Enzymatic and electron paramagnetic resonance studies
of anabolic pyruvate synthesis by pyruvate: ferredoxin
oxidoreductase from Hydrogenobacter thermophilus
Takeshi Ikeda
1,
*, Masahiro Yamamoto
1,
, Hiroyuki Arai
1
, Daijiro Ohmori
2
, Masaharu Ishii
1
and
Yasuo Igarashi
1
1 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
2 Department of Chemistry, School of Medicine, Juntendo University, Chiba, Japan
Introduction
Pyruvate: ferredoxin oxidoreductase (POR; pyruvate
synthase, EC 1.2.7.1) catalyzes the thiamine pyrophos-
phate (TPP)-dependent oxidative decarboxylation of
pyruvate to form acetyl-CoA and CO
2
. POR contains
one or multiple iron-sulfur clusters in addition to TPP
[1]; the two electrons that arise during oxidation of
pyruvate at the TPP site are sequentially transferred
via the iron-sulfur cluster(s) to external electron accep-
tors. The physiological electron acceptor is a small
iron-sulfur protein ferredoxin or FMN-containing

2
via the reductive tricarboxylic acid cycle. In this cycle,
POR acts as pyruvate synthase catalyzing the reverse reaction (i.e. reduc-
tive carboxylation of acetyl-CoA) to form pyruvate. The pyruvate synthesis
reaction catalyzed by POR is an energetically unfavorable reaction and
requires a strong reductant. Moreover, the reducing equivalents must be
supplied via its physiological electron mediator, a small iron-sulfur protein
ferredoxin. Therefore, the reaction is difficult to demonstrate in vitro and
the reaction mechanism has been poorly understood. In the present study,
we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxogluta-
rate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently
low-potential electrons to reduce ferredoxin, to drive the energy-demanding
pyruvate synthesis by POR. We demonstrate that H. thermophilus POR
catalyzes pyruvate synthesis from acetyl-CoA and CO
2
, confirming the
operation of the reductive tricarboxylic acid cycle in this bacterium. We
also measured the electron paramagnetic resonance spectra of the POR
intermediates in both the forward and reverse reactions, and demonstrate
the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thia-
mine pyrophosphate radical in both reactions. The reaction mechanism of
the reductive carboxylation of acetyl-CoA is also discussed.
Abbreviations
DTNB, 5,5¢-dithiobis-(2-nitrobenzoic acid); EPR, electron paramagnetic resonance; HE-TPP, 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-
thiamine pyrophosphate; LDH, lactate dehydrogenase; OGOR, 2-oxoglutarate: ferredoxin oxidoreductase; OR, 2-oxoacid oxidoreductase;
POR, pyruvate: ferredoxin oxidoreductase; TCA, tricarboxylic acid; TPP, thiamine pyrophosphate.
FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS 501
flavodoxin. By contrast to pyruvate dehydrogenase
multienzyme complex, which irreversibly catalyzes the
same reaction utilizing NAD

tive carboxylation of acetyl-CoA. Two [4Fe-4S] ferre-
doxins, Fd1 and Fd2, from this bacterium are
considered to serve as low-potential electron donors
for this key reaction [8].
POR is distributed among archaea, bacteria and
anaerobic protozoa, and is a member of the 2-oxoacid
oxidoreductase (OR) family, which catalyzes the oxida-
tive decarboxylation of 2-oxoacids to their acyl- or
aryl-CoA derivatives [9]. OR enzymes can be homodi-
meric [10,11], heterodimeric [12,13] or heterotetrameric
[14], depending on the organism. These three types of
OR are phylogenetically related and the heterotetra-
meric enzyme has been proposed to be the common
ancestor that underwent gene rearrangement and
fusion to generate homo- and heterodimeric ORs
[9,13,15]. We recently found novel heteropentameric
ORs [POR and 2-oxoglutarate: ferredoxin oxidoreduc-
tase (OGOR; 2-oxoglutarate synthase, EC 1.2.7.3)] in
H. thermophilus and its close relatives [16,17]; in these
organisms, the heteropentameric POR and OGOR
function as the key components of the reductive TCA
cycle and catalyze the anabolic reductive carboxylation
of acetyl-CoA and succinyl-CoA, respectively [17,18].
Four of the five subunits correspond to those of the
heterotetrameric ORs, suggesting that the heteropenta-
meric ORs might have evolved from an ancestral het-
erotetrameric enzyme by the acquisition of a unique
fifth polypeptide of unknown function. Sequence align-
ments suggest that H. thermophilus POR contains one
TPP and three [4Fe-4S]

reductive carboxylation of acetyl-CoA to form pyru-
vate. We also investigated the inter- and intramolecu-
lar electron transfer during the reductive carboxylation
by electron paramagnetic resonance (EPR) spectros-
copy to clarify its catalytic mechanism.
Results
In vitro assay for pyruvate synthesis by POR: the
coupled assay with OGOR and lactate
dehydrogenase (LDH; EC 1.1.1.27)
Because the synthesis of pyruvate from acetyl-CoA
and CO
2
is an energetically unfavorable reaction with
a reduction potential of )540 mV [22], this reaction
requires a strong reductant. Pyruvate dehydrogenase
multienzyme complex cannot catalyze the react-
ion because the requisite electron donor, NADH
(E
0
¢ = )320 mV), is much too weak an electron
source to drive the transformation. For the enzyme
assay, the reducing power must be supplied in vitro by
the physiological electron donor for POR, ferredoxin.
A possible strategy is to couple the POR reaction to a
Pyruvate synthesis by pyruvate oxidoreductase T. Ikeda et al.
502 FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS
ferredoxin-reducing enzyme. For example, POR from
Moorella thermoacetica biosynthesizes pyruvate using
ferredoxin reduced by CO dehydrogenase [23]; Chloro-
bium tepidum POR has been shown to catalyze pyru-

as a
result of NADH oxidation. The thermostable LDH from
a thermophilic bacterium Thermus caldophilus [28] was
used for the assay because the reaction was performed at
70 °C, which is the optimum temperature for H. thermo-
philus. Table 1 shows the overall reaction of this coupled
assay.
Using the coupled assay with OGOR and LDH,
H. thermophilus POR was found to catalyze the reduc-
tive carboxylation of acetyl-CoA. Indeed, acetyl-
CoA-dependent NADH oxidation was observed with
either reduced Fd1 or Fd2 as an electron donor
(Fig. 2). It was confirmed that pyruvate synthesis by
POR was rate-limiting in this coupled system. A slight
decrease in A
340
in the absence of acetyl-CoA was a
result of the spontaneous thermal degradation of
NADH [29]. The reductive carboxylation depended on
the presence of POR, OGOR, LDH, ferredoxin, 2-oxo-
glutarate and acetyl-CoA (data not shown), indicating
that the coupled assay shown in Fig. 1 proceeded as
expected. However, this reaction did not depend on
the presence of NaHCO
3
(CO
2
) and CoA (Fig. 2B).
Because CO
2

OGOR (oxidative
decarboxylation)
2-Oxoglutarate + CoA + 2 · Fd
ox
fi succinyl-CoA + CO
2
+2· Fd
red
LDH Pyruvate + NADH fi lactate + NAD
+
Total Acetyl-CoA + 2-oxoglutarate + NADH
fi lactate + succinyl-CoA + NAD
+
a
Protons are omitted from the reactions for simplicity. Fd
ox
,
oxidized ferredoxin; Fd
red
, reduced ferredoxin.
A
B
Fig. 2. Reductive carboxylation catalyzed by POR in the coupled
enzyme assay. The assay mixture contained 1 m
M acetyl-CoA,
10 m
M NaHCO
3
,10mM 2-oxoglutarate, 0.5 mM CoA, 0.2 mM
NADH, 1 mM fructose 1,6-bisphosphate, 10 mM MgCl

this assay, pyruvate synthesis by POR was inhibited in
the presence of excess CoA, which caused the reverse
reaction (oxidative decarboxylation of pyruvate). This
impasse prevented any further kinetic analysis of the
reaction.
In this assay, the reductive carboxylation activity of
POR was determined to be 0.23 UÆmg
)1
with 10 lm
Fd1, or 0.19 UÆmg
)1
with 10 lm Fd2. These values
were comparable to those of the oxidative decarboxyl-
ation of pyruvate with Fd1 or Fd2 as an electron
acceptor (0.55 UÆmg
)1
or 0.43 UÆmg
)1
, respectively;
data not shown), suggesting that H. thermophilus POR
functions as an active pyruvate synthase.
EPR measurements of POR during the oxidative
decarboxylation
The purified H. thermophilus POR showed an EPR sig-
nal (g
1,2,3
= 1.973, 2.012 and 2.024) attributed to the
oxidized S =1⁄ 2 [3Fe-4S]
1+
cluster [31] (Fig. 3A). In

C
D
E
F
Fig. 3. EPR spectra of H. thermophilus POR. The purified POR
was incubated with the components: (A) no substrate (as purified);
(B) dithionite, (C) pyruvate; (D) pyruvate and CoA; (E) Fd1, OGOR,
2-oxoglutarate and CoA; (F) acetyl-CoA, Fd1, OGOR, 2-oxoglutarate
and CoA. Instrument settings were: temperature, 10 °K; microwave
power, 100 lW for (A), 250 lW for (B, D–F) or 1 lW for (C); micro-
wave frequency, 9.024 GHz; modulation frequency, 100 kHz; mod-
ulation amplitude, 0.2 mT. The arrow indicates the signal of the
TPP radical intermediate generated during the reductive carboxyla-
tion of acetyl-CoA.
Pyruvate synthesis by pyruvate oxidoreductase T. Ikeda et al.
504 FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS
CoA. These results are consistent with a ping-pong cat-
alytic mechanism with pyruvate as the primary sub-
strate [35]. (Note that the microwave power for
Fig. 3C was 1 lW, 250-fold lower than that for the
others; the intensity of EPR signals is proportional to
the square root of the microwave power under nonsat-
urating conditions.) Although the [3Fe-4S]
1+
signal in
Fig. 3A disappeared at temperatures exceeding 30 °K,
the g = 2.0040 signal remained even at 70 °K (data
not shown), indicating that this signal was the result of
a TPP-radical intermediate. Indeed, this radical is pro-
posed to be the common intermediate in pyruvate

a reduced [4Fe-4S] signal, which was similar to that of
the dithionite-reduced POR (Fig. 3D), indicating the
second electron transfer from the radical to the [4Fe-
4S] cluster(s). The presence of both pyruvate and CoA
allows catalysis to proceed until all the oxygen is con-
sumed [40], preventing reoxidation of the reduced
[4Fe-4S] cluster(s). Because iron-sulfur clusters can
receive only one electron at a time, multiple clusters
should be reduced in this state. These electrons are
then readily released to external electron mediators.
Indeed, in the presence of Fd1, the rhombic
S =1⁄ 2 [4Fe-4S]
1+
signal of the reduced Fd1 [8] was
clearly observed (data not shown).
EPR measurements of POR during the reductive
carboxylation
Addition of 1 mm acetyl-CoA did not affect the EPR
signal of POR (data not shown), indicating that elec-
tron transfer from external electron donors is a key
step to initiate the carboxylation reaction. To supply
reducing equivalents to POR via ferredoxin, we
utilized OGOR as described above. In the presence of
2-oxoglutarate, CoA, OGOR and Fd1, the reduced
[4Fe-4S] signal of POR was observed, overlapping with
the rhombic [4Fe-4S]
1+
signal of Fd1 (g
z,y,x
= 2.08,

philus POR catalyzes pyruvate synthesis from acetyl-
CoA and CO
2
, by the coupled assay with OGOR and
LDH (Fig. 1). Although carboxylation activity is gen-
erally determined by monitoring the incorporation of
14
CO
2
to form [
14
C] pyruvate, OGOR catalyzes the
exchange reaction between CO
2
and the carboxyl
group of 2-oxoglutarate [44], and therefore interferes
with the detection of [
14
C] pyruvate. Instead, the rate
of pyruvate formation was determined by monitoring
the LDH-coupled oxidation of NADH to NAD
+
. The
coupled assay also demonstrated that Fd1 and Fd2
function as electron mediators for POR (and also for
OGOR) [45] in both the oxidative and reductive reac-
tions. These results corroborate the operation of the
reductive TCA cycle in H. thermophilus. Specifically,
two irreversible reactions in the oxidative TCA cycle,
oxidative decarboxylation of pyruvate and 2-oxogluta-

importance for the improvement of this coupled assay
and also with respect to obtaining a deeper under-
standing of the metabolism of H. thermophilus. Indeed,
this would enable the kinetic analysis of the POR reac-
tions in both directions. In particular, the reaction rate
under physiological intracellular concentrations of sub-
strates needs to be determined to demonstrate that
H. thermophilus POR functions toward pyruvate syn-
thesis in vivo.
To investigate the reaction mechanism of H. thermo-
philus POR, we measured the EPR spectra of the
enzyme in the presence of various combinations of sub-
strates. Intra- and intermolecular electron transfer dur-
ing the oxidative decarboxylation was essentially
consistent with the catalytic cycle proposed by Menon
and Ragsdale [36]. We further measured the EPR spec-
tra during the reductive carboxylation of acetyl-CoA,
using OGOR to reduce ferredoxin as in the coupled
assay. In the presence of the reduced Fd1 and acetyl-
CoA, the HE-TPP radical intermediate was formed
(Figs 3F and 4B), indicating the intermediacy of the
HE-TPP radical in both the oxidative and reductive
reactions. The results obtained also indicate that elec-
tron transfer from external ferredoxin to the enzyme is
an indispensable step to form the radical in the reductive
reaction. From the data obtained in the present study,
along with evidence available from the literature, we are
able to propose the catalytic mechanism of the reductive
carboxylation of acetyl-CoA (Fig. 5). (1) The TPP carb-
anion is generated by proton extraction from C2 carbon

above catalytic mechanism. Moreover, the investigation
of the reductive reaction using the coupled system
developed in this study is not only highly important
itself, but also would provide further insights into the
Pyruvate synthesis by pyruvate oxidoreductase T. Ikeda et al.
506 FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS
reverse, oxidative reaction and vice versa. Thus, further
studies on the POR reactions in both directions would
lead to a deeper understanding of the overall reaction
mechanism of this enzyme.
Materials and methods
Bacterial strains and growth conditions
Escherichia coli JM109 and BL21(DE3) were used as hosts
for derivatives of pUC19 and pET21c, respectively. E. coli
MV1184 was used as a host for the expression of T. caldo-
philus LDH. E. coli strains were grown in tryptic soy broth
or LB medium at 37 °C. When necessary, ampicillin
(100 lgÆmL
)1
) was added to the medium for plasmid
selection.
Heterologous expression and purification of POR,
OGOR and ferredoxins
Because H. thermophilus POR (UniProt accession numbers
Q9LBF7–Q9LBG1) is oxygen-sensitive, as is the case for
other ORs [51], the recombinant POR was expressed under
microaerobic conditions and purified under anaerobic con-
ditions as described previously [17]. In preparation for EPR
spectroscopy, dithionite was removed from the purification
buffers. H. thermophilus has two isozymes of OGOR, het-

N
N
NS
H
3
C
O
SCoA
TPP
C2-carbanion
NS
OHH
3
CH
3
C
NS
HO
SCoA
HE-TPP
radical
e
–
CoASH
OHH
3
C
NS
CO
2

′
-a 3
′
-b
e
–
Fig. 5. Proposed catalytic mechanism for the reductive carboxylation of acetyl-CoA catalyzed by POR. The HE-TPP radical is illustrated on
the basis of the model proposed by Barletta et al. [57] with the unpaired electron on the C2a carbon, although its chemical structure is still
controversial [37–39].
T. Ikeda et al. Pyruvate synthesis by pyruvate oxidoreductase
FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS 507
was calculated using an extinction coefficient of
6200 m
)1
Æcm
)1
. One unit of enzyme activity was defined as
the oxidation of 1 lmolÆmin
)1
of NADH.
POR enzyme assays
The oxidative decarboxylation activity of POR was assayed
at 70 °C by monitoring the ferredoxin-mediated reduction
of metronidazole [54]. The standard assay mixture con-
tained 20 mm pyruvate, 0.5 mm CoA, 10 lm ferredoxin,
0.1 mm metronidazole, 10 mm MgCl
2
,1mm dithiothreitol
and 0.5 mm TPP in 100 mm Hepes buffer (pH 8.0 at
20 °C). The decrease in A

by adding the NADH, acetyl-CoA and enzyme solutions to
the mixture, and the decrease in A
340
as a result of NADH
oxidation was measured. One unit of enzyme activity was
defined as the reduction of 1 lmolÆmin
)1
of NADH
(corresponding to the carboxylation of 1 lmolÆmin
)1
of
acetyl-CoA).
Quantification of CoA
The concentration of CoA was quantified using DTNB,
which reacts with free thiol groups (e.g. CoA-SH) to pro-
duce 2-nitro-5-thiobenzoate with an extinction coefficient of
13 600 m
)1
Æcm
)1
at 412 nm [56]. The assay mixture
contained 0.1 mm DTNB in 100 mm Tris–HCl buffer (pH
8.0). Measurement of A
412
was performed after the addition
of the sample solution.
EPR measurements
The enzyme solution was incubated with a substrate(s) in
an EPR sample tube at 70 °C for 5–10 min under a gentle
argon flow that had passed through a deoxidizing column

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