€
uber, 2003; Mayer et al., 2012a) as well as,
in one case, supported by experimental analysis
(McMillan et al., 2011).
Cytochrome-containing methanogens such as Methanosarcina acetivorans, Methanosarcina mazei, and Methanosarcina barkeri are evolutionarily more advanced and have
evolved in addition to Mtr a proton-motive electron
transport chain that includes cytochromes and the electron carrier methanophenazine (Deppenmeier, 2002b).
Therefore, cytochrome-containing methanogenic archaea
couple methanogenesis to the generation of a proton and
a sodium ion gradient at the same time (Fig. 1b; Schlegel
& M€
uller, 2013). The proton-motive electron transport chain involves a membrane-bound heterodisulfide
FEMS Microbiol Rev 38 (2014) 449–472
reductase (Hdr) anchored to the membrane by a cytochrome b, the soluble electron carrier methanophenazine,
and electron input modules that change with the growth
substrate. During methanogenesis from H2 + CO2, hydrogen is activated by a membrane-bound F420 nonreducing
hydrogenase (Vho) that also acts as an input module for
the electron transport chain (Vho–Hdr complex in
Fig. 1b). Altogether, four protons, two pumped and two
scalar ones, are finally on the outside of the membrane
(Deppenmeier, 2002b). During growth on methyl groupcontaining substrates, the methyl groups are oxidized to
CO2 with the deazaflavin cofactor F420 as an electron
acceptor. Reduced F420 is reoxidized by a membranebound F420H2 dehydrogenase that is, based on biochemical data and sequence analyses, very similar to complex I
of bacterial electron transport chains. Altogether four
protons are translocated (Deppenmeier, 2002b). The thermodynamically unfavorable, H2-dependent reduction of
ferredoxin is not catalyzed by a direct coupling mechanism via electron bifurcation, but by a secondary coupling mechanism using D~
lHþ as driving force, catalyzed
by the energy-converting hydrogenase (Ech; Welte et al.,
2010b), as we will see later.
Acetate is the worst substrate for methanogenesis with
a standard free energy change of only 36 kJ mol1 and
only used by cytochrome-containing methanogens. It is
converted according to:
CH3 COOH ! CO2 þ CH4
0
DG0 ¼ 36 kJ mol1
(15)
To conserve as much as possible of the little energy
available, acetotrophic methanogens have evolved sophisticated mechanisms. Acetate is cleaved to a methyl group
and CO. The methyl group is transferred onto tetrahydromethanopterin, and the subsequent reduction again
involves the sodium-motive Mtr. CO is oxidized to CO2
with subsequent reduction of ferredoxin. Reduced ferredoxin is the electron donor for heterodisulfide reduction,
but the electron input modules differ in methanogens.
M. mazei has an Ech-type hydrogenase, a remarkable and
unique coupling site in methanogens (Welte et al.,
2010a, b). This membrane integral enzyme contains iron–
sulfur centers as electron carriers and transfers electrons
from reduced ferredoxin to protons, thereby producing
H2. It is a small electron transport chain that uses protons as terminal electron acceptor and couples the exergonic electron transfer from reduced ferredoxin
(E0′ 500 mV) to protons (E0′ = 414 mV) to the
export of protons from the cytoplasm to the medium,
thus establishing a D~
lHþ across the membrane
(Hedderich, 2004; Fig. 2a). The reaction is reversible, and
the enzyme uses D~
lHþ to drive the endergonic reduction
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454
(a)
F. Mayer & V. Müller
(b)
Fig. 2. Energy conservation in
Methanosarcina mazei and Methanosarcina
acetivorans by aceticlastic methanogenesis. In
M. mazei (a), the Ech complex is used to
reduce protons to H2 gas with electrons
derived from reduced ferredoxin in a process,
which is coupled to the generation of a
transmembrane electrochemical proton
gradient. M. acetivorans (b) does not have
hydrogenases, but a Na+-translocating Rnf
complex. The electron transfer from ferredoxin
to methanophenazine is coupled to vectorial
Na+ transport. The subsequent heterodisulfide
reductase reaction is proton motive. The ion
stoichiometries depicted are mostly based on
thermodynamic calculations and have some
uncertainties.
of ferredoxin with H2, required in the first step of
o1
methanogenesis from H2 + CO2 in M. mazei G€
(Fig. 1a). This remarkable, energy-converting hydrogenase
may be regarded as the prototype of energy-converting
hydrogenases found in other energy-limited archaea discussed below. Hydrogen produced by Ech is then fed into
the proton-motive electron transport chain (Vho–Hdr
complex) by the membrane-bound F420 nonreducing
hydrogenase (Vho) described above (Fig. 2a; Deppenmeier et al., 1991; Welte et al., 2010a, b).
Methanosarcina acetivorans does not have a hydrogenase, and thus, hydrogen is not an intermediate in electron
transfer from reduced ferredoxin to the heterodisulfide
reductase (Hdr). Again, the diversity in this small group of
organisms is amazing. M. acetivorans employs a ferredoxin:methanophenazine oxidoreductase encoded by the
rnf genes (Li et al., 2006; Rohlin & Gunsalus, 2010). This
electron transfer protein complex contains several subunits
with flavins, iron–sulfur cluster, and a cytochrome c as an
electron carrier. The bacterial homologue (without cytochrome c) has been shown to be a vectorial Na+ pump
(Biegel & M€
uller, 2010; Biegel et al., 2011). In M. acetivorans, exergonic electron transfer from reduced ferredoxin
(E0′ 500 mV) to methanophenazine (E0′ = 165 mV)
is coupled to vectorial Na+ transport out of the cell
(Fig. 2b; Schlegel et al., 2012a). Reduced methanophenazine then donates the electrons to the heterodisulfide
reductase, and this reaction drives proton translocation
out of the cell (Schlegel et al., 2012a). Altogether, this
rather simple reaction sequence leading from acetate to
methane and carbon dioxide involves three coupling sites.
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Most interestingly, not only Mtr, but also Rnf translocates
Na+. That raises the question about the ion specificity of
the ATP synthase that is discussed below.
In sum, methanogens have evolved sophisticated coupling sites early in the history of life (Schlegel & M€
uller,
2013). First was the sodium-motive Mtr that, so far, has
only been found in methanogenic archaea. This reaction
lNaþ generation ( 2 Na+
uses 30 kJ mol1 to drive D~
translocated). Second, with the advent of the cytochromes, was the evolution of electron transfer chains
that are coupled to proton translocation. Remarkable are
also Rnf and Ech. Rnf is widespread in bacteria. In
anaerobes, it uses NAD+ as the electron acceptor,
whereas aerobes may use it to drive the endergonic reaction of ferredoxin with NADH at the expense of D~
lNaþ .
Rnf is the ancestor of the sodium-motive Nqr complex
found in some bacteria (Tokuda & Unemoto, 1984;
Juarez & Barquera, 2012). Ech is found in anaerobic
archaea and some bacteria as well, but also was the
ancestor of complex I of the respiratory chain (Biegel
et al., 2011).
Energy conservation by formate
oxidation in Thermococcus onnurineus
Thermococcus onnurineus is an anaerobic hyperthermophilic archaeon isolated from a deep-sea hydrothermal
vent within the PACMANUS field near the Manus Basin
in the Pacific Ocean. It grows optimally at pH 8.5 and
80 °C and requires complex substrates such as yeast
extract, peptone, casein, or starch (Bae et al., 2006). The
FEMS Microbiol Rev 38 (2014) 449–472
455
Archaeal life under extreme energy limitation
outstanding feature of T. onnurineus is its growth on
formate as carbon and energy source (Kim et al., 2010).
Due to the small energy change in the reaction,
HCOO þ H2 O ! HCO
3 þ H2
DG0 ¼ þ1:3 kJ mol1
(16)
it had been theoretically excluded that microorganisms
can grow by conversion of formate to carbon dioxide and
hydrogen. So far, growth under anaerobic conditions by
formate oxidation according to Eqn. (16) has only been
observed by syntrophic bacteria in mixed cultures. In
these consortia, a partner organism oxidizes hydrogen
and, thus, lowers the hydrogen partial pressure in the
environment and makes growth by formate oxidation
thermodynamically possible (Dolfing et al., 2008; Stams
& Plugge, 2009). Amazingly, T. onnurineus can do the
same type of metabolism in pure culture! At 80 °C, the
DG0 becomes slightly negative (2.6 kJ mol1). This is a
good example for a type of metabolism that is not possible under ambient, but only at higher temperatures. By
analyzing the concentrations of educts and products, the
DG of the reaction was calculated during growth on formate. These elegant studies revealed that T. onnurineus
does grow from 20 to 8 kJ mol1 (as calculated from
the measured concentrations of formate, HCO
3 , and H2),
the lowest value ever reported for an organism (Kim
et al., 2010). This finding challenges the current concept
of the minimal energy value that sustains life.
How is formate oxidation coupled to energy conservation? According to molecular and genetic studies, it is
postulated that formate is taken up by a formate transporter and oxidized to carbon dioxide and hydrogen by a
soluble formate dehydrogenase (Fdh) coupled to a proton-reducing hydrogenase (Kim et al., 2010; Fig. 3). The
genetic organization of this hydrogenase is similar to the
membrane-bound hydrogenase (Mbh) from Pyrococcus
furiosus (Schut et al., 2012). Both are more complex than
the Ech hydrogenase, but all are variations of one theme:
proton reduction to hydrogen gas coupled to proton
export from the cell, thus establishing a D~
lHþ . In analogy
to the formate hydrogen lyase system of Escherichia coli,
it is suggested that the electron is directly transferred
from the Fdh to the hydrogenase via an electron-transferring subunit. Although neither ion transport nor ion
dependence of these multisubunit hydrogenases have been
proven experimentally, it is argued that they are proton
pumps. Because DG values below 17 kJ mol1 are well
below the minimal amount of energy required to energize
the membrane at a D~
lHþ of 180 mV, it may be possible
that the D~
lHþ established is smaller than 180 mV.
Separated by two genes that encode a protein of
unknown function and a potential formate transporter
are genes that encode for subunits with similarities to
multisubunit Na+/H+ antiporter (Mrp). Such an Mrp-like
system was first discovered in the alkaliphilic Bacillus
halodurans and is necessary for pH homeostasis under
alkaline conditions in the habitat (Hamamoto et al.,
1994). Thus, it is postulated that the primary proton
potential is converted to a secondary sodium ion gradient
by these antiporters (Kim et al., 2010). This argument is
strengthened by the finding of a conserved Na+-binding
motif in subunit c of the ATP synthase. However, these
postulates have to be seen with caution because methanoarchaeal ATP synthases also have the Na+-binding motif
(M€
uller et al., 1999), but, so far, clear evidence for Na+
transport has not been obtained, and in one case (M. acetivorans), the enzyme was shown to translocate Na+ and
H+ concurrently (Schlegel et al., 2012b). Second, Na+/H+
antiporter-like modules are also present in complex I of
the respiratory chain of bacteria, and there is a clear evidence that complex I translocates protons (Efremov et al.,
Fig. 3. Model of energy conservation by formate oxidation in Thermococcus onnurineus. Formate is oxidized by the Fdh. Electrons are shuttled
directly to the membrane-bound hydrogenase (Mfh, similar to Mbh of the same organism and Pyrococcus furiosus), which drives electrogenic H+
translocation. The H+ gradient may be changed to a Na+ gradient by a Na+/H+ antiporter (Mrp), and the A1AO ATP synthase then uses this Na+
gradient for ATP synthesis (Kim et al., 2010). The figure shows the nature of the ions pumped, but not the stoichiometries of the reactions,
which so far are not known.
FEMS Microbiol Rev 38 (2014) 449–472
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
456
2010; Hunte et al., 2010; Brandt, 2011; Dr€
ose et al.,
2011). Clearly, the fascinating bioenergetics of T. onnurineus deserves much more attention in future studies.
Energy conservation in Pyrococcus
furiosus
Pyrococcus furiosus, a hyperthermophilic strictly anaerobic
archaeon that belongs to the phylum Euryarchaeota, conserves energy for growth by fermentation of carbohydrates and peptides to CO2, H2, and organic acids (e.g.
acetate) in the absence of elemental sulfur. In the presence of sulfur, P. furiosus rather reduces elemental sulfur
to H2S than producing H2 (Fiala & Stetter, 1986).
Therefore, it does not live under extreme energy limitations, but nevertheless is included in this review because
it is the paradigm for an anaerobe using a novel protontranslocating, proton-reducing electron transport chain.
Sugars are oxidized by a modified Embden–Meyerhof–
Parnas pathway. The important modification is a ferredoxin-dependent glyceraldehyde-3-phosphate dehydrogenase reaction coupled to ferredoxin reduction (GAP:
ferredoxin oxidoreductase, GAPOR). The organism also
does not employ a pyruvate dehydrogenase that would
reduce NAD+, but a pyruvate:ferredoxin oxidoreductase
F. Mayer & V. Müller
instead (Sapra et al., 2003). Why is this important? The
redox potential of ferredoxin (E0′ 500 mV) is low
enough to transfer electrons directly to protons
(E0′ = 414 mV), thereby producing hydrogen gas. Thus,
the reducing equivalents are literally ‘blown away’ as
hydrogen. This allows oxidation of glucose to two acetates with the production of 2 mol of ATP by substratelevel phosphorylation (Fig. 4). At a first glance, the invention of the GAPOR looks like a waste of energy because
one ‘substrate-level phosphorylation’ ATP is lost. But
‘blowing away’ electrons as hydrogen eliminates the need
to produce reduced end products such as ethanol and
allows acetate production by the ADP-forming acetylCoA synthetase (ACD) that also produces ATP (Br€asen
et al., 2008). Thus, there is no loss of ATP synthesized by
substrate-level phosphorylation. Even better, if hydrogen
production from reduced ferredoxin is coupled to D~
lHþ
generation, more ATP can be synthesized. This is where
the multisubunit, Mbh comes into play.
Hydrogen is evolved by hydrogenases, and the genome
of P. furiosus encodes three different hydrogenases. Two
of them (SH-I and SH-II) are soluble, consisting of four
subunits each, and are extremely thermostable (Bryant &
Adams, 1989; Ma et al., 2000; Robb et al., 2001). For
example, purified SH-I only loses 50% of activity after
Fig. 4. Model of energy conservation in Pyrococcus furiosus. Enzymes involved in energy conservation are a Mbh, an Mrp-like Na+/H+ antiporter
module, and a Na+-dependent A1AO ATP synthase. P. furiosus ferments different carbohydrates and peptides and produces organic acids, for
example, acetate, CO2, and H2. All oxidative steps of this modified Embden-Meyerhof-Parnas pathway, the reactions catalyzed by the
glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR) and the pyruvate:ferredoxin oxidoreductase (POR), use ferredoxin as an electron
acceptor. Hydrogen gas is produced by the Mbh, coupled to the oxidation of reduced ferredoxin and generation of a proton gradient. This
gradient may be changed to a Na+ gradient by the Mrp-like Na+/H+ antiporter module of the Mbh. This Na+ gradient is then used by the Na+dependent A1AO ATP synthase to synthesize ATP. The figure shows the nature of the ions pumped, but the stoichiometries of the reactions are
unknown. GAP, glyceraldehyde-3-phosphate; 3-PG, 3-phosphoglycerate; ACD, ADP-forming acetyl-CoA synthetase.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 38 (2014) 449–472
Archaeal life under extreme energy limitation
12 h of incubation at 80 °C (Ma & Adams, 2001; Jenney
& Adams, 2008). The third hydrogenase, encoded in the
genome of P. furiosus, is membrane-bound (Mbh). The
Mbh operon contains 14 genes (mbhA–mbhN), but a
purified Mbh complex had only two subunits (MbhK
and MbhL), in which subunit MbhL has the catalytic
activity (Jenney & Adams, 2008; Schut et al., 2012). It is
thought that this Mbh uses reduced ferredoxin, generated during glycolysis at the GAPOR (Mukund &
Adams, 1995; van der Oost et al., 1998) and the POR
reactions (Blamey & Adams, 1993), as an electron donor
and transfers electrons to protons, thereby producing
hydrogen gas. This exergonic reaction is used to pump
ions out of the cell, and the electrochemical ion gradient
established is then used to drive ATP synthesis (Fig. 4).
In principle, the reaction is similar to the Ech-catalyzed
reaction. Measurements of ATP synthesis and hydrogen
production in inverted membrane vesicles of P. furiosus
clearly showed that hydrogen production is coupled to
ATP synthesis (Sapra et al., 2003). Although the coupling ion was not addressed in this study, it was hypothesized that protons were translocated. However, with the
finding that the ATP synthase of P. furiosus uses Na+
(Pisa et al., 2007), the question about the ion translocated by the Mbh arose. The Mbh operon (14 genes in
total) encodes eight proteins (MbhA–MbhH) that are
similar to subunits MrpB–MrpG of the Mrp-type Na+/
H+ antiporter (Schut et al., 2012). Therefore, it might be
possible that the Mbh of P. furiosus, like the one of
T. onnurineus, consists of two modules: one is for
hydrogenase activity to produce a proton gradient, and
the other module, the Mrp-type Na+/H+ antiporter module, changes the proton gradient to a Na+ gradient
(Schut et al., 2012). The Na+ gradient can then be
utilized by the Na+-dependent A1AO ATP synthase of
P. furiosus (Pisa et al., 2007) to synthesize ATP at high
temperatures. There is also a striking sequence identity
of Mbh to complex I of respiratory chains. Its soluble
domain, consisting of modules N and Q, catalyzes
NADH oxidation and transfers electrons via a chain of
iron–sulfur center to the membrane. The soluble domain
is indeed similar to soluble hydrogenases. The membrane-embedded domain, which is called P-module or
pump module, contains two main modules, PP and PD,
with a row of four Na+/H+-antiporter-similar subunits,
which are connected by a long helical ‘transmission element’ (Efremov et al., 2010; Hunte et al., 2010; Brandt,
2011; Dr€
ose et al., 2011). These Na+/H+ antiporter-like
modules are the predicted sites of H+ translocation. In
complex I of E. coli and Rhodothermus marinus, not only
an export of H+ was observed, but also a Na+ transport
in the other direction was observed. One may work as a
H+ pump and the other as Na+/H+ antiporter. But this
FEMS Microbiol Rev 38 (2014) 449–472
457
Na+/H+ antiport activity does not seem to be a general
property of complex I because for Paracoccus denitrificans, only H+ translocation was observed. No Na+ transport in the opposite direction could be shown (Batista
et al., 2010; Batista & Pereira, 2011).
Is this system of energy conservation, composed of
Mbh, Mrp-type Na+/H+ antiporter module, and A1AO
ATP synthase also present in other hyperthermophiles?
Yes, such an Mrp–Mbh system is also genetically encoded
in the genera Thermococcus, Desulfurococcus, Thermosphaera, Ignisphaera, and Staphylothermus (Schut et al.,
2012), all hyperthermophilic archaea. It might be possible
that such a system of energy conservation is an adaptation of hyperthermophilic archaea to environments with
high temperatures.
Energy conservation in Ignicoccus
hospitalis and Nanoarchaeum equitans
Ignicoccus hospitalis is a hyperthermophilic, strictly anaerobic crenarchaeon isolated from a submarine hydrothermal system at the Kolbeinsey Ridge. It grows optimally at
90 °C and has a chemolithoautotrophic metabolism
(Paper et al., 2007). Together with Nanoarchaeum equitans cells, which are small cocci with only 400 nm in
diameter, I. hospitalis forms an ‘intimate association’, the
only known stable association between two archaea
(Huber et al., 2002; Jahn et al., 2008). Species of the
genus Ignicoccus are so far the only described members of
archaea who have an unusual cell architecture as well as
an unusual energy conservation mechanism (Fig. 5). They
are the only archaea that have an inner membrane and
an outer cellular membrane. These two membranes
enclose an intermembrane compartment with a width up
to 500 nm (Huber et al., 2000, 2012; Rachel et al., 2002).
First insights into the unusual energy conservation mechanisms in Ignicoccus cells came from immuno electron
microscopy of ultrathin sections of I. hospitalis cells. With
the use of antibodies against the A1AO ATP synthase of
I. hospitalis, the enzyme was detected exclusively in the
outer cellular membrane (K€
uper et al., 2010). This was a
major breakthrough into a new era of bioenergetics,
because till then, only cytoplasmic membranes have been
described as harboring ATP synthases (Lewalter & M€
uller,
2006), and it was believed that outer membranes are
generally ‘non-energy-conserving’ (Nicholls & Ferguson,
1992). However, not only an A1AO ATP synthase is essential to drive ATP synthesis, but also an ion gradient
across the membrane is necessary. This ion gradient is
produced by the H2:sulfur oxidoreductase reaction, in
which sulfur is reduced with H2 to H2S (Dirmeier et al.,
1998). The reduction of sulfur with hydrogen is not an
exclusive feature of Ignicoccus, but has been observed
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Published by John Wiley & Sons Ltd. All rights reserved
458
F. Mayer & V. Müller
before in archaea and bacteria (Hedderich et al., 1999).
The free energy change in the reaction
H2 þ S ! HS þ Hþ
0
DG0 ¼ 28 kJ mol1
(17)
is large enough to allow for proton translocation (Thauer
et al., 1977; Amend & Shock, 2001). However, the mechanism of membrane energization is obscure. If the hydrogenase and sulfur reductase face to the outside, it must
involve an active transport of protons from the inside to
the outside because there is no net charge generation in
the redox reaction. The c subunit of the A1AO ATP synthase of I. hospitalis has no conserved Na+-binding motif.
This is consistent with proton-driven ATP synthesis and
the presence of a primary proton gradient across the
membrane. Consistent with the finding that the outermost
membrane is the place of energy conservation (K€
uper
et al., 2010), an ATP-consuming key enzyme of autotrophic lifestyle was found in the intermembrane compartment. The AMP-forming acetyl-CoA synthetase uses the
ATP by the A1AO ATP synthase for activation of acetate
and conversion of acetate to acetyl-CoA, the primary
acceptor molecule of CO2 fixation in I. hospitalis (Mayer
et al., 2012b).
Nanoarchaeum equitans, the small riding companion of
I. hospitalis has apparently, as deduced from its genome
sequence, no genetic capability for energy conservation
(Giannone et al., 2011). Thus, the working hypothesis is
that it gains ATP from its host. Even if this is correct,
every living cell requires an energized membrane. In
principle, this can be achieved by a proton-translocating
ATP synthase/ATPase at the expense of ATP. In fact, this
is not uncommon and observed in many strictly fermenting bacteria. However, the genome of N. equitans harbors
only a subset of genes that are usually required to encode
a functional ATP synthase/ATPase (Lewalter & M€
uller,
2006; Giannone et al., 2011). Whether N. equitans has the
most rudimentary and simplest ATPase known to date is
discussed below.
ATP synthases: key enzymes in cellular
bioenergetics
Despite all the differences in the ways the electrochemical
ion gradient is generated, the common feature of archaea
(and other life forms) is an enzyme that synthesizes ATP,
according to Eqn. (1), at the expense of the transmembrane electrical ion gradient: the ATP synthase (M€
uller
et al., 1999; M€
uller & Gr€
uber, 2003). All present rotary
ATP synthases/ATPases arose from a common ancestor
and evolved into the three classes known to date: the
F1FO ATP synthase present in bacteria, mitochondria, and
chloroplasts; the V1VO ATPase present in eukaryotes; and
the A1AO ATP synthase present in archaea (Fig. 6; Cross
& Taiz, 1990; Nelson, 1992; M€
uller & Gr€
uber, 2003; Cross
& M€
uller, 2004). Their common feature is a structure
Fig. 5. Model of energy conservation in Ignicoccus hospitalis. This archaeon has two membranes, an outer cellular membrane and an inner
membrane. Protein complexes for energy conservation are localized in the outer cellular membrane, so far exclusively. For energy conservation,
the H2:sulfur oxidoreductase reduces sulfur with H2 to H2S. This reaction is coupled to the generation of an electrochemical ion (most likely
proton) potential by an unknown mechanism. It may involve a proton-motive Q-cycle or equivalent. Then, the proton gradient is used by the
A1AO ATP synthase to synthesize ATP. This ATP is used, for example, by the AMP-forming acetyl-CoA synthetase (ACS) for the activation of
acetate and the conversion of acetate to acetyl-CoA, the primary acceptor molecule of CO2 fixation in I. hospitalis. The figure shows the nature
of the ions pumped, but not the stoichiometries of the reaction. Adapted from Huber et al. (2012).
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 38 (2014) 449–472
Archaeal life under extreme energy limitation
459
Fig. 6. Comparison of the three classes of ATP synthases/ATPases. The archaeal A1AO ATP synthase consists of nine subunits, whereas the wellstudied bacterial F1FO ATP synthase has only eight different subunits. The yeast V1VO ATPase has 14 different subunits. Same or similar subunits
of A1AO ATP synthases, F1FO ATP synthases, and V1VO ATPases are illustrated with the same color. The yeast V1VO ATPase has three different c
subunits (c, c′, and c″), encoded by different genes, that build the rotor. Subunit a of the yeast V1VO ATPase has two isoforms Vph1p (complex
targeting to the vacuole) and Stv1p (complex targeting to the Golgi apparatus), which are not mentioned in the figure. X1 = A1 or V1 or F1 and
XO = AO or FO or VO.
made of two rotary motors, the A1/F1/V1 and the AO/FO/VO
motors, connected by a central stalk and one to three
peripheral stalks. A1AO ATP synthases, F1FO ATP synthases, and V1VO ATPases catalyze a reaction that in vitro is
fully reversible. In the ATP hydrolysis mode, ATP hydrolyzed by the soluble motor, localized in the cytoplasm of
bacteria and archaea, drives rotation of the central stalk
that is physically connected to the membrane-embedded,
ion-translocating motor. Rotation of this motor is obligatorily coupled to the translocation of ions, in the hydrolysis mode from the inside to the outside. In the synthesis
mode, the electrochemical ion potential drives the influx
of ions into the motor, forcing it to rotate. Ions are
translocated from the outside to the inside, and the central stalk starts to rotate. Rotational energy is then transmitted to the soluble motor where it is used to synthesize
ATP (M€
uller & Gr€
uber, 2003). A major functional difference is that the predominant physiological function of
A1AO and F1FO ATP synthases is to synthesize ATP at the
expense of an electrochemical ion gradient, whereas the
V1VO ATPases are designed by nature to drive ion transport at the expense of ATP hydrolysis in organelles of
eukaryotes. ATP synthesis in eukaryotes is by F1FO ATP
synthases that reside in mitochondria and chloroplasts.
Interestingly, despite their major difference in function,
the A1AO ATP synthase is evolutionarily more related to
V1VO ATPases than to F1FO ATP synthases. This is consistent with the phylogenetic tree of life that shows a
rather late divergence of archaea and eukarya from a
common ancestor. It should be noted in this connection
that the ‘tree of life’ has actually become a ‘bush of life’.
FEMS Microbiol Rev 38 (2014) 449–472
Genes are transferred between the domains of life by horizontal gene transfer. This also holds true for archaea and
bacteria (Averhoff, 2009). Actually, a number of bacteria
have ‘stolen’ the A1AO ATP synthase genes from archaea.
This is true for Thermus thermophilus and Enterococcus hirae and may be also true for other bacteria, as evident
from genome sequences. ATPase genes from bacteria
annotated as V1VO ATPase genes are actually A1AO ATP
synthase genes! As long as these wrong annotations are
not taken out of data bases, they will continue due to the
use of annotation programs.
How is the rotational energy used for ATP synthesis in
the A1/F1 domain? The A1 domain consists of three alternating A and B subunits and the F1 domain of three
alternating a and b subunits (Table 1). All subunits contain the Walker motifs A and B, which are located at the
boundary surface of each subunit. These Walker motifs
have conserved amino acids for the binding of nucleotides (Boyer, 1997). The three catalytic centers for ATP
synthesis and ATP hydrolysis are built predominantly by
amino acids of subunit A in A1AO ATP synthases and by
amino acids from subunit b in F1FO ATP synthases
(Weber et al., 1995; Weber & Senior, 1996; Kumar et al.,
2010). The Walker A motif, also named P-loop (Walker
et al., 1982), has a hairpin structure with the consensus
sequence (GXXXXGKT), which is directly involved in
coordination of the c-phosphoryl group of the nucleotide
(Hanson & Whiteheart, 2005). Similar to B subunits of
V1VO ATPases, the B subunits of A1AO ATP synthases
(functionally equivalent to the a subunits of F1FO ATP
synthases) do not contain a conserved P-loop motif
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
460
F. Mayer & V. Müller
Table 1. Comparison of subunit composition of A1AO ATP synthases,
V1VO ATPases, and F1FO ATP synthases*
A1AO ATP synthase
(nine subunits)†
V1VO ATPase
(14 subunits)‡
F1FO ATP synthase (eight
subunits)§
H
a
c
E
C
F
A
B
D
–
–
–
–
–
G
a
c, c′, c″
E
d
F
A
B
D
C
H
–
–
e
–
a
c
–
–
e
b
a
c
–
–
d
b
–
*Homologous subunits are in the same row.
†
Subunit composition of the A1AO ATP synthase from Pyrococcus
furiosus.
‡
Subunit composition of the V1VO ATPase from yeast.
§
Subunit composition of the F1FO ATP synthase from Escherichia coli.
(Olendzenski et al., 1998), but they are able to selectively
bind ADP and ATP (Weber et al., 1995; Weber & Senior,
1996; Kumar et al., 2008). The Walker B motif is
involved in coordination of the nucleotides and contains
a glutamate, which activates a water molecule, which is
essential for the cleavage of the phosphoanhydride bond
during ATP hydrolysis (Abrahams et al., 1994). ATP is
synthesized at the boundary surface of subunits A and B
(a and b in F1FO ATP synthases) in a ‘binding change’mechanism using the rotational energy from the membrane-embedded motor and the central stalk. In the ‘open
state’, the boundary surface between subunits A and B (a
and b in F1FO ATP synthases) is open for the binding of
ADP and Pi. After the binding of ADP and Pi, the central
stalk is rotating by 120°, and ATP is synthesized from
ADP and Pi, which is called the ‘tight state’. A further
rotation of the central stalk by 120° leads to the ‘loose
state’ in which the ATP is released from the boundary
surface. After a rotation of the central stalk of another
120°, the boundary surface is again in the ‘open state’.
With this mechanism, three ATPs can be synthesized by
one rotation of the membrane-embedded motor (Boyer,
1989; Yoshida et al., 2001; Nakanishi-Matsui et al., 2010).
ATP synthesis is reversible in vitro, but it is important
to note that this is different in vivo. For example, a
thiol modification as observed in plants turns off the
‘hydrolysis mode’ at night (Hisabori et al., 2002). Moreover, energy conservation in eukaryotes is in mitochondria or chloroplasts and catalyzed by F1FO ATP synthases.
In contrast, energization of membranes of eukaryal
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
compartments such as the vacuole or the endoplasmic
reticulum is by the generation of an ion gradient across
the organelle membranes by the action of rotary ATP hydrolases (the V1VO ATPases). Nature evolved those
enzymes to rest in the ‘hydrolysis mode’ by changing the
number of ion-binding sites in the membrane-embedded
rotor.
The number of ion-binding sites in the rotor is determined by the primary structure of the c subunit and the
number of c subunits in the ring (Fig. 7). In its simplest
form, the c subunit contains two transmembrane helices
connected by a small, cytoplasmic loop. Each c subunit
has one ion-binding site, and a carboxylate as in a glutamate or aspartate residue is essential to bind and translocate H+. The H+ is bound between the carboxyl
oxygen of a conserved carboxylate and the backbone carbonyl of another amino acid, for example phenylalanine
(Pogoryelov et al., 2009). Very few c subunits, notably
from anaerobic bacteria and archaea, bind Na+ instead
uller et al., 2001). The experiof H+ (Dimroth, 1992; M€
mentally determined Na+-binding site from Ilyobacter
tartaricus contains the before-mentioned carboxylate
(Glu65) and four more amino acid residues that coordinate the Na+ (Murata et al., 2005b; Meier et al., 2009),
notably Gln32, Val63, Ser66, and Thr67 (over a buried
structural water molecule). Tyr70 is forming a hydrogen
bond with Glu65 and stabilizes the geometry of the ion
coordination shell, but is not directly involved in Na+
coordination. Noteworthy, only Gln32, Glu65, and Ser66
are sufficiently conserved in evolution and found in all
methanogens, Pyrococci, and Thermococci. (However, it
should be noted that Gln32 can be changed to Glu,
Glu65 to Asp, and Ser66 often to Thr.) Therefore, the
switch from a sodium ion (P. furiosus) to a proton-binding site (Spirulina platensis) is just by a change in three
residues within the large complex of altogether 6657 residues (as calculated for the A1AO ATP synthase from
P. furiosus)!
The individual c subunits assemble to a ring that
rotates against the stator subunit a. Not only the ionbinding site with its conserved carboxylate, but also a
conserved arginine of the membrane-embedded part of
subunit a is essential for ion translocation (Fig. 8). It is
suggested that the deprotonated carboxylate of the c subunit is forming a salt bridge with the positively charged
arginine of subunit a and keeps the carboxylate in the
so-called ‘unlocked conformation’ (state 2). An ion (H+
or Na+), transported by the first ion channel of subunit a
from the periplasm to the hydrophilic a/c interface will
compete with this ion pair, which will allow the protonation of (or binding of Na+ to) the carboxylate (state 3).
A c subunit with a protonated (Na+-bound) carboxylate
can enter the lipid interface in this ‘ion-locked conformaFEMS Microbiol Rev 38 (2014) 449–472
Archaeal life under extreme energy limitation
461
Fig. 7. Rotor subunits and rotor composition in archaeal A1AO ATP synthases. A1AO ATP synthases have a higher diversity in c subunits than
F1FO ATP synthases or V1VO ATPases. Most archaea, like Methanosarcina mazei, have a c subunit with one hairpin and one ion-binding site (E,
glutamate residue). Methanothermobacter thermautotrophicus has a c subunit consisting of two hairpins with two ion-binding sites. The c
subunit of Methanocaldococcus jannaschii has a triplicated hairpin with two ion-binding sites, and the c subunit of Methanopyrus kandleri
consists of 13 hairpins with 13 ion-binding sites. Based on a hypothetical c ring composed of 24 transmembrane helices, the number of
monomers decreases with the temperature from 12 to 1 in the different species. The ion/ATP stoichiometry was calculated using the hypothetical
c ring composition.
tion’ (state 0). After rotation of the c ring and re-entering
the hydrophilic a/c interface from the other side, the
carboxylate in the ‘ion-locked conformation’ rotates outwards and releases the ion, with the help of the electrostatic influence of the arginine, over the second ion
channel of subunit a into the cytoplasm (state 1). Afterward, the carboxylate is in the ‘unlocked conformation’
(state 2) again and opened for re-entering of the next ion
(Pogoryelov et al., 2009). The rotational movement of the
c ring is coupled to the rotation of the central stalk,
which transmits this rotational energy into the soluble
domain (Fig. 8).
As mentioned above, the number of ion-binding sites in
the rotor is determined by the primary structure of the c
subunit and the number of c subunits in the ring (M€
uller,
2004). For example, if the c ring has 12 monomers, with
two transmembrane helices each, the c ring has 12 ionbinding sites. Because the soluble motor has three ATPhydrolyzing/ATP-synthesizing centers, the ion-to-ATP
ratio is four. One way to change this ratio is to change the
number of ion-binding sites in the c ring either by changing
the stoichiometry of monomers in the ring or by changing
the primary structure of the monomers. Actually, the c subunit of V1VO ATPases arose by gene duplication of an
ancestral 8-kDa c subunit gene followed by fusion of the
products. A c subunit twice as big has no consequences for
the efficiency of the enzyme (Jones & Fillingame, 1998).
FEMS Microbiol Rev 38 (2014) 449–472
However, one ion-binding site was lost during the duplication event. Thus, if the same number of transmembrane
helices is compared, the rotor of V1VO ATPases has only
half the number of ion-binding sites. This is nature’s very
clever way to lock the enzyme in the ‘hydrolysis mode’ in
vivo. ATP synthesis is thermodynamically impossible, but
the enzyme is a very good ion pump: because it translocates
fewer ions, it is able to generate steeper gradients (Nelson,
1992; c.f. Eqn. 11).
Up to the discovery of the A1AO ATP synthases, there
was a clear difference in structure and function of ATP
synthases/ATPases. The evolution of a duplicated c subunit with only half the number of ion-binding sites was
seen as the starting point for the evolution of ATPases
locked in the hydrolysis mode (V1VO ATPases). Later,
additional subunits were evolved to reflect the need for
the interaction of the enzyme with a complex regulatory
network in the eukaryotic cell (Wieczorek et al., 2000;
Kane & Smardon, 2003; Forgac, 2007). With the
improvement in DNA sequencing technology, genome
sequences from archaea became available. These data
revealed that the A1AO ATP synthases form a phylogenetically distinct group of enzymes. Moreover, these data
showed that A1AO ATP synthases have an extraordinary
variety in the size of c subunits as well as the number of
ion-binding sites per monomer (M€
uller, 2004), questioning the suggested evolution of ATP synthases/ATPases.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
462
Structure of the A1AO ATP synthase
The first insights into the structure of the A1AO ATP synthases came from analyses of electron micrographs of
enzymes isolated from hyperthermophiles. The global
structures of the A1AO ATP synthases from M. jannaschii
Fig. 8. Proposed mechanism of ion translocation in the membraneembedded domain of the ATP synthase. Individual c subunits
assemble to a ring that rotates against the stator subunit a. It is
suggested that the deprotonated carboxylate of the c subunit is
forming a salt bridge with the positively charged arginine of subunit a
and keeps the carboxylate in the ‘unlocked conformation’ (state 2).
An ion (H+ or Na+) transported by the first ion channel of subunit a
will compete with this ion pair and allows the protonation of (or
binding of Na+ to) the carboxylate (state 3). A c subunit with a
protonated (Na+ bound) carboxylate can enter the lipid interface in
the ‘ion-locked conformation’ (state 0). After rotation of the c ring
and re-entering the hydrophilic a/c interface from the other side, the
carboxylate in the ‘ion-locked conformation’ rotates outwards and
releases the ion over the second ion channel of subunit a into the
cytoplasm (state 1). Afterward, the carboxylate is in the ‘unlocked
conformation’ (state 2) again and opened for re-entering of the next
ion (Pogoryelov et al., 2009).
F. Mayer & V. Müller
and P. furiosus at a resolution of 1.8 and 2.3 nm,
respectively, showed features similar to F1FO ATP synthases, but also some unexpected characteristics (Coskun
et al., 2004; Vonck et al., 2009), which might be an adaptation to the high temperatures of their environments. In
general, the A1AO ATP synthase has a bipartite structure,
consisting of an A1 domain and AO domain, which form
a pair of coupled rotary motors connected with one central and two peripheral stalks (Fig. 9). Analyses of the
molecular mass of the A1AO ATP synthase of P. furiosus
by laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) revealed a molecular mass of
730 10 kDa and a stoichiometry of the enzyme complex of A3B3CDE2FH2ac10. The soluble A1 domain has the
catalytic activity, and the hydrophobic membrane-embedded AO domain is responsible for ion translocation across
the membrane. The A1 domain, often also described as
catalytic head domain, shows pseudo-3-fold symmetry
and clear asymmetry because of the presence of the central stalk (Vonck et al., 2009) and has a strong resemo1
blance to the isolated A1 domain from M. mazei G€
(Coskun et al., 2004). The head domain has a central cavity, which is surrounded by three alternating A and B
subunits, where A is the catalytic subunit comparable to
the b subunit in F1FO ATP synthases (Fig. 10). Subunit A
can be clearly identified in the 3D reconstruction by
its spike-like structures, and the gen atpA contains a 270bp-long intein, which makes atpA from archaea longer
than its homologues (Bowman et al., 1988; Zimniak
et al., 1988; Hirata et al., 1990; Puopolo et al., 1991; Vonck et al., 2009). The asymmetric head domain is connected to the central stalk, which is composed of subunits
C, D, and F. Subunit C has a funnel-shape structure,
composed of three domains, and is directly placed on top
of the membrane-embedded c ring (Bernal & Stock, 2004;
Iwata et al., 2004; Murata et al., 2005a; Vonck et al.,
2009). Furthermore, a knob-like structure over subunit C
Fig. 9. 3D reconstruction of the A1AO ATP synthase from Pyrococcus furiosus. Surface representation of the ATP synthase in top view, bottom
view, and side view orientation. The global structure was obtained by single particle analyses and 3D reconstruction of the A1AO ATP synthase
from P. furiosus. Main features of this new class of A1AO ATP synthases are a collar-like structure (subunit a) and two peripheral stalks (each
composed of an EH complex). Scale bar represents 100 A (Vonck et al., 2009).
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 38 (2014) 449–472
463
Archaeal life under extreme energy limitation
(a)
(e)
(b)
(f)
(c)
(g)
(d)
(h)
Fig. 10. Global structure of the A1AO ATP synthase from Pyrococcus furiosus. Fitting of known X-ray structures into the electron density map of
the A1AO ATP synthase from P. furiosus. (a–f) Cut open views of the map. (a–d) Vertical views of the map, cut at the levels indicated in (e). (a)
Fitting of A3B3, subunit A (blue) from Pyrococcus horikoshii (PDB: 1VDZ) and subunit B (green) from Methanosarcina mazei (PDB: 2C61). (b)
Fitting of the F subunit (purple) from P. furiosus (PDB: 2QAI). (c) Fitting of the C subunit (turquoise) from Thermus thermophilus (PDB: 1R5Z). (d)
Fitting of the 20-hairpin A-type c ring (gold) from Enterococcus hirae (PDB: 2BL2). (e, f) Two perpendicular views along the membrane plane. The
EC domain (pink) from P. horikoshii is fitted to the knobs (PDB: 2DM9). (g) All fitted subunits in the same orientation as (f). (h) Model for the
stator with the a subunit in yellow and the EH dimers in pink (Vonck et al., 2009).
is visible in the 3D reconstruction. This is subunit F,
which has a globular N-terminal domain and an elongated C-terminal part, which extends deep into the A1
head domain and interacts there with subunit B (Gayen
et al., 2007; Vonck et al., 2009). The third subunit of the
central stalk is D, which has a high a-helical content and
can be cross-linked to the inside of subunit B (Xu et al.,
1999; Arata et al., 2002). Unfortunately, subunit D could
not be detected in the 3D reconstruction of the A1AO
ATP synthase of P. furiosus, because of the low resolution
of 2.3 nm of the density map (Vonck et al., 2009).
New structural features are a collar-like structure
located perpendicular to the c ring and two peripheral
stalks (Fig. 10). In contrast, the F1FO ATP synthases of
bacteria, mitochondria, and chloroplasts have only one
peripheral stalk and no collar. Each of the two peripheral
stalks of A1AO ATP synthases consists of a dimer of subunits E and H (Vonck et al., 2009). As shown for the
A1AO ATP synthase of T. thermophilus, the EH heterodimer is formed by two distinct domains, a globular head
domain and a 140-
A-long right-handed coiled coil, a protein fold, which is quite unusual in nature (Lee et al.,
2010). This EH dimer is connected to the A1 domain.
Fluorescence correlation spectroscopy showed that subunit E of the EH dimer is binding stronger to subunit B
FEMS Microbiol Rev 38 (2014) 449–472
than to the catalytic subunit A (Hunke et al., 2011). The
stalks are mounted to the collar, located perpendicular to
the c ring that has two long crossed a-helices with four
bab-motifs. It might be possible that these four motifs
are the connecting sites for the peripheral stalks to subunit a and therefore to the AO domain. Subunit a has
also a membrane-embedded part, which is composed of
7–8 transmembrane a-helices (Vonck et al., 2009). These
a-helices are in close contact with the c ring, and they
presumably form two ‘ion channels’ to load the c ring
with coupling ions over the first channel and to remove
the ion again from the c ring over the second ion channel
(Lau & Rubinstein, 2012).
Rotor composition and coupling
efficiencies in A1AO ATP synthases
An outstanding feature of A1AO ATP synthases is their
difference in size of c subunits and the number of iontranslocating residues (M€
uller, 2004). Most archaea have
the typical 8-kDa c subunit, like F-type ATP synthases
(Inatomi et al., 1989; Wilms et al., 1996; Ihara et al.,
1997; Steinert et al., 1997), but some archaea have very
unusual c subunits. Species of the genera Pyrococcus
(experimentally proved) and Thermococcus (deduced from
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
464
DNA data) have a 16-kDa c subunit with four transmembrane helices, but only one ion-binding site (Mayer et al.,
2012a). In contrast, the 16-kDa c subunits from the
methanogens M. thermautotrophicus (experimentally;
Ruppert et al., 2001) and Methanosphaera stadtmanae
(deduced) have two ion-binding sites per monomer
(M€
uller, 2004; Fricke et al., 2006). M. jannaschii has a
triplicated c subunit (experimentally) with a molecular
mass of around 21 kDa and two ion-binding sites; the
third one was lost during evolution (Ruppert et al.,
1999). Also M. kandleri is very special, because it is the
only archaeon that has a c ring consisting of only one c
subunit with 13 hairpins and therefore 13 ion-binding
sites (Slesarev et al., 2002; M€
uller, 2004), as deduced from
the DNA sequence. What are the physiological consequences of the different numbers of ion-binding sites per
c ring? Obviously, the ion-to-ATP ratio changes and this
is an important, if not the most important, parameter to
adapt ATP synthesis to low-energy environments. According to Eqn. (12), a phosphorylation potential (DGp) of
~ +50 to +70 kJ mol1 would be sustained by the use of
n = 2.9–4.0 ions/ATP at a physiological electrochemical
ion potential of 180 mV (D~
lion ; Sch€afer et al., 1999).
Three ATP-synthesizing subunits in A1 and 12 ion-translocating subunits in the c ring calculate actually to four
ions per ATP. In this example, the loss of one ion-binding site in every second hairpin of the rotor would lower
the number of ions by half. The resulting value for ‘n’ of
two is not enough to synthesize ATP at a phosphorylalHþ
tion potential of ~ +50 to +70 kJ mol1 at a given D~
of 180 to 200 mV, and thus, the enzyme is no longer
able to pump ions against this potential and thus unable
to synthesize ATP.
Apparently, the ATP synthase of P. furiosus is an ATP
synthase that operates in the synthesis mode, although
it has a c subunit with only one ion-binding site in
four transmembrane helices (Mayer et al., 2012a) that
would prevent the enzyme from working as ATP synthase. Moreover, the function as ATP synthase is of
physiological importance because the ferredoxin-driven
H+ translocation by the Mbh enables additional membrane energization (see Fig. 4) that should be and is
used for ATP synthesis (Sapra et al., 2003). But how
can this apparent contradiction be solved? The answer
is rather simple: by adding more c subunits and thus
more ion-binding sites to the c ring. Indeed, the c ring
of P. furiosus contains 10 c subunits, each having one
ion-binding site in four transmembrane helices (Vonck
et al., 2009). This gives an ion/ATP ratio of 3.3. The
ratio is not unusual and, for example, also predicted for
yeast ATP synthase (Petersen et al., 2012) or the Na+
F1FO ATP synthase from Acetobacterium woodii (Fritz
et al., 2008).
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
F. Mayer & V. Müller
The deduction of the number of ion-binding sites is
not unambiguous for the other species. For example,
M. jannaschii has a c subunit containing three hairpins,
but only two ion-binding sites. If we take four subunits
to make the c ring, we have 12 transmembrane helices
with eight ion-binding sites; if we take 5, we will have 15
transmembrane helices and 10 ion-binding sites; six gives
18; and seven gives 21 with 12 or 14 ion-binding sites.
These values are all within the physiological range.
Clearly, the variation in c subunits of archaeal A1AO ATP
synthases has to be of evolutionary advantage and may
reflect an adaptation to the environment these archaea
live in.
Adaptations of A1AO ATP synthases to
life under extreme conditions
How proteins adapt to high temperature has been the
subject of many excellent reviews (Somero, 1995; Macario
et al., 1999; Robb & Clark, 1999; Stetter, 1999; Sterner &
Liebl, 2001). However, one should keep in mind that
most of the studies have been carried out with soluble
enzymes and not with membrane proteins, which are
more exposed to the ‘heat’ than soluble proteins that may
be protected by, for example, solutes (Martins & Santos,
1995; Martins et al., 1997). Among the membrane protein
complexes, the ATP synthases are special because they
work by a rotational mechanism. Because a number of
archaea live at the thermodynamic limit of life, the coupling mechanism has to be very tight, and any leakage of
the coupling ion through the motor has to be avoided.
To date, different features may classify as adaptations of
A1AO ATP synthases to high temperatures. Some of these
features were apparently evolved very early in the evolution, but then kept also by mesophiles.
The stator of A1AO ATP synthases is built by subunit a
on which two peripheral stalks are mounted that keep the
A3B3 subassembly in place. Ion transport drives rotation
of the central stalk within the static A3B3 subassembly.
X-ray structures of subunit E from Pyrococcus horikoshii
showed that the 112-
A-long N-terminal a-helix is
S-shaped like the backbone of a human being and would
facilitate the storage of elastic energy (Balakrishna et al.,
2012). The A1 domain is placed asymmetrically on the
central stalk, which causes a wobbling of the head
domain during rotation of the central stalk. Both peripheral stalks, consisting of EH dimers, are stabilizing the
‘wobbling head’ and are therefore essential to counteract
the torque generated by the central stalk during ATP
hydrolysis and ATP synthesis (Stewart et al., 2012).
The most important difference to other ATP synthases
is the length of the c subunits. Most mesophilic archaea
have a 8-kDa c subunit with two transmembrane helices,
FEMS Microbiol Rev 38 (2014) 449–472
465
Archaeal life under extreme energy limitation
and the copy number in the ring is not known, but in
F1FO ATP synthases, this varies from 8 to 15 (Mitome
et al., 2004; Meier et al., 2005; Pogoryelov et al., 2009;
Vollmar et al., 2009; Preiss et al., 2010; Watt et al., 2010).
Let us assume that the rotor of the ATP synthases consists of 8–15 noncovalently bound c subunits that form a
ring that rotates against the a subunit that is coupled to
ion translocation, one ion per two transmembrane helices. It is easily conceivable that thermal fluctuations will
result in less-stable interactions of c subunits in the ring.
One way to circumvent the problem is to covalently link
individual c subunits in the ring, and the more the covalent bonds, the higher is its stability (Fig. 7). Indeed,
while most mesophiles have a c subunit with two transmembrane helices, Methanothermobacter and Methanosphaera as well as Pyrococcus and Thermococcus species have
a c subunit with four transmembrane helices, and Methanocaldococcus species have a c subunit with six transmembrane helices. In M. kandleri, the c ring is most
likely made by just one c subunit that has 26 predicted
transmembrane helices (M€
uller, 2004).
Not only the size of the c subunits, but also the
coupling ion used may be seen as an adaptation to high
temperatures and for low-energy environments. Methanogens, for example, live at the thermodynamic limit of life
(Deppenmeier & M€
uller, 2008). In the cytochrome-free
methanogens, Mtr is the only coupling site, and the entire
pathway yields only two ions (Na+) translocated per mol
methane formed, equivalent to 0.5 mol of ATP per mol
CH4 (Thauer et al., 2008). Any loss of ions back into the
cell by leakage would be detrimental. Biological membranes are leakier for protons than for sodium ions, and
this effect is even bigger at high temperatures (van de
Vossenberg et al., 1995). Thus, a sodium bioenergetics is
advantageous and is seen as the primary event in the evolution of bioenergetics (Mulkidjanian et al., 2008; Poehlein et al., 2012). Indeed, cytochrome-free methanogens
rely on sodium bioenergetics (Thauer et al., 2008). Mtr is
the only coupling site in the metabolism of methanogens,
and it is a Na+ pump. Thus, the ATP synthase should be
highly Na+ specific. This is obviously the case (McMillan
et al., 2011; Mayer et al., 2012a). The cytochrome-containing methanogens have a mixed H+ and Na+ potential,
and as an adaptation to these two ion gradients, the ATP
synthase is concurrently driven by Na+ and H+ gradients
(Schlegel et al., 2012b).
The ATP synthase of P. furiosus requires Na+ for ATP
hydrolysis (Pisa et al., 2007), Na+ interacts with the c
subunit (Mayer et al., 2012a), and a sodium ion-binding
motif is conserved in subunit c. This would argue that
the Mbh antiporter module generates a Na+ gradient. The
same bioenergetic scenario may be present in the close
relative T. onnurineus.
FEMS Microbiol Rev 38 (2014) 449–472
A1AO ATP synthases have undergone multiple variations
as adaptation to their environment, but the biggest change
may be encountered in N. equitans. A search of the genome sequence revealed only genes encoding subunits A, B,
D, a, and c. Apparently, subunits C, F, E, and H are not
encoded (Waters et al., 2003). Although this has to be
interpreted with caution, this may also be seen as an adaptation to the energy metabolism of N. equitans. It lacks
respiratory enzymes that energize its membranes, and
apparently, the A1AO ATPase/ATP synthase is the only ion
pump present. The lack of subunits may be a result of a
transition from an ATP synthase to an ATP-driven ion
pump. Unfortunately, the cells are hard to grow, and purification and biochemical analysis of the enzyme have not
been possible yet due to a lack of biomass.
Concluding remarks
Apparently, anaerobic archaeal life under extreme energy
limitations has evolved sophisticated mechanisms to conserve even very little energy increments. Thermodynamics
does not allow a direct coupling via substrate-level phosphorylation, but allows for membrane energization reactions. These involve enzymes such as methyltransferases
that use an exergonic (DG0′ = 30 kJ mol1) methyl
transfer reaction to generate a D~
lNaþ . However, this type
of reaction is restricted to methanogenic archaea. A more
widespread reaction is the reversible conversion of hydrogen according to Eqn. (18):
Ferredoxinred þ 2Hþ þ n ionin
$ Ferredoxinox þ H2 þ n ionout
(18)
Exergonic electron flow from reduced ferredoxin to H+
(E = 414 mV) is used to generate a D~
lion (Buckel &
Thauer, 2013). This type of reaction is catalyzed by the
Ech hydrogenase and the much more complex Mbhs of
Pyrococcus or Thermococcus. The Ech hydrogenase is a
proton pump, and the complexity of the Mbh may arise
from the fusion of the hydrogenase module to a Na+/H+
antiporter module.
A similar type of reaction is catalyzed by the Rnf complex that is found in some archaea, but very widespread
in bacteria (Biegel et al., 2011). There, the electron acceptor of the ferredoxin-fueled electron transportation is
uller, 2010) or methanophenNAD+ (bacteria; Biegel & M€
azine (Methanosarcina sp.; Schlegel et al., 2012a). Thus,
the common principle is the use of a ferredoxin-fueled
electron transport chain inherent to one or more protein
complexes. Electron acceptors are NAD+ (bacteria), methanophenazine or protons.
A second principle observed in archaea that live under
extreme energy limitations is the use of Na+ instead of
0′
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
466
H+ as coupling ion. A sodium ion-based bioenergetics
was expected to be present in organisms that live at high
NaCl concentrations, but astonishingly, none of the
known halophilic or moderately halophilic archaea and
bacteria has a sodium ion-based primary bioenergetics.
They all have a secondary Na+ potential built by Na+/H+
antiporter used for various purposes such as pH regulation or flagellar rotation.
The only exception are some marine bacteria that have
evolved a Na+-pumping NADH:ubiquinone oxidoreductase (Nqr; Tokuda & Unemoto, 1984), but that also have
proton-motive complexes III and IV of their respiratory
chains. Flagellar rotation (Imae & Atsumi, 1989), but not
ATP synthesis is driven by D~
lNaþ . This was always discussed as an adaptation to the marine environment, but in
addition these environments are somewhat energy-limited
because the pH in these ecosystems is well above 8. These
bacteria and other alkaliphiles have a low D~
lHþ due to an
inverse DpH, and the secondary sodium ion potential
helps to move and take up nutrients (Krulwich et al.,
1996). How ATP is synthesized by this low D~
lHþ is still a
mystery (Meier et al., 2007; Matthies et al., 2009; Hicks
et al., 2010).
Apparently, the only primary sodium bioenergetics is
found in anaerobes, bacteria, and archaea, the latter have
been described here. Why is this? To answer this question, we have to have a look at the ecosystem: anaerobic
environments often have high concentrations of organic
acids such as acetate, propionate, or butyrate. These act
as protonophores that enter the cell in the protonated
form by diffusion and dissociate inside the cells, due to a
higher pH in the cytoplasm. Thus, organic acids act as
‘proton ferries’. The ion pumped out sneaks back in
through the back door and cannot be used for ATP synthesis. If you live on a diet, you better watch out that
your food does not diffuse away. Organic acids cannot
act as ‘Na+ ferries’, and this is why, some anaerobes have
a sodium ion-based bioenergetics.
But, it can get worse. Membranes are, in general, leakier to protons than to sodium ions (Lane & Martin,
2012). This difference increases at high temperature.
Thus, Thermococcus that lives at 8 to 20 kJ mol1
(Kim et al., 2010) has to avoid back leakage of ions into
the cytoplasm, and sodium ions are clearly superior over
H+ in this regard.
The third principle is that the ATP synthases are
adapted to life under extreme energy limitation. If the
primary gradient is a sodium ion gradient, then the ATP
synthase should be driven by D~
lNaþ . That is indeed
observed. Only a few (three) subtile changes in amino
acids are required to change the ion specificity. Apart
from the use of Na+, the A1AO ATP synthase shows
remarkable structural adaptations: an exciting variety of c
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
F. Mayer & V. Müller
subunits that most likely lead to rotors with different
coupling efficiencies. The general trend is that the ion/
ATP stoichiometry is decreased. Why and how can this
be of physiological importance? Considering life at only
17 kJ mol1, only one ion can be translocated across
the membrane, and several rounds of metabolism are
required to pump out the number of ions required to
make one ATP. The lower the ion/ATP stoichiometry, the
less is the number of substrates converted to make an
ATP. Of course, this comparison is odious because ATP
synthesis only works if the phosphorylation potential is
much below +60 kJ mol1.
This will bring us to the fourth principle. Maybe the
phosphorylation potential in these archaea is indeed considerably lower than +60 kJ mol1. Data are not available,
but in one case (Jetten et al., 1991), one can calculate,
based on the measurement of the nucleotides, a DGP of
around +40 kJ mol1. Describing the basis for life at thermodynamic equilibrium is a challenge for further studies.
Be it Thermococcus or anaerobic methane oxidizers that
both live at around equilibrium (Kim et al., 2010; Holler
et al., 2011), the thermodynamics and, more important,
mechanistic basis of energy conservation in these creatures
are not understood. In the last decades, the basis of energy
conservation in bacterial and mitochondrial electron transport chains has been deciphered down to the atomistic
level. The principles are well understood. Now, it is the
time to discover the beauty of diversity in microbial bioenergetics in unusual organisms such as the archaea. Much
can be learned, also about the origin of bioenergetics.
Acknowledgements
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (through SFB 807).
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