Phil. Trans. R. Soc.

B (2011) 366, 2949–2958

doi:10.1098/rstb.2011.0135

Review

Energy flows, metabolism and translation

Robert Pascal* and Laurent Boiteau
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université Montpellier 1 and 2 – CNRS,
CC DSBC 1706 – Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
Thermodynamics provides an essential approach to understanding how living organisms survive in
an organized state despite the second law. Exchanges with the environment constantly produce large
amounts of entropy compensating for their own organized state. In addition to this constraint on
self-organization, the free energy delivered to the system, in terms of potential, is essential to under-
stand how a complex chemistry based on carbon has emerged. Accordingly, the amount of free
energy brought about through discrete events must reach the strength needed to induce chemical
changes in which covalent bonds are reorganized. The consequence of this constraint was scruti-
nized in relation to both the development of a carbon metabolism and that of translation. Amino
acyl adenylates involved as aminoacylation intermediates of the latter process reach one of the
higher free energy levels found in biochemistry, which may be informative on the range in which
energy was exchanged in essential early biochemical processes. The consistency of this range with
the amount of energy needed to weaken covalent bonds involving carbon may not be accidental
but the consequence of the abovementioned thermodynamic constraints. This could be useful in
building scenarios for the emergence and early development of translation.
Keywords: carbon metabolism; aminoacyl adenylates; aminoacylation; chemiosmosis;
methanogenesis; heterotrophic hypothesis

1. INTRODUCTION pathways. Building an inventory of processes capable

Cells are characterized by the presence of genetic
information, of a metabolism and of compartments;
there has been an ongoing debate on the features
that came first [1 – 3]. This debate has also been com-
plicated by an excessive simplification of positions [4].
But the simultaneous requirement of two or all of the
sub-systems can be considered a likely possibility as
well [5]. Independent of that choice, a metabolic con-
tribution cannot be precluded as the presence of
genetic material or that of membrane components
requires synthetic pathways supporting, for example,
a preparatory metabolism variant of the genetic polymer
first option [4]. Therefore, the chemical free energy
released or used in these pathways represents an essen-
tial component in the majority of the hypotheses on
the origin of life. This is obviously the case for the che-
moautotrophic hypothesis [6,7], in which the
formation of organic matter relies on mineral sources
of energy. In the earlier heterotrophic hypothesis for
the origin of life [8,9], the energy brought about by
organic molecules has been considered as an essential
factor in addition to their role as building blocks. It is
likely that some of these molecules have constituted
the starting material yielding some of the high-energy
intermediates (thioesters, acyl phosphates, acyl aden-
ylates, phosphoenol pyruvate, aminoacyl adenylates)
that are nowadays involved in the main biochemical
* Author for correspondence (rpascal@univ-montp2.fr).
One contribution of 17 to a Discussion Meeting Issue ‘The chemical
origins of life and its early evolution’.
of providing energy to early living organisms is then
a major goal in the origin of life studies. Considering
thermodynamic capabilities of inorganic reactions
has commonly been used in the analysis of hydrother-
mal pathways, mostly performed by geochemists, but
it is less spontaneous for organic chemists who are
used to the fact that thermodynamic constraints are
often of little help in the analysis of organic reactions.
This overlook of thermodynamic constraints has
chemical grounds. It is related to the tendency of
carbon to form covalent bonds with other elements
and then to the fact that organic reactions usually
require these bonds to be cleaved to some extent at
the transition state, which raises high kinetic barriers
that partly reflect the strength of the full bond and
allow reaction products to be determined in many
instances by kinetics rather than thermodynamics. It
is worth noticing that this mere importance of kinetic
barriers in determining organic reactivity has also
been proposed as a key factor to induce self-organiz-
ation as it is a prerequisite for efficient catalytic or
autocatalytic pathways [10,11]. It may eventually be
helpful in understanding why life, defined as a state
of matter in which stability is governed by dynamics
[12], is based on carbon. However, even if many
organic reactions are under kinetic control, the fact
remains that thermodynamic constraints apply and
can be crucial in identifying prebiotic pathways. More-
over, life presents very specific thermodynamic
features useful in understanding the specificity of
living organisms [13]. Lokta [14] initially pointed
out this peculiarity and considered that

2949 This journal is q 2011 The Royal Society

2950 R. Pascal & L. Boiteau Review. Energy flows
thermodynamics is not sufficient to drive evolution,
and proposed to set natural selection as an additional
physical principle [15] in a way similar to the des-
cription proposed by Pross [12]. Anyway, the
developments of thermodynamics have shown that
complex dissipative structures can develop in systems
maintained away from equilibrium [16] and flows of
energy that are essential to living organisms may also
have played a role in the emergence of life [17].
An inventory of processes leading to the presence of
organic matter on the primitive Earth has been made
in the past, including synthesis driven by ultraviolet
light or electric discharges in a reducing atmosphere
and the delivery by impacts [18]. Since, at that time,
a non-reducing atmosphere was considered as likely,
an emphasis was made on the latter process. But,
new pieces of information have been published that
suggest an active atmospheric synthesis owing to a
more reducing character than previously believed
[19]. Moreover, even neutral atmospheres have been
reconsidered and have been shown to produce an
unexpected yield of amino acids when submitted to
electric discharges [20]. Other sources of organic
matter have been proposed as the result of redox gra-
dients that are present at the interface of the mantle
with oceans in hydrothermal systems. It is obvious
that the kind of chemical intermediates and products
that can be obtained would depend on the process
involved. It is then appealing to compare the energetic
properties of the most ancient biochemical intermedi-
ates to the possibilities of different prebiotic pathways.
The existence of a driving force for the transition
from non-living to living is suggested by thermodyn-
amic considerations. Without underestimating the
role of natural selection as a trigger for life’s origin, it
is likely that energy sources could be an important
factor in selecting early biochemical pathways and,
on the other hand, by analysing the potential dynamics
and the thermodynamics of early biochemistry, we
may infer some conclusions on both the nature of
free energy sources capable of driving the emergence
of life as we know it and how subsequent processes
were able to guide evolution. Therefore, this work is
aimed at determining the kind of chemistry that
could arise from these energy sources and their
respective limitations. We report here the progress
made in these investigations, which led us to several
conclusions supporting the heterotrophic nature of
early living organisms and provided new insight into
the chemistry that led to the emergence of the trans-
lation apparatus and the emergence of nucleoside
triphosphates as energy carriers. The suggested picture
is in agreement with the principle of evolutionary con-
tinuity in which random changes were selected as a
result of their ability to give a selective advantage
to the corresponding organisms at every stage of
evolution from the very beginning.
2. THE IMPORTANCE OF ENERGY IN LIFE
EMERGENCE
The idea that physical laws provide a driving force for
the emergence of dissipative structures, which near
bifurcation points depend on the behaviour of
Phil. Trans. R. Soc. B (2011)
energy inactivated
products/heat
chemical carriers (ΔGc)
photons… ΔS<0
high-energy
ΔS0
metabolites
ΔGm
living organism/
chemical system
Scheme 1. Self-organization in chemical systems induced by
a flow of energy and a local decrease in entropy compensa-
ted by an overall increase. The formation of high-energy
metabolites/intermediates is dependent upon the supply of
energy (as chemical carriers or under physical forms) into
the system and on its degradation into high entropy forms.
For closed systems, at the steady state, the same amounts
of energy are entering and leaving the system but energy is
released in a degenerate form characterized by high entropy.
Self-organization in this kind of system can depend upon the
occurrence of autocatalytic pathways, self-replication pro-
cesses or metabolic cycles increasing the rate of decay of
high-energy reactants and constituting dissipative structures.
Except unstable intermediates with very short lifetimes, the
free energy potential of the metabolites accumulating in sig-
nificant concentration is usually limited by the energy of
chemical carriers (reactants): 2DGm , –DGc, demonstrat-
ing that the incoming energy source determines the energy
of proto-metabolic intermediates. Generating metabolites
with higher energy potentials would require the presence of
a thermodynamic machine (such as a heat engine capable
of extracting work or heat from high-entropy energy forms
using an entropy sink), the complexity of which is highly
unlikely for primitive living forms. Therefore, the energy
flow constitutes the driving force and the nature of the
energy source is likely to have decisive consequences on the
kind of metabolism accessible from the self-organization
process.
fluctuations involving a limited number of events
occurring at the molecular scale rather than on macro-
scopic properties [16], has emerged in the second half
of last century. At a sufficient distance from equili-
brium, the evolution of a system may not obey linear
laws but follow nonlinear dynamics characterized by
the formation of these dissipative structures. Most
chemical and biochemical reactions (except those
having very low barriers or when reactants are at con-
centrations very close to equilibrium) satisfy the
criteria of distance from equilibrium [16] so that, in
chemistry, the occurrence of dissipative structures is
mainly governed by the availability of feedback mech-
anisms able to amplify fluctuations, which are usually
the result of autocatalysis or self-reproduction of mol-
ecules. As life is rooted in the properties of molecules,
this view means that amplification processes are
needed to reach the micro- or macroscopic size of
living organisms or cells starting from singularities
occurring at scales smaller by orders of magnitude.
These processes need the system to be maintained
away from equilibrium by an energy flow (scheme 1).
In addition to these requirements on the dynamics of
the processes, it must also be considered that the
nature of the system involved, composed of organic
molecules, has decisive consequences. Indeed, even
in the absence of oxygen, the large majority of

metabolites and biopolymers are generally not thermo-
dynamically stable [21] and, depending on the
conditions, can be converted into inorganic carbon,
CH4 or CO2. As a result, driving the system towards
equilibrium (for instance, by heating and then increas-
ing the rates of irreversible processes) will not
ultimately give a complex mixture of organic molecules
as products. Then, for both thermodynamic and
dynamic reasons, self-organization in biomolecular
systems requires a flow of energy [13,14]. In real irre-
versible systems, energy flows from sub-systems having
high potential towards those of low potential and is
usually released as heat and radiated at low frequen-
cies. As the life form that we know on the Earth is
mostly based on carbon chemistry and covalent
bonds, the amount of free energy needed to induce
self-organization in chemical systems very likely
approaches the order of magnitude needed to destabi-
lize covalent bonds (representing at least a significant
fraction of the corresponding bonding free energy, e.g.
350 kJ mol21 for a C – C bond). This value is consistent
with the free energy potential of many high-energy bio-
chemical metabolites, 50– 70 kJ mol21 [11], meaning
that energy must have been brought about at a molecu-
lar scale by carriers capable of delivering quanta
of energy in the corresponding range to trigger the
emergence of life (scheme 1).1
This free energy requirement is fulfilled by electro-
magnetic radiations with wavelengths close to the
range of visible light emitted by the Sun, a blackbody
heated at ca 6000 K. But, as previously mentioned
by others [17], a repeated absorption of photons by
early biological systems seems impracticable, though
more or less direct mechanisms can be responsible
for the photochemical generation of high-energy mole-
cules. As a matter of fact, ions, atoms or radicals can
be generated by photolysis, and recombination may
lead to activated molecules with a sufficient lifetime
(isolated in a free energy well by high enough kinetic
barriers) so that they can reach the ground or a
location in which a more complex chemistry can take
place [11]. Considering a range of lifetimes from 1 s
to 1 year leads to 85 – 120 kJ mol21 height for the kin-
etic barrier, which also matches the above-deduced
amount corresponding to a fraction of the free
energy of a covalent bond.
(a) The energy quality requirements for life
In any discussion of metabolisms available for the
origin of life, it is essential to take into account that
a consequence of the second law is that free energy
requirements cannot be fully understood at a macro-
scopic—extensive—scale, but are associated with
discrete chemical events occurring at a molecular
scale. As a matter of fact, providing a determined
amount of energy either as a single energy carrier or
as an identical quantity shared by a huge number of
molecules is not equivalent. Moreover, this non-equiv-
alence corresponds actually to an entropy production
that is precisely what is needed for compensating the
loss of entropy associated with self-organization. It
has then been suggested that a minimal amount of
free energy per chemical event, called the minimum
Phil. Trans. R. Soc. B (2011)
Review. Energy flows R. Pascal & L. Boiteau 2951
biological energy quantum, is needed to support life
[23]. The relevance of a minimal quantum of free
energy in biology has been debated and it has been
proposed that in certain environments, this need can
be overcome by the cooperation of different forms of
life based on complementary metabolisms [24]. But
complex associations of different species based on
different metabolisms are unlikely for emerging living
forms so that the existence of a minimum early bio-
chemical energy quantum remains relevant in this
context [11]. In fact, the formation of one adenosine
triphosphate (ATP) molecule from a proton gradient
is in principle capable of taking advantage of a mini-
mum amount of energy corresponding to ca one-
third of the free energy content of ATP (i.e. ca
20 kJ mol21 as more than three proton translocations
are needed to build one ATP from adenosine di-
phosphate (ADP) and inorganic phosphate). This
amount has been called a biological minimal quantum
of energy [23]; it is related to the requirement for a dis-
crete minimum value in energy delivered by unit event
below which function is not possible. As far as early
biochemical pathways are concerned, it is then reason-
able to consider that the minimum amount of energy
possible to integrate in the metabolism was much
higher for less evolved organisms.
(b) Energy and carbon metabolism
The indication that aldehydes are essential com-
ponents for the origin of life, unambiguous from
their role in the formose reaction [25] or in the for-
mation of amino acids [26], can also be appreciated
by an analysis of potential carbon metabolisms
[27,28]. Actually, the first attempt to introduce ther-
modynamic data in the discussion of possible early
carbon metabolisms has been made by Urey [29];
the corresponding data are quoted in the top left
part of table 1. The first conclusion inferred from
those data is the instability of organics in the presence
of oxygen (second column), which is the driving force
for the respiratory metabolism of contemporary living
organisms. But, under anoxic condition, organic mol-
ecules become much less unstable. This is consistent
with the formation of organics in the reduced environ-
ments found in the interstellar medium, on other
bodies of the solar system (Titan) or even in hydro-
thermal systems present in mid-ocean ridges.
Considering only one-carbon derivatives (CO2,
formic acid, formaldehyde, methanol and methane),
as in the original work of Urey, leads to the conclusion
that methanogenesis is the easiest pathway for the
autotrophic generation of energy from inorganic
sources. Though it requires (directly or indirectly)
the reduction of carbon dioxide in four steps, only
the last two are strongly exergonic, leaving unresolved
the question of the formation of significant amounts of
formaldehyde (and sugars). But expanding Urey’s
work by taking into account a more complex carbon
chemistry, including carbohydrate ( – CHOH – ),
aliphatic ( – CH2 – ) carbons, as well as graphite (inor-
ganic carbon) (table 1), it appears that several other
processes are capable of providing energy to the
metabolism. A chemistry based on exergonic reactions
2952 R. Pascal & L. Boiteau Review. Energy flows

Table 1. Standard free energy of reactions (DrG8 in kJ mol21) of simple carbon derivatives susceptible to giving rise to early
metabolisms. Values in parentheses are published by Urey in 1952 [29].

oxidation with reduction with disproportion to reduction with carbonization to

O2 to CO2 H2 to CH4 CH4, CO2 and H2 to -CH2-a conversion to graphite, H2O
compound and H2O and H2O H2O and H2O -CHOH-b,c and H2

CO2 (g) 0 2130 (2131) 0 274 þ6 n.a.

HCOOH (l) 2270 (2296) 2163 (2189) 266 (291) 2107 227 n.a.
CH2O (g) 2529 (2522) 2185 (2178) 2120 (2113) 2129 249 2135
CH3OH (l) 2702 (2707) 2121 (2126) 288 (293) 265 þ15 270
CH4 (g) 2818 (2818) 0 0 n.a. þ136 þ50
–CHOH – (l)b 2480 2136 271 280 0 286
–CH2 – (l)a 2630 249 þ4d 0 þ80 þ1
C (graphite) 2394 250 þ15d þ6 þ86 0
a
As a methylene group in pentanoic acid compared with butanoic acid, DfG8(– CH2 – ) ¼ 21 kJ mol21.
b
As –CHOH– group in glycerol compared with ethylene glycol, DfG8(– CHOH– ) ¼ 2151 kJ mol21.
c
By hydration, hydrogenation or dehydrogenation involving H2 and/or H2O as reagents or products as necessary.
d
Water is consumed (instead of being produced).

can be built from carbohydrates (–CHOH–)n, whereas
inorganic carbon and hydrocarbons appear to constitute
thermodynamic ends (no exergonic reaction possible
except hydrogenation under strongly reducing condi-
tions). Although they behave as energetic biomolecules
through respiratory metabolism, lipids made of aliphatic
hydrocarbons (–CH2 –)n are non-reactive end-products
in anoxic environments, which is consistent with the
long-term stability of oil as well as coal in sediments.
In addition to this nature of waste in an energy producing
pathway, fatty acids present the strong advantage of self-
aggregating into lipid bilayers and constitute boundaries
for early living entities [30,31], whereas carbonization or
methanogenesis leads to products that are useless for
living organisms. The potency of carbohydrate trans-
formations has already been emphasized in the
literature [24,27,28,32] and is clearly shown in table 1,
as well as the difficulty in their synthesis, strongly favour-
able starting from formaldehyde only. The need of
abundant abiotic sources of formaldehyde, or an equiv-
alent capable of generating carbohydrates, may then be
considered as an energetic constraint on the origin of
life, which is consistent with an atmospheric or extra-
terrestrial delivery to the early Earth [33]. The fact
that carbonyl carbon is reduced more easily than it
is formed from oxidized precursors (table 1) confirms
that the formation of substantial amounts of formal-
dehyde and formose products by reduction of CO2
requires very selective pathways that may be accessible
to living organisms having developed very specific
catalysts, but may be difficult for early life in the environ-
ment of hydrothermal systems. This inadequacy is
also consistent with the well-known high kinetic reactiv-
ity of the carbonyl group [34], which is the basis of
many organic synthesis routes but which also increases
the susceptibility of this group to reducing agents
including hydride.
(c) Heterotrophy
The line of reasoning developed above suggests that
the most important organic constituents of the first
living entities were not formed from CO2 by a direct
reduction that could hardly provide biochemical
Phil. Trans. R. Soc. B (2011)
metabolites and free energy simultaneously. Before
examining the possibility that the anabolism of auto-
trophic early living organisms may also have coupled
the thermodynamically unfavourable formation of
important biochemical constituents with the con-
sumption of chemical energy sources, we can
emphasize that this mere observation is consistent
with the heterotrophic hypothesis [8,9]. The metab-
olism of early living entities could then have been
dependent on the transformation of high-energy
organic molecules formed in the environment that
could be converted into metabolic end-products,
through processes able, at the same time, to provide
the amount of energy needed for the metabolism.
The class of molecules formed in this way includes
low-molecular weight reactants with double or triple
bonds (formaldehyde, hydrogen cyanide, cyanic acid,
urea, cyanamide, cyanoacetylene, etc.) that have
been observed in the interstellar medium [35]. For
instance, it has been demonstrated for years that mol-
ecules of this kind can sustain a very rich prebiotic
chemistry leading to the formation of building blocks
such as sugars [25] and amino acids [26]. But a
much more sophisticated chemistry has been observed
from these intermediates: they are additionally able to
activate phosphate [36], to phosphorylate nucleosides
and activate nucleotides [37], or even to directly drive
the formation of activated nucleotides [38]. Although,
they are found in interstellar media, it is unlikely that
high-energy intermediates survived for long periods
in the parent bodies of meteorites, so that their
source has to be found close to the location where
life emerged. Photolysis, lightning in reducing [39]
or neutral atmospheres [20], or impacts [18] have
been proposed as likely processes for the formation
of high-energy intermediates. Activated species built
in this way result from the recombination of atoms,
ions and radicals transiently formed during events
occurring during short timescales followed by associat-
ive processes leading to metastable molecules [40]
with lifetimes sufficient to reach locations where self-
organization can take place [11]. Ultraviolet light is
in principle sufficient to break most components of
the atmosphere although many reactions require

short wavelengths. Other processes such as impact
and lightning (leading to temperatures locally exceed-
ing 10 000 K) are also able to break most atmospheric
molecules through transient heating and lead sub-
sequently to efficient recombination processes
occurring at lower temperatures. Molecules formed
in this way are, in principle, able to supply energy,
though their role has been challenged because they
could hardly accumulate in favourable locations. But
it seems difficult to find alternative pathways that
could deliver organic species capable of performing
this task. This conclusion does not mean that hydro-
thermal formation of organic molecules was devoid
of any utility for the origin of life but that other sources
leading to high-energy intermediates were also needed.
(d) Adenosine triphosphate and chemiosmosis
Owing to its prominence in the metabolism of extant
living organisms, chemiosmosis is a potential pathway
for building the ATP needed for the survival of early
living organisms. The synthesis of ATP (or any other
alternative exchangeable energy carrier) through a
pathway independent of the formation of organic
carbon would be advantageous because of its sub-
sequent use in the development of an organic
chemistry starting from inactivated carbon sources
and reducing agents or sources of inactivated organic
matter (brought to the Earth by external delivery or
formed in hydrothermal vents). This kind of metab-
olism, constituting an anabolism, might have been
based on a redox disequilibrium present in hydrother-
mal environments. It is often considered that the
reducing power of rocks reaching the surface in
ocean ridges may have generated a redox gradient at
the contact with the ocean, which has a more oxidized
state (for recent examples, see [41,42]). Chemiosmo-
sis may then constitute a unique pathway to collect
the energy available from the redox processes that are
available from the hydrothermal environments, and
ATP produced in this way could have provided energy
to anabolic processes. This sophisticated process [43]
proceeds in two well-separated stages with a transient
storage of energy under the physical form of a proton
concentration gradient (scheme 2).
In the first stage, the proton concentration gradient
across the membrane is transiently generated through
an electron transfer cascade coupling the translocation
of as many protons as possible across the membrane to
the transfer of electrons between redox centres of the
electron transfer chain. In a second stage, ATP is syn-
thesized at the expense of the pH gradient using
membrane ATP-synthase, which couples proton trans-
location back to the cytoplasmic compartment to ATP
synthesis. Peter Mitchell proposed the chemiosmotic
theory [43] to explain how ATP is formed by respir-
ation, through oxidative phosphorylation, which is
now universally accepted. Photophosphorylation, the
process by which ATP is synthesized in photosynthetic
organisms, proceeds in a very similar way as well as
other redox metabolisms developed in bacteria and
archaea [44] that involve final electron acceptors
other than oxygen and donors other than the redox
cofactors generated by glycolysis (NADH and
Phil. Trans. R. Soc. B (2011)
Review. Energy flows R. Pascal & L. Boiteau 2953
E
e–
[H+] [H+]
e–
[H+] [H+]
e–
[H+] [H+]
ATP
ΔG [H+] [H+]
ADP
Scheme 2. The principle of generation of ATP through a
chemiosmosis pathway. The electron transfer cascade associ-
ated with redox reactions is coupled to the translocation of
protons towards the exterior of the cell, which generates a
proton concentration gradient. This gradient is used in a
second stage by membrane ATP-synthase (the proton
pump) to synthesize ATP by coupling phosphorylation of
ADP with the re-entry of Hþ in the cytoplasmic compartment.
FADH2) [44]. All these processes have very acute
requirements to be efficient: firstly, a membrane that
is impermeable to protons, secondly, a series of mem-
brane proteins capable of coupling electron transfers
to proton translocations, and finally, membrane ATP-
synthase that is by itself a very complex cellular machine
[45]. Performing only one of these tasks would hardly
be achievable by an emerging living organism, which
raises a series of unanswered questions:
— How could an impermeable membrane be formed
whereas fatty acid-made membranes are notoriously
leaky [46], except in the absence of alkali-metal
cations or other permeable cations [47]?
— How could the free energy from several events,
each involving the translocation of a proton
through the membrane, be harvested to build an
ATP molecule from ADP and inorganic phosphate
without a highly evolved molecular machine?
Otherwise, a pH gradient value exceeding the pos-
sibilities of simple vesicles would be needed to
build ATP in a single step and maintain its concen-
tration to a significant level in a primitive cell. The
fact that membrane ATP-synthases are complex
proteins made of several subunits renders the pro-
blem even more complex as, in this hypothesis,
ATP synthesis should have evolved before trans-
lation, which requires this task to be performed
by a ribozyme or an unknown catalyst.
It is then likely that chemiosmosis emerged later than
translation, rendering highly speculative the hypotheses
supporting that early living organisms exploited inor-
ganic electron donors for their metabolism [24]. The
reasons why evolution led cells to use this complex
mechanism are far from being fully understood.
However, compared with a direct conversion, the bio-
chemical solution presents decisive advantages. The
first one may be the high rates of proton transfers,
2954 R. Pascal & L. Boiteau Review. Energy flows
O R1
O–
N N
H H
O R2
Kpep
±H2O
Scheme 3. Formation of a peptide bond from two peptide segments.
which can reach the diffusion limit in aqueous solution
[48] and are potentially capable of being concerted [49]
with electron transfers, which may be used to get energy
from transient conformational states of membrane pro-
teins involved in the electron transfer cascade. A second
one may be related to the possibility of adaptation of a
wide range of redox potentials to the generation of a
single universal energy currency, namely ATP, which
can then be used to bring about energy in a diversity
of endergonic metabolic transformations. Both oxi-
dative phosphorylation and photophosphorylation can
then be considered as unlikely systems to get energy
for primitive cells.
3. FREE ENERGY REQUIREMENTS FOR THE
EMERGENCE OF TRANSLATION
(a) Bioenergetics of peptide formation
The formation of an amide bond from a peptide seg-
ment with a free C-terminal carboxylic acid and the
N-terminal amine of a second one (scheme 3) is not
very far from thermodynamic equilibrium (Kpep 
0.1 M21) at moderate pH [50].
Therefore, in water and under physiological con-
ditions, moderately activated esters such as aminoacyl-
transfer RNAs (aa-tRNA, DG80 ¼ 235 kJ mol21 [51])
are fully adapted to peptide biosynthesis provided that
the aminolysis step (peptide bond formation) can take
advantage of a catalyst capable of approximating reac-
tants, a role of entropy trap fulfilled by the ribosome
[52]. But, aa-tRNAs are not the only intermediates of
protein biosynthesis as amino acids are universally acti-
vated as adenylates (aa-AMP) before aminoacylation of
tRNA. The development of aminoacyl-tRNA synthe-
tases (aaRS) was one of the first processes needed to
establish an RNA–protein world and the corresponding
genes are among the most conserved ones [53].
(b) A driving force for the evolution of
translation?
Harry Noller [54] pointed out that finding a driving
force supporting a scenario for the development of
translation in an RNA world is not easy. The emergence
of enzymes that stabilize the intermediates of the trans-
lation process is especially puzzling in this discussion as
translation could not develop from pre-existing folded
proteins. For instance, understanding why adenylates
have been selected as intermediates cannot be easily
understood as the equilibrium for their formation
from the ATP in solution is highly unfavourable ([aa-
AMP][PPi]/[ATP][AA] ¼ 3.5  1027 [55]), and they
would not be formed in significant equilibrium
Phil. Trans. R. Soc. B (2011)
O
H3N
O R1 O
H
N
N N
H H
O R2
concentration from ATP unless a specific stabilization
is present as in the active site of aaRS. But the develop-
ment of the translation machinery required the
availability of a source of energy capable of promoting
the formation of aa-tRNA (or any of its precursors as
for example aminoacylated RNA mini-helices [56]).
The fact that the free energy content of aa-AMP is far
beyond that of ATP by as much as ca 37 kJ mol21 [55]
suggests that ATP was not the early activating agent
for amino acids [50]. A first explanation may be that
ATP was settled as a universal currency, whereas
tRNA amino-acid esters formed in a completely inde-
pendent way and had already been selected as
intermediates. A pathway leading from ATP to aa-
tRNA via aa-AMP may have afterwards improved this
system as a result of an unexpected event in evolution
(exaptation), for example through catalytic promiscuity.
Otherwise, the aaRS function may have emerged as
ribozymes in an RNA world with both an adenylation
domain capable of catalysing the formation of aa-
AMP (the thermodynamically favourable reverse reac-
tion of pyrophosphate with adenylate to give ATP is
not spontaneous in water) while stabilizing this inter-
mediate and abilities in the aminoacylation reaction.
Additionally, these catalysts should have to hinder
the spontaneous reaction with carbon dioxide known
to consume aminoacyl adenylates and related mixed
anhydrides [57]. The alternative is to consider that
adenylates (or similar mixed anhydrides formed from
nucleotides) were formed abiotically and available for
early living organisms; in this view, the role of ATP in
adenylate formation must have emerged later [57].
This view is supported by the fact that amino acid N-
carboxy anhydrides (NCA, scheme 4) can be abiotically
formed [50,58,59] and have been shown to spontaneou-
sly react to give mixed anhydride with phosphate
and nucleotides [60,61] with no need of enzyme at
neutral pH.
It is important to notice that as soon as NCA are con-
sidered as prebiotically relevant, peptides must have
been formed spontaneously as NCA polymerize easily.
Living organisms depending only on RNA for genetic
continuity and lacking encoded proteins (in agreement
with the RNA world hypothesis) would only have to
improve this process to take advantage of the formation
of non-coded random peptides, without the need of
specifically built machinery. It must be emphasized
that, like aa-AMP, the large majority of carboxy-acti-
vated amino acids, including thioesters, would have
been converted into NCA (scheme 4) [62] because of
the reaction of the amino group with CO2 as a result
of the abundance of CO2 and bicarbonate on the early
 Review. Energy flows R. Pascal & L. Boiteau 2955

O
OH
R1
NH2 activating agent
CO2
O O R1
CO2 H
X O fast N
R1 R1 N
H
NH2 N O O
H

O
OH
R1 CO2 fast slow
HN X
O O O
O P O R2
R1 OH
NH2

Scheme 4. NCA can be formed starting from sufficiently activated amino acid derivatives including mixed anhydrides with
phosphate and nucleotides. Peptide formation from a carboxy activated amino acid usually takes place through the interme-
diacy of NCA.

O B
O

O B O O
O NH2
O P
O B O O
O HO R1
NCA O OH
O OH O P O
O P O O
OH H 2N B
O O O
R1
O O
P
O O
Scheme 5. Intramolecular aminoacyl transfer from nucleotide phosphate mixed anhydrides occurring from mixed anhydrides
of 30 -phosphorylated nucleosides. Spontaneous aminoacylation of nucleotides by NCA is taking place as well as cyclization.

Earth [63]. Moreover, additional experiments [57] have
shown that aa-AMP like other amino acid phosphate-
mixed anhydrides do not give rise to efficient peptide
chain formation except in the presence of carbon diox-
ide demonstrating that peptides are mainly formed
through the intermediacy of NCA, which removes
any evolutionary advantage of adenylates as peptide
precursors. Actually, living organisms have probably
developed mechanisms to avoid the formation of NCA
in the cell in order to prevent uncontrolled aminoacyla-
tion of proteins so that NCA have presently no identified
role in cells. Finally, NCA have also demonstrated
abilities in aminoacylating nucleotides by reaction with
a 30 -phosphorylated end [60] (scheme 5).
4. A SCENARIO FOR THE EMERGENCE
OF BOTH AMINOACYLATION AND ADENOSINE
TRIPHOSPHATE
An appealing scenario to explain the selection of aden-
ylates by evolution in spite of their ineffectiveness as
Phil. Trans. R. Soc. B (2011)
peptide precursors is to consider that their initial role
lay in the conversion of the free energy available
from amino acid chemistry as NCA into a form
useful for nucleic acid chemistry. The spontaneous
reactions of NCA in aqueous solution leading both
to phosphate-mixed anhydride and aminoacylated
esters (scheme 5) constitute a chemical substratum
from which an early translation apparatus may have
evolved through selective recognition of amino acid
side-chains by RNA and ribosomal activity may have
developed. Independent of this process, any activated
form of nucleotides (mixed anhydrides, pyrophos-
phates, or triphosphates) may have been useful for
RNA replication and polymerization, providing a selec-
tive advantage to the NCA-driven pathway in an RNA
world. Afterwards, the selective advantage of translated
proteins may have driven evolution, and the develop-
ment of protein enzymes capable of sequestrating and
stabilizing adenylates would have been a key impro-
vement as well as their interconversion into more
stable phosphoanhydrides having an increased lifetime
2956 R. Pascal & L. Boiteau Review. Energy flows

initial stage NCA RNA-bound

random peptides
RNA2 5¢p-RNA1
RNA2-RNA1 RNA3
HO 2¢,3¢ RNA3
PPi aa-p-RNA1
ppp-RNA1 peptide-O 2¢,3¢

RNA world:

evolution
ribozyme-mediated mixed anhydride stabilization.
Catalysis of phosphoanhydride formation.
Selectivity in peptide formation.

intermediate stage NCA RNA3

Peptide-O 2¢,3¢
improvements in 5¢p-RNA1 RNA3
RNAsynthesis HO 2¢,3¢

PPi RNA3
ppp-RNA1 aa-p-RNA1 aa-O 2¢,3¢
ribozyme primitive ribosomal activity
RNA–protein world:
evolution

availability of independent pathways for ATP synthesis.

aaRS protein family replaces ribozymes and
improves amino acid specificity.

late stage loss of the useless

NCA
NCA pathways
RNA3
PPi
aa-AMP HO 2¢,3¢ RNA3
ATP proteins
aaRS aa-O 2¢,3¢

reversion of reactant flow due to the availability of ATP

Scheme 6. A scenario for the evolution of amino acid activation pathway starting from an NCA-driven nucleotide activation
towards an ATP-driven protein formation.

inducing the possibility of free energy exchanges
between different metabolic pathways (scheme 6).
5. CONCLUSIONS
Considering thermodynamics constraints leads to a
better understanding of the driving forces by which
life may have arisen or evolved during its early steps.
The most important one is the need for an energy
flow continuously compensating for the loss of entropy
associated with self-organization, which requires the
input of low-entropy energy sources (light for instance)
and high-entropy energy (heat or radiation at long
wavelengths) flowing out of the system. Natural selec-
tion may arise in those systems as soon as structures
that are capable of self-reproduction emerge, so that
the ones that replicate faster from precursors and
energy tend to predominate owing to their kinetic
behaviour, and as soon as their variability is not limited.
The consequences of the need for an energy flow have
been discussed with respect to carbon metabolism
and translation. Both processes are consistent with a
heterotrophic origin in which early life depended on
the availability of energy-rich building blocks. A hetero-
trophic supply of formaldehyde may have been useful
for conversion into carbohydrates and aliphatic chains
through processes releasing energy and leading to
lipidic end-products capable of constituting a boundary.
The analysis of amino acid activation shows that
high-energy mixed anhydrides were involved early,
which suggests that the driving force for the emergence
of translation was the delivery of energy from amino
acid chemistry to nucleotide chemistry. This role of
amino acid derivatives cannot be deduced from present
biochemical pathways but provides an explanation both
of the emergence of ATP as a universal biochemical
currency and of translation.
This work was supported by EPOV interdisciplinary
programme of the CNRS and COST action CM0703.
NOTE
1
The occurrence of very specific enzyme catalysts capable of enhan-
cing reaction rates with proficiencies that can exceed factors of 1019
[22] by stabilizing transition states and unstable intermediates con-
ceals the need of such amounts of free energy in contemporary
biochemistry, which was not the case in prebiotic chemical systems
as catalytic efficiencies were probably limited.
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