Simultaneous synthesis of thioesters and iron-sulfur clusters in water: two universal components of energy metabolism

Article abstract of DOI:10.1039/d0cc04078a

Thioesters are important intermediates in both synthetic organic and biosynthetic reaction pathways. Here we show that thioesters can be synthesized in an aqueous reaction between thioacetate and thiols. The reaction can be coupled to a second reaction between sulfide and either ferrous or ferric iron, which drives the reaction forward. We furthermore demonstrate that sulfide released during thioester formation can be used in the synthesis of peptide bound [Fe-S] clusters, which like thioesters, are ancient components of metabolism. Together our results reveal a primordial linkage between high-energy ester formation and redox chemistry.

Full text of DOI:10.1039/d0cc04078a

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Simultaneous synthesis of thioesters and iron-sulfur clusters in
water: two universal components of energy metabolism
Sebastian A. Sanden a,b*, Ruiqin Yi a, Masahiko Hara a,b and Shawn E. McGlynn a,c,d
*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Thioesters are important intermediates in both synthetic organic, extension algorithms have indicated the possibility of a phosphate-
and biosynthetic reaction pathways. Here we show that thioesters free core metabolic network, where coupling of endergonic reactions
can be synthesized in an aqueous reaction between thioacetic and to thioesters is sufficient for driving components of the reductive-
thiols. The reaction can be coupled to a second reaction between TCA cycle, amino acid biosynthesis, and the hydroxypropionate bi-
sulfide and either ferrous or ferric iron, which drives the reaction cycle.8 An early thioester enabled metabolism may have been
forward. We furthermore demonstrate that sulfide released during privileged compared to those using phosphoesters, due solubility
thioester formation can be used in the synthesis of peptide bound issues of phosphates in the ferruginous Archean ocean.9
[Fe-S] clusters, which like thioesters, are ancient components of Several prebiotically relevant synthetic routes for thioesters have
metabolism. Together our results reveal a primordial linkage been reported, for example the synthesis of iminothioesters from
between high-energy ester formation and redox chemistry.
malononitrile, which can subsequently hydrolyze producing a
thioester,10 thioester synthesis from acetaldehyde and a thiol via UV-
irradiation4, through the exergonic dehydration of glyceraldehyde11,
with carbonyldiimidazole as a condensing agent12, and also from CO
under hydrothermal conditions13. Thioacids are possible precursors
to thioesters, and can be synthesized by thiolysis of nitriles in water,
with subsequent hydrolysis of the produced thioamide,14 or
produced by the reaction of primary amines with carbonyl sulfide.15
Thioacetic acid (TAA) in particular has been found to be an efficient
acetylating agent for amino acids producing peptide bonds in the
presence of an oxidant.14,16 In addition to these synthetic reactions,
thioacetate would also have likely had important roles in energy
transfer reactions in a prebiotic metabolism: if thioesters could be
produced from thioacetate in non-peptide forming reactions, they
might be able to form a link between anabolic peptide forming
reactions and catabolic energy harvesting reactions. However,
information on direct thioester formation from thioacetate in
aqueous solution is lacking.
In this work, thioacetate was investigated as a thiol-acylating agent,
and the reaction yields and rates of thioacetate with two different
thiols were surveyed at different pH. Promotion of the reaction by
sequestering sulfide in iron sulfide was investigated, as was the
utilization of liberated sulfide in the formation of peptide bound iron
sulfur clusters. Thioester formation from thioacetate and a thiol
could potentially occur with or without ferrous iron, and in an
oxidative process in the presence of ferric iron (Scheme 1). 16,17 Given
that the free energy of hydrolysis of thioacetic acid has been
calculated to be -15.2 kJ mol-1 whereas thioester hydrolysis is
generally more negative (ΔG0’Hyd = -35.3 kJ mol-1 for methyl-
thioacetate), 18 we first considered if thiol-acetylation via thioacetic
A key question of the emergence of life is to understand how
prebiotically available molecules could have been assembled into
self-replicating systems. Biology today replicates by coupling
otherwise energetically unfavorable, endergonic reactions to energy
yielding reactions, but how such an energy coupling scheme may
have worked in the earliest life remains unclear. A large component
of the energetic cost of cell replication is derived from
polymerization reactions, where monomers must be activated so
that dehydration can proceed in water.1,2 These reactions are
typically powered by the hydrolysis of phosphoesters such as
adenosine triphosphate (ATP), which today are sustainably
regenerated in the cell mainly through electron transfer processes in
metabolism.3 Thioesters are a frequently invoked alternative to ATP
in abiotic processes: (i) Both have similar standard free energies of
hydrolysis, making them exchangeable from an energetic
perspective.4 (ii) Since thioester formation often precedes
phosphoester formation in metabolism, for example in glycolysis5
6
and in the Wood–Ljungdahl pathway, it is tempting to consider
them as primordial, having operated before phosphoesters in
metabolisms.7 (iii) Recent computational studies employing network
a. Earth Life Science Institute, Tokyo Institute of Technology
2-12-1 I7E Ookayama, Meguro, Tokyo 152-8550, Japan.
b. School of Materials and Chemical Technology, Tokyo Institute of Technology
4259 G1-7 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8502, Japan.
c. Center for Sustainable Resource Science, RIKEN
2-1 Hirosawa. Wako, Saitama 351-0198, Japan.
d. Blue Marble Space Institute of Science, Seattle, Washington 98154, United States
Email: mcglynn@elsi.jp, sanden@elsi.jp
Electronic supplementary information (ESI) available: Experimental details,
and Fig. S1–S12 mentioned in the text.
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acid could be enhanced by coupling to a redox reaction with Fe3+ or with FeCl3 at pH 7.5 showed a similar trend (Fig. 2b).
View Article Online
DOI: 10.1039/D0CC04078A
with non-redox formation of iron(II) sulfide (ΔG0 = -93.6 kJ mol-1), 19
with sulfide originating from TAA as shown in Scheme 1. In such a
way, low energy TAA could be converted into a high energy thioester,
representing a possible route of prebiotic energy conservation
between organic chemistry and mineral formation.
/
mM Fe2+ / Fe3+
O
0
5
0
O
S
O-
O
O
O-
5
Et N
/
mM
3
S
+
S-
HS
S
O
5.5 7.5
O
pH
/
, 70°C
5 mM
5 / 2.3 mM
O
O
1/2
Fe 2+
RS -
2+
Fe
S
S
FeS
3+
Fe
+
S 0
O
H2S
O
S-
RS-
SR
RS -
OH
S-
FeS
2+
Fe
SR
Scheme 1 Overview of the proposed reaction mechanisms of thioester
synthesis from thioacetate in iron independent and iron dependent reactions.
On the top, oxidation of thioacetate to diacetyldisulfide by Fe3+ precedes
thioester formation, and is followed by formation of the thioester along with
FeS and S0 with hydrogen disulfide as the leaving group.20 In the middle, the
non-iron dependent reaction involves degassing of H2S, and on the bottom,
sulfide is sequestered through the formation of iron sulfide (FeS) with Fe2+.
Figure 1 Time course measurement of the synthesized CoM thioester
(AcCoM) from 5 mM thioacetate at 70°C expressed in percent yield in respect
to 5 mM CoM as starting material (Panel a and b). Each time point for the
respective experiment shows data from experimental duplicates and a
regression with the least square fit. The red trace shows the reactions in
which FeCl3 was added, blue with FeCl2 and black with no iron. Panel c and d
had an initial concentration of 2.3 mM thioacetic acid and 5 mM of thiol and
The reaction between TAA and a model thiol compound of
mercaptoethanesulfonate (also known as Coenzyme M, CoM) at
70°C follows initially pseudo-zeroth order rate kinetics (Fig. 1). The
addition of 5 mM Fe2+ does not alter the initial reaction rate
significantly, but ferric iron (5 mM) increases the reaction rate by a
factor of 3 at pH 5.5, and shows a 1.6-times faster reaction at pH 7.5.
During separate experiments which ran for several days (Fig. S11,
ESI), reactions with the addition of Fe3+ at pH 5.5 exhibited maximum
thioester yields of 39% at 1.7 days, compared to 35% yield at 4.8 days
with the addition of Fe2+ (the percent yields are given with respect to
the starting thiol concentration, and are averages from duplicate
experiments measured by UPLC, with ranges in % yield between
duplicates given in Table S2, ESI). With no metal addition, the
maximum yield observed was 29%. At pH 7.5, the highest thioester
yields were 19% in the presence of 5 mM Fe3+ at 0.9 days, 13% with
Fe2+ at 1.7 days, and 9% at 0.9 days with no added metal.
Triethylamine has been employed previously during the acetylation
of alcohols and is expected to act as an auxiliary base.21 With an
altered experimental setup using lower concentrations of starting
materials (Fig. 1c), we found that the addition of triethylamine
accelerated the formation of thioesters with and without the
addition of Fe2+/Fe3+ (Fig. 1c and d).
Using a tertiary amine containing thiol (2-diethylamino)ethanethiol;
Et3NS) as the starting compound, a drastic increase in reaction rate
was observed (Fig. 2), and the reaction characteristics varied
between the two pH conditions investigated. The highest yields were
observed at pH 5.5, the highest concentration of acetylated Et3NS
(AcEt3NS) was measured after 1h reaction time (within the time
frame of the experiment and analysis via UPLC) at both pH values in
the case of FeCl3 or no metal addition. At pH 5.5, the addition of Fe2+
changed the production curve markedly, and in the case of ferric iron
or no addition, the hydrolysis of the thioester followed pseudo
zeroth order kinetics within the first 10h. The hydrolysis of the
thioester methyl-thioacetate was previously determined to follow a
pseudo first order18,22 but for AcEt3NS only the times series obtained
Fe2+/Fe3+. Panel
d contained 5mM triethylamine in addition to the
components in c. The y-axis scale is different for panels c and d where lower
concentrations were used compared to a and b. Additional reaction rate
measurements at lower TAA concentrations are given in Fig. S12, ESI.
In aqueous solution, thioester synthesis competes with the
hydrolysis of both the starting compound TAA and the product
thioester. An investigation of the hydrolysis rates of methyl-
thioacetate at different pH found the highest stability of the thioester
at pH 4,22 which would favor a more acidic pH for a higher yield of
acetylated CoM and Et3NS (AcCoM and AcEt3NS). In line with this,
only traces of the expected thioester could be found in a 100 mM
carbonate buffer at pH 10, suggesting rapid hydrolysis of any AcCoM
produced.
At longer time periods, the concentration of AcCoM decreased only
marginally at pH 5.5 over the course of five days compared to pH 7.5
where up to half of the thioester produced hydrolyzed (Fig. S11, ESI).
At the lower pH, the reaction equilibrium is expected to shift towards
thioester formation, due to the continuous removal of H2S to the
vapor phase (pKa = 7). This – in addition to differences in stability
against hydrolysis – could account for the highest yields obtained for
AcEt3NS: in the absence of Fe2+/Fe3+, a threefold higher AcEt3NS
concentration was measured at pH 5.5 (21% yield) than at pH 7.5
(6%) within the first hour of the experiment (Fig. 2a). For AcCoM, the
highest yields are found at longer time intervals, and the highest
average thioester concentration was also associated with the lower
pH either with or without the addition of iron (Fig. S11, ESI).
Importantly, heating a pH 5.5 acetate buffer with Et3NS at 70°C did
not yield any detectable thioester, indicating that acetate in solution
did not lead to acetylation of the thiol.
Other than sulfide removal by degassing, the formation of FeS by
reaction with ferrous iron provides another way to sequester sulfide
and shift the reaction equilibrium towards the formation of
thioesters. Using Et3NS, a black iron sulfide precipitate is formed
2 | J. Name., 2012, 00, 1-3
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form in plausible prebiotic conditions requires research into the
O
View Article Online
DOI: 10.1039/D0CC04078A
O
mM Fe2+ / Fe3+
5.5 7.5
0
5
/
NH+
NH+
30
availability of thiols, ferric iron, and sulfide. Since thioester
formation from thioacetate involves the release of sulfide, we
investigated peptide bound [Fe-S] cluster synthesis during thioester
forming reactions.
+
S-
S
HS
pH
/
, 70°C
5 mM
5 mM
Using a previously characterized peptide as a model [4Fe-4S] cluster
binding sequence,31 a reaction with Et3NS, thioacetate, and FeCl3 at
70°C produced an absorbance spectrum characteristic of [Fe-S]
clusters (Fig. 3, Fig. S10). Absorbance features at ~384nm, and
447nm, which are characteristic of the ligand to metal charge
transfer bands in iron-sulfur clusters,32 bleached with the addition
of dithionite, demonstrating redox activity (Fig. S10, ESI). Water
soluble thiols such as 2-mercaptoethanol can serve as ligands for
[4Fe4S] clusters produced from FeCl3 and Na2S,33 but while working
on a similar timescale as with the peptide, no appreciable amount of
[FeS] clusters were self-assembled from reactions that lacked the
peptide (Fig. 3). Instead, in the absence of the peptide an increase in
absorbance across the spectrum after 6.5h was observed, likely
resulting from FeS nanoparticle derived turbidity34 followed by
precipitation by 15h (Fig. S10, ESI).
After 6.5h, the measured yield of AcEt3NS was 1.2% compared to
2.3% under the same reaction conditions but without the addition of
the peptide. Thioacetate can thus serve as a source of both thioesters
and sulfide in [Fe-S] cluster formation. Besides the prebiotic
implications of this, molecular release of sulfide from thioacetate
may be of use in laboratory chemical reconstitution schemes, where
sulfide is usually added slowly in a dropwise manner.35
Figure 2
Time course measurement of the production of the AcEt3NS
thioester at 70°C is displayed as % yield in respect to the initial amount of
Et3NS at pH 5.5 (a) and pH 7.5. (b) with 5mM Fe2+ (blue), Fe3+ (red) or no metal
added (black). Duplicates were performed for each reaction and are shown at
the time point.
within a few hours of reaction at 70°C when either Fe2+ or Fe3+ were
in the reaction. At pH 5.5 and with the addition of FeCl2, the highest
yields of thioester increased by 9% and 5% for Et3NS and AcCoM
respectively (Fig. 2b, Fig. S11, ESI). At pH 7.5, a 4% yield increase of
AcCoM was observed with the addition of FeCl2, which at these lower
yields with respect to the starting thiol corresponds to a 1.4-fold
concentration increase in respect to the no metal addition (Fig. S11,
ESI). However, at pH 7.5 thioester formation was insignificantly
increased by FeCl2 using the Et3NS thiol. In the presence of ferric iron,
the highest yield of AcEt3NS of 48% was obtained after 1h reaction at
pH 5.5. Indeed, Fe3+ was associated with the maximum yields
observed in all of the experiments (Fig. 1, Fig. 2, S11, S12). The
enhancement of both reaction rate and yield with Fe3+ suggests that
the oxidation of thioacetate is involved as previously reported in the
case of amines and peptide bond formation16,23 and depicted in
Scheme 2 in the case of thiols.
S
S
Fe
S
+
750µM Ferredoxin maquette,
300µM Fe3+
O
O
Fe
S
NH+
300µM
NH+
Fe
S
+
S-
300µM
-S
S
S
S
pH 7.5, 70°C
Fe
S
2 Fe2+
O
O
O
O
O
O
2 Fe3+
HS S-
2
+
+
2
+
S-
S
S
SR
SR
S
S-
-SR
-SR
Scheme 2 Proposed reactions scheme for oxidation of thioacetate by Fe3+
to diacetyldithiol and subsequent thioester formation. The leaving hydrogen
persulfide likely reacts with Fe2+ produced in the reaction to form FeS and
elemental sulfur as reported earlier.20
The energetic efficiency of thioester formation can be assessed by
considering the reaction thermodynamics: Acetic anhydride, a
commonly used compound for acetylation in organic synthesis,24 has
a standard free energy of hydrolysis of -65.9 kJ mol-1,25 whereas
thioacetate amounts to only -15.2 kJ mol-1. 18 In the case of thioester
formation from iminothioesters10 and nitriles, the standard free
energy of hydrolysis of acetonitrile and hydrogen cyanide to the
corresponding amide is calculated to be -27.0 kJ mol-1 and -69.3 kJ
mol-1 respectively.26 With fair yields of thioesters obtained at mild
conditions in water, thioacetate thus presents an energy efficient
route for the acetylation of thiols. As the toxic sulfide produced
during the acetylation by thioacetate can be sequestered away as
FeS, this reaction scheme might be applicable to green chemistry due
to its energy efficiency and the usage of water as the sole solvent.
Peptide bound [Fe-S] clusters have prominent and diverse roles in
biological metabolism27,28 and likely emerged early in metabolism
bound by short peptides.29,30 Understanding how [Fe-S] clusters may
Figure 3 UV-Vis spectrum of the reaction of 300 µM Et3NS, thioacetate and
FeCl3 in the presence of 750 µM Ferredoxin peptide maquette at 70°C after
6.5h (yellow), after 15h (red) and without the maquette after 15h (blue).
Iron sulfur derivatives have featured prominently in origins of life
research36,37 and both synthetic [Fe-S] clusters and iron sulfide
minerals have been shown experimentally to facilitate CO2
reduction. Reductive carboxylations and amino acid synthesis have
been performed with synthetic [Fe-S] clusters38 and pyruvate has
been synthesized from CO2 on a greigite (Fe3S4) working electrode39
and in reactions involving metallic iron and also iron minerals and
hydrogen.40,41 With the availability of pyruvate for prebiotic
chemistry assured, a synergistic process between thioesters and Fe-
S clusters can be conceived regarding the work presented here. The
enzyme pyruvate ferredoxin-oxidoreductase (PFOR) catalyzes the
synthesis of the thioester acetyl-coenzyme A (CoA) from pyruvate via
oxidative decarboxylation and uses [4Fe-4S] clusters as electron
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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mediators.6 If the acetylation of a thiol by thioacetate and the
concomitant formation of [4Fe-4S] clusters were to be connected to
the reconstitution of a proto-PFOR, a catalytic feedback could be
envisioned, in which continued thioester formation could now be
synthesized from both thioacetate and prebiotically available
pyruvate. Thus, thioacetate and abiotically produced pyruvate could
work synergistically to produce high energy thioesters through two
different reactions in an early metabolism.
PFOR, [Fe-S] clusters, and thioesters adopt a central position in the
metabolism of bacteria and archaea, connecting the Wood-Ljungdahl
(WL) CO2 fixation pathway with the tricarboxylic acid cycle.42 The WL
pathway itself is thought to be the most ancient autotrophic
pathway,43 and in concert with a complete rTCA cycle as is found in
Thermovibrio Ammonificans,44 a hybrid WL-rTCA pathway may have
been a robust primordial metabolism due to redundancy in the
formation of thioesters as a central metabolite.45
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S.A.S. acknowledges the funding for a graduate scholarship received from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of
Japan. S.E.M. acknowledges support by NSF award # 1724300. The authors
thank Robert K. Szilagyi and Christopher Butch for helpful discussions and
Kumiko Nishiuchi for assistance with the mass spectrometry.
Conflicts of interest
There are no conflicts to declare.
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4 | J. Name., 2012, 00, 1-3
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Journal Name
COMMUNICATION
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DOI: 10.1039/D0CC04078A
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DOI: 10.1039/D0CC04078A
Simultaneous synthesis of thioesters and iron-sulfur clusters in water: two
universal components of energy metabolism
Sebastian A. Sanden*, Ruiqin Yi, Masahiko Hara and Shawn E. McGlynn*
Table of Contents Entry
SR
RS
S
Fe
S
[FeS] cluster
binding Peptide
O
O
Fe
S
S
Fe3+
+
-S R1
R1
+
+
S-
S
Fe
RS
Fe
SR
Thioesters and peptide ligated [Fe-S] clusters can be synthesized simultaneously from thioacetic
acid in an aqueous one-pot reaction.