Tài liệu Báo cáo khoa học: Enzymatic and electron paramagnetic resonance studies of anabolic pyruvate synthesis by pyruvate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus doc

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

Keywords
Hydrogenobacter thermophilus; iron-sulfur
cluster; pyruvate: ferredoxin oxidoreductase;
reductive tricarboxylic acid cycle; thiamine
pyrophosphate
Correspondence
M. Ishii, Department of Biotechnology,
Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Fax: +81 3 5841 5272
Tel: +81 3 5841 5143
E-mail:
Present address
*Research Institute for Nanodevice and Bio
Systems, Hiroshima University, Japan
Institute of Biogeoscience, Japan Agency
for Marine-Earth Science and Technology
(JAMSTEC), Kanagawa, Japan
(Received 11 September 2009, revised 17
November 2009, accepted 19 November
2009)
doi:10.1111/j.1742-4658.2009.07506.x
Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thia-
mine pyrophosphate-dependent oxidative decarboxylation of pyruvate to
form acetyl-CoA and CO
2
. The thermophilic, obligate chemolithoauto-
trophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6,
assimilates CO

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

+
as an electron acceptor
in mitochondria and respiratory bacteria, POR can
also catalyze the reverse reaction (i.e. the reductive car-
boxylation of acetyl-CoA) provided that a sufficiently
low-potential electron donor is available. The reverse
reaction (pyruvate synthesis) is a central step for some
autotrophic bacteria because it serves to assimilate
CO
2
into cell carbon [1].
Hydrogenobacter thermophilus TK-6 is a faculta-
tive aerobic, thermophilic, obligate chemolithoauto-
trophic hydrogen-oxidizing bacterium [2]. The optimum
growth temperature range for H. thermophilus TK-6 is
70–75 °C. Phylogenetic analyses of 16S ribosomal
RNA sequences have shown that the genus Hydroge-
nobacter is a member of the deepest branching order in
the domain Bacteria [3]. H. thermophilus assimilates
CO
2
via the reductive tricarboxylic acid (TCA) cycle
[4], which is one of the microbial CO
2
fixation path-
ways [5]. The reductive TCA cycle is a reversal of the
oxidative TCA cycle, and is an endergonic anabolic
pathway that requires reducing equivalents to complete
the cycle [6,7]. POR is one of the key enzymes of the
reductive TCA cycle, and catalyzes the anabolic reduc-

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]

2+ ⁄ 1+
clusters per catalytic unit
[17]; the enzyme contains all of the motifs required for
cofactor binding that were identified in the crystal
structure of the homodimeric Desulfovibrio africanus
POR [19].
In most cases, the enzyme activity of POR is assayed
by monitoring the reduction of an artificial electron
carrier, methyl viologen, during the oxidative decar-
boxylation of pyruvate. However, when the anabolic
role of the novel heteropentameric POR in H. thermo-
philus is considered, the reverse reaction [i.e. the reduc-
tive carboxylation of acetyl-CoA (pyruvate synthesis)]
needs to be assayed. However, this reverse reaction has
proven more difficult to study because a strong reduc-
tant is required to drive the reaction. Hence, the cata-
lytic mechanism of the reductive carboxylation
catalyzed by POR is poorly understood, whereas that
of the oxidative decarboxylation has been intensively
investigated [20,21]. In the present study, we developed
an assay system to demonstrate the reductive carboxyl-
ation catalyzed by H. thermophilus POR with reduced
ferredoxin as an electron donor. Specifically, we uti-
lized another OR-family enzyme, OGOR from H. ther-
mophilus, to supply reduced ferredoxin for anabolic
pyruvate synthesis mediated by POR. OGOR also cat-
alyzes the oxidative decarboxylation of 2-oxoglutarate
using (oxidized) ferredoxin as an electron acceptor. By
coupling the OGOR decarboxylation, we demonstrate
that H. thermophilus POR catalyzes the anabolic

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-

vate synthesis mediated by ferredoxin reduced by the
light-driven reactions of spinach chloroplasts or Chloro-
bium reaction centers [24,25]. However, H. thermophilus
does not possess these ferredoxin-reducing systems.
In this bacterium, reducing equivalents are derived
from hydrogen oxidization catalyzed by multiple
hydrogenases [26], although the physiological electron
transfer pathway(s) from hydrogen to ferredoxin has
not yet been clarified. Instead, we utilized OGOR from
this bacterium to reduce the low-potential ferredoxin
(Fig. 1). The oxidative decarboxylation of 2-oxogluta-
rate catalyzed by OGOR generates low-potential
electrons that reduce ferredoxins as follows: 2-oxoglut-
arate + CoA fi succinyl-CoA + CO
2
+H
+
+2e
)
,
E
0
¢ = )520 mV [27]. The reactions catalyzed by POR
and OGOR were further coupled to the LDH reaction to
detect pyruvate formation spectrophotometrically
(Fig. 1). Pyruvate generated by coupling the POR and
OGOR reactions was reductively converted to lactate
with NADH as an electron donor. Thus, the rate of pyru-
vate formation was monitored as the decrease in A
340

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

was produced from 2-oxoglutarate by
OGOR in this coupled assay, the addition of NaHCO
3
was not necessarily required for the total reaction
(Fig. 1, dashed arrow). By contrast to CO
2
, CoA was
an essential substrate to initiate this coupled reaction;
nevertheless, the reaction actually proceeded without
the addition of CoA with a higher reaction rate than
Fig. 1. Schematic representation of the coupled enzyme assay.
The reductive carboxylation catalyzed by POR was coupled with
the OGOR and LDH reactions. Fd
ox
, oxidized ferredoxin; Fd
red
,
reduced ferredoxin.
Table 1. Enzymatic reactions.
Enzyme Reaction catalyzed by the enzyme
a
POR (reductive
carboxylation)
Acetyl-CoA + CO
2
+2· Fd
red
fi
pyruvate + CoA + 2 · Fd
ox

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

2
,1mM di-
thiothreitol, 0.5 m
M TPP, 0.03 U of OGOR, 0.2 U of LDH and
10 l
M Fd1 (A) or Fd2 (B) in 100 mM Hepes buffer (pH 8.0). Open
circles, complete reaction; filled circles, acetyl-CoA omitted; open
squares, NaHCO
3
omitted; filled squares, CoA omitted.
T. Ikeda et al. Pyruvate synthesis by pyruvate oxidoreductase
FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS 503
that of the complete reaction (Fig. 2B, filled square).
This was the result of a trace amount of CoA in the
assay mixture. CoA quantification by the 5,5¢-
dithiobis-(2-nitrobenzoic acid) (DTNB) assay showed
that 100 mm acetyl-CoA stock solution contained
2.4 mm CoA, corresponding to 24 lm CoA in the stan-
dard assay mixture (i.e. without the addition of CoA
solution). The K
m
value for CoA of the OGOR
enzyme is reported to be 80 lm [30]. Thus, decarboxyl-
ation of 2-oxoglutarate can proceed as a result of CoA
contamination of the acetyl-CoA solution. When the
reaction commences, CoA is regenerated by the reduc-
tive carboxylation of acetyl-CoA (Fig. 1, dashed
arrow). The reaction rate of this coupled assay was
significantly affected by the concentration of CoA
(Fig. 2B). Although CoA was an essential substrate of

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

the dithionite-reduced state, the [3Fe-4S] signal disap-
peared and a new signal attributed to the reduced
S =1⁄ 2 [4Fe-4S]
1+
clusters was observed (Fig. 3B).
This new signal is an overlap of a major signal with
g-values of 1.910, 1.922 and 2.040 and a minor signal
(approximately 4% of the major signal; determined by
spectral simulation) with g-values of 1.880, 2.003 and
2.020 (note that the minor feature around g = 2.00 in
Fig. 3B was a result of this minor signal and not a
TPP radical intermediate described later). The relative
amount of the [3Fe-4S]
1+
cluster in Fig. 3A was
< 10% that of the [4Fe-4S]
1+
clusters in Fig. 3B
(determined by comparing the double integrals of the
EPR spectra), indicating that a substoichiometric
amount of the [3Fe-4S] cluster was derived from the
oxygen-sensitive [4Fe-4S] by partial oxidative damage,
as reported for other ORs [14,32–34]. In the presence
of 20 mm pyruvate, POR showed a strong sharp signal
centered at g = 2.0040 (Fig. 3C), whereas 0.5 mm
CoA did not affect the signal of POR (data not
shown), indicating that the oxidative decarboxylation
reaction begins with the binding of pyruvate, but not
A
B

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

decarboxylation catalyzed by all PORs [36], which is
generated by the binding of pyruvate to TPP and the
resultant decarboxylation. Although the chemical
structure of this intermediate is still controversial [37–
39], it is often referred to as a 2-(1-hydroxyethyl)- or
2-(1-hydroxyethylidene)-TPP (HE-TPP) radical. The
hyperfine structure of the radical was determined in
detail at 70 °K, at which the EPR signals of iron-sulfur
clusters are not detectable (Fig. 4A). The hyperfine
splitting pattern is essentially the same as reported in
other studies [11,36–38,40,41]. The HE-TPP radical is
generated by one-electron transfer; one of the two elec-
trons that are generated during decarboxylation of
pyruvate remains on the TPP intermediate, and the
other electron moves to the intramolecular iron-sulfur
cluster [42]. Consistent with this mechanism, the oxi-
dized [3Fe-4S]
1+
signal in Fig. 3A disappeared in
Fig. 3C, indicating that the cluster was reduced by this
electron. However, no reduced [4Fe-4S] signal was
detectable in Fig. 3C. This is probably because a
reduced [4Fe-4S] cluster(s) can be reoxidized by a trace
amount of oxygen [42]. Because the redox potential
of [3Fe-4S] clusters is generally much higher than
that of [4Fe-4S] clusters ()150  )100 mV versus
)650  )250 mV) [43], the [3Fe-4S] cluster in the
enzyme was not reoxidized. Upon further addition of
CoA, the signal of the HE-TPP radical markedly
decreased, accompanied by concomitant formation of

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,

1.94 and 1.92) [8] (Fig. 3E). Upon further addition of
acetyl-CoA, a signal with g = 2.0040 was observed
(Fig. 3F, arrow). (It was confirmed that this signal was
not a result of the OGOR intermediate.) The hyperfine
structure of this signal shows essentially the same
hyperfine splitting pattern as that of the HE-TPP radi-
cal intermediate observed during the decarboxylation
of pyruvate (Fig. 4), indicating that the HE-TPP radi-
cal was a common intermediate in both the oxidative
and reductive reactions. The HE-TPP radical was
formed only after the reduction of the iron-sulfur clus-
ters of the enzyme, suggesting that the electrons sup-
plied via external ferredoxin molecules played an
important role in forming the radical intermediate
from TPP with bound acetyl-CoA.
A
B
Fig. 4. Hyperfine structures of the EPR signal of the TPP radical
intermediates. (A) incubated with pyruvate and CoA (three scans);
(B) incubated with acetyl-CoA, Fd1, OGOR, 2-oxoglutarate and CoA
(five scans). Instrument settings were: temperature, 70 °K; micro-
wave power, 1 lW for (A) or 100 lW for (B); modulation amplitude,
0.02 mT for (A) or 0.2 mT for (B); other settings were as described
in Fig. 3. The higher power and wider modulation were used for (B)
to increase sensitivity because the amount of the radical intermedi-
ate was much less than for (A).
T. Ikeda et al. Pyruvate synthesis by pyruvate oxidoreductase
FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS 505
Discussion
In the present study, we demonstrate that H. thermo-

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-

rate, are anabolically reversed by POR and OGOR,
respectively, as suggested by our early work [4], with
Fd1 and Fd2 acting as physiological electron donors.
However, because this assay is a complex system
involving four proteins, kinetic analysis was not possi-
ble. The substrates, CoA and CO
2
, were involved in
the two reactions catalyzed by POR and OGOR
(Fig. 1, dashed arrows). In particular, CoA was a sub-
strate of OGOR as well as a product of POR, and sig-
nificantly affected the reaction rate of pyruvate
synthesis. These problems were derived from the fact
that POR and OGOR are similar OR-family enzymes,
both reversibly catalyzing the CoA-dependent
oxidative decarboxylation of 2-oxoacids. For further
analysis, an alternative enzyme that can generate low-
potential electrons to reduce ferredoxin is required.
Thus far, two other enzymes that utilize ferredoxin as
an electron mediator have been purified from H. ther-
mophilus: ferredoxin-NADP
+
reductase (EC 1.18.1.2)
[46] and ferredoxin-dependent glutamate synthase
(EC 1.4.7.1) [47]. However, the midpoint potentials of
the half reactions catalyzed by these enzymes are
higher than that mediated by OGOR, and are there-
fore unsuitable for the reduction of ferredoxin. Thus,
the identification and characterization of enzymes that
transfer electrons to ferredoxins in vivo is of particular

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

atom of the thiazolium ring by the tautomeric 4¢ imino
group of the 4¢-aminopyrimidine ring [48], as is the case
for all TPP-dependent enzymes; this process is also com-
mon to the oxidative decarboxylation catalyzed by this
enzyme. (2) The nucleophilic TPP C2-carbanion attacks
the carbonyl carbon of acetyl-CoA (as it attacks the car-
bonyl carbon of pyruvate in the oxidative decarboxyl-
ation) to form a transient tetrahedral intermediate. (3)
The tetrahedral intermediate undergoes CoA release
and one-electron transfer to the adduct of TPP to form
the HE-TPP radical intermediate. It is not known
whether CoA release and one-electron reduction occur
in a stepwise manner [possibly forming the acetyl-TPP
as an intermediate (3¢-a)] or simultaneously. However,
we believe the latter process is more likely because dur-
ing the oxidative decarboxylation the binding of CoA
appears to be tightly coupled to electron transfer from
the HE-TPP radical [49]. (4) The generated HE-TPP
radical is reduced to the HE-TPP C2a carbanion by a
second electron transfer and then (5 and 6) the resultant
carbanion attacks CO
2
, which might be tightly bound to
the active site of the enzyme [37], to form pyruvate.
These latter steps (4, 5 and 6) correspond to the
exchange reaction between CO
2
and the carboxyl group
of pyruvate catalyzed by this enzyme [50].
Further studies are being planed to confirm the

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-

erodimeric Kor (UniProt accession numbers Q9AJL9 and
Q9AJM0) and heteropentameric For (UniProt accession
numbers Q93RA0–Q93RA4) [16,30]. Because the former is
much more active than the latter, we utilized the recombi-
nant Kor in the present study. Kor, Fd1 (UniProt accession
number Q75VV9) and Fd2 (UniProt accession number
Q4R2T6) were heterologously expressed and purified as
described previously [8,52].
Heterologous expression and purification of LDH
The plasmid, p8T4, carrying the gene encoding T. caldophi-
lus LDH (UniProt accession number P06150) [53] was a
kind gift from Professor Hayao Taguchi (Tokyo University
of Science). LDH was heterologously expressed and purified
to apparent homogeneity (M. Aoshima, A. Nishiyama and
Y. Igarashi, unpublished results). The enzyme activity of
the recombinant LDH was assayed at 70 ° C by monitoring
the lactate-dependent NADH oxidation as the decrease in
A
340
. The standard assay mixture contained 1 mm lactate,
0.2 mm NADH and 1 mm fructose 1,6-bisphosphate (an
allosteric effector of T. caldophilus LDH) [28] in 100 mm
Hepes buffer (pH 8.0 at 20 °C). The oxidation of NADH
NS
H
H
N
H
3
C

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

H
3
C
NS
HO
COO
–
H
3
C
O
COO
–
e
–
HE-TPP
C2
α
-carbanion
2
3
4
56
1
NS
OH
3
C
CoASH
3

′
-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

320
was measured under an argon
atmosphere. The reduction of metronidazole was calculated
using an extinction coefficient of 9300 m
)1
Æcm
)1
. One unit
of enzyme activity was defined as the reduction of 2 lmolÆ-
min
)1
of metronidazole (corresponding to the decarboxyl-
ation of 1 lmolÆmin
)1
of pyruvate on the assumption that
the bleaching of the chromophore is a one-electron process)
[55]. The reductive carboxylation activity of POR was
determined at 70 ° C by the coupled assay with OGOR and
LDH (see Results). The standard assay mixture contained
1mm acetyl-CoA, 10 mm NaHCO
3
,10mm 2-oxoglutarate,
0.5 mm CoA, 0.2 mm NADH, 1 mm fructose 1,6-bisphos-
phate, 10 mm MgCl
2
,1mm dithiothreitol, 0.5 mm TPP,
0.03 U of OGOR, 0.2 U of LDH and 10 lm ferredoxin
(Fd1 or Fd2) in 100 mm Hepes buffer (pH 8.0). The assay
mixture without NADH and acetyl-CoA was incubated at
70 °C under an argon atmosphere. The reaction was started

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

(Gasclean GC-RP; Nikka Seiko, Tokyo, Japan). The reac-
tion was stopped by immersion of the tube in liquid nitro-
gen. EPR spectra were measured on a JES-FA300
spectrometer (JEOL, Tokyo, Japan) using a cylindrical
cavity (TE
101
mode). The measurement temperature was
controlled with a JEOL ES-CT470 cryostat system and
a digital temperature indicator ⁄ controller model 9650
(Scientific Instruments, West Palm Beach, FL, USA). The
magnetic field was calibrated with a JEOL NMR field
meter ES-FC5. The g-values were determined by spectral
simulation using JEOL anisimu ⁄ fa software, version 2.0.0.
Acknowledgements
The authors thank Professor Hayao Taguchi (Tokyo
University of Science) for the gift of the plasmid carry-
ing the LDH gene from T. caldophilus; Dr Miho
Aoshima and Ms Ayako Nishiyama for the preparation
of the recombinant LDH; and Dr Ki-Seok Yoon (Iba-
raki University) for helpful discussions. This research
was supported in part by Grants-in-Aid for Scientific
Research from the Japan Society for the Promotion of
Science.
References
1 Kerscher L & Oesterhelt D (1982) Pyruvate: ferredoxin
oxidoreductase – new findings on an ancient enzyme.
Trends Biochem Sci 7, 371–374.
2 Kawasumi T, Igarashi Y, Kodama T & Minoda Y
(1984) Hydrogenobacter thermophilus gen. nov., sp.
nov., an extremely thermophilic, aerobic, hydrogen-oxi-

dizing bacterium. Int J Syst Bacteriol 34, 5–10.
3 Pitulle C, Yang Y, Marchiani M, Moore ERB, Siefert
JL, Aragno M, Jurtshuk P Jr & Fox GE (1994) Phylo-
genetic position of the genus Hydrogenobacter. Int J
Syst Bacteriol 44, 620–626.
4 Shiba H, Kawasumi T, Igarashi Y, Kodama T &
Minoda Y (1985) The CO
2
assimilation via the reduc-
tive tricarboxylic acid cycle in an obligately autotrophic,
aerobic hydrogen-oxidizing bacterium, Hydrogenobacter
thermophilus. Arch Microbiol 141, 198–203.
5Hu
¨
gler M, Huber H, Stetter KO & Fuchs G (2003)
Autotrophic CO
2
fixation pathways in archaea
(Crenarchaeota). Arch Microbiol 179, 160–173.
6 Evans MCW, Buchanan BB & Arnon DI (1966) A new
ferredoxin-dependent carbon reduction cycle in a photo-
synthetic bacterium. Proc Natl Acad Sci USA 55,
928–934.
7 Buchanan BB & Arnon DI (1990) A reverse KREBS
cycle in photosynthesis: consensus at last. Photosynth
Res 24, 47–53.
8 Ikeda T, Yamamoto M, Arai H, Ohmori D, Ishii M &
Igarashi Y (2005) Two tandemly arranged ferredoxin
genes in the Hydrogenobacter thermophilus genome:
comparative characterization of the recombinant

[4Fe-4S] ferredoxins. Biosci Biotechnol Biochem 69,
1172–1177.
Pyruvate synthesis by pyruvate oxidoreductase T. Ikeda et al.
508 FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS
9 Adams MWW & Kletzin A (1996) Oxidoreductase-type
enzymes and redox proteins involved in fermentative
metabolisms of hyperthermophilic archaea. Adv Protein
Chem 48, 101–180.
10 Drake HL, Hu S-I & Wood HG (1981) Purification of
five components from Clostridium thermoaceticum which
catalyze synthesis of acetate from pyruvate and methyl-
tetrahydrofolate. Properties of phosphotransacetylase.
J Biol Chem 256, 11137–11144.
11 Pieulle L, Guigliarelli B, Asso M, Dole F, Bernadac A
& Hatchikian EC (1995) Isolation and characterization
of the pyruvate-ferredoxin oxidoreductase from the
sulfate-reducing bacterium Desulfovibrio africanus.
Biochim Biophys Acta 1250, 49–59.
12 Kerscher L & Oesterhelt D (1981) Purification and proper-
ties of two 2-oxoacid: ferredoxin oxidoreductases from
Halobacterium halobium. Eur J Biochem 116, 587–594.
13 Zhang Q, Iwasaki T, Wakagi T & Oshima T (1996)
2-oxoacid: ferredoxin oxidoreductase from the thermo-
acidophilic archaeon, Sulfolobus sp. strain 7. J Biochem
120, 587–599.
14 Blamey JM & Adams MWW (1994) Characterization of
an ancestral type of pyruvate ferredoxin oxidoreductase
from the hyperthermophilic bacterium, Thermotoga
maritima. Biochemistry 33, 1000–1007.
15 Kletzin A & Adams MWW (1996) Molecular and phy-

logenetic characterization of pyruvate and 2-ketoisoval-
erate ferredoxin oxidoreductases from Pyrococcus
furiosus and pyruvate ferredoxin oxidoreductase from
Thermotoga maritima. J Bacteriol 178, 248–257.
16 Yun N-R, Yamamoto M, Arai H, Ishii M & Igarashi Y
(2002) A novel five-subunit-type 2-oxoglutarate: ferre-
doxin oxidoreductase from Hydrogenobacter thermophi-
lus TK-6. Biochem Biophys Res Commun 292, 280–286.
17 Ikeda T, Ochiai T, Morita S, Nishiyama A, Yamada E,
Arai H, Ishii M & Igarashi Y (2006) Anabolic five sub-
unit-type pyruvate: ferredoxin oxidoreductase from
Hydrogenobacter thermophilus TK-6. Biochem Biophys
Res Commun 340, 76–82.
18 Deckert G, Warren PV, Gaasterland T, Young WG,
Lenox AL, Graham DE, Overbeek R, Snead MA,
Keller M, Aujay M et al. (1998) The complete genome
of the hyperthermophilic bacterium Aquifex aeolicus.
Nature 392, 353–358.
19 Chabrie
`
re E, Charon M-H, Volbeda A, Pieulle L,
Hatchikian EC & Fontecilla-Camps J-C (1999) Crystal
structures of the key anaerobic enzyme pyruvate:
ferredoxin oxidoreductase, free and in complex with
pyruvate. Nat Struct Biol 6, 182–190.
20 Ragsdale SW (2003) Pyruvate ferredoxin oxidoreductase
and its radical intermediate. Chem Rev 103, 2333–2346.
21 Tittmann K (2009) Reaction mechanisms of thiamin
diphosphate enzymes: redox reactions. FEBS J 276,
2454–2468.

22 Thauer RK, Jungermann K & Decker K (1977) Energy
conservation in chemotrophic anaerobic bacteria.
Bacteriol Rev 41, 100–180.
23 Furdui C & Ragsdale SW (2000) The role of pyruvate
ferredoxin oxidoreductase in pyruvate synthesis during
autotrophic growth by the Wood-Ljungdahl pathway.
J Biol Chem 275, 28494–28499.
24 Yoon K-S, Hille R, Hemann C & Tabita FR (1999)
Rubredoxin from the green sulfur bacterium Chloro-
bium tepidum functions as an electron acceptor for
pyruvate ferredoxin oxidoreductase. J Biol Chem 274,
29772–29778.
25 Yoon K-S, Bobst C, Hemann CF, Hille R & Tabita FR
(2001) Spectroscopic and functional properties of novel
2[4Fe-4S] cluster-containing ferredoxins from the green
sulfur bacterium Chlorobium tepidum. J Biol Chem 276,
44027–44036.
26 Ueda Y, Yamamoto M, Urasaki T, Arai H, Ishii M &
Igarashi Y (2007) Sequencing and reverse transcription-
polymerase chain reaction (RT-PCR) analysis of four
hydrogenase gene clusters from an obligately autotroph-
ic hydrogen-oxidizing bacterium, Hydrogenobacter
thermophilus TK-6. J Biosci Bioeng 104, 470–475.
27 Tersteegen A & Hedderich R (1999) Methanobacterium
thermoautotrophicum encodes two multisubunit mem-
brane-bound [NiFe] hydrogenases. Transcription of the
operons and sequence analysis of the deduced proteins.
Eur J Biochem 264, 930–943.
28 Taguchi H, Yamashita M, Matsuzawa H & Ohta T
(1982) Heat-stable and fructose 1,6-bisphosphate-

activated l-lactate dehydrogenase from an extremely
thermophilic bacterium. J Biochem 91, 1343–1348.
29 Robb FT, Park J-B & Adams MWW (1992) Character-
ization of an extremely thermostable glutamate dehy-
drogenase: a key enzyme in the primary metabolism of
the hyperthermophilic archaebacterium, Pyrococcus
furiosus. Biochim Biophys Acta 1120, 267–272.
30 Yoon K-S, Ishii M, Igarashi Y & Kodama T (1996)
Purification and characterization of 2-oxoglutarate:
ferredoxin oxidoreductase from a thermophilic, obli-
gately chemolithoautotrophic bacterium, Hydrogeno-
bacter thermophilus TK-6. J Bacteriol 178, 3365–3368.
31 Beinert H & Thomson AJ (1983) Three-iron clusters
in iron-sulfur proteins. Arch Biochem Biophys 222,
333–361.
32 Blamey JM & Adams MWW (1993) Purification and
characterization of pyruvate ferredoxin oxidoreductase
from the hyperthermophilic archaeon Pyrococcus
furiosus. Biochim Biophys Acta 1161, 19–27.
33 Mai X & Adams MWW (1994) Indolepyruvate
ferredoxin oxidoreductase from the hyperthermophilic
archaeon Pyrococcus furiosus. A new enzyme
involved in peptide fermentation. J Biol Chem 269,
16726–16732.
T. Ikeda et al. Pyruvate synthesis by pyruvate oxidoreductase
FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS 509
34 Ozawa Y, Nakamura T, Kamata N, Yasujima D,
Urushiyama A, Yamakura F, Ohmori D & Imai T
(2005) Thermococcus profundus 2-ketoisovalerate
ferredoxin oxidoreductase, a key enzyme in the archaeal

energy-producing amino acid metabolic pathway.
J Biochem 137, 101–107.
35 Uyeda K & Rabinowitz JC (1971) Pyruvate-ferredoxin
oxidoreductase. IV. Studies on the reaction mechanism.
J Biol Chem 246, 3120–3125.
36 Menon S & Ragsdale SW (1997) Mechanism of the
Clostridium thermoaceticum pyruvate: ferredoxin oxido-
reductase: evidence for the common catalytic intermedi-
acy of the hydroxyethylthiamine pyrophosphate radical.
Biochemistry 36, 8484–8494.
37 Chabrie
`
re E, Verne
`
de X, Guigliarelli B, Charon M-H,
Hatchikian EC & Fontecilla-Camps JC (2001)
Crystal structure of the free radical intermediate of
pyruvate: ferredoxin oxidoreductase. Science 294 ,
2559–2563.
38 Mansoorabadi SO, Seravalli J, Furdui C, Krymov V,
Gerfen GJ, Begley TP, Melnick J, Ragsdale SW & Reed
GH (2006) EPR spectroscopic and computational char-
acterization of the hydroxyethylidene-thiamine pyro-
phosphate radical intermediate of pyruvate: ferredoxin
oxidoreductase. Biochemistry 45, 7122–7131.
39 Frank RAW, Kay CWM, Hirst J & Luisi BF (2008)
Off-pathway, oxygen-dependent thiamine radical in the
Krebs cycle. J Am Chem Soc 130, 1662–1668.
40 Cammack R, Kerscher L & Oesterhelt D (1980) A sta-
ble free radical intermediate in the reaction of 2-oxo-

acid: ferredoxin oxidoreductases of Halobacterium
halobium. FEBS Lett 118 , 271–273.
41 Bock A-K, Scho
¨
nheit P & Teixeira M (1997) The iron-
sulfur centers of the pyruvate: ferredoxin oxidoreduc-
tase from Methanosarcina barkeri (Fusaro). FEBS Lett
414, 209–212.
42 Kerscher L & Oesterhelt D (1981) The catalytic mecha-
nism of 2-oxoacid: ferredoxin oxidoreductases from
Halobacterium halobium. One-electron transfer at two
distinct steps of the catalytic cycle. Eur J Biochem 116,
595–600.
43 Sticht H & Ro
¨
sch P (1998) The structure of iron-sulfur
proteins. Prog Biophys Mol Biol 70, 95–136.
44 Gehring U & Arnon DI (1972) Purification and proper-
ties of a-ketoglutarate synthase from a photosynthetic
bacterium. J Biol Chem 247, 6963–6969.
45 Yamamoto M, Ikeda T, Arai H, Ishii M & Igarashi Y
(2009) Carboxylation reaction catalyzed by 2-oxogluta-
rate: ferredoxin oxidoreductases from Hydrogenobacter
thermophlius. Extremophiles doi: 10.1007/s00792-009-
0289-4.
46 Ikeda T, Nakamura M, Arai H, Ishii M & Igarashi Y
(2009) Ferredoxin-NADP
+
reductase from the thermo-
philic hydrogen-oxidizing bacterium, Hydrogenobacter

thermophilus TK-6. FEMS Microbiol Lett 297, 124–130.
47 Kameya M, Ikeda T, Nakamura M, Arai H, Ishii M &
Igarashi Y (2007) A novel ferredoxin-dependent gluta-
mate synthase from the hydrogen-oxidizing chemoauto-
trophic bacterium Hydrogenobacter thermophilus TK-6.
J Bacteriol 189, 2805–2812.
48 Schneider G & Lindqvist Y (1998) Crystallography and
mutagenesis of transketolase: mechanistic implications
for enzymatic thiamin catalysis. Biochim Biophys Acta
1385, 387–398.
49 Furdui C & Ragsdale SW (2002) The roles of coenzyme
A in the pyruvate: ferredoxin oxidoreductase reaction
mechanism: rate enhancement of electron transfer from
a radical intermediate to an iron-sulfur cluster.
Biochemistry 41, 9921–9937.
50 Yoon K-S, Ishii M, Kodama T & Igarashi Y (1997)
Carboxylation reactions of pyruvate: ferredoxin oxido-
reductase and 2-oxoglutarate: ferredoxin oxidoreductase
from Hydrogenobacter thermophilus TK-6. Biosci
Biotechnol Biochem 61, 510–513.
51 Yoon K-S, Ishii M, Kodama T & Igarashi Y (1997)
Purification and characterization of pyruvate: ferredoxin
oxidoreductase from Hydrogenobacter thermophilus
TK-6. Arch Microbiol 167, 275–279.
52 Yamamoto M, Arai H, Ishii M & Igarashi Y (2003) Char-
acterization of two different 2-oxoglutarate: ferredoxin
oxidoreductases from Hydrogenobacter thermophilus
TK-6. Biochem Biophys Res Commun 312, 1297–1302.
53 Koide S, Iwata S, Matsuzawa H & Ohta T (1991) Crys-
tallization of allosteric l-lactate dehydrogenase from

Thermus caldophilus and preliminary crystallographic
data. J Biochem 109, 6–7.
54 Chen J-S & Blanchard DK (1979) A simple hydroge-
nase-linked assay for ferredoxin and flavodoxin. Anal
Biochem 93, 216–222.
55 Moreno SNJ, Mason RP, Muniz RPA, Cruz FS &
Docampo R (1983) Generation of free radicals from
metronidazole and other nitroimidazoles by Tritricho-
monas foetus. J Biol Chem 258, 4051–4054.
56 Ellman GL (1959) Tissue sulfhydryl groups. Arch
Biochem Biophys 82, 70–77.
57 Barletta G, Chung AC, Rios CB, Jordan F & Schlegel
JM (1990) Electrochemical oxidation of enamines
related to the key intermediate on thiamin diphosphate
dependent enzymatic pathways: evidence for one-elec-
tron oxidation via a thiazolium cation radical. JAm
Chem Soc 112, 8144–8149.
Pyruvate synthesis by pyruvate oxidoreductase T. Ikeda et al.
510 FEBS Journal 277 (2010) 501–510 ª 2009 The Authors Journal compilation ª 2009 FEBS